博莱霉素-Ce(Ⅲ)对DNA的切断机理初探

根据外切核酸酶Ⅲ酶解博莱霉素-Ce(Ⅲ)［BLMA5-Ce(Ⅲ)］作用过的双链直线型DNA时, 酶解速率明显增大, 酶解产物除5′-dAMP、5′-dGMP、5′-dCMP和5′-dTMP 4种单核苷酸外, 还有其他成分存在的实验事实, 推测出BLMA5-Ce(Ⅲ)在DNA双链的特定部位沿5′→3′的方向切断磷酸二酯键, 使DNA的双链上形成多个暴露的3′-OH末端.     

Extensive Sequence Conservation Among Insect,Nematode, and Vertebrate Vitellogenins Reveals Ancient Common Ancestry

The eggs of most oviparous animals are provisioned with a class of protein called vitellogenin (Vg) which is stored as the major component of yolk. Until recently, deduced amino acid sequences were available only from vertebrate and nematode Vgs, which proved to be homologous. The sequences of several insect Vgs are now known, but early attempts at pairwise alignments with vertebrate and nematode Vgs have been problematic, leading to conflicting conclusions about how closely insect Vgs are related to the others. In this paper we demonstrate that insect Vg sequences can be confidently aligned with one another along their entire lengths and with multiple vertebrate and nematode Vg sequences along most of their spans. Although divergence is high, conservation among insect, vertebrate, and nematode Vg sequences is widespread with a preponderance of glycine, proline, and cysteine residues among strictly conserved amino acids, establishing conclusively that Vgs from the three phyla are homologous. Areas of least-certain alignment are primarily in and around insect and vertebrate polyserine domains which are not homologous. Phylogenetic reconstructions of Vgs based on sequence identities indicate that the insect lineage is the most diverged and that the mammalian serum protein, apolipoprotein B-100, arose from a Vg ancestor after the nematode/vertebrate divergence. Received: 6 May 1996 / Accepted: 27 September 1996     

Characterization of Calpain-Mediated Proteolysis of GluR1 Subunits of α-Amino-3-Hydroxy-5-Methylisoxazole-4-Propionate Receptors in Rat Brain

Abstract: Previous results have indicated that GluR1 subunits of α-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors are targets of calpain. In the present study, we determined the effects of calpain treatment of synaptic membranes on GluR1 subunits using western blots with antibodies directed against the C-terminal (C-Ab) and the N-terminal (N-Ab) domains of the proteins, and compared them with the effects of calcium treatment of frozen-thawed brain sections. Calpain treatment of synaptic membranes resulted in a large decrease in the GluR1 band (105 kDa) labeled with C-Ab and in the formation of a doublet band labeled with N-Ab due to the appearance of a new species of GluR1 (98 kDa). These effects were blocked almost completely by calpain inhibitors. Calpain-induced changes in GluR1 immunological properties were not associated with modifications of [³H]AMPA or 6-cyano-7-[³H]nitroquinoxaline-2,3-dione ([³H]CNQX) binding. Treatment of frozen-thawed brain sections with concentrations of calcium as low as 0.2 m M resulted in a large decrease in the 105-kDa GluR1 band and in the concurrent appearance of the 98-kDa band. This treatment was associated with increased [³H]AMPA and [³H]CNQX binding. These results suggest that there exist several types/states of GluR1 subunits exhibiting different sensitivities to calpain. Our data also indicate the existence of additional calcium-dependent processes regulating the characteristics of receptors in intact tissues.     

Sequential action of six virus-encoded DNA-packaging RNAs during phage phi29 genomic DNA translocation.

A 120-base pRNA encoded by bacteriophage b29 has a novel and essential role in genomic DNA packaging. Six DNA-packaging RNAs (pRNAs) were bound to the sixfold symmetrical portal vertex of procapsids during the DNA translocation process and left the procapsid after the DNA-packaging reaction was completed, suggesting that the pRNA participated in the translocation of genomic DNA into procapsids. To further investigate the mechanism of DNA packaging, it is crucial to determine whether these six pRNA molecules work as an integrated entity or each pRNA acts as a functional individual. If pRNAs work individually, then do they work in sequence with communication or in random order without interaction? Results from compensation and complementation analysis did not support the integrated model. Computation of the probability of combination between wild-type and mutant pRNAs and experimental data of competitive inhibition excluded the random model while favoring the proposal that the six pRNAs functioned sequentially. Sequential action of the pRNA also explains why the pRNA is so sensitive to mutation, since the effect of a pRNA mutation will be amplified by 6 orders of magnitude after six consecutive steps, resulting in the observed complete loss of DNA-packaging activity caused by small alterations. When any one of the six pRNAs was replaced with an inactive one, complete blockage of DNA packaging resulted, strongly supporting the speculation that individual pRNAs, presumably together with other components such as the packaging ATPase gp16, take turns mediating successive steps of packaging. Although the data provided here could not exclude the integrated model completely, there is no evidence so far to argue against the model of sequential action.     

