An experimental comparison of intracellular ice formation and freeze-thaw survival of hela S-3 cells

A small-volume fluorescent dye viability assay has been successfully applied to a conduction cryomicroscope freezing-thawing stage as a means of determining post-thaw survival of the nucleated mammalian cell HeLa S-3. The survival signature for HeLa S-3 cells has been determined, revealing an optimum cooling rate of −30 °C/min where the maximum survival is 30%. No cells survive for cooling rates greater than −128 °C/min and the decreased survival at supraoptimal cooling rates coincides with a linear increase in the percentage of cells containing intracellular ice from 0% at −16 °C/min to 100% at −128 °C/min.Although no data were taken to identify increased salt concentration as the mechanism responsible for cell injury at suboptimal cooling rates, the post-thaw leakage of intracellular fluorescent dye at these rates takes approximately 4–10 min as opposed to instantaneous release of dye for cells which contain ice at the high cooling rates. This indicates two modes of damage.Cell number density has been identified as an important parameter in freezing studies since survival can be enhanced at slow rates by packing cells together in groups. Packing also causes a greater fraction of the cells in a sample to have intracellular ice present, thus decreasing survival at the faster rates. These responses can be explained by assuming that the outer cells in a group protect the inner ones from solution damage at slow rates, yet restrict water flux from the inner cells at faster rates, causing an increased likelihood of intracellular ice formation. Both of these results are consistent with the dual-mechanism freezing damage theory proposed by Mazur.     

Oncostatin M receptor-beta mutations underlie familial primary localized cutaneous amyloidosis

Familial primary localized cutaneous amyloidosis (FPLCA) is an autosomal-dominant disorder associated with chronic skin itching and deposition of epidermal keratin filament-associated amyloid material in the dermis. FPLCA has been mapped to 5p13.1–q11.2, and by candidate gene analysis, we identified missense mutations in the OSMR gene, encoding oncostatin M-specific receptor β (OSMRβ), in three families. OSMRβ is a component of the oncostatin M (OSM) type II receptor and the interleukin (IL)-31 receptor, and cultured FPLCA keratinocytes showed reduced activation of Jak/STAT, MAPK, and PI3K/Akt pathways after OSM or IL-31 cytokine stimulation. The pathogenic amino acid substitutions are located within the extracellular fibronectin type III-like (FNIII) domains, regions critical for receptor dimerization and function. OSM and IL-31 signaling have been implicated in keratinocyte cell proliferation, differentiation, apoptosis, and inflammation, but our OSMR data in individuals with FPLCA represent the first human germline mutations in this cytokine receptor complex and provide new insight into mechanisms of skin itching.     

Genetic diversity in selected, historical US sugarbeet germplasm and Beta vulgaris ssp. maritima

 Diversity among sugar beet accessions released over the first 50 years of public breeding in the United States was examined to ascertain a baseline of genetic diversity and to gauge the effect of breeding on the loss or gain of diversity over this time period. Accessions were chosen as released germplasm from the major breeding stations contributing to the US germplasm pool and their presumed ancestors from Europe, including representatives for the wild forms Beta vulgaris ssp. maritima. Sixty nine polymorphic RAPD fragments were used for gene frequency analysis, and heterozygosity was determined within and among groups of accessions related either by breeding station or simply inherited agronomic characters for monogerm seed and restoration of fertility in a cytoplasmic male-sterile background. In general, heterozygosity within releases declined with time, but total genetic diversity in the US germplasm pool remained constant. Breeding for the agronomic characters had a marked influence in reducing diversity. Received: 20 October 1998 / Accepted: 28 October 1998     

Implication of new WHO growth standards on identification of risk factors and estimated prevalence of malnutrition in rural Malawian infants

Background

The World Health Organization (WHO) released new Child Growth Standards in 2006 to replace the current National Center for Health Statistics (NCHS) growth reference. We assessed how switching from the NCHS to the newly released WHO Growth Standards affects the estimated prevalence of wasting, underweight and stunting, and the pattern of risk factors identified.

Methodology/Principal Findings

Data were drawn from a village-informant driven Demographic Surveillance System in Northern Malawi. Children (n = 1328) were visited twice at 0–4 months and 11–15 months. Data were collected on the demographic and socio-economic environment of the child, health history, maternal and child anthropometry and child feeding practices. Weight-for-length, weight-for-age and length-for-age were derived in z-scores using the two growth references. In early infancy, prevalence estimates were 2.9, 6.1, and 8.5 fold higher for stunting, underweight, and wasting respectively using the WHO standards compared to NCHS reference (p<0.001 for all). At one year, prevalence estimates for wasting and stunting did not differ significantly according to reference used, but the prevalence of underweight was half that with the NCHS reference (p<0.001). Patterns of risk factors were similar with the two growth references for all outcomes at one year although the strength of association was higher with WHO standards.

