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The use of reproductive technology to service a preference for male offspring has created an artificial gender imbalance, notably in Asian countries. The social effects of this large surplus of young men are not yet clear, but concerted action might be necessary to address the problemOne of the problems of sexual reproduction, especially in predominantly monogamous species that pair ‘for life'', is to ensure a balance between the birth rate of males and females. In humans, this balance has been remarkably even, but the past few decades have seen a substantial shift towards men, notably in some Asian countries. The reason, however, is not biological; there has simply been a cultural preference for sons in the affected societies, which together with recent availability of prenatal sex-selection technologies has led to widespread female feticide. The result has been a huge excess of males in several countries. Whilst it is not yet fully clear how a surplus of millions of men will affect these societies—perhaps even leading to civil unrest—some countries have already taken steps to alleviate the problem by addressing the underlying cultural factors. However, the problem is about to come to a crisis point, as a large surplus of men reach reproductive age. It will take many decades to reach a balanced representation of both sexes again.The sex ratio at birth (SRB) is defined as the number of boys born to every 100 girls. It is remarkably consistent in human populations, with around 103–107 male babies for every 100 female ones. John Graunt first documented this slight excess of male births in 1710 for the population of London, and many studies have since confirmed his finding [1]. Higher mortality from disease, compounded by the male tendency towards risky behaviours and violence, means that the initial surplus of boys decreases to roughly equal number of males and females during the all-important reproductive years in most populations.Researchers have studied a large number of demographic and environmental factors that could affect the SRB, including family size, parental age and occupation, birth order, race, coital rate, hormonal treatments, environmental toxins, several diseases and, perhaps most intriguingly, war [2,3,4]. It is well documented that wars are associated with a small increase in the sex ratio. This phenomenon occurs both during the war and for a short period afterwards. The best examples of this were reported for the First and Second World Wars in both the USA and Europe, and for the Korean and Vietnam Wars in the USA [5,6]. However, these findings were not reproduced in the more recent Balkan Wars and the Iran–Iraq war [7]. There have been several biological explanations for these increases. It has been proposed, for example, that the stress of war adversely affects the viability of XY-bearing sperm. Alternatively, a higher frequency of intercourse after prolonged separation during times of war is thought to lead to conception earlier in the menstrual cycle, which has been shown to result in more males [4,8]. There have been evolutionary explanations, such as the loss of large numbers of men in war leading to an adaptive correction of the sex ratio [4,9]. Nonetheless, the real causes of the altered SRB during war remain elusive: all of the discussed biological and social factors have been shown to cause only marginal deviations from the normal sex ratio.Whilst war has only slightly shifted SRB towards more male babies and only for a limited time period, cultural factors, namely a strong preference for sons, has been causing large distortions of gender balance during the past decades. Son preference is most prevalent in a band of countries from East Asia through to South Asia and the Middle East to North Africa [9]. For centuries, sons have been regarded as more valuable, because males can earn higher wages especially in agrarian economies, they generally continue the family line, are recipients of inheritance and are responsible for their parents in illness and old age. By contrast, daughters often become members of the husband''s family after marriage, no longer having responsibility for their biological parents [10]. There are also location-specific reasons for son preference: in India, the expense of the dowry, and in South Korea and China, deep-rooted Confucian values and patriarchal family systems [11].… cultural factors, namely a strong preference for sons, has been causing large distortions of gender balance during the past decadesUntil recently, son preference was manifest post-natally through female infanticide, abandonment of newborn girls, poorer nutrition and neglect of health care, all causing higher female mortality [12]. Studies have shown that unequal access to health care is the most important factor in differential gender mortality [13,14], especially in countries where health care costs are borne by the family [15]. As early as 1990, the Indian economist Amaryta Sen estimated that differential female mortality had resulted in around 100 million missing females across the developing world with the overwhelming majority of these in China, India, Pakistan and Bangladesh [16].
Open in a separate windowAdapted from Zhu et al 2009 [18].South Koreans are inclined to use sex selection, even in their first pregnancy, as there is a traditional preference for the first-born to be a son. This tendency towards sex selection rises for third and fourth births as parents try to ensure they produce a son. In the peak years of the early 1990s, when the overall SRB was 114, the sex ratio for fourth births was 229 [17].… it is clear that large parts of China and India will have a 15–20% excess of young men during the next 20 yearsSince prenatal sex determination only became accessible during the mid-1980s, and even later still in rural areas, the large cohorts of surplus young men have only now started to reach reproductive age. The consequences of this male surplus in the all-important reproductive age group are therefore still speculative and the existing literature about the consequences of distorted sex ratios is predominantly theoretical with few hypothesis-testing investigations [24,25]. In addition, most research focuses on countries in which sex ratios differ only marginally from biological norms [26]; few researchers have systematically examined the massive sex ratio distortion in China and India. However, it is clear that large parts of China and India will have a 15–20% excess of young men during the next 20 years. These men will be unable to get married, in societies in which marriage is regarded as virtually universal, and where social status depends, in large part, on being married and having children. An additional problem is the fact that most of these men will come from the lowest echelons of society: a shortage of women in the marriage market enables women to ‘marry-up'', inevitably leaving the least desirable men with no marriage prospects [27]. As a result, most of these unmarriageable men are poor, uneducated peasants.One hypothesis assumes that not being able to meet the traditional expectations of marriage and childbearing will cause low self-esteem and increased susceptibility to psychological difficulties, including suicidal tendencies [28]. A recent study using in-depth interviews with older unmarried men in Guizhou province, in south west China, found that most of these men have low self-esteem, with many describing themselves as depressed, unhappy and hopeless [29].The combination of psychological vulnerability and sexual frustration might lead to aggression and violence. There is empirical