Comparative studies on the adhesiveness of granulocytes of guinea pig and man

Summary The high affinity of granulocytes of guinea pig and man to glass surfaces is modified by serum. Native serum contains both an adherence-promoting activity, which is related to complement, and components which reduce the adhesiveness of granulocytes. These components are stable at 56°C for 30 min and are tightly bound to the glass surface. -Lipoproteins are candidates for this adherence reducing ability of serum. Adherence promotion by native serum is mediated by coating the glass surface with C3b/C3bi. Human granulocytes from the peripheral blood adhered to glass surfaces coated by native human or guinea pig serum with C3b/C3bi to almost the same extent as in the presence of native serum, but on guinea pig granulocytes elicited in the peritoneal cavity, a cell surface metalloproteinase degraded the C3b/C3bi, thus reducing the adhesiveness of these cells. This proteinase was inhibited by MgEDTA, DTT, and 1,10-phenanthroline, whereby the high adhesiveness of granulocytes was restored to C3b/C3bi-coated glass.Abbreviations BA benzamidine hydrochloride - BTS Bacillus thuringiensis subtoxicus - DTT dithiothreitol - EAC -amino-caproic acid - gp guinea pig - LDL low density lipoproteins - SEM scanning electron microscopy     

The duplication in Charcot-Marie-Tooth disease type 1a spans at least 1100 kb on chromosome 17p11.2

Summary Recently, it has been shown that Charcot-Marie-Tooth disease type 1a (CMT1a) is linked with a duplication of a DNA segment that is detected by probe VAW409R3, and that is located on chromosome 17p11.2. Here, we show that this duplication also contains VAW412R3a, but not A10-41 and EW503. Accounting for the duplication in recombination analysis, we found recombinants between CMT1a and EW301 and EW502, but not with A10-41, VAW409R3, and VAW412R3. Using pulsed-field gel electrophoresis analysis, we estimated the minimal size of the duplicated region in CMT1a patients to be 1100 kb.     

DNase I sensitivity of nuclear DNA measured by flow cytometry

The DNase I digestion kinetics of DNA in isolated nuclei (from HeLa or murine mammary carcinoma, 67 cells) were assayed flow cytometrically by measuring the changes in ethidium bromide (EtBr) fluorescence following various digestion time intervals. The DNase I digestion curve was characterized by an initial 25-30% increase in fluorescence upon addition of the enzyme, a rapid reduction in fluorescence to approximately 50-55% in 30 minutes, and a limit digest of 45-50% beyond 45 minutes. Throughout digestion, the DNA histogram retained its characteristic bimodal shape, showing that histogram rearrangement was not responsible for the changes in EtBr fluorescence. Irradiation with 5 X 10(6) rads (137Cs-gamma-rays) or exposure to 50 mM EDTA caused an increase in EtBr fluorescence similar to that caused by DNase I, suggesting that DNA nicking and/or chromatin loosening were responsible for this increase. Residual DNA assayed by the solubilization of 14C-TdR (thymidine)-labeled DNA indicated a similar kinetic pattern without the initial increase. However, at the limit digest, the fraction of DNA remaining trichloroacetic acid (TCA) insoluble (10%) was smaller than that measured by loss of EtBr fluorescence (50% of initial, 40% of maximum). Part of this difference was due to the presence of TCA soluble DNA trapped within the nuclear matrix (15-20%). This trapped DNA was released when the digested nuclei were exposed to 0.5-1.0 M NaCl just prior to EtBr staining. Exposure of HeLa cells to three agents that are believed to cause changes in chromatin structure resulted in alterations in the DNase I digestion kinetics measured flow cytometrically.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence studies of Hoechst 33342 with supercoiled and relaxed plasmid pBR322 DNA

The fluorescence properties of Hoechst 33342 (HO 33342) were examined with plasmid pBR322 in the supercoiled (Form I) or relaxed covalently closed circular (Form Io) conformation in order to determine whether qualitative or quantitative differences in fluorescence properties might provide an assay for topological states of DNA. It was found that HO 33342 exhibited a 30% greater fluorescence intensity with Form I pBR322, independent of the dye or DNA concentration. As the dye to DNA ratio was increased, a red shift of approximately 8 nm was observed for HO 33342 complexed with Form I or Form Io. The red shift in fluorescence emission occurred at higher HO 33342 concentrations with Form I vs. Form Io DNA; however, when Form I and Form Io were mixed in various proportions, neither the fluorescent intensity differences nor the HO 33342 concentration at which the wavelength shift occurred could be used to quantitate the relative proportions of topological states present. These results suggest that although the fluorescence properties of HO 33342 complexed with Form I DNA are different than those of HO 33342 complexed with Form Io DNA, the fluorescence assay is not sufficiently sensitive to quantitatively discriminate among a mixture of DNA in various topological states.     

