Using the TAP component of the antigen-processing machinery as a molecular adjuvant

We hypothesize that over-expression of transporters associated with antigen processing (TAP1 and TAP2), components of the major histocompatibility complex (MHC) class I antigen-processing pathway, enhances antigen-specific cytotoxic activity in response to viral infection. An expression system using recombinant vaccinia virus (VV) was used to over-express human TAP1 and TAP2 (VV-hTAP1,2) in normal mice. Mice coinfected with either vesicular stomatitis virus plus VV-hTAP1,2 or Sendai virus plus VV-hTAP1,2 increased cytotoxic lymphocyte (CTL) activity by at least 4-fold when compared to coinfections with a control vector, VV encoding the plasmid PJS-5. Coinfections with VV-hTAP1,2 increased virus-specific CTL precursors compared to control infections without VV-hTAP1,2. In an animal model of lethal viral challenge after vaccination, VV-hTAP1,2 provided protection against a lethal challenge of VV at doses 100-fold lower than control vector alone. Mechanistically, the total MHC class I antigen surface expression and the cross-presentation mechanism in spleen-derived dendritic cells was augmented by over-expression of TAP. Furthermore, VV-hTAP1,2 increases splenic TAP transport activity and endogenous antigen processing, thus rendering infected targets more susceptible to CTL recognition and subsequent killing. This is the first demonstration that over-expression of a component of the antigen-processing machinery increases endogenous antigen presentation and dendritic cell cross-presentation of exogenous antigens and may provide a novel and general approach for increasing immune responses against pathogens at low doses of vaccine inocula.     

Reduction of ventricular hypertrophy and fibrosis in spontaneously hypertensive rats by L-arginine

The purpose of this experiment was to explore long-term L-arginine administration on ventricular hypertrophy and cardiac fibrosis in spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) rats. Twenty-four rats of each strain at eight wks of age were divided into two groups--one receiving L-arginine and the other vehicle for twelve wks. Arterial pressure (AP) and heart rate were monitored. At 20 wks of age, the rats' rings of thoracic aorta were isolated to record isometric tension. The study measured left ventricular weight (LVW), body weight (BW), left ventricular (LV) contents of cGMP, and collagen volume fraction (LVCVF). Histological examination of the LV tissue determined changes in cardiomyocytes. Administration of L-arginine did not alter the AP change in SHR, but reduced the AP in WKY after six wks. Our results showed a significantly higher LVW/BW ratio and LVCVF in vehicle-treated SHR compared to levels in corresponding WKY, whereas, the LV cGMP and nitrite/nitrate measurements were higher in vehicle-treated WKY than in SHR. L-Arginine treatment decreased LVW/BW ratio and LVCVF, while increasing the levels of LV cGMP and nitrite/nitrate only in SHR, consistent with histopathological examinations that showed L-arginine prevented cardiomyocytes from thickness and hypertrophy. Our results suggested that the mechanism of reduction in ventricular hypertrophy and fibrosis following long-term L-arginine administration in SHR may stem from increased myocardial nitric oxide-cGMP signaling, independent of AP and EDV of thoracic aorta.     

下丘脑减压区微量注射牛磺酸对血压的影响

实验采用微量注射和荧光分光光度测定的方法,探讨了下丘脑前部减压区牛磺酸对大鼠血压的影响及其可能的机制。结果显示：（１）下丘脑前部减压区微量注射牛磺酸可致大鼠血压降低;（２）侧脑室注射β受体阻断剂心得安可阻断牛磺酸的降压效应。而α受体阻断剂酚妥拉明对牛磺酸的降压效应无明显影响;（３）下丘脑前部减压区注射牛磺酸后,可使下丘脑去甲肾上腺素含量明显增高。     

Development and Use of Field Application Vectors To Express Nonadaptive Foreign Genes in Competitive Environments

Many potential applications of genetically engineered microorganisms in environmental and agricultural biotechnology involve introducing genetic capabilities into nonsterile competitive environments in which they provide no advantage to the host. Field application vectors have been designed for the purpose of creating a temporary niche for the host in such environments. This technique involves the addition to the target environment of a selective substrate readily utilizable by the host microorganism but unavailable to most indigenous species. Thirteen nonionic and anionic detergents, representing a wide range of structural complexities and molecular weights, were screened as potential selective substrates. Competition experiments in soil, using Warburg respirometry, indicated that isolates from six different detergent enrichment cultures were more active on their corresponding detergents than the indigenous microorganisms. Detergents of intermediate structural complexities and molecular weights were most effective for use as selective substrates. A field application vector that utilizes 1.0% Igepal CO-720 (detergent) as the selective substrate and Pseudomonas paucimobilis 1IGP4 as the host was tested for its ability to increase the presence of nonadaptive tetracycline resistance marker genes in soil. In soil amended with the selective substrate, strain 1IGP4 plate counts increased by three orders of magnitude and tetracycline-resistant transformant (pRK293) counts increased from 1.8 × 10⁶/g of soil to 4.3 × 10⁸/g in 2 days. Inoculation in the absence of substrate amendment or amendment with a nonselective substrate did not result in growth of strain 1IGP4. These results demonstrate the effectiveness of field application vectors for increasing the concentration of nonadaptive genes in competitive environments.     