Conclusions/Significance

Differences in prevalence estimates differed in magnitude but not direction from previous studies. The scale of these differences depends on the population''s nutritional status thus it should not be assumed a priori. The increase in estimated prevalence of wasting in early infancy has implications for feeding programs targeting lactating mothers and ante-natal multiple micronutrients supplementation to tackle small birth size. Risk factors identified using WHO standards remain comparable with findings based on the NCHS reference in similar settings. Further research should aim to identify whether the young infants additionally diagnosed as malnourished by this new standard are more appropriate targets for interventions than those identified with the NCHS reference.     

Glycine substitutions in the triple-helical region of type VII collagen result in a spectrum of dystrophic epidermolysis bullosa phenotypes and patterns of inheritance.

The dystrophic forms of epidermolysis bullosa (DEB) are characterized by fragility of the skin and mucous membranes. DEB can be inherited in either an autosomal dominant or autosomal recessive pattern, and the spectrum of clinical severity is highly variable. The unifying diagnostic hallmark of DEB is abnormalities in the anchoring fibrils, which consist of type VII collagen, and, recently, mutations in the corresponding gene, COL7A1, have been disclosed in a number of families. In this study, we report six families with glycine substitution mutations in the triple-helical region of type VII collagen. Among the six families, two demonstrated a mild phenotype, and the inheritance of the mutation was consistent with the dominantly inherited form of DEB. In the four other families, the mutation was silent in the heterozygous state but, when present in the homozygous state, or combined with a second mutation, resulted in a recessively inherited DEB phenotype. Type VII collagen is, therefore, unique among the collagen genes, in that different glycine substitutions can be either silent in heterozygous individuals or result in a dominantly inherited DEB. Inspection of the locations of the glycine substitutions along the COL7A1 polypeptide suggests that the consequences of these mutations, in terms of phenotype and pattern of inheritance, are position independent.     

Mechanisms of arsenic hyperaccumulation in Pteris vittata. Uptake kinetics,interactions with phosphate,and arsenic speciation

The mechanisms of arsenic (As) hyperaccumulation in Pteris vittata, the first identified As hyperaccumulator, are unknown. We investigated the interactions of arsenate and phosphate on the uptake and distribution of As and phosphorus (P), and As speciation in P. vittata. In an 18-d hydroponic experiment with varying concentrations of arsenate and phosphate, P. vittata accumulated As in the fronds up to 27,000 mg As kg(-1) dry weight, and the frond As to root As concentration ratio varied between 1.3 and 6.7. Increasing phosphate supply decreased As uptake markedly, with the effect being greater on root As concentration than on shoot As concentration. Increasing arsenate supply decreased the P concentration in the roots, but not in the fronds. Presence of phosphate in the uptake solution decreased arsenate influx markedly, whereas P starvation for 8 d increased the maximum net influx by 2.5-fold. The rate of arsenite uptake was 10% of that for arsenate in the absence of phosphate. Neither P starvation nor the presence of phosphate affected arsenite uptake. Within 8 h, 50% to 78% of the As taken up was distributed to the fronds, with a higher translocation efficiency for arsenite than for arsenate. In fronds, 49% to 94% of the As was extracted with a phosphate buffer (pH 5.6). Speciation analysis using high-performance liquid chromatography-inductively coupled plasma mass spectroscopy showed that >85% of the extracted As was in the form of arsenite, and the remaining mostly as arsenate. We conclude that arsenate is taken up by P. vittata via the phosphate transporters, reduced to arsenite, and sequestered in the fronds primarily as As(III).     

Alpha-chloralose as a capture and restraint agent of birds: therapeutic index determination in the chicken

The median effective dose for capture (ED50) and the median lethal dose (LD50) of alpha-chloralose given orally to domestic chickens (Gallus domesticus) were determined by probit analysis to be 45 mg/kg and 300 mg/kg, respectively. The therapeutic index (TI = LD50/ED50) was 6.7. This indicates that alpha-chloralose is only a marginally safe capture agent in domestic species and particularly in field applications involving other wild avian species in which the amount of the drug ingested by an individual bird is not controlled.     

Identification and Characterization of Lactococcal-Prophage-Carried Superinfection Exclusion Genes

Superinfection exclusion (Sie) proteins are prophage-encoded phage resistance systems. In this study, genes encoding Sie systems were identified on the genomes of Lactococcus lactis subsp. cremoris MG1363 and L. lactis subsp. lactis IL1403. These Sie systems are genetically distinct and yet were shown to act specifically against a particular subset of the 936 phage group. Each of the systems allows normal phage adsorption while affecting plasmid transduction and intracellular phage DNA replication, which points to the blocking of phage DNA injection as their common mode of action. Sie-specifying genes found on the MG1363 prophages are also present in various lactococcal strains, whereas the prophage-encoded Sie systems of IL1403 do not appear to be as widely disseminated.