Human immunodeficiency virus type 2 (HIV-2) seroprevalence and characterization of a distinct HIV-2 genetic subtype from the natural range of simian immunodeficiency virus-infected sooty mangabeys.

The extent of zoonotic infections in rural Sierra Leone, where both feral and pet sooty mangabeys harbor divergent members of the human immunodeficiency virus type 2 (HIV-2)-sooty mangabey simian immunodeficiency virus (SIVsm) family, was tested in blood samples collected from 9,309 human subjects in 1993. Using HIV-1- and HIV-2-specific enzyme immunoassays and confirmatory Western blot analysis to test for antibodies to SIVsm-related lentiviruses, we found only nine subjects (0.096%) who tested positive for HIV: seven tested positive for HIV-1 and two tested positive for HIV-2. Compared with other rural West African communities, Sierra Leone displayed the lowest seroprevalence (0.021%) of HIV-2 infection yet reported, much lower than the previously reported seroprevalence in SIVsm-infected feral and household pet sooty mangabeys. Heteroduplex analysis demonstrated that two of the newly found HIV-1 strains belonged to subtype A, the most common HIV-1 subtype in Africa, but this is the first report of subtype A in Sierra Leone. The two HIV-2-infected individuals harbored two distinct HIV-2 strains, designated 93SL1 and 93SL2. Phylogenetic analysis indicated that HIV-2 93SL1 is a member of HIV-2 subtype A, the first strain of this HIV-2 subtype found in Sierra Leone. In contrast, HIV-2 93SL2 belongs to none of the five previously characterized HIV-2 subtypes (A to E) but is a new subtype, herein designated F, having the most divergent transmembrane sequences yet reported for HIV-2. The fact that both of the two most divergent HIV-2 subtypes known, E and F, are rare and found as single occurrences in persons from Sierra Leone may be related to the fact that this small region of West Africa also contains free-living and household pet sooty mangabeys with highly divergent variants of SIVsm. This finding provides support for the hypotheses that new HIV-2 subtypes result from independent cross-species transmission of SIVsm to the human population and that these single-occurrence transmission events had not spread widely into the population by 1993.     

Characterization of humoral and CD4+ cellular responses after genetic immunization with retroviral vectors expressing different forms of the hepatitis B virus core and e antigens.

The humoral and CD4+ cellular immune responses in mice following genetic immunization with three retroviral vectors encoding different forms of hepatitis B virus core antigen (HBcAg) and e antigen (HBeAg) were analyzed. The retroviral vectors induced expression of intracellular HBcAg (HBc[3A4]), secreted HBeAg (HBe[5A2]), or an intracellular HBcAg-neomycin phosphoryltransferase fusion protein (HBc-NEO[6A3]). Specific antibody levels and immunoglobulin G isotype restriction were highly dependent on both the host major histocompatibility complex and the transferred gene. Humoral and CD4+ cellular HBcAg and/or HBeAg (HBc/eAg)-specific immune responses following retroviral vector immunization were of a lower magnitude but followed the same characteristics compared with those after immunization with HBc/eAg in adjuvant. Two factors influenced the humoral responses. First, in vivo depletion of CD8+ cells in HBc-NEO[6A3]-immunized H-2k mice abrogated both HBcAg-specific antibodies and in vitro-detectable cytotoxic T lymphocytes. Second, priming of H-2b mice with an HBc/eAg-derived T-helper (Th) peptide in adjuvant prior to retroviral vector immunization greatly enhanced the HBc/eAg-specific humoral responses to all three vectors, suggesting that insufficient HBc/eAg-specific CD4+ Th-cell priming limits the humoral responses. In conclusion, direct injection of retroviral vectors seems to be effective in priming HBc/eAg-specific CD8+ but comparatively inefficient in priming CD4+ Th cells and subsequently specific antibodies. However, the limited HBc/eAg-specific CD4+ cell priming can effectively be circumvented by prior administration of a recombinant or synthetic form of HBc/eAg in adjuvant.     

Episodic evolution mediates interspecies transfer of a murine coronavirus.

