Effect of Environmental Conditions on Extracellular Protease Activity in Lignolytic Cultures of Phanerochaete chrysosporium

Two different types of extracellular protease activity were identified in the culture fluid of Phanerochaete chrysosporium wild-type BKM-F grown in submerged batch culture on N-limited media. The first activity, which appears to be inherent to the active growth phase, displayed a maximum on day 2 and decreased to a very low level on day 4. The second activity, which appeared at day 8 following the peak of ligninase activity, seems to be characteristic of late secondary metabolism and is stimulated by carbon starvation. Cultures started with half the amount of glucose of other cultures showed a remarkably earlier development of secondary activity. In contrast, the fed-batch addition of glucose started when ligninase activity was at a maximum (day 6) completely repressed secondary protease activity and enhanced ligninase production. The addition of exogenous veratryl alcohol increased the level of secondary protease activity, whereas the oxygen supply pattern significantly affected both the time course and the level of overall proteolytic activity. The addition of phenylmethylsulfonyl fluoride to growing cultures (0, 1, or 6 days) diminished overall protease activity, while it significantly enhanced ligninase activity. In all cases, the time courses of protease and ligninase activities were negatively correlated, indicating that protease activity promotes the decline of ligninase activity in batch culture.     

Study of spatiotemporal regulation of kinase signaling using genetically encodable molecular tools

Precise spatiotemporal organization and regulation of signal transduction networks are essential for cellular response to internal and external cues. To understand how this biochemical activity architecture impacts cellular function, many genetically encodable tools which regulate kinase activity at a subcellular level have been developed. In this review, we highlight various types of genetically encodable molecular tools, including tools to regulate endogenous kinase activity and biorthogonal techniques to perturb kinase activity. Finally, we emphasize the use of these tools alongside biosensors for kinase activity to measure and perturb kinase activity in real time for a better understanding of the cellular biochemical activity architecture.     

Phospholipase of rat intestine: mode of action]

Phospholipase activities of rat intestinal mucosa homogenate have been determined from lysophosphatidylcholines [14C] and phosphatidylcholines [-3H-14C]. In the presence of phosphatidylcholines, at pH 6.5, the homogenate has a phospholipase B activity. At pH 8.5, a phospholipase A2 activity was shown. In the presence of lysophospatidylcholines, at pH 6.5, we notice a lysophospholipase A1 activity. A kinetic study of the reactions allows us to separate the activity B into a phospholipase A2 activity and a lysophospholipase A1 activity. Thus, it appears that the total phospholipase activity of rat intestinal mucosa would results from a phospholipase A2 activity and a lysophospholipase A1 activity.     

The effect of progesterone on the activity-wheel running of ovariectomized rats

Progesterone treatment failed to significantly depress activity-wheel activity elicited by food deprivation in either ovariectomized or ovariectomized estrogen-treated rats. These results indicate: (1) that the lack of effect of progesterone on the activity of ovariectomized rats is not due only to their already low level of activity; and (2) that the depressant effect of progesterone on activity in the estrogen-treated ovariectomized rat is, to a large extent, a specific inhibition of estrogen facilitation of activity, rather than a nonspecific activity depressant effect.     

