A new strategy for the detection of subtelomeric rearrangements

We present a new strategy for the detection of subtelomeric rearrangements. This approach is based on two hybridizations with different probe sets. The first set consists of microdissected subtelomeric probes (each 5-10 megabases in size) labeled combinatorially employing 7 different fluorochromes. With this set, subtelomeric interchromosomal exchanges can be detected in a 24-color experiment. The second set comprises a second generation of subtelomeric PAC-, P1- and BAC-clones. Probes for p- and q-arms are labeled with two different colors. This second set detects small deletions; in addition it provides regional information, so that translocated material identified by the first probe set can be assigned to the p- or q-arm of a chromosome. The test has been evaluated in a blind study on a series of subtle translocations and deletions.     

Phosphorylation of Synaptic Membrane Glycoproteins: The Effects of Ca2+ and Calmodulin

Synaptic membranes were incubated with [gamma-32P]ATP, and glycoproteins were isolated by affinity chromatography on concanavalin A agarose. Glycoproteins accounted for 1.5-2.5% of the total 32P incorporated into synaptic membrane proteins. Ca2+ and calmodulin enhanced the phosphorylation of synaptic membrane glycoproteins approximately threefold. In the presence of Ca2+ and calmodulin, the rate of glycoprotein dephosphorylation was also increased three- to four-fold. Gel electrophoretic analysis identified several synaptic membrane glycoproteins that incorporated 32P, with the most highly labeled glycoprotein under basal phosphorylating conditions having an apparent Mr of 205,000 (gpiii). Ca2+ and calmodulin produced a marked increase in the phosphorylation of a glycoprotein with an apparent Mr of 180,000 (gpiv) and lesser increases in the labeling of three other glycoproteins. Membranes that had been labeled with [gamma-32P]ATP were extracted with Triton X-100 under conditions that yield a detergent-insoluble residue enriched in postsynaptic structures. The Triton X-100 insoluble residue accounted for 20-25% of the 32P associated with synaptic membrane glycoproteins. Gpiv and other glycoproteins, the phosphorylation of which was stimulated by calmodulin, were located exclusively in the Triton X-100 insoluble residue, whereas gpiii and other calmodulin-insensitive glycoproteins partitioned predominantly into the Triton X-100-soluble fraction. Phosphopeptide maps and phosphoamino acid analysis of gpiv isolated from synaptic membranes and a postsynaptic glycoprotein of apparent Mr of 180,000 (gp180) isolated from synaptic junctions indicated that the former protein was identical to the previously identified postsynaptic-specific gp180. In addition to phosphoserine and phosphothreonine, gpiv also contained phosphotyrosine, identifying it as a substrate for tyrosine-protein kinase as well as for Ca2+/calmodulin-dependent protein kinase.     

Limited Nucleotide Changes in the Rev Response Element (RRE) during HIV-1 Infection Alter Overall Rev-RRE Activity and Rev Multimerization

HIV-1 Rev and the Rev response element (RRE) enable a critical step in the viral replication cycle by facilitating the nuclear export of intron-containing mRNAs, yet their activities have rarely been analyzed in natural infections. This study characterized their genetic and functional variation in a small cohort of HIV-infected individuals. Multiple Rev and RRE sequences were obtained using single-genome sequencing (SGS) of plasma samples collected within 6 months after seroconversion and at a later time. This allowed the identification of cognate sequences that were linked in vivo in the same viral genome and acted together as a functional unit. Phylogenetic analyses of these sequences indicated that 4/5 infections were founded by a single transmission event. Rev and RRE variants from each time point were subjected to functional analysis as both cognate pairs and as individual components. While a range of Rev-RRE activities were seen, the activity of cognate pairs from a single time point clustered to a discrete level, which was termed the set point. In 3/5 patients, this set point changed significantly over the time period studied. In all patients, RRE activity was more sensitive to sequence variation than Rev activity and acted as the primary driver of the cognate set point. Selected patient RREs were also shown to have differences in Rev multimerization using gel shift binding assays. Thus, rather than acting as a simple on-off switch or maintaining a constant level of activity throughout infection, the Rev-RRE system can fluctuate, presumably to control replication.     

Cryopreservation of skin using a murine model: validation of a prognostic viability assay

In order to evaluate the many variables that can affect cryopreservation success, a simple, highly reproducible model system is required. We have evaluated the use of tetrazolium reductase activity as a prognostic indicator of skin viability in an inbred murine model. Two inbred hairless mouse strains were characterized in studies on autografting and allografting following different skin-storage protocols. Skin tetrazolium reductase (TR) activity correlated well with oxygen consumption, and with graft success--the ultimate performance criterion--following varying degrees of cryogenic injury. The assay was shown to be highly reproducible. In a series of factorial experiments the only factors affecting TR activity were those concerning the mouse donors, i.e., mouse strain, age, sex, and body area. The effects of these factors on TR activity were fully characterized.     

Diet preferences of warblers for specific fatty acids in relation to nutritional requirements and digestive capabilities

During energy-demanding periods of the annual cycle such as migration or during cold days in winter, birds store fat comprised mostly of 16- or 18-carbon unsaturated fatty acids. In such situations, birds may feed selectively on foods with specific fatty acids that enable efficient fat deposition. We offered wild-caught yellow-rumped warblers Dendroica coronata paired choices between semi-synthetic diets that differed only in their fatty acid composition. Warblers strongly preferred diets containing long-chain (18:1; carbon atoms:double bonds) unsaturated, unesterified fatty acids to diets containing long-chain saturated, unesterified fatty acids (18:0) and they preferred diets containing mono-unsaturated fats (18:1) to diets containing poly-unsaturated fats (18:2). The preference for diets containing long-chain unsaturated fatty acids to diets containing long-chain saturated fatty acids was consistent in birds tested one week after capture at 21°C, one month after capture when cold-acclimated (1°C), and six weeks after capture at 21°C. Birds acclimated to a diet with 50% of the fat comprised of unesterified stearic acid (18:0) lost mass and reduced their food intake when we reduced ambient temperature from 21°C to 11°C over three days. We conclude that especially in energy-demanding situations there are limits to the yellow-rumped warblers' ability to assimilate some long-chain saturated fatty acids and that this digestive constraint can explain in part why yellow-rumped warblers prefer diets containing long-chain unsaturated fatty acids to diets containing long-chain saturated fatty acids.     

American and korean ginseng tissue cultures: Growth,chemical analysis,and plantlet production

Summary Roots, stems, or leaves of American (Panax quinquefolium) and Korean (Panax ginsing) ginseng were grown as callus or supension tissue cultures. Tissue cultures ofP. ginseng would occasionally form plantlets. The fundamental chemical composition, inorganic analysis, and saponin (panaquilin) content of American and Korean ginseng plants and tissue cultures were determined. The crude saponin content is very similar to, but approximately one-half (1.3%, fresh weight) of that present in ginseng roots. Two-dimensional thin layer chromatographic analysis revealed minor differences in the panaquilins present in American and Korean ginseng tissue cultures. The sapogenin, panaxadiol, was isolated from Korean ginseng callus.