support for this prediction: gender is a well-established individual-level correlate of crime, especially violent crime [30,31]. A consistent finding across cultures is that most crime is perpetrated by young, single males, of low socioeconomic status [32]. A particularly intriguing study carried out in India in the early 1980s showed that the sex ratio at the state level correlated strongly with homicide rates, and the relationship persisted after controlling for confounders such as urbanization and poverty [33]. The authors had expected to find that the high sex ratio would lead to increased violence against women, but their conclusion was that high sex ratios are a cause of violence of all types in society.However, no other study has found similar results. The study mentioned above from rural Guizhou, for example, could find no evidence that unmarried men were especially prone to violence and aggression. Rather, the men were characterized as shy and withdrawn, rather than aggressive [29]. In addition, reports of crime and disorder are not higher in areas with a known excess of young, single men. This might be because there is not yet a large enough crucial mass of unmarriageable men to have an impact, or assumptions about male aggression do not apply in this context.A consistent finding across cultures is that most crime is perpetrated by young, single males, of low socioeconomic statusIn China and parts of India, the sheer numbers of single men have raised other concerns. Because these men might lack a stake in the existing social order, it is feared that they will bind together in an outcast culture, turning to antisocial behaviour and organized crime [34], thereby threatening societal stability and security [35]. Some theorize that it could lead to intergroup conflict and civil war could erupt [32]; other authors go further, predicting that such men will be attracted to military-type organizations, potentially triggering large-scale domestic and international conflicts [36]. However, there is no evidence yet to support these scenarios. Crime rates are relatively low in India and China compared with other countries [37]. Such outcomes are probably multifactorial in their causes, and therefore the role of sex imbalance is difficult to determine.An excess of men, however, should be beneficial for women, especially in those Asian societies in which women have traditionally low social status. In fact, much of the literature on sex ratios has focused on women''s status and role in society, and on mating strategies; but again the literature has come from scenarios in which the sex ratio is only marginally distorted [38,39]. It is intuitive to see that women are a valuable commodity when sex ratios are high [40,41]. Because women generally prefer long-term monogamous relationships [42], it is predicted that monogamy will be more prevalent in high sex ratio societies, with less premarital and extramarital sex [43], lower divorce rates [38,24] and less illegitimacy [31]. In India and China, tradition militates against some of these eventualities; for example, divorce and illegitimacy are rare in both countries, owing to the traditional values of these societies. But other effects can be explored. If women are more highly valued, it is predicted that they will have higher self-esteem, resulting in lower rates of depression and suicide [24]. In China, where suicide rates in rural women have been among the highest in the world [28], women now show improved self-esteem and self-efficacy: 47% of university graduates are female and women account for 48% of the labour force [19].However, this increase in the value of women could also have paradoxically adverse effects on women, especially in rural societies. Benefits might accrue to men, such as fathers, husbands, traffickers and pimps, who control many female lives [35]. Increases in prostitution, kidnapping and trafficking of women in China have already been attributed to high sex ratios [44]. Hudson and Den Boer [36] cite the increase in kidnapping and trafficking of women, which has been reported from many parts of Asia, and the recent large increases in dowry prices in parts of India.Despite the negative and potentially damaging culturally driven use of prenatal sex selection, there might be some positive aspects of easy access to this technology. First, access to prenatal sex determination probably increases the proportion of wanted births, leading to less discrimination against girls and lower postnatal female mortality. India, South Korea and China have all reported reductions in differential mortality [45]. Second, it has been argued that an imbalance in the sex ratio could be a means to reduce population growth [46]. Third, the improved status of women should result in reduced son preference with fewer sex-selective abortions and an ultimate rebalancing of the sex ratio [4].Other consequences of an excess of men have been described, but the evidence for causation is limited. Much has been made of the impact on the sex industry. It is assumed that the sexual needs of large numbers of single men will lead to an expansion of the sex industry, including the more unacceptable practices of coercion and trafficking. During the past 20 years the sex industry has in fact expanded in both India and China [47,48], but the role that the high sex ratio has played is impossible to isolate. The marked rise in the number of sex workers in China, albeit from a low baseline, has been attributed more to a relaxation in sexual attitudes, increased inequality, and much greater mobility in the country, than an increase in the sex ratio. For example, the sex ratio is close to normal in border areas of Yunnan Province, where there is known to be the highest number of sex workers [49].Similarly, it is impossible to say whether gender imbalance is a contributory factor to the reported, largely anecdotal, increases in trafficking for the sex industry and for marriage. Most unmarried men in China and India are in the poorest echelons of society, and thus unable to buy a bride. In addition, trafficking is probably far more common in parts of Eastern Europe and Africa where the sex ratio is normal [50]. Several commentators have suggested that an excess of men might encourage an increase in homosexual behaviour [17]. This is clearly highly contentious, and begs questions about the aetiology of sexual orientation. However, if this leads to increased tolerance towards homosexuality in societies where homophobia is still highly prevalent, it is perhaps a positive consequence of the high sex ratio.There is clear concern at the governmental level about high sex ratios in the affected countries. In 2004, clearly risible with hindsight, China set a target to lower the SRB to normal levels by 2010 [51]. The Chinese government expressed concerns recently about the potential consequences of excess men for societal stability and security [52]. In the short term, little can be done to address the problem. There have been some extreme suggestions, for example recruiting men into the armed forces and posting them to remote areas [35], but such suggestions are clearly not feasible or realistic.However, much can be done to reduce sex selection, which would have clear benefits for the next generation. There are two obvious policy approaches: to outlaw sex selection, and to address the underlying problem of son preference. In China and India, laws forbidding infanticide and sex selection exist. It is therefore perplexing that sex-selective abortion is carried out, often quite openly, by medical personnel in clinics and hospitals that are often state-run and not in back-street