Wheel-Running Activity Patterns of Five Species of Desert Rodents

In contrast to the extensive laboratory data on activity patterns in rodent species inhabiting temperate zones, much less is known about the activity patterns of desert rodents. In order to address this issue, we measured wheel-running activity patterns in males and females of five species of wild-trapped desert rodents (Dipodillus dasyurus, Gerbillus andersoni, Gerbillus pyramidum, Meriones shawi, and Acomys cahirinus) in long 'summer-like' and short, 'winter-like' day lengths. The specific goals of the present study were to characterize activity patterns in several desert rodent species in the laboratory and to determine if activity patterns are expressed in a seasonal or sexually dimorphic manner. Specifically, wheel-running was measured for 11 weeks in long days followed by 11 weeks in short days to test for photoperiodic entrainment as well as responsiveness to changes in the light-dark cycle. All animals exhibited rhythmic patterns of wheel-running with consistent onsets and offsets that had well-defined relations with the light-dark cycle. All individuals of G. andersoni showed nocturnal activity patterns. Most individuals of G. pyramidum had nocturnal activity patterns, but some individuals showed a short bout of activity at the beginning of the light period. Most individuals of D. dasyurus and M. shawi showed bimodal (i.e., nocturnal and diurnal) activity patterns, although some showed markedly nocturnal activity patterns. There was no sexual dimorphism in wheel running activity rhythms in any of the species examined. As expected, decreases in day length resulted in an overall increase in the duration of activity in all species. Collectively, these data provide an initial characterization of activity patterns within desert rodents in a controlled laboratory setting.     

玉米感染禾谷镰刀菌后PAL、POD活性和同工酶谱的变化

为了明确苯丙氨酸解氨酶活和过氧化物酶活与玉米对镰刀菌茎腐病的抗性关系,探讨玉米对镰刀菌茎腐病的抗病机制及其在玉米抗病性鉴定中的利用,用禾谷镰刀菌孢子悬浮液对抗病品种陕单931和感病品种西农11号在抽雄初期进行接种,并于接种后测定茎秆髓部组织内的PAL,POD活性变化以及POD同工酶谱的变化。结果表明,玉米植株受镰刀菌侵染后,抗病品种的PAL酶活上升快,活性强,且形成两个活性高峰,高活性时间持续长;感病品种PAL酶活上升慢,活性相对较弱,且只形成一个峰,高活性持续时间短。抗病品种POD酶活峰值高,感病品种峰值低;抗病品种高酶活持续的时间长,感病品种高酶活持续的短。POD同工酶谱研究表明,抗、感品种POD同工酶带都有增多。抗病品种增5条,感病品种新增2条。PAL活性变化、POD活性变化及同工酶谱酶活变化与其对茎腐病的抗性有密切的关系,可作为抗病育种的生理生化指标。     

Effects of nitric oxide on growth of maize seedling leaves in the presence or absence of ultraviolet-B radiation

The leaves of maize seedlings were used to measure leaf biomass including leaf length, width and weight, and to examine the relationship between nitric oxide (NO) synthase activity in microsomes and cytosol to the exo- and endo-beta-glucanase activity during growth. It was found that ultraviolet-B radiation (UV-B radiation) strongly induced nitric oxide synthase (NOS) activity but caused both a decrease of leaf biomass and exo- or endo-beta-glucanase activity. In contrast, the NOS inhibitor and NO donor largely decreased the activity of NOS in non-irradiated seedlings. The inhibitor also reduced exo- and endo-beta-glucanase activity and leaf biomass while the donor increased the enzyme activity and leaf biomass under normal conditions. Alternatively, under ultraviolet-B, the additional inhibitor of NOS and NO donor appeared to compromise the effects of ultraviolet-B on glucanase activity and leaf biomass, making the relationship between NOS activity and glucanase activity negatively correlated. This suggests that the changes of NOS activity showed a positive correlation to glucanase activity and leaf biomass in the absence of ultraviolet-B, but a negative correlation to ultraviolet-B irradiation and NO donor treatment alone. It is assumed that exo- and endogenous NO is responsible for the up-regulation of regular growth and development without ultraviolet-B. Under UV-B radiation, however, it might function as a signaling molecule of ultraviolet-B inhibiting leaf growth of maize seedlings to carry out stress-signaling transduction.     

