Amino acid substitution analysis of E. coli thymidylate synthase: The study of a highly conserved region at the N-terminus

Amino acid substitution analysis within a highly conserved region of Escherichia coli thymidylate synthase (TS), using suppression of amber mutations by tRNA suppressors, has yielded a bank of 124 new mutationally altered TS proteins. These mutant proteins have been used to study the structure-function relationship of the Escherichia coli TS protein at the N-terminus corresponding to residues 20 through 35. This region contains a block of amino acids whose sequence has been well conserved among other known TS proteins from various organisms. Positions 20 through 25 contain a surface loop structure and positions 26 through 35 encompass a β-strand. We find that residues surrounding a β-bulge structure within the β-strand are particularly sensitive to amino acid substitution, suggesting that this structure is maintained by a highly ordered packing arrangement. Three residues in the surface loop that are present at the base of the substrate binding pocket are also sensitive to amino acid substitution. The remainder of the conserved sites, including those at the dimer interface, are tolerant to most, if not all, of the substitutions tested. © 1992 Wiley-Liss, Inc.     

Gastric effects of thyrotropin-releasing hormone microinjected into the dorsal vagal nucleus in cats

We investigated the gastric acid secretory and motility responses to microinjection of thyrotropin-releasing hormone (TRH) into the dorsal motor nucleus of the vagus (DMV) in anesthetized cats. Gastric acid output was collected every 15 min through a gastric cannula after saline flush and titrated to pH 7.0. Antral and corpus contractions were continuously recorded by extraluminal force transducers. TRH dissolved in 200 nl of saline and microinjected unilaterally into the DMV induced a dose-dependent (50-200 ng) increase in gastric acid secretion. The acid secretory response began in the first 15 min collection and lasted 45 min. TRH frequently increased the force of contractions of the antrum and corpus within one minute of microinjection. The minimal effective dose for eliciting increased motility was lower than for inducing acid secretion. These results demonstrate that TRH acts in the DMV of cats to stimulate gastric acid secretion and contractions.     

Paracrinology of growth regulation

Embryonic and fetal growth is dependent on genetic factors and epigenetic factors such as peptide growth factors. We describe here the interactions of several peptide growth factors during the growth and function of two cell types, growth plate chondrocytes from the ovine fetus and astroglial cells from the newborn rat cerebral cortex. Isolated chondrocytes released two endogenous growth factors, basic fibroblast growth factor (bFGF) and insulin-like growth factor II (IGF II). Although the latter was released in greater abundance, as detected by radioimmunoassay, exogenous bFGF was more than a thousand fold more active as a mitogen. Insulin was also able to increase chondrocyte replication at physiological concentrations, and bFGF, insulin and IGFs were additive in their effects on DNA and protein synthesis. Transforming growth factor beta (TGF beta), which is abundant in bone, had little effect on chondrocyte DNA or total protein synthesis alone, but blocked the stimulatory actions of insulin and IGFs on these parameters. However, TGF beta when alone or in combination caused an increase in the collagen: non collagenous protein ratio of new proteins synthesized by chondrocytes. Adult rat brain is a rich source of IGF II, and both IGF I and II are present during neurogenesis and gliagenesis in the fetal and neonatal rat respectively. We have cultured astroglial cells isolated from neonatal rat cerebral cortex to examine the production and interaction of peptide growth factors during their growth. Isolated astroglial cells contained mRNAs encoding both IGF I and II but abundance was not regulated by other hormones or growth factors. Using affinity cross-linking we found that cultured cells also released two species of IGF binding protein (IGF-BP) of 33 kDa and 38 kDa. Northern blot analysis using homologous cDNA probes showed that astroglial cells expressed IGF-BP2 and BP3, but little BP1. Both IGF I and II were mitogenic for astroglial cells, as was insulin at physiologic concentrations. Exogenous IGF-BP2 was able to modulate the mitogenic actions of exogenous IGF I. These two very different cell models show many similarities of endogenous growth control. Both release IGFs and IGF-BPs which regulate mitogenic rate. In addition, in both insulin functions as a growth factor at physiologic concentrations. These findings suggest common principles governing embryonic and fetal growth and development. Studies have shown that fetal and neonatal growth is independent of regulation by classic hormones (e.g. growth hormones) synthesized by the mother or the fetus. It is believed that embryonic and fetal growth is controlled by two major mechanisms, namely, (i) the genetic factors as determined by the embryonic and fetal genome, and (ii) the epigenetic and environmental factors that alter the expression of the embryonic or fetal genome.(ABSTRACT TRUNCATED AT 400 WORDS)     

Recombinant human granulocyte macrophage colony-stimulating factor (rhGM-CSF) induces human macrophage production of transforming growth factor-alpha

Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) is known as an inducer of proliferation and functional activation of myeloid cells. This study was carried out to characterize the effect of purified recombinant human GM-CSF (rhGM-CSF) on induction of TGF-alpha in macrophages. Using Northern blot analysis and immunoassays, we show here that rhGM-CSF markedly stimulates production of TGF-alpha messenger RNA and protein in normal tonsil macrophages. The findings are consistent with macrophages being a normal inducible source of TGF-alpha which may be an important mediator of various activities of GM-CSF both in hematopoietic and non-hematopoietic cells.     

Regional differences in retinoid release from embryonic neural tissue detected by an in vitro reporter assay.

