Thyroid cryotherapy in an experimental rat model-topography of temperature during therapy and functional results

The aim of this study was to investigate post cryotherapy thyroid function status of normal rat thyroid tissue and to determine the topography of temperature of cryotreated tissues and of tissues adjacent to them. Nitrous oxide cryotherapy was performed in 40 male Wistar rats. They were divided into four groups of 10. In group I, the right thyroid lobe was subjected to cryotherapy and the left lobe was not frozen. In group II, both thyroid lobes were cryotreated. In group III, the right lobe was frozen and the left lobe was excised. In group IV, the thyroid was subjected to neither cryotherapy nor surgery. During cryotherapy, the temperature in various places of the thyroid and in the surrounding tissues was measured. Serum thyrotropin concentrations were determined before an experiment and 4 weeks after in all rats. The results of temperature measurements proved that it is possible to limit cryotherapy to certain areas of thyroid tissue and to spare the neighboring tissues, because they are not subjected to temperatures that are damaging. The effectiveness of cryotherapy was confirmed by functional effect. Cryotherapy changed function of thyroid tissue. There was a statistically significant difference between mean baseline and follow-up concentrations in rats of groups II and III. In both groups hypothyroidism occurred post cryotherapy.     

The Heterogeneity of Structural and Functional Photosynthetic Characteristics of Mesophyll Chloroplasts in Various Parts of Mature or Senescing Leaf Blade of Two Maize (Zea Mays L.) Genotypes

Differences in ultrastructural parameters of mesophyll cell (MC) chloroplasts, contents of photosynthetic pigments, and photochemical activities of isolated MC chloroplasts were studied in the basal, middle, and apical part of mature or senescing leaf blade of two maize genotypes. A distinct heterogeneity of leaf blade was observed both for structural and functional characteristics of chloroplasts. In both mature and senescing leaves the shape of MC chloroplasts changed from flat one in basal part of leaf to nearly spherical one in leaf apex. The volume density of granal thylakoids decreased from leaf base to apex in both types of leaves examined, while the amount of intergranal thylakoids increased in mature leaves but decreased in senescing leaves. The most striking heterogeneity was found for the quantity of plastoglobuli, which strongly increased with the increasing distance from leaf base. The differences in chloroplast ultrastructure were accompanied by differences in other photosynthetic characteristics. The Hill reaction activity and activity of photosystem 1 of isolated MC chloroplasts decreased from leaf base to apex in mature leaves. Apical part of senescing leaf blade was characterised by low contents of chlorophyll (Chl) a and Chl b, whereas in mature leaves, the content of Chls as well as the content of total carotenoids (Car) slightly increased from basal to apical leaf part. This was reflected also in the ratio Chl (a+b)/total Car; the ratio of Chl a/b did not significantly differ between individual parts of leaf blade. Both genotypes examined differed in the character of developmental gradient observed along whole length of leaf blade.     

A predominantly indigenous paternal heritage for the Austronesian-speaking peoples of insular Southeast Asia and Oceania

Modern humans reached Southeast Asia and Oceania in one of the first dispersals out of Africa. The resulting temporal overlap of modern and archaic humans-and the apparent morphological continuity between them-has led to claims of gene flow between Homo sapiens and H. erectus. Much more recently, an agricultural technology from mainland Asia spread into the region, possibly in association with Austronesian languages. Using detailed genealogical study of Y chromosome variation, we show that the majority of current Austronesian speakers trace their paternal heritage to Pleistocene settlers in the region, as opposed to more-recent agricultural immigrants. A fraction of the paternal heritage, however, appears to be associated with more-recent immigrants from northern populations. We also show that the northern Neolithic component is very unevenly dispersed through the region, with a higher contribution in Southeast Asia and a nearly complete absence in Melanesia. Contrary to claims of gene flow (under regional continuity) between H. erectus and H. sapiens, we found no ancestral Y chromosome lineages in a set of 1,209 samples. The finding excludes the possibility that early hominids contributed significantly to the paternal heritage of the region.     