establishments [20]. Enforcement of the law should therefore be straightforward—as the lessons from South Korea demonstrate. In the late 1980s, alarming rises in the SRB, because of easy access to sex-selective abortion, caused the government to act decisively. Eight physicians in Seoul, who had performed sex determination, had their licenses suspended in 1991 leading to a fall in the SRB from 117 to 113 in the following year. Following this success, laws forbidding sex selection were enforced across the country. This was combined with a widespread and influential public awareness campaign, warning of the dangers of distorted sex ratios, focusing especially on the shortage of brides. The results led to a gradual decline in the SRB from 116 in 1998 to 110 in 2009 [11].An excess of men […] should be beneficial for women, especially in those Asian societies in which women have traditionally low social statusThe lessons are clear. The fact that in China and India sex-selective abortion is still carried out with impunity—by licensed medical personnel and not even in backstreet establishments—makes the failure of the government to enforce the law all the more obvious. One of the problems is that although sex-selective abortion is illegal, abortion itself is readily available, especially in China, and it is often difficult to prove that an abortion has been carried out to select the sex of the child, as opposed to family planning reasons.To successfully address the underlying issue of son preference is, of course, hugely challenging, and requires a multi-faceted approach. Evidence from areas outside Asia strongly supports the idea that a higher status for women leads to less traditional gender attitudes and lower levels of son preference [52]. Laws in China and India have made important moves towards gender equality in terms of social and economic rights. These measures, together with socio-economic improvements and modernization, have improved the status of women and are gradually influencing traditional gender attitudes [44].The recognition that intense intervention would be necessary to change centuries-long traditions in China led to the Care for Girls campaign, instigated by the Chinese Population and Family Planning Commission in 2003. It is a comprehensive programme of measures, initially conducted in 24 counties in 24 provinces, which aims to improve perceptions of the value of girls and emphasizes the problems that young men face in finding brides. In addition, there has been provision of a pension for parents of daughters in rural areas. The results have been encouraging: in 2007, a survey showed that the campaign had improved women''s own perceived status, and that stated son preference had declined. In one of the participating counties in Shanxi Province, the SRB dropped from 135 in 2003 to 118 in 2006 [53].Surveys of sex preference are encouraging. In 2001, a Chinese national survey found that 37% of the female respondents—predominantly younger, urban women—claimed to have no gender preference for their offspring, 45% said the ideal family consisted of one boy and one girl, and the number expressing a preference for a girl was almost equal to those who wanted a boy [54]. A study conducted ten years later in three Chinese provinces showed that around two-thirds of adults of reproductive age classify themselves as gender indifferent; of the remainder, 20% said they would prefer to have a girl, with just 12% admitting to wanting a boy [52].Other policy measures that can influence social attitudes include equal social and economic rights for males and females—for example, in relation to rights of inheritance—and free basic health care to remove the financial burden of seeking health care for daughters. Neither of these has yet been implemented. However, another suggestion that special benefits be given to families with no sons to ensure protection in old age has been introduced in some Chinese provinces.Despite the grim outlook for the generation of males entering their reproductive years over the next two decades, the future is less bleak. The global SRB has probably already peaked. In South Korea, the sex ratio has already declined markedly and China and India are both reporting incipient declines: in China the SRB for 2010 was reported as 118 down from the peak of 121 in 2005, and, importantly,14 provinces with high sex ratios are beginning to show a downward trend [19]. India is now reported to have an SRB of around 109, down from a peak of around 111 in 2005 [21]. Whilst the combination of these incipient declines in SRB, and the changing attitudes towards the imperative to have sons, are encouraging, they will not start to filter through to the reproductive age group for another two decades. In China and India the highest sex ratio cohorts have yet to reach reproductive age, so the situation will get worse before it gets better. Normal sex ratios will not be seen for several decades.?
Open in a separate windowTherese HeskethOpen in a separate windowJiang Min Min 相似文献
Science & Society Series on Sex and Science
Sex is the greatest invention of all time: not only has sexual reproduction facilitated the evolution of higher life forms, it has had a profound influence on human history, culture and society. This series explores our attempts to understand the influence of sex in the natural world, and the biological, medical and cultural aspects of sexual reproduction, gender and sexual pleasure.To make matters worse, during the 1980s, diagnostic ultrasound technology became available in many Asian countries, and the opportunity to use the new technology for prenatal sex selection was soon exploited. Indeed, the highest SRBs are seen in countries with a combination of son preference, easy access to sex-selection technologies and abortion, and a small family culture. The latter is important because where larger families are the norm, couples will continue to have children until they have a boy. If the couple plan, or are legally restricted, as in China, to only one or two children, they will use sex selection to ensure the birth of a son [17]. This combination has resulted in serious and unprecedented sex ratio imbalances that are now affecting the reproductive age groups in several countries, most notably China, South Korea and parts of India.South Korea was the first country to report a very high SRB, because the widespread uptake of sex-selection technology preceded other Asian countries. The sex ratios started to rise in the mid-1980s in cities; ultrasound was already widely available even in rural areas by 1990 [17]. By 1992, the SRB was reported to be as high as 125 in some cities.South Korea was the first country to report a very high SRB, because the widespread uptake of sex-selection technology preceded other Asian countriesChina soon followed. Here, the situation was further complicated by the one-child policy introduced in 1979. This has undoubtedly contributed to the steady increase in the reported SRB from 106 in 1979 to 111 in 1990, 117 in 2001, 121 in 2005 and as high as 130 in some rural counties [18]. The latest figures for 2010 report an SRB of 118 [19] (National Bureau of Statistics of China 2011), the first drop in three decades, suggesting an incipient downturn. However, the number of excess males in the reproductive age group will continue to increase for at least another two decades. Because of China''s huge population, these ratios translate into massive numbers: in 2005, an estimated 1.1 million excess males were born across the country and the number of males under the age of 20 might exceed females by around 30 million [18].These overall figures conceal wide