Scavenging of Hydrogen Peroxide in the Endosperm of Ricinus communis by Ascorbate Peroxidase

The fat-storing endosperm of Ricinus communis L. was found tocontain an ascorbate peroxidase (EC 1.11.1.11 [EC] ), which is nearlyas active as catalase (EC 1.11.1.6 [EC] ) in degradation of hydrogenperoxide (H₂O₂) at its physiological concentrations. This ascorbateperoxidase probably functions together with monodehydroascorbatereductase (EC 1.6.5.4 [EC] ) or dehydroascorbate reductase (EC 1.8.5.1 [EC] )and glutathione reductase (EC 1.6.4.2 [EC] ) to remove the H₂O₂ producedduring the transformation of fat to carbohydrate in the glyoxysomes.The activities of these enzymes as well as the content of ascorbateand glutathione increase parallel to the activities of glyoxysomalmarker enzymes during the course of germination. Inhibitionof catalase by aminotriazole results in increases of the ascorbateperoxidase activity and of the glutathione content. All fourenzymes are predominantly localized in the cytosol of the Ricinusendosperm with low activities found in the plastids and themitochondria. The results suggest, that the ascorbate-dependentH₂O₂ scavenging pathway, which has been shown to be responsiblefor the reduction of photosynthetically derived H₂O₂ in thechloroplasts, operates also in the Ricinus endosperm. (Received June 5, 1990; Accepted July 31, 1990)     

Chromosomal assignment of the human smg GDP dissociation stimulator gene to human chromosome 4q21-q25

The 3-end of the cDNA encoding the smg GDP dissociation stimulator (smg GDS) protein shares 100% homology with the previously published expressed sequence tag 00038 site. This site extends the 3-end of the smg GDS gene by 212 bp. It has been localized to human chromosome 4. Here, we have refined the localization of smg GDP to human chromosome 4q21-q25 using a mapping panel of rodent/human somatic cell hybrids containing different parts of chromosome 4. This chromosomal localization of smg GDP to 4q21-25 overlaps with a region of allele loss in primary hepatocellular carcinoma (4q13-q26).HGM symbol: RAP1GDS1     

New methods for estimating the numbers of synonymous and nonsynonymous substitutions

New methods for estimating the numbers of synonymous and nonsynonymous substitutions per site were developed. The methods are unweighted pathway methods based on Kimura's two-parameter model. Computer simulations were conducted to evaluate the accuracies of the new methods, Nei and Gojobori's (NG) method, Miyata and Yasunaga's (MY) method, Li, Wu, and Luo's (LWL) method, and Pamilo, Bianchi, and Li's (PBL) method. The following results were obtained: (1) The NG, MY, and LWL methods give overestimates of the number of synonymous substitutions and underestimates of the number of nonsynonymous substitutions. The major cause for the biased estimation is that these three methods underestimate the number of synonymous sites and overestimate the number of nonsynonymous sites. (2) The PBL method gives better estimates of the numbers of synonymous and nonsynonymous substitutions than those obtained by the NG, MY, and LWL methods. (3) The new methods also give better estimates of the numbers of synonymous and nonsynonymous substitutions than those obtained by the NG, MY, and LWL methods. In addition, estimates of the numbers of synonymous and nonsynonymous sites obtained by the new methods are reasonably accurate. (4) In some cases, the new methods and the PBL method give biased estimates of substitution numbers. However, from the number of nucleotide substitutions at the third position of codons, we can examine whether estimates obtained by the new methods are good or not, whereas we cannot make an examination of estimates obtained by the PBL method. (5) When there are strong transition/transversion and nucleotide-frequency biases like mitochondrial genes, all of the above methods give biased estimates of substitution numbers. In such cases, Kondo et al.'s method is recommended to be used for estimating the number of synonymous substitutions, although their method cannot estimate the number of nonsynonymous substitutions and is time-consuming. These results, particularly result (1), call for reexaminations of some genes. This is because evolutionary pictures of genes have often been discussed on the basis of results obtained by the NG, MY, and LWL methods, which are favorable for the neutral theory of molecular evolution.     

Effects of cadmium,copper, and zinc on growth and thiol content of aquatic hyphomycetes

The effects of cadmium, copper, and zinc on the growth of ten strains ofaquatic hyphomycetes were investigated. On a solid medium, Cd and Cu reducedradial growth of most strains by 50% at concentrations between150–400 µM; in a liquid medium, the strains were more sensitive.The inhibitory effects of zinc were much less severe. Two isolates(Articulospora tetracladia and Tetracladium marchalianum) from a copper-minestream were more resistant against copper than conspecific strains from anon-polluted stream. Heliscus lugdunensis and Varicosporium elodeaeresponded to Cd exposure, but not to Cu or Zn exposure, by increasedsynthesis of SH-containing compounds. Glutathione levels showed a unimodalresponse to increasing Cd and Zn exposure. With copper, glutathionedecreased at intermediate levels of contamination. In the presence of Cd, H. lugdunensis synthesized several unknown sulfur-rich substances that wereabsent or produced at reduced rates in control cultures.