Distribution of polarizing activity and potential for limb formation in mouse and chick embryos and possible relationships to polydactyly

A central feature of the tetrapod body plan is that two pairs of limbs develop at specific positions along the head-to-tail axis. However, the potential to form limbs in chick embryos is more widespread. This could have implications for understanding the basis of limb abnormalities. Here we extend the analysis to mouse embryos and examine systematically the potential of tissues in different regions outside the limbs to contribute to limb structures. We show that the ability of ectoderm to form an apical ridge in response to FGF4 in both mouse and chick embryos exists throughout the flank as does ability of mesenchyme to provide a polarizing region signal. In addition, neck tissue has weak polarizing activity. We show, in chick embryos, that polarizing activity of tissues correlates with the ability either to express Shh or to induce Shh expression. We also show that cells from chick tail can give rise to limb structures. Taken together these observations suggest that naturally occurring polydactyly could involve recruitment of cells from regions adjacent to the limb buds. We show that cells from neck, flank and tail can migrate into limb buds in response to FGF4, which mimics extension of the apical ectodermal ridge. Furthermore, when we apply simultaneously a polarizing signal and a limb induction signal to early chick flank, this leads to limb duplications.     

Calcium-dependent translocation of S100A11 requires tubulin filaments

Protein translocation between different subcellular compartments might play a significant role in various signal transduction pathways. The S100 family is comprised of the multifunctional, small, acidic proteins, some of which translocate in the form of vesicle-like structures upon increase in intracellular Ca(2+) levels. Previously, cells were fixed before and after calcium activation in order to examine the possible relocation of S100 proteins. In this study, we were able to track the real-time translocation. We compared the localization of endogenous S100A11 to that of the S100A11-green fluorescent protein. The application of thapsigargin, an agent increasing intracellular Ca(2+) levels, resulted in the relocation of the S100A11. In contrast, addition of EGTA, which specifically binds Ca(2+), either inhibited the ongoing process of translocation or prevented its induction. Since translocation was not affected by treatment with brefeldin A, it appears that S100A11 relocates in an endoplasmic reticulum-Golgi-independent pathway. Furthermore, the depolymerization of actin filaments by amlexanox did not affect the capacity of S100A11 to translocate. However, the time course treatment with demecolcine, which depolymerizes tubulin filaments, resulted in cease of translocation, suggesting that the tubulin network is required for this process.     

CD40 ligand trimer and IL-12 enhance peripheral blood mononuclear cells and CD4+ T cell proliferation and production of IFN-gamma in response to p24 antigen in HIV-infected individuals: potential contribution of anergy to HIV-specific unresponsiveness

It has been suggested that CD4+ T cell proliferative responses to HIV p24 Ag may be important in the control of HIV infection. However, these responses are minimal or absent in many HIV-infected individuals. Furthermore, while in vitro and in vivo responses to non-HIV recall Ags improve upon administration of highly active antiretroviral therapy, there does not appear to be a commensurate enhancement of HIV-specific immune responses. It is possible that CD4+ p24-specific T cells are deleted early in the course of infection. However, it is also possible that a discrete unresponsiveness, or anergy, contributes to the lack of proliferation to p24. To evaluate the possible contribution of unresponsiveness to the lack of CD4+ T cell proliferation to p24 in HIV-infected individuals, we attempted to overcome unresponsiveness. CD40 ligand trimer (CD40LT) and IL-12 significantly increased PBMC and CD4+ T cell proliferative responses to p24 Ag in HIV-infected, but not uninfected, individuals. No increase in proliferative response to CMV Ag was observed. CD40LT exerted its effect through B7-CD28-dependent and IL-12- and IL-15-independent mechanisms. Finally, the increase in proliferation with CD40LT and IL-12 was associated with an augmented production of IFN-gamma in most, but not all, individuals. These data suggest the possible contribution of HIV-specific unresponsiveness to the lack of CD4+ T cell proliferation to p24 Ag in HIV-infected individuals and that clonal deletion alone does not explain this phenomenon. They also indicate the potential for CD40LT and IL-12 as immune-based therapies for HIV infection.     

