Differences in Anionic Dependence of the Synaptic Efflux of d-Aspartic Acid and γ-Aminobutyric Acid

The synaptosomal effluxes of D-aspartate and gamma-aminobutyric acid (GABA) induced by a substitution of the Cl- ions with propionate in the incubation medium were measured in preparations of hippocampal tissue homogenates. The efflux of aspartate was significantly higher than spontaneous efflux at 125 mM Cl- (normal = 144 mM) and was increased with decreasing Cl- concentration. GABA efflux was much less sensitive to a reduction in Cl- concentration than D-aspartate. The efflux was Ca2+-dependent in both cases.     

Microsatellite markers for Bistorta vivipara (Polygonaceae)

? Premise of the study: Using genomic shotgun 454 sequencing, 50 candidate microsatellite markers were targeted for the arctic-alpine polyploid perennial herb Bistorta vivipara to distinguish between individual genets and ramets within a population. ? Methods and Results: Out of the 50 markers, 31 were polymorphic for seven test samples. We have developed a multiplex protocol for 16 of these microsatellite markers. ? Conclusions: Our results show that the microsatellite markers provide a powerful tool for the research on genetic variation of B. vivipara.     

Effect of zirconium(IV) complexes on the thermal and enzymatic stability of type I collagen

Understanding the mechanism of stabilization of collagen is an important area of research. Metal ions are known to interact with collagen and bring about the stability of the same. In the present investigation, the interaction of zirconium(IV) complexes with collagen was studied. The effect of zirconium(IV) complexes, namely zirconium oxychloride and zirconium oxalate on the enzymatic and thermal stability of collagen was investigated. Zirconium has been found to increase the hydrothermal stability of the rat tail tendon (RTT) collagen fibers to about 8-10 degrees C more than that of the native collagen. The order of stabilization of zirconium(IV) complexes is zirconium oxychloride>zirconium oxalate. This could be due to the differences in the type of interaction with collagen, which is also reflected in the differences in the conformational changes of collagen brought about by the two complexes. Zirconium oxychloride, which forms tetrameric species in solution, has been shown to have better crosslinking with collagen as seen from viscometry studies and hence provides better enzymatic stability to collagen than zirconium oxalate, which largely forms monomeric species in solution.     

Parental Perceptions of Quality of Life in Children on Long-Term Ventilation at Home as Compared to Enterostomy Tubes

Objective

Health related quality of life (HRQL) of children using medical technology at home is largely unknown. Our aim was to examine the HRQL in children on long-term ventilation at home (LTHV) in comparison to a cohort using an enterostomy tube.

Study Design

Participants were divided into three groups: 1) LTHV without an enterostomy tube (LTHV cohort); 2) Enterostomy tube (GT cohort); 3) LTHV with an enterostomy tube (LTHV+GT cohort). Caregivers of children ≥ 5 years and followed at SickKids, Toronto, Canada, completed three questionnaires: Health Utilities Index 2/3 (HUI2/3), Caregiver Priorities Caregiver Health Index (CPCHILD), and the Paediatric Quality of Life Inventory (PedsQL). The primary outcome was the difference in utility (HUI2/3) scores between the cohorts.

Results

One hundred and nineteen children were enrolled; 47 in the LTHV cohort, 44 in the GT cohort, and 28 in the LTHV+GT cohort. In univariate analysis, HUI2 mean (SE) scores were lowest for the GT cohort, 0.4 (0.04) followed by the LTHV+GT, 0.42 (0.05) and then the LTHV cohort, 0.7 (0.04), p = 0.001. A similar trend was seen for the HUI3 mean (SE) scores: GT cohort, 0.1 (0.06), followed by the LTHV +GT cohort, 0.2 (0.08) and then the LTHV cohort, 0.5 (0.06), p = 0.0001. Technology cohort, nursing hours and the severity of health care needs predicted HRQL as measured by the HUI2/3.

Conclusion

The HRQL of these children is low. Children on LTHV had higher HRQL than children using enterostomy tubes. Further work is needed to identify modifiable factors that can improve HRQL.     