The Role of Physical Activity in Harm Reduction among Betel Quid Chewers from a Prospective Cohort of 419,378 Individuals

ObjectiveTo assess the benefits of regular exercise in reducing harms associated with betel quid (BQ) chewing.MethodsThe study cohort, 419,378 individuals, participated in a medical screening program between 1994 and 2008, with 38,324 male and 1,495 female chewers, who consumed 5–15 quids of BQ a day. Physical activity of each individual, based on “MET-hour/week”, was classified as “inactive” or “active”, where activity started from a daily 15 minutes/day or more of brisk walking (≥3.75 MET-hour/week). Hazard ratios for mortality and remaining years in life expectancy were calculated.ResultsNearly one fifth (18.7%) of men, but only 0.7% of women were chewers. Chewers had a 10-fold increase in oral cancer risk; and a 2-3-fold increase in mortality from lung, esophagus and liver cancer, cardiovascular disease, and diabetes, with doubling of all-cause mortality. More than half of chewers were physically inactive (59%). Physical activity was beneficial for chewers, with a reduction of all-cause mortality by 19%. Inactive chewers had their lifespan shortened by 6.3 years, compared to non-chewers, but being active, chewers improved their health by gaining 2.5 years. The improvement, however, fell short of offsetting the harms from chewing.ConclusionsChewers had serious health consequences, but being physically active, chewers could mitigate some of these adverse effects, and extend life expectancy by 2.5 years and reduce mortality by one fifth. Encouraging exercise, in addition to quitting chewing, remains the best advice for 1.5 million chewers in Taiwan.     

Absence of MMACHC in peripheral retinal cells does not lead to an ocular phenotype in mice

Combined methylmalonic aciduria with homocystinuria (cblC type) is a rare disease caused by mutations in the MMACHC gene. MMACHC encodes an enzyme crucial for intracellular vitamin B₁₂ metabolism, leading to the accumulation of toxic metabolites e.g. methylmalonic acid (MMA) and homocysteine (Hcy), and secondary disturbances in folate and one-carbon metabolism when not fully functional. Patients with cblC deficiency often present in the neonatal or early childhood period with a severe multisystem pathology, which comprises a broad spectrum of treatment-resistant ophthalmological phenotypes, including retinal degeneration, impaired vision, and vascular changes. To examine the potential function of MMACHC in the retina and how its loss may impact disease, we performed gene expression studies in human and mouse, which showed that local expression of MMACHC in the retina and retinal pigment epithelium is relatively stable over time. To study whether functional MMACHC is required for retinal function and tissue integrity, we generated a transgenic mouse lacking Mmachc expression in cells of the peripheral retina. Characterization of this mouse revealed accumulation of cblC disease related metabolites, including MMA and the folate-dependent purine synthesis intermediates AICA-riboside and SAICA-riboside in the retina. Nevertheless, fundus appearance, morphology, vasculature, and cellular composition of the retina, as well as ocular function, remained normal in mice up to 6 or 12 months of age. Our data indicates that peripheral retinal neurons do not require intrinsic expression of Mmachc for survival and function and questions whether a local MMACHC deficiency is responsible for the retinal phenotypes in patients.     

Rational design to improve thermostability and specific activity of the truncated Fibrobacter succinogenes 1,3-1,4-β-d-glucanase

1,3-1,4-β-D-Glucanase has been widely used as a feed additive to help non-ruminant animals digest plant fibers, with potential in increasing nutrition turnover rate and reducing sanitary problems. Engineering of enzymes for better thermostability is of great importance because it not only can broaden their industrial applications, but also facilitate exploring the mechanism of enzyme stability from structural point of view. To obtain enzyme with higher thermostability and specific activity, structure-based rational design was carried out in this study. Eleven mutants of Fibrobacter succinogenes 1,3-1,4-β-D-glucanase were constructed in attempt to improve the enzyme properties. In particular, the crude proteins expressed in Pichia pastoris were examined firstly to ensure that the protein productions meet the need for industrial fermentation. The crude protein of V18Y mutant showed a 2 °C increment of Tm and W203Y showed ～30% increment of the specific activity. To further investigate the structure-function relationship, some mutants were expressed and purified from P. pastoris and Escherichia coli. Notably, the specific activity of purified W203Y which was expressed in E. coli was 63% higher than the wild-type protein. The double mutant V18Y/W203Y showed the same increments of Tm and specific activity as the single mutants did. When expressed and purified from E. coli, V18Y/W203Y showed similar pattern of thermostability increment and 75% higher specific activity. Furthermore, the apo-form and substrate complex structures of V18Y/W203Y were solved by X-ray crystallography. Analyzing protein structure of V18Y/W203Y helps elucidate how the mutations could enhance the protein stability and enzyme activity.     