Flow cytometric analysis of microorganisms

The application of flow cytometry to microorganisms is as old as the technique itself, but it has historically been underexploited for microbial applications. This is now being reversed and microbiologists are ideally placed to benefit from recent technological advances. While earlier papers demonstrated the use of flow cytometry for studies of viability and taxonomy, recent developments in bioinformatics and reporter gene technologies are leading to novel applications in microbiology. Variants of green fluorescent protein have been used for the study of conditional microbial gene regulation in medically important host-pathogen interactions and fluorescence-activated cell sorting is being applied to the isolation of novel mutants in directed evolution studies. This paper reviews the reasons for the delay in the application of flow cytometry to microbial problems, the range of applications, and their limitations and considers the progress made in developing new strategies for use in microbiological investigations.     

Analysis of the N-acetylneuraminic acid and N-glycolylneuraminic acid contents of glycoproteins by high-pH anion-exchange chromatography with pulsed amperometric detection

Presence or absence of N-acetylneuraminic acid (Neu5Ac) can change a sialylated glycoprotein's serum half-life and possibly its function. We evaluated the linearity, sensitivity, reproducibility, and accuracy of a HPAEC/PAD method to determine its suitability for routine simultaneous analysis of Neu5Ac and N-glycolylneuraminic acid (Neu5Gc). An effective internal standard for this analysis is 3-deoxy-d-glycero-d- galacto-2-nonulosonic acid (KDN). We investigated the effect of the Au working electrode recession and determined that linear range and sensitivity were dependent on electrode recession. Using an electrode that was 350 &mgr;m recessed from the electrode block, the minimum detection limits of Neu5Ac, KDN, and Neu5Gc were 2, 5, and 2 pmol, respectively, and were reduced to 1, 2, and 0.5 pmol using a new electrode. The response of standards was linear from 10 to 500 pmol (r2>0.99) regardless of electrode recession. When Neu5Ac, KDN, and Neu5Gc (200 pmol each) were analyzed repetitively for 48 h, area RSDs were <3%. Reproducibility was unaffected when injections of glycoprotein neuraminidase and acid digestions were interspersed with standard injections. Area RSDs of Neu5Ac and Neu5Gc improved when the internal standard was used. We determined the precision and accuracy of this method for both a recessed and a new working electrode by analyzing Neu5Ac and Neu5Gc contents of bovine fetuin and bovine and human transferrins. Results were consistent with published values and independent of the working electrode. The sensitivity, reproducibility, and accuracy of this method make it suitable for direct routine analysis of glycoprotein Neu5Ac and Neu5Gc contents.      

A comparison of methods for direct gene transfer into maize (Zea mays L.)

Summary Techniques for transforming intact tissues of cereals were evaluated for their efficacy in transforming immature embryos and Type II callus of maize (Zea mays L.). The techniques used were particle bombardment, tissue electroporation, tissue electrophoresis, and silicon carbide fibers. Each method was assessed in terms of transient β-glucuronidase (GUS) expression. High levels of GUS expression were observed in A188 Type II callus using both tissue electroporation and particle bombardment, with means of 417.8 and 954.5 blue expression units (beu) per g fresh weight (FW) callus, respectively. Only particle bombardment resulted in high transient gene expression in immature embryos, with a mean transformation frequency of 34.8 b.e.u. per embryo. Very low levels of GUS expression were achieved with silicon carbide-mediated gene transfer, even when employing tissues used in the original publication (Black Mexican Sweet suspension cells). GUS expression was not obtained following tissue electrophoretic gene delivery.     

