Maximum shortening velocity of smooth muscle: zero load-clamp vs. afterloaded method

Previous reports from this laboratory of force-velocity relationships of canine tracheal smooth muscle (TSM) have presented maximum shortening velocities (Vmax) mathematically derived from the linearized transformation of the Hill equation (A. V. Hill, Proc. Roy. Soc. London, Ser. B., 126:136-195, 1938). Recent technical advances enable us to measure Vmax directly using an electromagnetic lever system that can instantaneously clamp to a zero load, thus we compared values of Vmax derived mathematically and those directly measured on the same TSM strips. Derived Vmax values from afterloaded isotonic shortening curves for loads greater than preload were 0.328 +/- 0.021 optimal length (lO)/s and were not significantly different from zero load-clamp measurements of 0.301 +/- 0.022 lO/s from the same (n = 15) muscles. These data indicate that Vmax values mathematically derived for TSM from conventional isotonic afterloaded force-velocity curves are valid estimates of zero load velocity, because they were not significantly different from values obtained by direct measurement using the zero load-clamp technique.     

Serological similarity of flagellar and mitotic microtubules

An antiserum to flagellar axonemes from sperm of Arbacia punctulata contains antibodies which react both with intact flagellar outer fibers and with purified tubulin from the outer fibers. Immunodiffusion tests indicate the presence of similar antigenic determinants on outer-fiber tubulins from sperm flagella of five species of sea urchins and a sand dollar, but not a starfish. The antibodies also react with extracts containing tubulins from different classes of microtubules, including central-pair fibers and both A- and B-subfibers from outer fibers of sperm flagella, an extract from unfertilized eggs, mitotic apparatuses from first cleavage embryos, and cilia from later embryos. Though most tubulins tested share similar antigenic determinants, some clear differences have been detected, even, in Pseudoboletia indiana, between the outer-fiber tubulins of sperm flagella and blastular cilia. Though tubulins are "actin-like" proteins, antitubulin serum does not react with actin from sea urchin lantern muscle. On the basis of these observations, we suggest that various echinoid microtubules are built of similar, but not identical, tubulins.     

High-yield trapping of EGF-induced receptor dimers by chemical cross-linking

The binding of epidermal growth factor (EGF) to its plasma membrane receptor results in the stimulation of a tyrosyl residue-specific protein kinase, which has been shown to be part of the receptor. The mechanism by which EGF binding give rise to the stimulation of kinase activity is not understood in detail; however, a number of recent studies have implicated receptor dimerization or oligomerization in this process. We prepared Triton X-100 extracts of A431 cells in which the concentration of EGF receptors was on the order of 10(-7) M. When samples of the extracts were incubated with or without EGF and then treated with the high-yield cross-linking reagent bis(sulfosuccinimidyl)suberate (BS3), covalent receptor dimers could be detected in high yield in samples that had been treated with both EGF and BS3, whereas only monomeric receptor was detected in untreated samples or in samples that had been treated with either EGF or BS3. The yield of receptor dimers trapped by cross-linking correlated with the stimulation of autophosphorylation by EGF and with the concentration of EGF present. EGF-induced receptor dimers were also efficiently cross-linked in highly purified receptor preparations, suggesting that EGF-induced dimerization is a process intrinsic to the receptor, requiring no additional accessory proteins.     

Cloning and characterization of cDNAs encoding equine infectious anemia virus tat and putative Rev proteins.

We isolated and characterized six cDNA clones from an equine infectious anemia virus-infected cell line that displays a Rev-defective phenotype. With the exception of one splice site in one of the clones, all six cDNAs exhibited the same splicing pattern and consisted of four exons. Exon 1 contained the 5' end of the genome; exon 2 contained the tat gene from mid-genome; exon 3 consisted of a small section of env, near the 5' end of the env gene; and exon 4 contained the putative rev open reading frame from the 3' end of the genome. The structures of the cDNAs predict a bicistronic message in which Tat is encoded by exons 1 and 2 and the presumptive Rev protein is encoded by exons 3 and 4. tat translation appears to be initiated at a non-AUG codon within the first 15 codons of exon 1. Equine infectious anemia virus-specific tat activity was expressed in transient transfections with cDNA expression plasmids. The predicted wild-type Rev protein contains 30 env-derived amino acids and 135 rev open reading frame residues. All of the cDNAs had a frameshift in exon 4, leading to a truncated protein and thus providing a plausible explanation for the Rev-defective phenotype of the original cells. We used peptide antisera to detect the faulty protein, thus confirming the cDNA sequence, and to detect the normal protein in productively infected cells.     

Cell surface expression of human cytomegalovirus (HCMV) gp55-116 (gB): use of HCMV-recombinant vaccinia virus-infected cells in analysis of the human neutralizing antibody response.

Cell surface expression of the human cytomegalovirus (HCMV) major envelope glycoprotein complex, gp55-116 (gB), was studied by using monoclonal antibodies and an HCMV gp55-116 (gB) recombinant vaccinia virus. HCMV-infected human fibroblasts and recombinant vaccinia virus-infected HeLa cells expresses three electrophoretically distinct proteins of Mr 170,000, 116,000, and 55,000 on their surface. These species have been previously identified within infected cells and purified virions. Two unique neutralizing epitopes were shown to be present on the cell surface gp55-116 (gB). Utilizing HeLa cells infected with the gp55-116 recombinant vaccinia virus as a specific immunosorbent, we have shown that approximately 40 to 70% of the total serum virus-neutralizing activity of a group of individuals with past HCMV infections was directed against this single envelope glycoprotein. The implications of this finding for vaccine development are discussed.     

Physical mapping of the von Recklinghausen neurofibromatosis region on chromosome 17

The von Recklinghausen neurofibromatosis (NF1) locus has been linked to chromosome 17, and recent linkage analyses place the gene on the proximal long arm. NF1 probably resides in 17q11.2, since two unrelated NF1 patients have been identified who possess constitutional reciprocal translocations involving 17q11.2 with chromosomes 1 and 22. We have used a somatic-cell hybrid from the t(17;22) individual, along with other hybrid cell lines, to order probes around the NF1 locus. An additional probe, 17L1, has been isolated from a NotI linking library made from flow-sorted chromosome 17 material and has been mapped to a region immediately proximal to the translocation breakpoint. While neither NF1 translocation breakpoint has yet been identified by pulse-field gel analysis, an overlap between two probes, EW206 and EW207, has been detected. Furthermore, we have identified the breakpoint in a non-NF1 translocation, SP-3, on the proximal side of the NF1 locus. This breakpoint has been helpful in creating a 1,000-kb pulsed-field map, which includes the closely linked NF1 probes HHH202 and TH17.19. The combined somatic-cell hybrid and pulsed-field gel analysis we report here favors the probe order D17Z1-HHH202-TH17.19-CRYB1-17L1-NF1- (EW206, EW207, EW203, L581, L946)-(ERBB2, ERBA1). The agreement in probe ordering between linkage analysis and physical mapping is excellent, and the availability of translocation breakpoints in NF1 should now greatly assist the cloning of this locus.     

我国微茎类吸虫研究:ⅩⅢ.微茎类吸虫成虫结构特征排序

对我国52种微茎类吸虫的18项成虫形态学特征进行主成分分析,结果表明:卵巢位置、子宫延伸位置等7项性状对第一主成分贡献较大,提示描述器官位置的指标是重要的分类依据。52个虫种在前三个主成分上的排序图显示应将其划分成4个亚科。