variations across the country (Fig 1): the SRB is higher than 130 in a strip of heavily populated provinces from Henan in the north to Hainan in the south, but close to normal in the large sparsely populated provinces of Xinjiang, Inner Mongolia and Tibet. Some are sceptical about these high SRB figures or have suggested that, under the constraints of the one-child policy, parents might fail to register a newborn girl, so that they might go on to have a boy [20]. However, recent evidence shows that such under-registration explains only a small proportion of missing females and that sex-selective abortion undoubtedly accounts for the overwhelming majority [18].Open in a separate windowFigure 1Sex ratio at birth for China''s provinces in 2005.There are marked regional differences in SRB in India. Because incomplete birth registrations make the SRB difficult to calculate accurately, the closely related ratio of boys to girls under the age of six is used, showing distinct regional differences across the country with much higher levels in the north and west. According to the most recent census in 2010, the SRB for the whole country was 109, a marginal increase on the previous census in 2001, which showed an SRB of 108. These national figures, however, hide wide differences from a low SRB of 98 in the state of Kerala to 119 in Haryana State. The highest SRBs at district level for the whole of India are in two districts of Haryana state, where the SRBs are both 129 [21]. The Indian figures contrast with the Chinese in two ways: nowhere in China is the sex ratio low, and in India the sex ratio is higher in rural than urban areas, whereas the reverse is true for China [22].A consistent pattern in all three countries is a clear trend across birth order, that is first, second and subsequent children, and the sex of the preceding child. This is driven by the persistence of the ‘at least one boy'' imperative in these cultures. Where high fertility is the norm, couples will continue to reproduce until they have a boy. Where couples aim to restrict their family size, they might be content if the first child is a girl, but will often use sex selection to ensure a boy in the second pregnancy. This was shown in a large Indian study: the SRB was 132 for second births with a preceding girl, and 139 for third births with two previous girls. By contrast, the sex ratios were normal when the first born was a boy [23].The sex ratio by birth order is particularly interesting in China (18].Table 1
Sex ratio at birth for China''s provinces in 2005.| Total | First order | Second order | Third order | |
|---|---|---|---|---|
| Total | 120 | 108 | 143 | 157 |
| Urban | 115 | 110 | 138 | 146 |
| Rural | 123 | 107 | 146 | 157 |
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One of the greatest challenges in microbial ecology remains to link the metabolic activity of individual cells to their taxonomic identity and localization within environmental samples. Here we combined mass-spectrometric imaging (MSI) through (matrix-assisted) laser desorption ionization time-of-flight MSI ([MA]LDI-TOF/MSI) with fluorescence in situ hybridization (FISH) to monitor antibiotic production in the defensive symbiosis between beewolf wasps and ‘Streptomyces philanthi'' bacteria. Our results reveal similar distributions of the different symbiont-produced antibiotics across the surface of beewolf cocoons, which colocalize with the producing cell populations. Whereas FISH achieves single-cell resolution, MSI is currently limited to a step size of 20–50 μm in the combined approach because of the destructive effects of high laser intensities that are associated with tighter laser beam focus at higher lateral resolution. However, on the basis of the applicability of (MA)LDI-MSI to a broad range of small molecules, its combination with FISH provides a powerful tool for studying microbial interactions in situ, and further modifications of this technique could allow for linking metabolic profiling to gene expression.Ecological analyses of microbial metabolites have thus far been hampered by the difficulty of localizing and quantifying these compounds in situ and tying their production to subpopulations or even single cells of individual microbial taxa. However, recent advances in mass-spectrometric imaging (MSI) techniques provide excellent tools to monitor metabolic processes and chemical communication in an ecological context (Svatoš, 2010, 2011). For example, matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) has successfully been employed to observe antagonistic interactions between Streptomyces and Bacillus strains in vitro (Yang et al., 2009). The analysis of microbial interactions in situ, however, requires the combination of metabolic profiling with taxonomic identification and localization of the involved microorganisms. Previous studies employing microautoradiography or high-resolution secondary ion mass spectrometry in combination with in situ hybridization have provided insights into the metabolism of individually identified bacterial cells in environmental samples (Orphan et al., 2001; Kindaichi et al., 2004; Behrens et al., 2008; Musat et al., 2008). However, the need for isotopic labeling limits the application of these techniques to a subset of biological questions.Here we combine MSI with fluorescence in situ hybridization (FISH) for simultaneous metabolite profiling and taxonomic identification of bacteria, using the defensive symbiosis between beewolf wasps and Streptomyces bacteria as a model. Beewolves of the genera Philanthus, Trachypus and Philanthinus (Hymenoptera, Crabronidae) cultivate ‘Streptomyces philanthi'' in specialized antennal gland reservoirs (Kaltenpoth et al., 2006; Goettler et al., 2007; Kaltenpoth et al., 2014) and secrete the bacteria into their subterranean brood cells before oviposition (Kaltenpoth et al., 2010a). Later, the larva incorporates the symbionts into the cocoon silk, where the streptomycetes produce a cocktail of at least nine different antibiotics (Kroiss et al., 2010) and thereby protect the larva against pathogenic fungi and bacteria during the long (up to 9 months) and vulnerable phase of hibernation (Kaltenpoth et al., 2005; Koehler et al., 2013). Previous studies using MSI revealed that the antibiotics abound on the outer surface of the cocoon, while they are virtually absent from the inner surface (Kroiss et al., 2010).We used (matrix-assisted) laser desorption ionization time-of-flight MSI ([MA]LDI-TOF/MSI) to visualize the abundance of two different antibiotics (piericidin A1 and B1) and subsequently localized the symbionts producing these compounds on beewolf cocoons using FISH. Pieces of beewolf cocoons were fixed to MALDI target plates without any pre-treatment using double-sided adhesive tape, with the outer cocoon surface facing upward. In order to allow for later alignment of ion-intensity maps and FISH images, the cocoon pieces were surrounded by thin paint markings (Edding751, 1–2 mm tip width, white), applied with the tip of a needle. This marker was chosen because it yielded characteristic signals in (MA)LDI-MS (measured at m/z 322.5±0.5) and also showed fluorescence at 640 nm, the excitation wavelength of the fluorescent dye used for FISH (that is, Cy5). MSI was carried out without any pretreatment of the samples (Hoelscher et al., 2009; Kroiss et al., 2010), or after application of 2,5-dihydroxybenzoic acid matrix by sublimation (Svatoš and Mock, 2013). A MALDI micro MX mass spectrometer (Waters, Milford, MA, USA) equipped with a nitrogen laser (337 nm) was used in the reflectron mode and positive polarity for data acquisition as previously reported (Kroiss et al., 2010). The step size in both x and y directions was set to 50 μm corresponding to 508 dots per inch resolution. Two-dimensional ion-intensity maps were reconstructed using the spectral data for the respective potassium adduct ions of piericidin A1 (PA1, m/z 454±0.5 [M+K]+) and piericidin B1 (PB1, m/z 468±0.5 [M+K]+) with the BioMAP software (Novartis Institutes for BioMedical Research, Basel, Switzerland). After (MA)LDI imaging, samples were subjected to FISH with the ‘S. philanthi''-specific probe SPT177-Cy5 (Kaltenpoth et al., 2005, 2006) as described previously (Kaltenpoth et al., 2010b). Fluorescence images were recorded on a Zeiss AxioImager Z.1 (Zeiss, Jena, Germany) using both the mosaic and z-stack options for obtaining high-resolution images with increased focusing depth. Overlays of (MA)LDI and FISH images were achieved in Adobe Photoshop CS5 Extended 12.0 by using the pen markings as a guide.