Expression of a chitinase transgene in rose (Rosa hybrida L.) reduces development of blackspot disease (Diplocarpon rosae Wolf)

Blackspot, caused by the Ascomycete fungus Diplocarpon rosae, is the most widespread and pernicious disease of cultivated roses. While some species of rose possess resistance to D. rosae, none of the modern-day rose cultivars are fully resistant to the pathogen. In the current study, Biolistic gene delivery was used to introduce a rice gene, encoding a basic (Class I), chitinase into embryogenic callus of the blackspot-susceptible rose (Rosa hybrida L.) cv. Glad Tidings. The plasmid used for transformation carried the neomycin phosphotransferase (nptII) gene facilitating the selection and regeneration of transgenic plants on medium containing 250 mg/l kanamycin. Southern analysis confirmed integration of 2–6 copies of the chitinase gene into the rose genome; gene expression was confirmed by enzyme assay. Bioassays demonstrated that expression of the chitinase transgene reduced the severity of blackspot development by 13–43%. This degree of resistance to the pathogen correlated with the level of chitinase expression in the transgenic rose plants. The introduction of disease defence genes into rose provides a method of producing blackspot-resistant rose cultivars sought by breeders and growers.     

Is the Association between Vitamin D and Cardiovascular Disease Risk Confounded by Obesity? Evidence from the Andhra Pradesh Children and Parents Study (APCAPS)

Background

Evidence of an association between serum vitamin D and cardiovascular disease risk is inconsistent and comes predominantly from studies in high-income settings. We assessed the association between serum levels of 25-hydroxyvitamin D₃ (25(OH)D) and cardiovascular disease risk factors in a population of young Indian adults.

Methods

Cross-sectional analyses of data from APCAPS (Andhra Pradesh Children and Parents Study); a prospective birth cohort study in rural south India. Participants were 1038 (40.3% females) adults aged 18-24 years. Main outcome measures were blood pressures, fasting serum lipids (cholesterols and triglycerides), fasting glucose, insulin, measures of arterial stiffness (aortic augmentation index and aortic pulse wave velocity (aPWV)), carotid intima-media thickness, body mass index (BMI) and body fat (dual X-ray absorptiometry).

Results

Vitamin D deficiency (≤20ng/ml) was observed in 41.1% of this lean (mean BMI: 19.5) and active (mean minutes of moderate or vigorous physical activity per day: 186) population. Vitamin D deficiency was associated with higher median body fat in both males (15.9% body fat in vitamin D deficient males vs. 14.6% in non-deficient males, p<0.05) and females (29.1% body fat in vitamin D deficient females vs. 27.8% in non-deficient females, p<0.05) but no associations were observed between vitamin D deficiency and mean BMI or median fat mass index (FMI). Except a weak inverse association with fasting insulin in males, there was no clear association between serum vitamin D levels and cardiovascular disease risk factors in fully adjusted models.

Conclusions

We did not find clear evidence for an association between serum vitamin D levels and cardiovascular disease risk factors. Our results, consistent with the limited evidence from randomised trials of vitamin D supplementation and Mendelian randomisation experiments, suggest that the postulated link between serum vitamin D and cardiovascular disease may be non-causal. Instead, it may be attributable to confounding by lifestyle factors such as obesity and physical inactivity which may provide more fruitful targets for cardiovascular disease prevention.     

Prediction of human core body temperature using non-invasive measurement methods

The measurement of core body temperature is an efficient method for monitoring heat stress amongst workers in hot conditions. However, invasive measurement of core body temperature (e.g. rectal, intestinal, oesophageal temperature) is impractical for such applications. Therefore, the aim of this study was to define relevant non-invasive measures to predict core body temperature under various conditions. We conducted two human subject studies with different experimental protocols, different environmental temperatures (10 °C, 30 °C) and different subjects. In both studies the same non-invasive measurement methods (skin temperature, skin heat flux, heart rate) were applied. A principle component analysis was conducted to extract independent factors, which were then used in a linear regression model. We identified six parameters (three skin temperatures, two skin heat fluxes and heart rate), which were included for the calculation of two factors. The predictive value of these factors for core body temperature was evaluated by a multiple regression analysis. The calculated root mean square deviation (rmsd) was in the range from 0.28 °C to 0.34 °C for all environmental conditions. These errors are similar to previous models using non-invasive measures to predict core body temperature. The results from this study illustrate that multiple physiological parameters (e.g. skin temperature and skin heat fluxes) are needed to predict core body temperature. In addition, the physiological measurements chosen in this study and the algorithm defined in this work are potentially applicable as real-time core body temperature monitoring to assess health risk in broad range of working conditions.