Glomerular podocytes in cultured rat kidney slices

Summary The ultrastructure of rat glomerular epithelial cells (podocytes) in kidney slices in vitro was examined using qualitative and quantitative electron microscopy. The kidney slices were cultured in Medium 199 with Hanks' salts in a 5% CO₂/95% O₂ environment for up to 14 days. Few changes in podocyte ultrastructure occurred in the first 12 h of culture, but by 24 h cell bodies were rounded, microvilli were present on all podocyte surfaces, and some foot processes had been replaced by flattened expanses of cytoplasm. These changes were more pronounced by 3 days, when some podocytes had developed pseudopodal extensions and appeared to be migrating from glomeruli onto the slice surface. Podocytes could still be identified after 8, 10 and 14 days of culture, although relatively few glomeruli remained at 14 days. Morphometric methods were used to analyse podocyte shape, volume and surface area during the first 4 days of culture. The most significant change involved loss of foot processes: the number of filtration slits per 100 m of basement membrane decreased from 211.8 ± 15.0 (mean ± SD) at the commencement of culture, to 55.3 ± 22.6 after 2 days (P < 0.001). These data provide baseline information for in vitro studies on the effects of nephrotoxins on podocytes.     

Heat-soaked PCR: an efficient method for DNA amplification with applications to forensic analysis.

Heat-soaked PCR (HS-PCR) is a method for enhancing amplification performed by heating the DNA sample at 94 degrees C in 90 microliters 1.1 x buffer for 30 min. A 10-microliters bolus of concentrated (10x) deoxynucleotides, Taq DNA polymerase and primers prepared without buffer is then added just prior to thermal cycling. We have investigated the application of this method in a variety of forensically important DNA samples and compared it with regular PCR (R-PCR). DNA samples extracted from bone, postmortem tissues, bloodstains and hair contained low concentrations of human DNA or were contaminated with either non- human DNA or hemoglobin degradation products. Optimal conditions for HS-PCR were determined for the 3' ApoB VNTR locus and applied to a centromeric repeat element and to a single-copy locus. HS-PCR consistently and reproducibly enhanced product yield and specificity over R-PCR at all three loci in the entire set of DNA samples. HS-PCR was also effective in overcoming the inhibitory effect of hemoglobin at concentrations that fully impeded R-PCR.     

Diurnal beta-endorphin changes in human cerebrospinal fluid

Plasma and cerebrospinal fluid (CSF) beta-endorphin levels were determined by a RIA method in seven hydrocephalic male patients. The samples were simultaneously collected every two hours from 8 AM to 12 midnight and every hour from 1 AM to 7 AM. In both plasma and CSF beta-endorphin levels showed significant time-related variations during the 24 hour period. These results suggest the existence of diurnal CSF beta-endorphin variations analogous to those observed in plasma.     

Human keratinocyte growth factor activity on proliferation and differentiation of human keratinocytes: differentiation response distinguishes KGF from EGF family.

Human keratinocyte growth factor (KGF) is an epithelial cell specific mitogen which is secreted by normal stromal fibroblasts. In the present studies, we demonstrate that KGF is as potent as EGF in stimulating proliferation of primary or secondary human keratinocytes in tissue culture. Exposure of KGF- or EGF-stimulated keratinocytes to 1.0 mM calcium, an inducer of differentiation, led to cessation of cell growth. However, immunologic analysis of early and late markers of terminal differentiation, K1 and filaggrin, respectively, revealed striking differences in keratinocytes propagated in the presence of these growth factors. With KGF, the differentiation response was associated with expression of both markers whereas their appearance was retarded or blocked by EGF. TGF alpha, which also interacts with the EGF receptor, gave a similar response to that observed with EGF. These findings functionally distinguish KGF from the EGF family and support the role of KGF in the normal proliferation and differentiation of human epithelial cells.     

The lepidopteran mitochondrial control region: structure and evolution

For several species of lepidoptera, most of the approximately 350-bp mitochondrial control-region sequences were determined. Six of these species are in one genus, Jalmenus; are closely related; and are believed to have undergone recent rapid speciation. Recent speciation was supported by the observation of low interspecific sequence divergence. Thus, no useful phylogeny could be constructed for the genus. Despite a surprising conservation of control-region length, there was little conservation of primary sequences either among the three lepidopteran genera or between lepidoptera and Drosophila. Analysis of secondary structure indicated only one possible feature in common--inferred stem loops with higher-than-random folding energies-- although the positions of the structures in different species were unrelated to regions of primary sequence similarity. We suggest that the conserved, short length of control regions is related to the observed lack of heteroplasmy in lepidopteran mitochondrial genomes. In addition, determination of flanking sequences for one Jalmenus species indicated (i) only weak support for the available model of insect 12S rRNA structure and (ii) that tRNA translocation is a frequent event in the evolution of insect mitochondrial genomes.      