Daily activity profiles over the lifespan of female medflies as biomarkers of aging and longevity

The relationship between the early-age activity of Mediterranean fruit flies (medflies) or other fruit flies and their lifespan has not been much studied, in contrast to the connections between lifespan and diet, sexual signaling, and reproduction. The objective of this study is to assess intra-day and day-to-day activity profiles of female Mediterranean fruit flies and their role as biomarker of longevity as well as to explore the relationships between these activity profiles, diet, and age-at-death throughout the lifespan. We use advanced statistical methods from functional data analysis (FDA). Three distinct patterns of activity variations in early-age activity profiles can be distinguished. A low-caloric diet is associated with a delayed activity peak, while a high-caloric diet is linked with an earlier activity peak. We find that age-at-death of individual medflies is connected to their activity profiles in early life. An increased risk of mortality is associated with increased activity in early age, as well as with a higher contrast between daytime and nighttime activity. Conversely, medflies are more likely to have a longer lifespan when they are fed a medium-caloric diet and when their daily activity is more evenly distributed across the early-age span and between daytime and nighttime. The before-death activity profile of medflies displays two characteristic before-death patterns, where one pattern is characterized by slowly declining daily activity and the other by a sudden decline in activity that is followed by death.     

Neuroregulation of nonexercise activity thermogenesis and obesity resistance

High levels of spontaneous physical activity in lean people and the nonexercise activity thermogenesis (NEAT) derived from that activity appear to protect lean people from obesity during caloric challenge, while obesity in humans is characterized by dramatically reduced spontaneous physical activity. We have similarly demonstrated that obesity-resistant rats have significantly greater spontaneous physical activity than obesity-prone rats, and that spontaneous physical activity predicts body weight gain. Although the energetic cost of activity varies between types of activity and may be regulated, individual level of spontaneous physical activity is important in determining propensity for obesity. We review the current status of knowledge about the brain mechanisms involved in controlling the level of spontaneous physical activity and the NEAT so generated. Focus is on potential neural mediators of spontaneous physical activity and NEAT, including orexin A (also known as hypocretin 1), agouti-related protein, ghrelin, and neuromedin U, in addition to brief mention of neuropeptide Y, corticotrophin releasing hormone, cholecystokinin, estrogen, leptin, and dopamine effects on spontaneous physical activity. We further review evidence that strain differences in orexin stimulation pathways for spontaneous physical activity and NEAT appear to track with the body weight phenotype, thus providing a potential mechanistic explanation for reduced activity and weight gain.     

生物絮凝剂絮凝活性评价方法的探讨

对生物絮凝剂絮凝活性的评价方法进行了探讨。研究发现,在固定絮凝剂添加量的情况下仅仅比较絮凝率大小,从而对絮凝活性进行评价的方法是不科学的。絮凝剂絮凝活性应以絮凝率和絮凝剂最适添加量两个指标为其活性评价参数,在有较高絮凝率的情况下,絮凝剂添加量越少则絮凝活性越高。     

Enhancement of cytotoxicity of active macrophages by mycoplasma: role of mycoplasma-associated induction of tumor necrosis factor-alpha (TNF-alpha) in macrophages

Tumor necrosis factor-alpha (TNF-alpha)-inducing activity of several mycoplasmas including Mycoplasma pneumoniae, a causative agent in human respiratory infectious diseases, was investigated. Purified peritoneal macrophages from BALB/c mice markedly enhanced their cytotoxic activity to Meth A cells, when cultured with either viable or non-viable mycoplasmas. The supernatants of the macrophage culture with mycoplasmas, M. pneumoniae and Acholeplasma laidlawii, showed the potent cytotoxic activity to TNF-alpha-sensitive L cells but not to TNF-alpha-insensitive L cells. Addition of anti-TNF-alpha antiserum inhibited completely the cytotoxic activity of these supernatants, indicating that a major part of the cytotoxic activity might be due to TNF-alpha. Various other mycoplasmas, either glucose- or arginine-utilizing species, as far as tested showed also the potent activity to produce TNF-alpha. These results strongly suggest the possibility that mycoplasmas possess the activity of TNF-alpha induction which might be responsible for a part of enhancement of cytotoxic activity of macrophages and resistance to infection with mycoplasmas in vivo.     