(MA)LDI-MSI revealed a patchy distribution of antibiotics across the outer cocoon surface of European beewolves. The two measured antibiotic substances showed very similar distributions (Figures 1a–j), suggesting that both compounds—as well as possibly the other seven antibiotics produced by the symbionts on the beewolf cocoon that could not be measured here because of their low concentrations—are produced by individual bacterial cells or subpopulations of cells. This is supported by MSI with a high-resolution atmospheric pressure scanning microprobe (AP-SMALDI-MSI) of PA1 and PB1 produced by ‘S. philanthi'' on beewolf cocoons and in vitro, which confirmed the colocalization of both antibiotics (Figures 1k–n and Supplementary Figure S1, for experimental procedures see Supplementary Online Material). Thus, different symbiont subpopulations apparently do not specialize in the production of individual compounds, but instead produce a mixture of antibiotics.Open in a separate windowFigure 1(MA)LDI-FISH of antibiotics produced by symbiotic ‘Streptomyces philanthi'' bacteria on a beewolf cocoon (Philanthus triangulum) and in vitro. Ion-intensity maps of (a) the paint marker for alignment of LDI and FISH pictures (m/z 322.5); inset: image of the cocoon piece surrounded by white paint markings on the LDI target plate, (b) piericidin A1 (PA1, m/z 454.5 [M+K]+) and (c) piericidin B1 (PB1, m/z 468.5 [M+K]+). (d–f) The same maps, overlayed with a FISH micrograph of the cocoon piece. Symbiont cells were labeled with the fluorescent oligonucleotide probe SPT177-Cy5. (g–h) Magnifications of (e, f), respectively, with individual bacterial cells visible. (i–j) MALDI-FISH of (i) PA1 (m/z 454.5 [M+K]+) and (j) PB1 (m/z 468.5 [M+K]+) on another cocoon piece. (k–n) AP-SMALDI imaging of antibiotics produced by ‘S. philanthi'' in vitro. (k) Light microscopic image of an ‘S. philanthi'' colony, (l) PA1 (m/z 416.27 [M+H]+), (m) PB1 (m/z 430.25 [M+H]+), (n) overlay of PA1 (green) and PB1 (red).On the cocoon, FISH allowed for the visualization of individual symbiont cells, which were abundant across the entire cocoon surface and often occurred in highest densities along the outer cocoon threads (Supplementary Figure S2). The presence of the matrix had no influence on the efficiency of FISH after MSI (data not shown). The alignment of ion-intensity maps with FISH images revealed high concentrations of antibiotics around some subpopulations of cells, whereas other cell aggregations were surrounded by much lower amounts of antibiotics (Figures 1g–j), highlighting the possibility for cheating in the symbiosis. However, the current limitations in sensitivity and lateral resolution of MALDI-MSI do not permit the visualization of compounds on the single-cell level (~1 μm) and thereby may obscure fine-scale patterns of antibiotic production. This is supported by AP-SMALDI-MSI of beewolf cocoons at two different resolutions (step sizes 20 and 5 μm, Supplementary Figure S1): Whereas the high-resolution measurement revealed high concentrations of antibiotics along the outer cocoon threads, which agrees with the FISH experiments showing a similar pattern of symbiont cell densities (Supplementary Figure S2), this pattern was not as apparent at lower resolutions (Supplementary Figure S1). However, the high laser intensities required for a step size of 5 μm were destructive for the samples; therefore, subsequent FISH experiments could not be performed.The combination of (MA)LDI imaging and FISH provides a powerful tool for tying metabolite profiling to taxonomic identification in environmental samples. However, the laser intensity needs to be carefully adjusted for the desired application to achieve maximum sensitivity and resolution while at the same time conserving the structure of the sample. This problem can be circumvented by using desorption electrospray ionization (DESI) imaging, which we also successfully combined with FISH in preliminary experiments (data not shown). Still, the limitations of both (MA)LDI and DESI in lateral resolution and sensitivity currently prohibit single-cell resolution of metabolic profiling and restrict the technique to mapping the distribution of metabolites on the subpopulation level (Svatoš, 2011). Thus, the exploration of complex environmental samples is at present limited to microbial communities with distinct spatial structure. However, the major strength of MSI-FISH is its broad applicability to a wide range of small molecules as well as proteins (Svatoš, 2010). Therefore, (MA)LDI-FISH and DESI-FISH allow for addressing a multitude of questions in microbial ecology, ranging from interactions in mixed-species biofilms or cross-feeding associations to the chemical basis and dynamics of mutualistic and antagonistic encounters. As several signal enhancement techniques for FISH have been developed (for example, Schönhuber et al., 1997; Zwirglmaier, 2005), modifications of the approach described here could also be employed to tie metabolic profiling by (MA)LDI or DESI imaging to the presence (Moraru et al., 2010) or expression (Pernthaler and Amann, 2004) of particular genes of interest in microbial communities or eukaryotic tissues. Future studies should explore the possibility for using oligonucleotide labels and hybridization protocols that allow for simultaneous MSI of labeled cells and metabolites of interest, which would obviate the necessity for subsequent FISH and thereby circumvent the problems with high laser intensities. Alternatively, new MALDI matrices capable of dissipating the high ultraviolet-laser intensities and thus preventing DNA damage could be developed.
Open in a separate windowAbbreviations: DESI, desorption electrospray ionization; EL-FISH, element labeling fluorescence in situ hybridization; FISH, fluorescence in situ hybridization; HISH, halogen in situ hybridization; (MA)LDI, (matrix-assisted) laser desorption/ionization; SIMS, secondary ion mass spectrometry. 相似文献
Table 1
Comparison of established methods for linking localization and taxonomic identification of microbes to the production of particular metabolites of interest in environmental samples| Metabolite imaging method | Bacterial visualization method | Metabolite imaging | Reference | ||||
|---|---|---|---|---|---|---|---|
| Equipment costs | Need for labeling | Versatility (compounds) | Lateral resolution | Sensitivity | |||
| Microautoradiography | FISH | $ | Yes | Low | High | Medium | Kindaichi et al., 2004 |
| nanoSIMS | FISH/HISH/EL-FISH | $$$$ | Yes | Low | High | High | Orphan et al., 2001; Behrens et al., 2008; Musat et al., 2008; Li et al., 2008 |
| (MA)LDI | FISH | $$$ | No | Very high | Medium | Medium | This study |
| DESI | FISH | $$ | No | Very high | Low | Low | This study |
17.