Improved cryopreservation of bovine preimplantation embryos cultured in chemically defined medium

The aim of this study was to examine the effects of modifications to a standard slow freezing protocol on the viability of in vitro produced bovine embryos. Bovine oocytes were matured, fertilized with frozen-thawed semen, and presumptive zygotes cultured in defined two-step culture media. The standard freezing medium was 1.5M ethylene glycol (EG), 0.1M sucrose, 10% fetal bovine serum (FBS) in Dulbecco's phosphate buffered saline (D-PBS). A preliminary trial showed that in vitro produced embryos cryopreserved in this medium had a survival rate of 74.6% at 24h and 53.5% at 48 h post-thaw. Experiment 1 studied the effects of omitting the sucrose supplement or replacing it with 0.1M xylose. In Experiment 2, the effects of partial (0%, 25% or 50%) or total (100%) replacement of sodium chloride with choline chloride in the cryopreservation medium were examined (the medium with 100% replacement was designated CJ1). The effects of replacing the 10% FBS with 0.4% BSA or 0.4% lipid-rich BSA (Albumax I) in CJ1 was studied in Experiment 3. In Experiment 4, pregnancy/calving rates following the post-thaw transfer of in vitro produced embryos cryopreserved in the standard freezing medium were compared with those of in vitro and in vivo produced embryos cryopreserved in the improved medium (Albumax I in CJ1). Supplementation of the cryopreservation medium with 0.1M sucrose resulted in higher post-thaw survival rates at 24 h (71.3% versus 53.5 and 51.7%; P<0.05), 48 h (51.1% versus 45.3 and 40.2%), and 72 h (34.0% versus 24.4 and 23.0%) than 0.1M xylose or no supplement, respectively, in Experiment 1. Experiment 2 showed that embryos cryopreserved in the standard medium had poorer survival rates at 24 h (72.8% versus 86.5%; P<0.05), 48 h (53.1% versus 66.3%) or 72 h (28.4% versus 44.9%) than those frozen in CJ1. The post-thaw survival rate of embryos frozen in medium supplemented with Albumax I was better than that for the FBS or BSA supplements at 24h (92.0% versus 90.7 and 87.3%), 48 h (87.3% versus 76.9 and 70.9%; P<0.05), and 72 h (70.4% versus 49.1 and 46 4%; P<0.05; Experiment 3). In Experiment 4, in vitro produced embryos cryopreserved in CJ1 medium supplemented with Albumax I resulted in higher pregnancy rates at Day 35 (31.9% versus 22.9%) and Day 60 (24.1% versus 14.3%) of gestation, and calving rates (22.6% versus 10.0%; P<0.05) than similar embryos frozen in the standard medium. However, in vivo produced embryos cryopreserved in Albumax I in CJ1 resulted in higher pregnancy rates at Day 35 (50.7%; P<0.05) and Day 60 (45.1%; P<0.05) of gestation, and calving rate (43.7%; P<0.05). It was concluded that modification of the freezing medium by addition of lipid-rich BSA and replacing sodium chloride with choline chloride improves the post-thaw survival of in vitro produced embryos, and their viability post-transfer.     