Characterization of the cellulolytic activity of a Bacillus isolate.

A group I Bacillus strain, DLG, was isolated and characterized as being most closely related to Bacillus subtilis. When grown on any of a variety of sugars, the culture supernatant of this isolate was found to possess cellulolytic activity, as demonstrated by degradation of trinitrophenyl-carboxymethyl cellulose. Growth in medium containing cellobiose or glucose resulted in the greatest production of cellulolytic activity. The cellulolytic activity was not produced until the stationary phase of growth, and the addition of glucose or cellobiose to a culture in this phase had no apparent effect on enzyme production. Fractionation of the culture supernatant showed that the molecular weight of the enzymatic activity was less than 100,000. Maximum cellulolytic activity in assays was observed at pH 4.8 and at 58C, although maximum thermal stability of the activity. Kinetic experiments suggested that more than one enzyme was acting upon trinitrophenyl-carboxymethyl cellulose. Exocellular protein produced by this Bacillus isolate showed roughly one-fifth the cellulolytic activity displayed by Trichoderma reesei C30 on noncrystalline, cellulosic substrates. In contrast to T. reesei cellulase, the Bacillus enzymatic activity showed no ability to degrade crystalline forms of cellulose, nor was cellobiase activity detectable.     

Transport activity of the sodium bicarbonate cotransporter NBCe1 is enhanced by different isoforms of carbonic anhydrase

Transport metabolons have been discussed between carbonic anhydrase II (CAII) and several membrane transporters. We have now studied different CA isoforms, expressed in Xenopus oocytes alone and together with the electrogenic sodium bicarbonate cotransporter 1 (NBCe1), to determine their catalytic activity and their ability to enhance NBCe1 transport activity. pH measurements in intact oocytes indicated similar activity of CAI, CAII and CAIII, while in vitro CAIII had no measurable activity and CAI only 30% of the activity of CAII. All three CA isoforms increased transport activity of NBCe1, as measured by the transport current and the rate of intracellular sodium rise in oocytes. Two CAII mutants, altered in their intramolecular proton pathway, CAII-H64A and CAII-Y7F, showed significant catalytic activity and also enhanced NBCe1 transport activity. The effect of CAI, CAII, and CAII mutants on NBCe1 activity could be reversed by blocking CA activity with ethoxyzolamide (EZA, 10 μM), while the effect of the less EZA-sensitive CAIII was not reversed. Our results indicate that different CA isoforms and mutants, even if they show little enzymatic activity in vitro, may display significant catalytic activity in intact cells, and that the ability of CA to enhance NBCe1 transport appears to depend primarily on its catalytic activity.     

A kinetic description of antifreeze glycoprotein activity

The antifreeze glycoproteins (AFGP) of polar fish have the ability to depress the freezing temperature of water approximately 500 times the amount expected based on the number of AFGP molecules in solution; yet AFGP solutions have a purely colligative melting point depression. The difference of solution melting and freezing temperatures is the antifreeze activity of AFGP. One characteristic of AFGP activity that requires further examination is the effect of concentration on antifreeze activity, especially whether the activity saturates at high concentrations or the measured activity increases ad infinitum. This study first surveys the activity of the various antifreeze components from both Pagothenia borchgrevinki and the Arg-containing antifreeze glycoprotein from Eleginus gracilis (EgAF). It was found that all AFGP components examined have a plateau in activity at high concentration, but the actual value of the plateau activity differs between the different length AFGP components and between AFGP and EgAF. While the low molecular weight components of both AFGP and EgAF lose activity at deep supercooling, at high concentration activity is restored. The activity data is then shown to fit a reversible kinetic model of AFGP activity, and the coefficients obtained are used to compare the activity differences between AFGP components and between AFGP and EgAF. The model is also shown to describe the activity of the antifreeze protein of the fish Pseudopleuronectes americanus and the thermal hysteresis protein of the insect, Tenebrio molitor.     

