Heat Shock RNA Levels in Brain and Other Tissues After Hyperthermia and Transient Ischemia

A number of studies have demonstrated increased synthesis of heat shock proteins in brain following hyperthermia or transient ischemia. In the present experiments we have characterized the time course of heat shock RNA induction in gerbil brain after ischemia, and in several mouse tissues after hyperthermia, using probes for RNAs of the 70-kilodalton heat shock protein (hsp70) family, as well as ubiquitin. A synthetic oligonucleotide selective for inducible hsp70 sequences proved to be the most sensitive indicator of the stress response whereas a related rat cDNA detected both induced RNAs and constitutively expressed sequences that were not strongly inducible in brain. Considerable polymorphism of ubiquitin sequences was evident in the outbred mouse and gerbil strains used in these studies when probed with a chicken ubiquitin cDNA. Brief hyperthermic exposure resulted in striking induction of hsp70 and several-fold increases in ubiquitin RNAs in mouse liver and kidney peaking 3 h after return to room temperature. The oligonucleotide selective for hsp70 showed equivalent induction in brain that was more rapid and transient than observed in liver, whereas minimal induction was seen with the ubiquitin and hsp70-related cDNA probes. Transient ischemia resulted in 5- to 10-fold increases in hsp70 sequences in gerbil brain which peaked at 6 h recirculation and remained above control levels at 24 h, whereas a modest 70% increase in ubiquitin sequences was noted at 6 h. These results demonstrate significant temporal and quantitative differences in heat shock RNA expression between brain and other tissues following hyperthermia in vivo, and indicate that hsp70 provides a more sensitive index of the stress response in brain than does ubiquitin after both hyperthermia and ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)     

70-Kilodalton Heat Shock Protein Induction in Cerebellar Astrocytes and Cerebellar Granule Cells In Vitro: Comparison with Immunocytochemical Localization After Hyperthermia In Vivo

Induction of the 70-kDa heat shock protein, hsp70, was evaluated in cultured cerebellar astrocytes and granule cell neurons subjected to a hyperthermic stress, using a monoclonal antibody and an oligonucleotide probe that selectively recognize stress-inducible species of hsp70-related proteins and RNAs, respectively. Immunoblots of cultures enriched in either granule cells or astrocytes, and immunocytochemical localization studies in cocultures of these cell types, demonstrated that hsp70 induction was restricted to the astrocyte population. Amino acid incorporation experiments showed little difference in the loss and recovery of overall protein synthesis activity in these two cell types following transient hyperthermic stress. RNA blot hybridizations confirmed the preferential glial induction of hsp70. In vivo immunocytochemical studies in brains of adult rats following hyperthermia were consistent with earlier observations that suggested a primarily glial and vascular localization of the heat shock response in most brain regions, although the intense immunoreactivity in the cerebellar granule cell layer suggests that there is induction of hsp70 in these neurons under in vivo conditions. These results suggest the potential value of such defined cell cultures in identifying mechanisms responsible for differences in the heat shock response of various cell types in vitro, and in revealing factors that may account for the apparent absence of the stress response in cultured cerebellar granule cell neurons.     

Complete Genomic Sequence of the Human Retinoblastoma Susceptibility Gene

A 180,388-bp contig encompassing the human retinoblastoma gene was sequenced in its entirety. Partial analysis of the sequence revealed (1) a high (A + T)/(G + C) ratio and a high density of Line-1 (L1) repeat sequences, suggesting that the locus maps to G-bands 13q14.12 or 13q14.2; (2) Alu repeats that are asymmetrically oriented over a region extending 87 kb; (3) an overabundance of non-Alu-associated poly(A) tracts 10 bp or larger oriented in the antisense rather than the sense direction (36 vs 6); (4) an Alu sequence nested within an L1 repeat, indicating that the expansion of L1 repeats predates at least some of the Alu expansions; (5) at least three newly discovered microsatellite polymorphisms, one of which was subsequently found to be identical to a polymorphism in a microsatellite-based linkage map of the human genome published by another group; and (6) the basis of previously discovered intragenic RFLPs. This sequence should enhance studies of this locus and of the organization of the human genome.     