Mesenchymal stem cells (MSC) are adult-derived multipotent stem cells that have been derived from almost every tissue. They are classically defined as spindle-shaped, plastic-adherent cells capable of adipogenic, chondrogenic, and osteogenic differentiation. This capacity for trilineage differentiation has been the foundation for research into the use of MSC to regenerate damaged tissues. Recent studies have shown that MSC interact with cells of the immune system and modulate their function. Although many of the details underlying the mechanisms by which MSC modulate the immune system have been defined for human and rodent (mouse and rat) MSC, much less is known about MSC from other veterinary species. This knowledge gap is particularly important because the clinical use of MSC in veterinary medicine is increasing and far exceeds the use of MSC in human medicine. It is crucial to determine how MSC modulate the immune system for each animal species as well as for MSC derived from any given tissue source. A comparative approach provides a unique translational opportunity to bring novel cell-based therapies to the veterinary market as well as enhance the utility of animal models for human disorders. The current review covers what is currently known about MSC and their immunomodulatory functions in veterinary species, excluding laboratory rodents.Abbreviations: AT, adipose tissue; BM, Bone marrow; CB, umbilical cord blood; CT, umbilical cord tissue; DC, dendritic cell; IDO, indoleamine 2;3-dioxygenase; MSC, mesenchymal stem cells; PGE2, prostaglandin E2; VEGF, vascular endothelial growth factorMesenchymal stem cells (MSC, alternatively known as mesenchymal stromal cells) were first reported in the literature in 1968.39 MSC are thought to be of pericyte origin (cells that line the vasculature)21,22 and typically are isolated from highly vascular tissues. In humans and mice, MSC have been isolated from fat, placental tissues (placenta, Wharton jelly, umbilical cord, umbilical cord blood), hair follicles, tendon, synovial membrane, periodontal ligament, and every major organ (brain, spleen, liver, kidney, lung, bone marrow, muscle, thymus, pancreas, skin).23,121 For most current clinical applications, MSC are isolated from adipose tissue (AT), bone marrow (BM), umbilical cord blood (CB), and umbilical cord tissue (CT; 11,87,99 Clinical trials in human medicine focus on the use of MSC both for their antiinflammatory properties (graft-versus-host disease, irritable bowel syndrome) and their ability to aid in tissue and bone regeneration in combination with growth factors and bone scaffolds (clinicaltrials.gov).131 For tissue regeneration, the abilities of MSC to differentiate and to secrete mediators and interact with cells of the immune system likely contribute to tissue healing (Figure 1). The current review will not address the specific use of MSC for orthopedic applications and tissue regeneration, although the topic is covered widely in current literature for both human and veterinary medicine.57,62,90
Open in a separate windowOpen in a separate windowFigure 1.The dual roles of MSC: differentiation and modulation of inflammation.Long-term studies in veterinary species have shown no adverse effects with the administration of MSC in a large number of animals.9,10,53 Smaller, controlled studies on veterinary species have shown few adverse effects, such as minor localized inflammation after MSC administration in vivo.7,15,17,45,86,92,98 Private companies, educational institutions, and private veterinary clinics (including Tufts University, Cummins School of Veterinary Medicine, University of California Davis School of Veterinary Medicine, VetStem, Celavet, Alamo Pintado Equine Medical Center, and Rood and Riddle Equine Hospital) offer MSC as a clinical treatment for veterinary species. Clinical uses include tendon and cartilage injuries, tendonitis, and osteoarthritis and, to a lesser extent, bone regeneration, spinal cord injuries, and liver disease in both large and small animals.38,41,113 Even with this broad clinical use, there have been no reports of severe adverse effects secondary to MSC administration in veterinary patients. 相似文献
Table 1.
Tissues from which MSC have been isolated| Tissue source (reference no.) | |||||
| Species | Fat | Bone marrow | Cord blood | Cord tissue | Other |
| Cat | 134 | 83 | 56 | ||
| Chicken | 63 | ||||
| Cow | 138 | 12 | 108 | ||
| Dog | 97 | 3, 59 | 78, 119 | 139 | Periodontal ligament65 |
| Goat | 66 | 96 | 4 | ||
| Horse | 26, 130 | 37, 40, 123 | 67 | 130 | Periodontal ligament and gingiva88 |
| Nonhuman primate | 28, 54 | 5 | |||
| Pig | 135 | 114 | 70 | 14, 20, 91 | |
| Rabbit | 128 | 80 | 32 | Fetal liver93 | |
| Sheep | 84 | 95 | 42, 55 | ||
18.
19.
