Temperature dependence of fluid phase endocytosis coincides with membrane properties of pig platelets

In previous studies we have shown that platelets take up low molecular weight molecules from the medium by fluid phase endocytosis, a phenomenon that we previously have used to load trehalose into human platelets, after which we have successfully freeze-dried them. We now extend those findings to a species to be used in animal trials of freeze-dried platelets:pigs. Further, we report results of studies aimed at elucidating the mechanism of the uptake. Temperature dependence of fluid-phase endocytosis was determined in pig platelets, using lucifer yellow carbohydrazide (LY) as a marker. A biphasic curve of marker uptake versus temperature was obtained. The activation energy was significantly higher above 22 degrees C (18.7+/-1.8 kcal/mol) than below that critical temperature (7.5+/-1.5 kcal/mol). The activation energy of fluid phase endocytosis in human platelets was 24.1+/-1.6 kcal/mol above 15 degrees C. In order to establish a correlation between the effect of temperature on fluid phase endocytosis and the membrane physical state, Fourier transform infrared spectroscopy (FTIR) and fluorescence anisotropy experiments were conducted. FTIR studies showed that pig platelets exhibit a main membrane phase transition at approximately 12 degrees C, and two smaller transitions at 26 and 37 degrees C. Anisotropy experiments performed with 1,6 diphenyl-1,3,5 hexatriene (DPH) complemented FTIR results and showed a major transition at 8 degrees C and smaller transitions at 26 and 35 degrees C. In order to investigate the relative roles of known participants in fluid phase endocytosis, the effects of several chemical inhibitors were investigated. LY uptake was unaffected by colchicine, methylamine, and amiloride. However, disruption of specific microdomains in the membrane (rafts) by methyl-beta-cyclodextrin reduced uptake of LY by 35%. Treatment with cytochalasin B, which inhibits actin polymerization, reduced the uptake by 25%. We conclude that the inflection point in the LY uptake versus temperature plot at around 22 degrees C is correlated with changes in membrane physical state, and that optimal LY internalization requires an intact cytoskeleton and intact membrane rafts.     

Studies on the Phosphonate Isostere of Nucleoside 3′- and 2′-Phosphates as Precursors of the Related Oligonucleotides

Abstract

Synthesis of 2′,3′-dideoxy-3′-C-(dihydroxyphosphinylmethyl)-adenosine and -thymidine 5, as well as of 2′-deoxy-2′-C-(dihydroxyphosphinylmethyl)-adenosine and -thymidine 9 was accomplished with the use of the universal carbohydrate precursor 3-deoxy-1,2;5,6-di-O-isopropylidene-3-C-(mesyloxymethyl)-α-D-allofuranose (1).     

Effects of amino-acid substitutions in the Brome mosaic virus capsid protein on RNA encapsidation

Brome mosaic virus (BMV) packages its genomic RNAs (RNA1, RNA2, and RNA3) and subgenomic RNA4 into three different particles. However, since the RNAs in the virions have distinct lengths and electrostatic charges, we hypothesize that subsets of the virions should have distinct properties. A glutamine to cysteine substitution at position 120 of the capsid protein (CP) was found to result in a mutant virus named QC that exhibited a dramatically altered ratio of the RNAs in virions. RNA2 was far more abundant than the other RNAs, although the ratios could be affected by the host plant species. RNAs with the QC mutation were competent for replication early in the infection, suggesting that they were either selectively packaged or degraded after packaging. In support of the latter idea, low concentrations of truncated RNA1 that co-migrated with RNA2 were found in the QC virions. Spectroscopic analysis and peptide fingerprinting experiments showed that the QC virus capsid interacted with the encapsidated RNAs differently than did the wild type. Furthermore, wild-type BMV RNA1 was found to be more susceptible to nuclease digestion relative to RNA2 as a function of the buffer pH. Other BMV capsid mutants also had altered ratios of packaged RNAs.     

A new approach to cloning of tandem repetitive DNA sequences

A new approach to screening of the repeated human DNA sequences tandemly arranged in the genome is described. Efficiency of the developed approach for search of tandemly arranged DNA sequences is corroborated by the obtained experimental data.     

