Comparison of arbitrarily primed polymerase chain reaction and ribotyping for subtyping Actinobacillus actinomycetemcomitans

This study investigated the compatibility of arbitrarily primed polymerase chain reaction (AP-PCR) and ribotyping in the characterization of Actinobacillus actinomycetemcomitans , a major pathogen in the mixed anaerobic microflora of human periodontitis. AP-PCR was performed directly on lysed bacterial colonies using a random-sequence 10-base oligonucleotide primer. Ribotyping was carried out by using purified bacterial chromosomal DNA digested with BglI. DNA fragments were separated electrophoretically, blotted onto a nylon membrane and hybridized with the plasmid pKK3535 containing the rRNA operon of Escherichia coli. The two genetic methods were evaluated on isolates from single individuals and from family members. Twelve AP-PCR types and 47 ribotypes were distinguished among 76 A. actinomycetemcomitans isolates of different serotypes. AP-PCR typing and ribotyping gave compatible results in 18 of 20 comparisons. Although AP-PCR detected less genetic heterogeneity in A. actinomycetemcomitans than ribotyping, the rapid and relatively simple AP-PCR technique seems to be sufficiently discriminative to be used in large scale epidemiological studies which preclude the application of the more laborious ribotyping technique.     

南亚和东南亚巨蜥居群的种间亲缘和越南─新种描述

１９９２年１１月１５日从云南省河口县城自由市场的一位越南人手中买得一雌性巨蜥,经鉴定为一新种,定名为越南巨蜥Ｖａｒａｎｕｓｖｉｅｔｎａｍｅｎｓｉｓ。鉴别特征：新种背部鳞片光滑,仅在后半部插有极少的起棱鳞片,不同于Ｖ．ｎｅｂｕｌｏｓｕｓ（Ｂｏｕｌｅｎｇｅｒ,１８８５,１８９０;Ｔａｙｌｏｒ,１９６３）;新种不同于Ｖ．ｓａｌｖａｔｏｒ在于其背部没有眼斑状横斑带（ｏｃｅｌｌｉｍａｒｋｓ）（Ｇｕｅｎｔｈｅｒ,１８６４;Ｈａｒｒｉｓｏｎ等,１９５７;Ｐｏｐｅ,１９３５）;不同于Ｖ．ｉｒｒａｗａｒｄｉｃｕｓ（Ｙａｎｇ等,１９８７）,Ｖ．ｇｒｉｓｅｕｓ和Ｖ．ｍｏｎｉｔｏｒ（Ｓｍｉｔｈ,１９３５）在于有６─７个横向扩大的眼上鳞。文中运用了１５个外部性状对产于南亚,东南亚大陆及岛屿共９个种（含新种）的种间亲缘关系进行了研究,结果认为,种间亲缘关系的亲疏与分布地区相距的远近成正相关。     

新疆六个民族指纹白线的调查研究The hand Print of Six Nationalities in Xinjiang

本文首次报道了新家6各民族6134人（南3181人、女2953人）的指纹白线出现频率。结果表明,白线频率女高于男,民族间亦有差异,其中维吾尔、哈萨克、鸟孜别克族频率接近。塔吉克族居住高寒高原地区,属白色人种,频率明显低于以上3个民族,锡伯族和汉族生活习惯近似,且高于以上4个民族,作者认为,白线出现频率的高低与种族、性别、年龄和群体生长环境及生活习惯有关。We’ve studied 6134 individuals (male 3181, fimale 2953) of six nationalities in Xinjiang.The result is that the white line of hand print is associated with rae,sex,age,grow environments of groups and life habbits.The ifference of races is:The appearant rates of white line of Weivuerite,Hasakese and Wuzibese are near,Tajike’s rate is lower than xiboman’s and han are higher than others.     

A double hairpin structure is necessary for the efficient encapsidation of spleen necrosis virus retroviral RNA.