Phenotypic Plasticity in Photosynthetic Temperature Acclimation among Crop Species with Different Cold Tolerances

While interspecific variation in the temperature response of photosynthesis is well documented, the underlying physiological mechanisms remain unknown. Moreover, mechanisms related to species-dependent differences in photosynthetic temperature acclimation are unclear. We compared photosynthetic temperature acclimation in 11 crop species differing in their cold tolerance, which were grown at 15°C or 30°C. Cold-tolerant species exhibited a large decrease in optimum temperature for the photosynthetic rate at 360 μL L⁻¹ CO₂ concentration [Opt (A₃₆₀)] when growth temperature decreased from 30°C to 15°C, whereas cold-sensitive species were less plastic in Opt (A₃₆₀). Analysis using the C₃ photosynthesis model shows that the limiting step of A₃₆₀ at the optimum temperature differed between cold-tolerant and cold-sensitive species; ribulose 1,5-bisphosphate carboxylation rate was limiting in cold-tolerant species, while ribulose 1,5-bisphosphate regeneration rate was limiting in cold-sensitive species. Alterations in parameters related to photosynthetic temperature acclimation, including the limiting step of A₃₆₀, leaf nitrogen, and Rubisco contents, were more plastic to growth temperature in cold-tolerant species than in cold-sensitive species. These plastic alterations contributed to the noted growth temperature-dependent changes in Opt (A₃₆₀) in cold-tolerant species. Consequently, cold-tolerant species were able to maintain high A₃₆₀ at 15°C or 30°C, whereas cold-sensitive species were not. We conclude that differences in the plasticity of photosynthetic parameters with respect to growth temperature were responsible for the noted interspecific differences in photosynthetic temperature acclimation between cold-tolerant and cold-sensitive species.The temperature dependence of leaf photosynthetic rate shows considerable variation between plant species and with growth temperature (Berry and Björkman, 1980; Cunningham and Read, 2002; Hikosaka et al., 2006). Plants native to low-temperature environments and those grown at low temperatures generally exhibit higher photosynthetic rates at low temperatures and lower optimum temperatures, compared with plants native to high-temperature environments and those grown at high temperatures (Mooney and Billings, 1961; Slatyer, 1977; Berry and Björkman, 1980; Sage, 2002; Salvucci and Crafts-Brandner, 2004b). For example, the optimum temperature for photosynthesis differs between temperate evergreen species and tropical evergreen species (Hill et al., 1988; Read, 1990; Cunningham and Read, 2002). Such differences have been observed even among ecotypes of the same species (Björkman et al., 1975; Pearcy, 1977; Slatyer, 1977).Temperature dependence of the photosynthetic rate has been analyzed using the biochemical model proposed by Farquhar et al. (1980). This model assumes that the photosynthetic rate (A) is limited by either ribulose 1,5-bisphosphate (RuBP) carboxylation (A_c) or RuBP regeneration (A_r). The optimum temperature for photosynthetic rate in C₃ plants is thus potentially determined by (1) the temperature dependence of A_c, (2) the temperature dependence of A_r, or (3) both, at the colimitation point of A_c and A_r (Fig. 1; Farquhar and von Caemmerer, 1982; Hikosaka et al., 2006).Open in a separate windowFigure 1.A scheme illustrating the shift in the optimum temperature for photosynthesis depending on growth temperature. Based on the C₃ photosynthesis model, the A₃₆₀ (white and black circles) is limited by A_c (solid line) or A_r (broken line). The optimum temperature for the photosynthetic rate is potentially determined by temperature dependence of A_c (A), temperature dependence of A_r (B), or the intersection of the temperature dependences of A_c and A_r (C). When the optimum temperature for the photosynthetic rate shifts to a higher temperature, there are also three possibilities determining the optimum temperature: temperature dependence of A_c (D), temperature dependence of A_r (E), or the intersection of the temperature dependences of A_c and A_r (F). Especially in the case that the optimum temperature is determined by the intersection of the temperature dependences of A_c and A_r, the optimum temperature can shift by changes in the balance between A_c and A_r even when the optimum temperatures for these two partial reactions do not change.In many cases, the photosynthetic rate around the optimum temperature