抗菌肽改良设计及抗炎作用的研究进展

抗菌肽因其具有广谱抗菌活性、不容易引起抵抗性,被认为是先天免疫系统对抗微生物感染的多功能工具。然而,天然抗菌肽存在抗菌活性低、稳定性低、溶血性高等问题,使其较难应用于临床,所以研究人员对抗菌肽进行改良设计以期获得更高抗菌活性、更低溶血活性的新型抗菌肽。另外,天然抗菌肽作为一类免疫效应因子而被发现,其表现出的抑菌、免疫调节、内毒素中和等作用,使得研究人员对抗菌肽在抗炎作用的研究表现出极大的兴趣。就抗菌肽的药物设计方法及抗炎作用机制进行综述。     

Seasonal activity and spatial distribution of host-seeking adults of the tick Dermacentor variabilis

The seasonal temporal and spatial distribution and emergence of overwintering and summer cohorts of host-seeking American dog tick, Dermacentor variabilis (Say) (Acari: Ixodidae), adults were examined for 5 years in the Bluegrass region of Kentucky. Weekly drag cloth sampling was conducted from early March until late September 1992-1995 and early March until the end of May 1996. Overwintering adult cohorts became active in early to mid-April followed by bimodal host-seeking activity each year, with the duration and intensity of the first activity period being related to the previous year's summer cohort activity. The first activity peak extended from mid-April through late May and the second activity peak occurred in July. All host-seeking activity ceased by early August, except in 1995, when activity ended on 22 August. Laboratory survival of adults, collected weekly during 1993-1996, was used to estimate the emergence of summer adult cohorts. Summer adult cohorts became active in late May or early June and overlapped with overwintering adult host-seeking activity. The termination of host-seeking activity in overwintering adults could not be determined using the methods employed in this study and is most likely to have continued for the entire season. Host-seeking adults displayed contagious distribution patterns along trails bordering ecotone habitats, as determined by fitting a negative binomial frequency distribution to weekly sampling data. These patterns did not vary from year to year. In addition, the location of D. variabilis activity did not change from year to year, which suggests that microhabitats within apparently homogeneous environments enhance adult survival and lead to 'hot spots' of tick activity despite the ability of vertebrate hosts to disperse D. variabilis populations more widely. These data better define our understanding of D. variabilis adult seasonal and spatial host-seeking activity, estimate for the first time the intensity of summer cohort host-seeking activity, and relate summer cohort activity with spring activity the following year.     

高温胁迫下两种藓类植物过氧化物酶活性的变化

在不同的高温胁迫条件下 ,对湿地匍灯藓 (Plagiomnium acutum)和大羽藓 (Thuidium cymbifolium)过氧化物酶 (POD)活性及其与处理时间和处理温度的关系进行了初步研究。结果表明 ,在一定的温度范围内 ,随着温度的升高 ,POD活性增加 ,二者成线形关系。在一定温度条件下 ,一般随着处理时间的延长 ,POD活性增加。但是当超过一定的温度 (4 5～ 5 0°C)以及一定的处理时间 (4～ 6h) ,POD活性有所下降。结果还表明 ,湿地匍灯藓的POD活性显著高于大羽藓。而且在高温胁迫下 ,湿地匍灯藓 POD活性变化比大羽藓活跃 ,其变化幅度也比大羽藓大。     

Stimulation of dehydrogenase synthesis by cadmium in a cadmium-resistant strain of Saccharomyces cerevisiae.