We conducted a mutational analysis within the previously defined encapsidation sequence (E) for spleen necrosis virus (SNV), an avian retrovirus. We found that two regions are necessary for efficient SNV replication. The first region is a double hairpin structure as proposed by Konings et al. (1992, J. Virol., 66, 632-640); the second region is located downstream of the hairpins. We showed further that the double hairpin structure is required for efficient SNV RNA encapsidation. Our work is the first to demonstrate, via linker-scanning and site-directed mutagenesis, that a specific RNA secondary structure is required for the encapsidation of retroviral RNA. Analysis of a series of mutations within the E region indicates (i) that preserving the secondary structure of the two hairpins is important for efficient encapsidation and (ii) that the stem regions of the hairpins contain specific sequences critical for encapsidation. Within the hairpins, the presence of at least one of the two conserved GACG four-residue loops, but not the moderately conserved bulge sequence of the first hairpin, is crucial for function. The function of the hairpins is independent of the relative order of the two hairpins. However, the two hairpins are not redundant and are not functionally identical. Replacement of SNV double hairpin sequences with those of Moloney murine leukemia virus (M-MLV) has no detectable effect on the replication of SNV-based retrovirus vectors with reticuloendotheliosis virus strain A (REV-A) helper virus. Furthermore, replacement of the entire E sequence of SNV with that of Moloney murine sarcoma virus (M-MSV) and M-MLV results in retroviral vectors that replicate as well as SNV vectors with wild type SNV E. This result indicates that the encapsidation sequences of M-MSV/M-MLV and SNV are not virus specific and that, during packaging of SNV and MLV RNA with viral proteins from REV-A, the encapsidation sequences are recognized largely by their secondary or tertiary structures.     

Displacement of housekeeping proteasome subunits by MHC-encoded LMPs: a newly discovered mechanism for modulating the multicatalytic proteinase complex.

The degradation of cytoplasmic antigens to peptides presented by class I MHC molecules is thought to be mediated by the ubiquitin/proteasome pathway. Support for this view came from our observation that the subunit composition of proteasomes can be changed by interferon-gamma (IFN-gamma) treatment. Thereby two subunits, LMP2 and LMP7, which are encoded in the MHC class II region, are incorporated into the proteasomal complex, whereas other subunits disappear. In the experiments reported in this communication we studied the subunit changes occurring in cell lines where the expression of LMP2 or LMP7 can be regulated individually either by IFN-gamma induction or by applying a new system to control the expression of transfected LMPs. In both situations LMP2 induction leads exclusively to the disappearance of housekeeping subunit 2, whereas LMP7 affects only subunit 10. Subunit 2 was found to be 76% homologous to LMP2. Since incorporation of LMP2 into the proteasomal complex prevents processing of the subunit 2 precursor, we conclude that LMP2 displaces subunit 2 during assembly. Subunit displacement is most likely a general mechanism to modulate the catalytic activity of the proteasomal complex without changing its structure. Furthermore, the controlled incorporation of transfected subunits into the complex offers a new approach to study proteasome function in vivo.     

Functional evidence for a second tumor suppressor gene on human chromosome 17.

The development and progression of human tumors often involves inactivation of tumor suppressor gene function. Observations that specific chromosome deletions correlate with distinct groups of cancer suggest that some types of tumors may share common defective tumor suppressor genes. In support of this notion, our initial studies showed that four human carcinoma cell lines belong to the same complementation group for tumorigenic potential. In this investigation, we have extended these studies to six human soft tissue sarcoma cell lines. Our data showed that hybrid cells between a peripheral neuroepithelioma (PNET) cell line and normal human fibroblasts or HeLa cells were nontumorigenic. However, hybrid cells between the PNET cell line and five other soft tissue sarcoma cell lines remained highly tumorigenic, suggesting at least one common genetic defect in the control of tumorigenic potential in these cells. To determine the location of this common tumor suppressor gene, we examined biochemical and molecular polymorphic markers in matched pairs of tumorigenic and nontumorigenic hybrid cells between the PNET cell line and a normal human fibroblast. The data showed that loss of the fibroblast-derived chromosome 17 correlated with the conversion from nontumorigenic to tumorigenic cells. Transfer of two different chromosome 17s containing a mutant form of the p53 gene into the PNET cell line caused suppression of tumorigenic potential, implying the presence of a second tumor suppressor gene on chromosome 17.     

Induction versus progression of brain tumor development: differential functions for the pRB- and p53-targeting domains of simian virus 40 T antigen.