is limited by A_c, and thus the temperature dependence of A_c determines the optimum temperature for the photosynthetic rate (Hikosaka et al., 1999, 2006; Yamori et al., 2005, 2006a, 2006b, 2008; Sage and Kubien, 2007; Sage et al., 2008). As the temperature increases above the optimum, A_c is decreased by increases in photorespiration (Berry and Björkman, 1980; Jordan and Ogren, 1984; von Caemmerer, 2000). Furthermore, it has been suggested that the heat-induced deactivation of Rubisco is involved in the decrease in A_c at high temperature (Law and Crafts-Brandner, 1999; Crafts-Brandner and Salvucci, 2000; Salvucci and Crafts-Brandner, 2004a; Yamori et al., 2006b). Numerous previous studies have shown changes in the temperature dependence of A_c with growth temperature (Hikosaka et al., 1999; Bunce, 2000; Yamori et al., 2005). Also, the temperature sensitivity of Rubisco deactivation may differ between plant species (Salvucci and Crafts-Brandner, 2004b) and with growth temperature (Yamori et al., 2006b), which may explain variation in the optimum temperature for photosynthesis (Fig. 1, A and D).A_r is more responsive to temperature than A_c and often limits photosynthesis at low temperatures (Hikosaka et al., 1999, 2006; Sage and Kubien, 2007; Sage et al., 2008). Recently, several researchers indicated that A_r limits the photosynthetic rate at high temperature (Schrader et al., 2004; Wise et al., 2004; Cen and Sage, 2005; Makino and Sage, 2007). They suggested that the deactivation of Rubisco at high temperatures is not the cause of decreased A_c but a result of limitation by A_r. However, it remains unclear whether limitation by A_r is involved in the variation in the optimum temperature for the photosynthetic rate (Fig. 1, B and E).A shift in the optimum temperature for photosynthesis can result from changes in the balance between A_r and A_c, even when the optimum temperatures for these two partial reactions do not change (Fig. 1, C and F; Farquhar and von Caemmerer, 1982). The balance between A_r and A_c has been shown to change depending on growth temperature (Hikosaka et al., 1999; Hikosaka, 2005; Onoda et al., 2005a; Yamori et al., 2005) and often brings about a shift in the colimitation temperature of A_r and A_c. Furthermore, recent studies have shown that plasticity in this balance differs among species or ecotypes (Onoda et al., 2005b; Atkin et al., 2006; Ishikawa et al., 2007). Plasticity in this balance could explain interspecific variation in the plasticity of photosynthetic temperature dependence (Farquhar and von Caemmerer, 1982; Hikosaka et al., 2006), although there has been no evidence in the previous studies that the optimum temperature for photosynthesis occurs at the colimitation point of A_r and A_c.Temperature tolerance differs between species and, with growth temperature, even within species from the same functional group (Long and Woodward, 1989). Bunce (2000) indicated that the temperature dependences of A_r and A_c to growth temperature were different between species from cool and warm climates and that the balance between A_r and A_c was independent of growth temperature for a given plant species. However, it was not clarified what limited the photosynthetic rate or what parameters were important in temperature acclimation of photosynthesis. Recently, we reported that the extent of temperature homeostasis of leaf respiration and photosynthesis, which is assessed as a ratio of rates measured at their respective growth temperatures, differed depending on the extent of the cold tolerance of the species (Yamori et al., 2009b). Therefore, comparisons of several species with different cold tolerances would provide a new insight into interspecific variation of photosynthetic temperature acclimation and their underlying mechanisms. In this study, we selected 11 herbaceous crop species that differ in their cold tolerance (Yamori et al., 2009b) and grew them at two contrasting temperatures, conducting gas-exchange analyses based on the C₃ photosynthesis model (Farquhar et al., 1980). Based on these results, we addressed the following key questions. (1) Does the plasticity in photosynthetic temperature acclimation differ between cold-sensitive and cold-tolerant species? (2) Does the limiting step of photosynthesis at several leaf temperatures differ between plant species and with growth temperature? (3) What determines the optimum temperature for the photosynthetic rate among A_c, A_r, and the intersection of the temperature dependences of A_c and A_r?