Kuo-Liang Su Ya-Fan Liao Hui-Chih Hung Guang-Yaw Liu 《The Journal of biological chemistry》2009,284(39):26768-26777
Ornithine decarboxylase (ODC) is the first enzyme involved in polyamine biosynthesis, and it catalyzes the decarboxylation of ornithine to putrescine. ODC is a dimeric enzyme, whereas antizyme inhibitor (AZI), a positive regulator of ODC that is homologous to ODC, exists predominantly as a monomer and lacks decarboxylase activity. The goal of this paper was to identify the essential amino acid residues that determine the dimerization of AZI. The nonconserved amino acid residues in the putative dimer interface of AZI (Ser-277, Ser-331, Glu-332, and Asp-389) were substituted with the corresponding residues in the putative dimer interface of ODC (Arg-277, Tyr-331, Asp-332, and Tyr-389, respectively). Analytical ultracentrifugation analysis was used to determine the size distribution of these AZI mutants. The size-distribution analysis data suggest that residue 331 may play a major role in the dimerization of AZI. Mutating Ser-331 to Tyr in AZI (AZI-S331Y) caused a shift from a monomer configuration to a dimer. Furthermore, in comparison with the single mutant AZI-S331Y, the AZI-S331Y/D389Y double mutant displayed a further reduction in the monomer-dimer Kd, suggesting that residue 389 is also crucial for AZI dimerization. Analysis of the triple mutant AZI-S331Y/D389Y/S277R showed that it formed a stable dimer (Kd value = 1.3 μm). Finally, a quadruple mutant, S331Y/D389Y/S277R/E332D, behaved as a dimer with a Kd value of ∼0.1 μm, which is very close to that of the human ODC enzyme. The quadruple mutant, although forming a dimer, could still be disrupted by antizyme (AZ), further forming a heterodimer, and it could rescue the AZ-inhibited ODC activity, suggesting that the AZ-binding ability of the AZI dimer was retained.Polyamines (putrescine, spermidine, and spermine) have been shown to have both structural and regulatory roles in protein and nucleic acid biosynthesis and function (1–3). Ornithine decarboxylase (ODC,3 EC 4.1.1.17) is a central regulator of cellular polyamine synthesis (reviewed in Refs. 1, 4, 5). This enzyme catalyzes the pyridoxal 5-phosphate (PLP)-dependent decarboxylation of ornithine to putrescine, and it is the first and rate-limiting enzyme in polyamine biosynthesis (2, 3, 6, 7). ODC and polyamines play important roles in a number of biological functions, including embryonic development, cell cycle, proliferation, differentiation, and apoptosis (8–15). They also have been associated with human diseases and a variety of cancers (16–26). Because the regulation of ODC and polyamine content is critical to cell proliferation (11), as well as in the origin and progression of neoplastic diseases (23, 24), ODC has been identified as an oncogenic enzyme, and the inhibitors of ODC and the polyamine pathway are important targets for therapeutic intervention in many cancers (6, 11).ODC is ubiquitously found in organisms ranging from bacteria to humans. It contains 461 amino acid residues in each monomer and is a 106-kDa homodimer with molecular 2-fold symmetry (27, 28). Importantly, ODC activity requires the formation of a dimer (29–31). X-ray structures of the ODC enzyme reveal that this dimer contains two active sites, both of which are formed at the interface between the N-terminal domain of one monomer, which provides residues involved in PLP interactions, and the C-terminal domain of the other subunit, which provides the residues that interact with substrate (27, 32–41).ODC undergoes a unique ubiquitin-independent proteasomal degradation via a direct interaction with the regulatory protein antizyme (AZ). Binding of AZ promotes the dissociation of the ODC homodimers and targets ODC for degradation by the 26 S proteasome (42–46). Current models of antizyme function indicate that increased polyamine levels promote the fidelity of the AZ mRNA translational frameshift, leading to increased concentrations of AZ (47). The AZ monomer selectively binds to dimeric ODC, thereby inactivating ODC by forming inactive AZ-ODC heterodimers (44, 48–50). AZ acts as a regulator of polyamine metabolism that inhibits ODC activity and polyamine transport, thus restricting polyamine levels (4, 5, 51, 52). When antizymes are overexpressed, they inhibit ODC and promote ubiquitin-independent proteolytic degradation of ODC. Because elevated ODC activity is associated with most forms of human malignancies (1), it has been suggested that antizymes may function as tumor suppressors.In contrast to the extensive studies on the oncogene ODC, the endogenous antizyme inhibitor (AZI) is less well understood. AZI is homologous to the enzyme ODC. It is a 448-amino acid protein with a molecular mass of 50 kDa. However, despite the homology between these proteins, AZI does not possess any decarboxylase activity. It binds to antizyme more tightly than does ODC and releases ODC from the ODC-antizyme complex (53, 54). Both the AZI and AZ proteins display rapid ubiquitin-dependent turnover within a few minutes to 1 h in vivo (5). However, AZ binding actually stabilizes AZI by inhibiting its ubiquitination (55).AZI, which inactivates all members of the AZ family (53, 56), restores ODC activity (54), and prevents the proteolytic degradation of ODC, may play a role in tumor progression. It has been reported that down-regulation of AZI is associated with the inhibition of cell proliferation and reduced ODC activity, presumably through the modulation of AZ function (57). Moreover, overexpression of AZI has been shown to increase cell proliferation and promote cell transformation (58–60). Furthermore, AZI is capable of direct interaction with cyclin D1, preventing its degradation, and this effect is at least partially independent of AZ function (60, 61). These results demonstrate a role for AZI in the positive regulation of cell proliferation and tumorigenesis.It is now known that ODC exists as a dimer and that AZI may exist as a monomer physiologically (62). Fig. 1 shows the dimeric structures of ODC (Fig. 1A) and AZI (Fig. 1B). Although structural studies indicate that both ODC and AZI crystallize as dimers, the dimeric AZI structure has fewer interactions at the dimer interface, a smaller buried surface area, and a lack of symmetry of the interactions between residues from the two monomers, suggesting that the AZI dimer may be nonphysiological (62). In this study, we identify the critical amino acid residues governing the difference in dimer formation between ODC and AZI. Our preliminary studies using analytical ultracentrifugation indicated that ODC exists as a dimer, whereas AZI exists in a concentration-dependent monomer-dimer equilibrium. Multiple sequence alignments of ODC and AZI from various species have shown that residues 277, 331, 332, and 389 are not conserved between ODC and AZI (Open in a separate windowFIGURE 1.Crystal structure and the amino acid residues at the dimer interface of human ornithine decarboxylase (hODC) and mouse antizyme inhibitor (mAZI). A, homodimeric structure of human ODC with the cofactor PLP analog, LLP (Protein Data Bank code 1D7K). B, putative dimeric structure of mouse AZI (Protein Data Bank code 3BTN). The amino acid residues in the dimer interface are shown as a ball-and-stick model. The putative AZ-binding site is colored in cyan. This figure was generated using PyMOL (DeLano Scientific LLC, San Carlos, CA).
Open in a separate window 相似文献
TABLE 1
Amino acid residues at the dimer interface of human ODC and AZI| Human ODC | Residue | Human AZI |
|---|---|---|
| Nonconserved | ||
| Arg | 277 | Ser |
| Tyr | 331 | Ser |
| Asp | 332 | Glu |
| Tyr | 389 | Asp |
| Conserved | ||
| Asp | 134 | Asp |
| Lys | 169 | Lys |
| Lys | 294 | Lys |
| Tyr | 323 | Tyr |
| Asp | 364 | Asp |
| Gly | 387 | Gly |
| Phe | 397 | Phe |
20.