The ability of simian virus 40-encoded large T antigen to disrupt the growth control of a variety of cell types is related to its ability to interfere with certain cellular proteins, such as p53 and the retinoblastoma susceptibility gene product (pRB). We have used wild-type and mutant forms of T antigen in transgenic mice to dissect the roles of pRB, p53, and other cellular proteins in tumorigenesis of different cell types. In this study, using a cell-specific promoter to target expression specifically to brain epithelium (the choroid plexus) and to B and T lymphoid cells, we characterize the tumorigenic capacity of a T-antigen fragment that comprises only the amino-terminal 121 residues. This fragment (dl1137) retains the ability to interact with pRB and p107 but lacks the p53-binding domain. While loss of the p53-binding region results in loss of the capacity to induce lymphoid abnormalities, dl1137 retains the ability to induce choroid plexus tumors that are histologically indistinguishable from those induced by wild-type T antigen. Tumors induced by dl1137 develop much more slowly, however, reaching an end point at around 8 months of age rather than at 1 to 2 months. Analysis of tumor progression indicates that tumor induction by dl1137 does not require secondary genetic or epigenetic events. Rather, the tumor growth rate is significantly slowed, indicating that the T-antigen C-terminal region contributes to tumor progression in this cell type. In contrast, the pRB-binding region appears essential for tumorigenesis as mutation of residue 107, known to disrupt pRB and p107 binding to wild-type T antigen, abolishes the ability of the dl1137 protein to induce growth abnormalities in the brain.     

Autophosphorylation-dependent protein kinase predominantly phosphorylates Ser115, thein vivo site in brain myelin basic protein

In a previous report [Yanget al., (1987a),J. Biol Chem. 262, 7034–7040], a cyclic-AMP- and calcium-independent brain kinase which requires autophosphorylation for activity was identified as a very potent myelin basic protein (MBP) kinase. In this report, the phosphorylation sites of MBP by this autophosphorylation-dependent protein kinase (autokinase) are further determined by two-dimensional electrophoresis/thin-layer chromatography, phosphoamino acid analysis, high-performance liquid chromatography, tryptic peptide mapping, sequential manual Edman degradation, and direct peptide sequencing. Autokinase phosphorylates MBP on both threonine and serine residues. Three major tryptic phosphopeptide peaks were resolved by C₁₈-reversed phase highper-formance liquid chromatography. Sequential manual Edman degradation together with direct sequence analysis revealed that FS(p)WGAEGQKPGFGYGGR is the phosphorylation site sequence (molar ratio 1.0) for the first major phosphopeptide peak. When mapping with bovine brain MBP sequence, we finally demonstrate Ser¹¹⁵, one of thein vivo phosphorylation sites in MBP, as the major site phosphorylated by autokinase, implicating a physiologically relevant role of autokinase in the regulation of brain myelin function. By using the same approach, we also identified HRDT(p)GILDSLGR (molar ratio 0.9) and TT(p)HYGSLPQK (molar ratio 0.8) as the major phosphorylation site sequences in³²P-MBP phosphorylated by autokinase, further indicating that -Arg-XSer/Thr-(neutral amino acid)₃-(amino acid-containing hydroxyl group such as Ser/Glu/Asp)-(neutral amino acid)₂-may represent a unique consensus sequence motif specifically recognized by this autophosphorylation-dependent multisubstrate/ multifunctional protein kinase in the brain.     

Erythroid expression of the heme-regulated eIF-2 alpha kinase.

The role of heme-regulated eIF-2 alpha kinase (HRI) in the regulation of protein synthesis in rabbit reticulocytes is well documented. Inhibitors of protein synthesis with properties similar to those of HRI have been described in some nonerythroid cell types, but it has not yet been determined whether these eIF-2 alpha kinase activities are mediated by HRI or one or more as yet uncharacterized kinases. We have studied the expression of mRNA, polypeptide, and kinase activities of HRI in various tissues from both nonanemic and anemic rabbits. Our results indicate that HRI is expressed in an erythroid cell-specific manner. HRI is present in the bone marrow and peripheral blood of both nonanemic and anemic rabbits but not in any of the other tissues tested. HRI mRNA is present at low levels in uninduced mouse erythroleukemic (MEL) cells and human K562 cells and accumulates to higher levels upon induction. The accumulation of HRI mRNA in differentiating MEL cells is dependent upon the presence of heme. The addition of 3-amino-1,2,4-triazole (AT), an inhibitor of heme biosynthesis, to the induction medium markedly reduced HRI mRNA accumulation. Simultaneous addition of hemin and AT to the dimethyl sulfoxide induction medium largely prevented the inhibition of HRI mRNA induction by AT. These findings indicate that HRI is expressed in an erythroid cell-specific manner and that the major physiologic role of HRI is in adjusting the synthesis of globins to the availability of heme.