Creation of the library of Bordetella pertussis genome clones and detection of clones containing genes for the synthesis of antigenic determinants of the whooping cough microbe

Escherichia coli clones containing hybrid phasmids with the inserts of B. pertussis DNA were obtained with the use of a phasmid vector. The total amount of the clones thus obtained was 97,000, which considerably exceeded the volume of the clone library necessary for the detection of individual genes with probability approximating 1. The hybrid plasmids were shown to contain 6-19 kilobases. The screening of the clone library was carried out by means of the enzyme immunoassay (EIA). The assay was aimed at detecting clones containing the genes of the subunits of B. pertussis lymphocytosis-stimulating factor (LSF). The EIA techniques used in this investigation were based on the capacity of LSF for binding with fetuin. Six clones giving positive response were detected. These data suggest the presence and expression of the genes controlling the synthesis of the antigenic determinants of LSF in E. coli cells.     

Molecular and cytogenetic approach to the mapping of methylated polymorphic restriction fragments of human rRNA genes

Combined restriction with Bam H I and Sal I (or Hpa II) has revealed Bam H I fragment on a non-transcribed spacer of rRNA genes in one out of four individuals under study. Using Ag-staining and hybridization in situ, chromosome 13p+ enriched by inactive rRNA gene copies was found in the given individual. Since Sal I does not restrict methylated sequences and rRNA genes are repressed by methylation, it is concluded that methylated Banm I-restricted rRNA gene fragments of non-transcribed spacer are localized in chromosome 13p+ of the individual in question.     

Lipid and protein changes due to freezing in Dunning AT-1 cells

Defining the process of cellular injury during freezing, at the molecular level, is important for cryosurgical applications. This work shows changes to both membrane lipids and protein structures within AT-1 Dunning prostate tumor cells after a freezing stress which induced extreme injury and cell death. Cells were frozen in an uncontrolled fashion to -20 or -80 degrees C. Freezing resulted in an increase in the gel to liquid crystalline phase transition temperature (T(m)) of the cellular membranes and an increase in the temperature range over which the transition occurred, as determined by Fourier transform infrared spectroscopy (FTIR). Thin layer chromatography (TLC) analysis of total lipid extracts showed free fatty acids (FFA) in the frozen samples, indicating a change in the lipid composition. The final freezing temperature had no effect on the thermotropic response of the membranes or on the FFA content of the lipid fraction. The overall protein secondary structure as determined by FTIR showed only slight changes after freezing to -20 degrees C, in contrast to a strong and apparently irreversible denaturation after freezing to -80 degrees C. Taken together, these results suggest that the decrease in viability between control and frozen cells can be correlated with small changes in the membrane lipid composition and membrane fluidity. In addition, loss of cell viability is associated with massive protein denaturation as observed in cells frozen to -80 degrees C, which was not observed in samples frozen to -20 degrees C.     

4-Trifluoromethylumbelliferyl glycosides as new substrates for revealing diseases connected with hereditary deficiency of lysosome glycosidases.

The following glycosides of 4-trifluoromethylumbelliferone: alpha-D-mannopyranoside, alpha-L-fucopyranoside, alpha-D-glucopyranoside, beta-D-glucopyranoside, alpha-D-galactopyranoside, beta-D-galactopyranoside, alpha-L-iduronide and beta-D-glucuronide were studied. 4-Trifluoromethylumbelliferyl glycosides were shown to be substrates for glycosidases. Some of them were cleaved even better than the corresponding methylumbelliferyl glycosides. 4-Trifluoromethylumbelliferyl glycosides were applied for revealing the corresponding enzyme deficiencies upon diagnosis of Gaucher and Hurler diseases as well as GM1 gangliosidosis and alpha-mannosidosis. 4-Trifluoromethylumbelliferone released after enzymatic hydrolysis of 4-trifluoromethylumbelliferyl glycosides exhibits more contrast yellow fluorescence in UV-light than the blue one of methylumbelliferone upon exposure of enzyme activity on solid supports. Therefore 4-trifluoromethylumbelliferyl glycosides are convenient substrates for revealing glycosidase activity directly in tissue samples, e.g. in placenta, and thus for fast prenatal diagnosis of lysosomal diseases.     

The physical properties of the synovial fluid as a lubrication of joints

The properties of the synovial fluid and the results of the electrophysical studies of the fluid by the thermoelectret method are briefly described. The interrelation between current peaks on the thermostimulated current spectra of thermostimulated currents of the synovial fluid and the liguid-crystalline state of its components was established. The results can be used for developing the methods of diagnostics of synovial joint diseases and design of artificial joints.