Using indirect methods to understand the impact of forced migration on long-term under-five mortality

Despite the large numbers of displaced persons and the often-lengthy periods of displacement, little is known about the impact of forced migration on long-term under-five mortality. This paper looks at the Brass Method (and adaptations of this method) and the Preceding Birth Technique in combination with a classification of women by their migration and reproductive histories, in order to study the impact of forced migration on under-five mortality. Data came from the Demography of Forced Migration Project, a study on mortality, fertility and violence in the refugee and host populations of Arua District, Uganda and Yei River District, Sudan. Results indicate that women who did not migrate in a situation of conflict and women who repatriated before the age of 15, had children with the highest under-five mortality rates compared with women who were currently refugees and women who repatriated after the age of 15.     

Mechanisms of Topoisomerase I (TOP1) Gene Copy Number Increase in a Stage III Colorectal Cancer Patient Cohort

Background

Topoisomerase I (Top1) is the target of Top1 inhibitor chemotherapy. The TOP1 gene, located at 20q12-q13.1, is frequently detected at elevated copy numbers in colorectal cancer (CRC). The present study explores the mechanism, frequency and prognostic impact of TOP1 gene aberrations in stage III CRC and how these can be detected by fluorescent in situ hybridization (FISH).

Methods

Nine CRC cell line metaphase spreads were analyzed by FISH with a TOP1 probe in combination with a reference probe covering either the centromeric region of chromosome 20 (CEN-20) or chromosome 2 (CEN-2). Tissue sections from 154 chemonaive stage III CRC patients, previously studied with TOP1/CEN-20, were analyzed with TOP1/CEN-2. Relationships between biomarker status and overall survival (OS), time to recurrence (TTR) in CRC and time to local recurrence (LR; rectal cancer only) were determined.

Results

TOP1 aberrations were observed in four cell line metaphases. In all cell lines CEN-2 was found to reflect chromosomal ploidy levels and therefore the TOP1/CEN-2 probe combination was selected to identify TOP1 gene gains (TOP1/CEN-2≥1.5). One hundred and three patients (68.2%) had TOP1 gain, of which 15 patients (14.6%) harbored an amplification (TOP1/CEN-20≥2.0). TOP1 gene gain did not have any association with clinical endpoints, whereas TOP1 amplification showed a non-significant trend towards longer TTR (multivariate HR: 0.50, p = 0.08). Once amplified cases were segregated from other cases of gene gain, non-amplified gene increases (TOP1/CEN-2≥1.5 and TOP1/CEN-20<2.0) showed a trend towards shorter TTR (univariate HR: 1.57, p = 0.07).

Conclusions

TOP1 gene copy number increase occurs frequently in stage III CRC in a mechanism that often includes CEN-20. Using CEN-2 as a measurement for tumor ploidy levels, we were able to discriminate between different mechanisms of gene gain, which appeared to differ in prognostic impact. TOP1 FISH guidelines have been updated.     

Limited predictive ability of surrogate indices of insulin sensitivity/resistance in Asian-Indian men

Insulin resistance is highly prevalent in Asian Indians and contributes to worldwide public health problems, including diabetes and related disorders. Surrogate measurements of insulin sensitivity/resistance are used frequently to study Asian Indians, but these are not formally validated in this population. In this study, we compared the ability of simple surrogate indices to accurately predict insulin sensitivity as determined by the reference glucose clamp method. In this cross-sectional study of Asian-Indian men (n = 70), we used a calibration model to assess the ability of simple surrogate indices for insulin sensitivity [quantitative insulin sensitivity check index (QUICKI), homeostasis model assessment (HOMA2-IR), fasting insulin-to-glucose ratio (FIGR), and fasting insulin (FI)] to predict an insulin sensitivity index derived from the reference glucose clamp method (SI(Clamp)). Predictive accuracy was assessed by both root mean squared error (RMSE) of prediction as well as leave-one-out cross-validation-type RMSE of prediction (CVPE). QUICKI, FIGR, and FI, but not HOMA2-IR, had modest linear correlations with SI(Clamp) (QUICKI: r = 0.36; FIGR: r = -0.36; FI: r = -0.27; P < 0.05). No significant differences were noted among CVPE or RMSE from any of the surrogate indices when compared with QUICKI. Surrogate measurements of insulin sensitivity/resistance such as QUICKI, FIGR, and FI are easily obtainable in large clinical studies, but these may only be useful as secondary outcome measurements in assessing insulin sensitivity/resistance in clinical studies of Asian Indians.     