Assessment of counting efficiencies for labeled protein and other macromolecules in filter disk assays

A several-fold greater counting efficiency is observed for protein labeled with [³H]leucine than for free [³H]leucine using a conventional filter disk assay. A similar, though less marked, effect is noted for ¹⁴C-labeled molecules. These results are comparable to those reported by others for counting efficiencies of labeled DNA and deoxynucleotides and illustrate the generality of this effect with regard to macromolecules and their low-molecular weight precursors. This phenomenon, presumably due to differences in the distribution of large and small molecules within filters, gives rise to errors in the quantitation of macromolecule synthesis if a counting efficiency identical to that of the precursor is assumed to apply. A convenient method for determining counting efficiencies of various molecules bound to filters is presented which eliminates this problem.     

Simulation modeling reveals the evolutionary role of landscape shape and species dispersal on genetic variation within a metapopulation

Different shapes of landscape boundaries can affect the habitat networks within them and consequently the spatial genetic-patterns of a metapopulation. In this study, we used a mechanistic framework to evaluate the effects of landscape shape, through watershed elongation, on genetic divergence among populations at the metapopulation scale. Empirical genetic data from four, sympatric stream-macroinvertebrates having aerial adults were collected from streams in Japan to determine the roles of species-specific dispersal strategies on metapopulation genetics. Simulation results indicated that watershed elongation allows the formation of river networks with fewer branches and larger topographic constraints. This results in decreased interpopulation connectivity but a lower level of spatial isolation of distal populations (e.g. those found in headwaters) occurring in the landscapes examined. Distal populations had higher genetic divergence when their downstream-biased dispersal (relative to upstream- and/or overland-biased dispersal) was high. This underscores the importance of distal populations influencing genetic divergence at the metapopulation scale for species having downstream-biased dispersal. In turn, lower genetic divergence was observed under watershed elongation when the genetic isolation of distal populations was decreased in such species. This strong association between landscape shape and evolutionary processes highlights the importance of natural, spatial architecture in assessing the effectiveness of conservation and management strategies.     

Human Cdc14A becomes a cell cycle gene in controlling Cdk1 activity at the G2/M transition

Cdc14 belongs to a dual-specificity phosphatase family highly conserved through evolution that preferentially reverses CDK (Cyclin dependent kinases) –dependent phosphorylation events. In the yeast Saccharomyces cerevisiae, Cdc14 is an essential regulator of late mitotic events and exit from mitosis by counteracting CDK activity at the end of mitosis. However, many studies have shown that Cdc14 is dispensable for exiting mitosis in all other model systems analyzed. In fission yeast, the Cdc14 homologue Flp1/Clp1 regulates the stability of the mitotic inducer Cdc25 at the end of mitosis to ensure Cdk1 inactivation before cytokinesis. We have recently reported that human Cdc14A, the Cdc14 isoform located at the centrosomes during interphase, down-regulates Cdc25 activity at the G2/M transition to prevent premature activation of Cdk1-Cyclin B1 complexes and untimely entry into mitosis. Here we speculate about new molecular mechanisms for Cdc14A and discuss the current evidence suggesting that Cdc14 phosphatase plays a role in cell cycle control in higher eukaryotes.     

Genetic diversity of Cryptosporidium spp. in captive reptiles

The genetic diversity of Cryptosporidium in reptiles was analyzed by PCR-restriction fragment length polymorphism and sequence analysis of the small subunit rRNA gene. A total of 123 samples were analyzed, of which 48 snake samples, 24 lizard samples, and 3 tortoise samples were positive for Cryptosporidium: Nine different types of Cryptosporidium were found, including Cryptosporidium serpentis, Cryptosporidium desert monitor genotype, Cryptosporidium muris, Cryptosporidium parvum bovine and mouse genotypes, one C. serpentis-like parasite in a lizard, two new Cryptosporidium spp. in snakes, and one new Cryptosporidium sp. in tortoises. C. serpentis and the desert monitor genotype were the most common parasites and were found in both snakes and lizards, whereas the C. muris and C. parvum parasites detected were probably the result of ingestion of infected rodents. Sequence and biologic characterizations indicated that the desert monitor genotype was Cryptosporidium saurophilum. Two host-adapted C. serpentis genotypes were found in snakes and lizards.