Molecular mechanisms permitting the establishment and dissemination of a virus within a newly adopted host species are poorly understood. Mouse hepatitis virus (MHV) strains (MHV-A59, MHV-JHM, and MHV-A59/MHV-JHM) were passaged in mixed cultures containing progressively increasing concentrations of nonpermissive Syrian baby hamster kidney (BHK) cells and decreasing concentrations of permissive murine DBT cells. From MHV-A59/MHV-JHM mixed infection, variant viruses (MHV-H1 and MHV-H2) which replicated efficiently in BHK cells were isolated. Under identical treatment conditions, the parental MHV-A59 or MHV-JHM strains failed to produce infectious virus or transcribe detectable levels of viral RNA or protein. The MHV-H isolates were polytrophic, replicating efficiently in normally nonpermissive Syrian hamster smooth muscle (DDT-1), Chinese hamster ovary (CHO), human adenocarcinoma (HRT), primate kidney (Vero), and murine 17Cl-1 cell lines. Little if any virus replication was detected in feline kidney (CRFK) and porcine testicular (ST) cell lines. The variant virus, MHV-H2, transcribed seven mRNAs equivalent in relative abundance and size to those synthesized by the parental virus strains. MHV-H2 was an RNA recombinant virus containing a crossover site in the S glycoprotein gene. At the molecular level, episodic evolution and positive Darwinian natural selection were apparent within the MHV-H2 S and HE glycoprotein genes. These findings differ from the hypothesis that neutral changes are the predominant feature of molecular evolution and argue that changing ecologies actuate episodic evolution in the MHV spike glycoprotein genes that govern interspecies transfer and spread into alternative hosts.     

Effects of chronic exercise and deconditioning on platelet function in women

Wang, Jong-Shyan, Chauying J. Jen, and Hsiun-Ing Chen.Effects of chronic exercise and deconditioning on plateletfunction in women. J. Appl. Physiol.83(6): 2080-2085, 1997.To investigate the effects of chronicexercise and deconditioning on platelet function in women, 16 healthysedentary women were divided into control and exercise groups. Theexercise group cycled on an ergometer at 50% maximal oxygenconsumption for 30 min/day, 5 days/wk, for two consecutive menstrualcycles and then were deconditioned for three menstrual cycles. Duringthis period, platelet adhesiveness on a fibrinogen-coated surface,ADP-induced platelet aggregation and intracellular calciumconcentration elevation, guanosine 3,5-cyclic monophosphate (cGMP) content in platelets, and plasma nitric oxide metabolite levels were measured before and immediately after a progressive exercise test in the midfollicular phase. Ourresults indicated that, after exercise training,1) resting heart rates and bloodpressures were reduced, and exercise performance was improved;2) resting platelet function wasdecreased, whereas plasma nitrite and nitrate levels and platelet cGMPcontents were enhanced; and 3) thepotentiation of platelet function by acute strenuous exercise wasdecreased, whereas the increases in plasma nitrite and nitrate levelsand platelet cGMP contents were enhanced by acuteexercise. Furthermore, deconditioning reversed these training effects. This implies that training-induced platelet functional changes in women in the midfollicular phase may be mediatedby nitric oxide.

     

Inputs from upper airway affect firing of respiratory-associated midbrain neurons

Chen, Zibin, and Frederic L. Eldridge. Inputs fromupper airway affect firing of respiratory-associated midbrain neurons. J. Appl. Physiol. 83(1): 196-203, 1997.In 16 decerebrated unanesthetized cats, we studied effects ofneural inputs from upper airway on firing of 62 mesencephalic neuronsthat also developed respiratory-associated (RA) rhythmic firing whenrespiratory drive was high [Z. Chen, F. L. Eldridge, and P.G.Wagner. J. Physiol. (Lond.) 437:305-325, 1991] and on firing of 16 neurons that did notdevelop the rhythmic firing (non-RA neurons). Activity in RA neuronsincreased after mechanical expansion of pharynx (45% of those tested)or larynx (68%) and after stimulation of glossopharyngeal (50%) orsuperior laryngeal nerves (77%). The increased neuronal firingoccurred despite decreases or abolition of respiratory activity(expressed in phrenic nerve). Neuronal firing also increased aftermechanical stimulation of nasal mucosa (66%) or by jetsof air directed into the nares (48%) and after lightbrushing of nasal skin (~40%). Most stimuli led to decreased firingin a smaller number of neurons, and some neurons showed no response.None of the non-RA neurons developed an increase of firing after any ofthe stimuli, although one had decreased firing after stimulation of thesuperior laryngeal nerve. We conclude that inputs from the upper airwayand nasal skin have independent modulatory effects on the samemesencephalic neurons that are stimulated by ascending rhythmic RAinput from the medulla. These findings may have relevance to generationof the sensation of dyspnea.