A quantitative analysis of germ cells and the histone variants in the testes of vitamin A-deficient rats and during subsequent repletion with vitamin A

A quantitative analysis of the different types of germ cells present in the seminiferous tubules of vitamin A-deficient-retinoate maintained rats revealed that the number of pachytene spermatocytes and spermatogonia was greatly reduced in the deficient rats. Spermatids were virtually absent in the deficient tubules which contained mostly spermatogonia and preleptotene spermatocytes along with the Sertoli cells. There was no change in the number of Sertoli cells present in the tubules of deficient rats as compared to that of normal rats. Following supplementation of retinyl acetate to vitamin A-deficient-retinoate maintained rats, there was an immediate thinning of the germinal epithelium resulting from the sloughing off of the damaged spermatocytes which were beyond repair. However, after 12 days of vitamin A supplementation fresh batch of pachytene spermatocytes started appearing while by day 16 round spermatids could be seen. Analysis of the acid soluble proteins from nuclei on different types of Polyacrylamide gel electrophoretic systems has revealed that the levels of the testis specific histone variants Hlt, TH2A and TH2B, synthesized predominantly in the pachytene spermatocytes were greatly reduced in the testes of retinoate maintained rats. Following supplementation of retinyl acetate for either 4 days or 8 days the levels of these histone variants further decreased which correlated with the decrease in the number of pachytene spermatocytes. However, by day 12 of supplementation onwards, their levels started increasing and reached near normal levels by day 24 of vitamin A-supplementation     

Identification of Bilirubin Binding Site in Type I Collagen

The interaction of bilirubin with collagen in the significance of jaundice incidence have been previously reported and investigated. The novel peptide sequences containing bilirubin binding domain was identified and located to develop a basis for further studies investigating the interactions of collagen with bilirubin in the present study. In this study an intricate interaction between bilirubin and collagen was characterized and their binding domain has been established using in-gel digestion and LC–MS/MS analysis based on the collagen sequencing and peptide mass fingerprinting. The biotinylated bilirubin derivatives bind to α₁(I) chain but not to α₂(I) chains which clearly designates that bilirubin shows greater affinity to α₁ chains of collagen. The intact proteins collected after analyzing the resulting complex mixture of peptides was used for peptide mapping. Using the electrospray method, among the other peptide sequence information obtained, the molecular weight of collagen alpha-2(I) chain was obtained by locating a 130 kDa weight peptide sequences with greater pi value (9.14) with 1,364 amino acid residues and collagen alpha-1(I) chain with 1,463 amino acid residues with 138.9 kDa molecular weight. This information leads to locate the exact sequence of these helices focussing on the domain identification. The total charge of the peptide domain sequences infers that the bilirubin participates in the electrostatic mode of interaction with collagen peptide. Moreover, other modes of interactions such as hydrogen bonding, covalent interactions and hydrophobic interactions are possible.     

Mutation of the regulatory phosphorylation site of tobacco nitrate reductase results in high nitrite excretion and NO emission from leaf and root tissue

In wild-type Nicotiana plumbaginifolia Viv. and other higher plants, nitrate reductase (NR) is regulated at the post-translational level and is rapidly inactivated in response to, for example, a light-to-dark transition. This inactivation is caused by phosphorylation of a conserved regulatory serine residue, Ser 521 in tobacco, and interaction with divalent cations or polyamines, and 14-3-3 proteins. The physiological importance of the post-translational NR modulation is presently under investigation using a transgenic N. plumbaginifolia line. This line expresses a mutated tobacco NR where Ser 521 has been changed into aspartic acid (Asp) by site-directed mutagenesis, resulting in a permanently active NR enzyme [C. Lillo et al. (2003) Plant J 35:566–573]. When cut leaves or roots of this line (S₅₂₁) were placed in darkness in a buffer containing 50 mM KNO₃, nitrite was excreted from the tissue at rates of 0.08–0.2 mol (g FW)^–1 h^–1 for at least 5 h. For the control transgenic plant (C1), which had the regulatory serine of NR intact, nitrite excretion was low and halted completely after 1–3 h. Without nitrate in the buffer in which the tissue was immersed, nitrite excretion was also low for S₅₂₁, although 20–40 mol (g FW)^–1 nitrate was present inside the tissue. Apparently, stored nitrate was not readily available for reduction in darkness. Leaf tissue and root segments of S₅₂₁ also emitted much more nitric oxide (NO) than the control. Importantly, NO emission from leaf tissue of S₅₂₁ was higher in the dark than in the light, opposite to what was usually observed when post-translational NR modulation was operating.Abbreviations NR Nitrate reductase - NO Nitric oxide - Ser Serine - WT Wild type