Glutamine repeats and inherited neurodegenerative diseases: molecular aspects

Several dominantly inherited, late onset, neurodegenerative diseases are due to expansion of CAG repeats, leading to expansion of glutamine repeats in the affected proteins. These proteins are of very different sizes and, with one exception, show no sequence homology to known proteins or to each other; their functions are unknown. In some, the glutamine repeat starts near the N-terminus, in another near the middle and in another near the C-terminus, but regardless of these differences, no disease has been observed in individuals with fewer than 37 repeats, and absence of disease has never been found in those with more than 41 repeats. Protein constructs with more than 41 repeats are toxic to E. coli and to CHO cells in culture, and they elicit ataxia in transgenic mice. These observations argue in favour of a distinct change of structure associated with elongation beyond 37–41 glutamine repeats. The review describes experiments designed to find out what these structures might be and how they could influence the properties of the proteins of which they form part. Poly- -glutamines form pleated sheets of β-strands held together by hydrogen bonds between their amides. Incorporation of glutamine repeats into a small protein of known structure made it associate irreversibly into oligomers. That association took place during the folding of the protein molecules and led to their becoming firmly interlocked by either strand- or domain-swapping. Thermodynamic considerations suggest that elongation of glutamine repeats beyond a certain length may lead to a phase change from random coils to hydrogen-bonded hairpins. Possible mechanisms of expansion of CAG repeats are discussed in the light of looped DNA model structures.     

Food plant choices of two goldenrod beetles: Relation to plant quality

Summary The leaf beetles Trirhabda borealis and T. virgata are specialist herbivores of meadow goldenrods, Solidago spp., in central New York. Upon emergence, both larvae and adults must actively search for food plants, and must choose among the five goldenrod species (S. canadensis, S. gigantea, S. graminifolia, S. juncea and S. rugosa) that co-occur in old fields. The relationship between Trihabda foraging decisions and plant quality was examined by comparing food preferences in the field with the performance of beetles caged on each host. Trirhabda adults were highly selective in their use of food plants. Adults of T. borealis preferred a single host, S. canadensis, while T. virgata adults were most common on S. canadensis and S. gigantea. These preferences were not strictly related to variation in plant quality. In the laboratory, T. borealis performed equally well on four goldenrods (but completely failed to reproduce when fed S. graminifolia), and T. virgata performed equally well on all five hosts.Larval feeding preferences in each beetle species were broader, and were more in accord with subtle variation in plant quality. Newly-hatched T. borealis larvae readily colonized four hosts and rejected only S. graminifolia, which conferred the lowest survivorship and the slowest growth. Larvae of T. virgata accepted each host, even though growth rates were somewhat slower on S. graminifolia and S. juncea.Ontogenetic differences in host preference and host tolerance may have evolved because of the different host-finding abilities of each beetle stage. During host search, the sluggish, newly-emerged larvae may be more food-limited and accept even marginally inferior food plants. The relatively mobile adults are more discriminating and use a subset of suitable hosts. The intermingled dispersion of Solidago species in old fields results in frequent larval colonization of hosts seldom used by adults. In diverse plant communities, ultimate patterns of food plant choice can be a complex function of at least three factors: intrinisic plant quality, local plant dispersion, and the host-search abilities of the insect forager.     

Disinfection of bacteria in water systems by using electrolytically generated copper:silver and reduced levels of free chlorine

As an alternative disinfectant to chlorination, electrolytically generated copper:silver (400 and 40 micrograms/L copper and silver, respectively) with and without free chlorine (0.3 mg/L) was evaluated over a period of 4 weeks in indoor and outdoor water systems (100 L tap water with natural body flora and urine). Numbers of total coliform, pseudomonas, and staphylococci were all less than drinking water standards in systems treated with copper:silver and free chlorine and systems treated with free chlorine alone (1.0 mg/L). No significant differences (p less than or equal to 0.05) in bacterial numbers were observed between systems with copper:silver and free chlorine and those with free chlorine alone. Overall, free-chlorine treatments (0.3 or 1.0 mg/L) showed significantly lower heterotrophic plate numbers than those without free chlorine. When challenged with a natural Staphylococcus sp. isolate, water with copper:silver and free chlorine had a 2.4 log10 reduction in bacterial numbers within 2 min, while free chlorine alone or copper:silver alone showed 1.5 and 0.03 log10 reductions, respectively. Addition of copper:silver to water systems may allow the concentration of free chlorine to be reduced while still providing comparable sanitary quality of the water.