The activity of dehydrogenase in Saccharomyces cerevisiae was estimated by reduction of 2,3,5-triphenyltetrazolium chloride. By the adaptation of yeast to cadmium, the high activity of dehydrogenase was observed. Furthermore, the activity of dehydrogenase in Cd-resistant cells was increased by growing in medium containing CdSO4. However, the activity of dehydrogenase was inhibited by the addition of CdSO4 to the reaction mixture. The activity of dehydrogenase in Cd-sensitive cells was increased slightly by incubation with low concentrations of CdSO4. High activity of dehydrogenase in Cd-resistant cells was completely negated by the addition of cycloheximide to the incubation medium. The increase of dehydrogenase activity is due partly to de novo synthesis of protein.     

Clearing-factor lipase in adipose tissue. Distinction of different states of the enzyme and the possible role of the fat cell in the maintenance of tissue activity

1. Incubation of intact epididymal adipose tissue from fed rats at 37 degrees in an albumin solution at pH7.4 in vitro results in rapid loss of clearing-factor lipase activity until a low activity, stable to prolonged incubation, is attained. The clearing-factor lipase activity of intact tissue from starved rats, which is initially much less than that of tissue from fed rats, is mainly stable to incubation at 37 degrees . 2. Much of the clearing-factor lipase activity of intact epididymal adipose tissue from fed rats is inactivated by collagenase. The enzyme activity of intact tissue from starved rats is not inactivated by collagenase. 3. The clearing-factor lipase activity of fat cells isolated from the epididymal adipose tissue of fed rats is stable to prolonged incubation at 37 degrees . It represents only a small proportion of the total activity of the intact tissue. In starved rats, the isolated fat cells contain a much higher proportion of the activity of the intact tissue. Their activity is also stable at 37 degrees . 4. Incubation of isolated fat cells in a serum-based medium leads to a progressive rise in clearing-factor lipase activity. Actinomycin increases the extent of this rise in activity. No rise in clearing-factor lipase activity occurs when stromal-vascular cells isolated from epididymal adipose tissue are incubated in the medium. 5. The findings indicate that less than 20% of the activity of intact adipose tissue from fed rats is retained when fat cells are isolated from the tissue by collagenase treatment. The activity that is lost could be that which normally functions in the uptake of triglyceride fatty acids by the tissue.     

Ontogeny of cyclic AMP-dependent protein phosphokinase during hepatic development of the rat.

The ontogeny of protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) and cyclic AMP-binding activity in subcellular fractions of liver was examined during prenatal and postnatal development of the male rat. 1. Protein kinase activity and cyclic AMP-binding activity were found in the nuclear, microsomal, lysosomal-mitochondrial, and soluble liver fractions. 2. The protein kinase activity of the soluble (105 000 X g supernatant) fraction measured with histone F1 as substrate was stimulated by cyclic AMP. Cyclic AMP did not stimulate the protein kinase activity of the particulate fractions. 3. The protein kinase activity of all subcellular fractions increased rapidly from the activity observed in prenatal liver (3-4 days before birth) to reach maximal activity in 2-day-old rats. Thereafter, the protein kinase activity declined more slowly and regained the prenatal levels at 10 days after birth. 4. Considerable latent protein kinase activity was associated with liver microsomal fractions which could be activated by treatment of microsomes with Triton X-100. The latent microsomal protein kinase activity was highest in prenatal liver, at the time of birth, and 2 days after birth. During the subsequent postnatal development the latent microsomal protein kinase activity gradually declined to insignificantly low levels. 5. During the developmental period examined (4 days before birth to age 60-90 days) marked alterations of the cyclic AMP-binding activity were determined in all subcellular fractions of rat liver. In general, cytosol, microsomal, and lysosomal-mitochondrial cyclic AMP-binding activity was highest in 10-11 day-old rats. Nuclear cyclic AMP-binding activity was highest 3-4 days before birth and declined at birth and during the postnatal period. There was no correlation between the developmental alteration of cyclic AMP-binding activity and cyclic AMP dependency of the protein kinase activity in any of the subcellular fractions. This suggests that the measured cyclic AMP-binding activity does not reflect developmental alterations of the cyclic AMP-binding regulatory subunit of cyclic AMP-dependent protein kinase.