Concordant Evolution of a Symbiont with Its Host Insect Species: Molecular Phylogeny of Genus Glossina and Its Bacteriome-Associated Endosymbiont, Wigglesworthia glossinidia

Many arthropods with restricted diets rely on symbiotic associations for full nutrition and fecundity. Tsetse flies (Diptera: Glossinidae) harbor three symbiotic organisms in addition to the parasitic African trypanosomes they transmit. Two of these microorganisms reside in different gut cells, while the third organism is harbored in reproductive tissues and belongs to the genus Wolbachia. The primary symbiont (genus Wigglesworthia glossinidia) lives in differentiated epithelial cells (bacteriocytes) which form an organ (bacteriome) in the anterior gut, while the secondary (S) symbionts are present in midgut cells. Here we have characterized the phylogeny of Wigglesworthia based on their 16S rDNA sequence analysis from eight species representing the three subgenera of Glossina: Austenina (=fusca group), Nemorhina (=palpalis group), and Glossina (=morsitans group). Independently, the ribosomal DNA internal transcribed spacer-2 (ITS-2) regions from these species were analyzed. The analysis of Wigglesworthia indicated that they form a distinct lineage in the γ subdivision of Proteobacteria and display concordance with their host insect species. The trees generated by parsimony confirmed the monophyletic taxonomic placement of Glossina, where fusca group species formed the deepest branch followed by morsitans and palpalis groups, respectively. The placement of the species Glossina austeni by both the traditional morphological and biochemical criteria has been controversial. Results presented here, based on both the ITS-2 and the symbiont 16S rDNA sequence analysis, suggest that Glossina austeni should be placed into a separate fourth subgenus, Machadomyia, which forms a sister-group relationship with the morsitans group species. Received: 17 March 1998 / Accepted: 1 May 1998     

Substituted 4-hydroxyproline di- and tri-peptides as cytotoxic agents

Summary 4-Hydroxyproline di- and tri-peptides and N-cbz-hydroxypropylglycinamides were observed to be potent cytotoxic agents against the growth of suspended single cells, L-1210, Tmolt₃, and HeLa-S³. The agents were not as potent against the growth of cultured solid tumor cells. Selected derivatives were investigated for their mode of action in Tmolt₃ leukemia cells. The compounds selectively inhibited DNA synthesis at 50 and 100smM. The target site of action of the agents appeared to be the purinede novo pathway with marked inhibition of the activities of the two regulatory enzymes of the pathway, i.e. PRPP amido-transferase and IMP dehydrogenase. d[NTP] pools were reduced by the agents consistent with their overall reduction of DNA synthesis. Other marginally inhibited targets of the agents were r-RNA polymerase and TMP-kinase activities. The DNA molecule itself did not appear to be a target of these agents.     

Identification of the site of inhibition of oncogenic ras-p21-induced signal transduction by a peptide from a ras effector domain

We have previously found that a peptide corresponding to residues 35–47 of the ras-p21 protein, from its switch 1 effector domain region, strongly inhibits oocyte maturation induced by oncogenic p21, but not by insulin-activated cellular wild-type p21. Another ras–p21 peptide corresponding to residues 96–110 that blocks ras–jun and jun kinase (JNK) interactions exhibits a similar pattern of inhibition. We have also found that c-raf strongly induces oocyte maturation and that dominant negative c-raf strongly blocks oncogenic p21-induced oocyte maturation. We now find that the p21 35–47, but not the 96–110, peptide completely blocks c-raf-induced maturation. This finding suggests that the 35–47 peptide blocks oncogenic ras at the level of raf; that activated normal and oncogenic ras–p21 have differing requirements for raf-dependent signaling; and that the two oncogenic-ras-selective inhibitory peptides, 35–47 and 96–110, act at two different critical downstream sites, the former at raf, the latter at JNK/jun, both of which are required for oncogenic ras-p21 signaling.