Rajesh Kasiviswanathan Matthew J. Longley Sherine S. L. Chan William C. Copeland 《The Journal of biological chemistry》2009,284(29):19501-19510
Forty-five different point mutations in POLG, the gene encoding the catalytic subunit of the human mitochondrial DNA polymerase (pol γ), cause the early onset mitochondrial DNA depletion disorder, Alpers syndrome. Sequence analysis of the C-terminal polymerase region of pol γ revealed a cluster of four Alpers mutations at highly conserved residues in the thumb subdomain (G848S, c.2542g→a; T851A, c.2551a→g; R852C, c.2554c→t; R853Q, c.2558g→a) and two Alpers mutations at less conserved positions in the adjacent palm subdomain (Q879H, c.2637g→t and T885S, c.2653a→t). Biochemical characterization of purified, recombinant forms of pol γ revealed that Alpers mutations in the thumb subdomain reduced polymerase activity more than 99% relative to the wild-type enzyme, whereas the palm subdomain mutations retained 50–70% wild-type polymerase activity. All six mutant enzymes retained physical and functional interaction with the pol γ accessory subunit (p55), and none of the six mutants exhibited defects in misinsertion fidelity in vitro. However, differential DNA binding by these mutants suggests a possible orientation of the DNA with respect to the polymerase during catalysis. To our knowledge this study represents the first structure-function analysis of the thumb subdomain in pol γ and examines the consequences of mitochondrial disease mutations in this region.As the only DNA polymerase found in animal cell mitochondria, DNA polymerase γ (pol γ)3 bears sole responsibility for DNA synthesis in all replication and repair transactions involving mitochondrial DNA (1, 2). Mammalian cell pol γ is a heterotrimeric complex composed of one catalytic subunit of 140 kDa (p140) and two 55-kDa accessory subunits (p55) that form a dimer (3). The catalytic subunit contains an N-terminal exonuclease domain connected by a linker region to a C-terminal polymerase domain. Whereas the exonuclease domain contains essential motifs I, II, and III for its activity, the polymerase domain comprising the thumb, palm, and finger subdomains contains motifs A, B, and C that are crucial for polymerase activity. The catalytic subunit is a family A DNA polymerase that includes bacterial pol I and T7 DNA polymerase and possesses DNA polymerase, 3′ → 5′ exonuclease, and 5′-deoxyribose phosphate lyase activities (for review, see Refs. 1 and 2). The 55-kDa accessory subunit (p55) confers processive DNA synthesis and tight binding of the pol γ complex to DNA (4, 5).Depletion of mtDNA as well as the accumulation of deletions and point mutations in mtDNA have been observed in several mitochondrial disorders (for review, see Ref. 6). mtDNA depletion syndromes are caused by defects in nuclear genes responsible for replication and maintenance of the mitochondrial genome (7). Mutation of POLG, the gene encoding the catalytic subunit of pol γ, is frequently involved in disorders linked to mutagenesis of mtDNA (8, 9). Presently, more than 150 point mutations in POLG are linked with a wide variety of mitochondrial diseases, including the autosomal dominant (ad) and recessive forms of progressive external ophthalmoplegia (PEO), Alpers syndrome, parkinsonism, ataxia-neuropathy syndromes, and male infertility (tools.niehs.nih.gov/polg) (9).Alpers syndrome, a hepatocerebral mtDNA depletion disorder, and myocerebrohepatopathy are rare heritable autosomal recessive diseases primarily affecting young children (10–12). These diseases generally manifest during the first few weeks to years of life, and symptoms gradually develop in a stepwise manner eventually leading to death. Alpers syndrome is characterized by refractory seizures, psychomotor regression, and hepatic failure (11, 12). Mutation of POLG was first linked to Alpers syndrome in 2004 (13), and to date 45 different point mutations in POLG (18 localized to the polymerase domain) are associated with Alpers syndrome (9, 14, 15). However, only two Alpers mutations (A467T and W748S, both in the linker region) have been biochemically characterized (16, 17).During the initial cloning and sequencing of the human, Drosophila, and chicken pol γ genes, we noted a highly conserved region N-terminal to motif A in the polymerase domain that was specific to pol γ (18). This region corresponds to part of the thumb subdomain that tracks DNA into the active site of both Escherichia coli pol I and T7 DNA polymerase (19–21). A high concentration of disease mutations, many associated with Alpers syndrome, is found in the thumb subdomain.Here we investigated six mitochondrial disease mutations clustered in the N-terminal portion of the polymerase domain of the enzyme (Fig. 1A). Four mutations (G848S, c.2542g→a; T851A, c.2551a→g; R852C, c.2554c→t; R853Q, c.2558g→a) reside in the thumb subdomain and two (Q879H, c.2637g→t and T885S, c.2653a→t) are located in the palm subdomain. These mutations are associated with Alpers, PEO, mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), ataxia-neuropathy syndrome, Leigh syndrome, and myocerebrohepatopathy (POLG mutation Disease Genetics Reference G848S Alpers syndrome In trans with A467T, Q497H, T251I-P587L, or W748S-E1143G in Alpers syndrome 15, 35, 43–50 Leigh syndrome In trans with R232H in Leigh syndrome 49 MELAS In trans with R627Q in MELAS 38 PEO with ataxia-neuropathy In trans with G746S and E1143G in PEO with ataxia 50 PEO In trans with T251I and P587L in PEO 51, 52 T851A Alpers syndrome In trans with R1047W 48, 53 In trans with H277C R852C Alpers syndrome In trans with A467T 14, 48, 50 In cis with G11D and in trans with W748S-E1143G or A467T Ataxia-neuropathy In trans with G11D-R627Q 15 R853Q Myocerebrohepatopathy In trans with T251I-P587L 15 Q879H Alpers syndrome with valproate-induced hepatic failure In cis with E1143G and in trans with A467T-T885S 35, 54 T885S Alpers syndrome with valproate-induced hepatic failure In cis with A467T and in trans with Q879H-E1143G 35, 54