Retinoic acid and related retinoids have been suggested to contribute to the pattern of cell differentiation during vertebrate embryonic development. To identify cell groups that release morphogenetically active retinoids, we have developed a reporter assay that makes use of a retinoic acid inducible response element (RARE) to drive lacZ or luciferase reporter genes in stably transfected cell lines. This reporter gene assay allows detection of retinoids released from embryonic tissues over a range equivalent to that induced by femtomole amounts of retinoic acid. We have used this assay first to determine whether the floor plate, a cell group that has polarizing properties in neural tube and limb bud differentiation, is a local source of retinoids within the spinal cord. We have also examined whether the effects of exogenously administered retinoic acid on anteroposterior patterning of cells in the developing central nervous system correlate with differences in retinoid release from anterior and posterior neural tissue. We find that the release of morphogenetically active retinoids from the floor plate is only about 1.5-fold that of the dorsal spinal cord, which does not have neural tube or limb polarizing activity. These results suggest that the spatial distribution of retinoid release from spinal cord tissues differs from that of the neural and limb polarizing activity. This assay has also shown that retinoids are released from the embryonic spinal cord at much greater levels than from the forebrain. This result, together with previous observations that the development of forebrain structures is suppressed by low concentrations of retinoic acid, suggest that the normal development of forebrain structures is dependent on the maintenance of low concentrations of retinoids in anterior regions of the embryonic axis. This assay has also provided initial evidence that other embryonic tissues with polarizing properties in vivo release retinoids in vitro.     

Identification and characterization of an {alpha}-mannosidase from Trypanosoma cruzi

In this report we describe the first purification and characterizationof the acid -mannosidase from the human parasite Trypanosomacruzi. The purified enzyme exhibited a native mol. wt of 240000 Da and is apparently composed of four identical subunitsof mol. wt 58 000 Da. Each of the four subunits contains oneN-linked high-mannose-type oligosaccharide. The -mannosidaseexhibited a pH optimum of 3.5 and a pI of 5.9. This low pH optimumand the ability of swainsonine to inhibit its activity suggestthat the -mannosidase is a lysosomal enzyme. Antibodies againstthe T.cruzi enzyme did not react with mammalian lysosomal -mannosidaseand, conversely, antibody against a rat lysosomal -mannosidasedid not react with the T.cruzi enzyme. Thus, the T.cruzi enzymeappears to be distinct from its mammalian counterpart. -mannosidase lysosomal enzyme Trypanosoma cruzi     

单眼剥夺对金黄地鼠视觉中枢GABA神经元分布的影响

用免疫细胞化学技术观察了单眼剥夺后金黄地鼠视觉中枢GABA神经元分布的变化。结果表明:单眼剥夺后,金黄地鼠视皮层和上丘的GABA阳性神经元暂时性增多,但剥夺后六个月,其数目显著减少。在单眼剥夺前和剥夺后侧膝体中GABA阳性神经元数目没有明显差异。剥夺眼对侧视皮层GABA阳性神经元数比剥夺眼同侧视皮层GABA神经元数目少。单眼剥夺后视觉中枢GABA神经元类型及形态与剥夺前没有差别。晚期单眼剥夺也能引起视觉中枢GABA神经元数量和分布的变化。以上结果表明,单眼剥夺后视觉中枢抑制神经元的结构发生了变化。     

利用带有原核增强子样序列的新型载体在大肠杆菌中高效表达人α肿瘤坏死因子

将去除信号肽的人肿瘤坏死因子-α(Tumor necrosis factor-α,TNF-α)cDNA插入到带有原核增强子样序列Px的新型表达载体pBV320中,使TNF cDNA 5′端直接置于大肠杆菌trp启动子下游,采用37℃恒温培养,使TNF在大肠杆菌中获得了高效表达,表达活性达1.35(±0.17)×10~6U/L菌液。表达的TNF-α对L929细胞的毒性作用可被抗人肿瘤坏死因子-α的单克隆抗体所中和。表达菌裂解液作SDS-聚丙烯酰胺凝胶电泳,显示有一条分子量与TNF分子量吻合、约为17000道尔顿的蛋白带。利用DEAE-Sepharose阴离子交换层析及Sephacryl S-200凝胶过滤对上述重组人TNF-α进行纯化,获得电泳纯产品,比活性为1.48×10~6U/mg。     

Fetal and variant alpha-fetoproteins are encoded by mRNAs that differ in sequence at the 5' end

The temperature-sensitive RLA209-15 fetal rat hepatocyte line grown at the nonpermissive temperature (40 degrees C, normal phenotype) produces authentic rat alpha-fetoproteins (AFPs) of 69K and 73K (fetal AFPs) which are encoded by a 2.2-kb mRNA. These cells also produce low levels of a 1.7-kb AFP mRNA and a 65K variant AFP when grown at the permissive temperature (33 degrees C, transformed phenotype). Hybrid-selected translation demonstrates that the 1.7-kb AFP mRNA encodes the 65K variant AFP. Northern blot hybridization and S1 nuclease analyses indicate that the 1.7-kb mRNA lacks sequences present in the first seven 5' exons of the 2.2-kb AFP mRNA. However, the 1.7- and 2.2-kb AFP mRNAs share common sequences extending from the beginning of the eighth exon (corresponding to nucleotide 873 of the fetal AFP mRNA) to the 3' end. Primer extension analysis suggests that the 1.7-kb RNA contains additional sequences 5' to the common regions shared by both AFP mRNAs. We have previously shown that adult rat liver produces a 1.7-kb AFP mRNA; we now report the isolation of a cDNA (ARFP5) encoding this variant AFP mRNA from an adult rat liver cDNA library. Restriction endonuclease mapping and sequence analysis of ARFP5 confirm that the 1.7- and 2.2-kb AFP mRNAs share similar sequences at the 3' region (approximately 1.1 kb). However, ARFP5 contains an additional 90 bp variant AFP mRNA-specific 5' sequence which is located in the seventh intron of the rat AFP gene.(ABSTRACT TRUNCATED AT 250 WORDS)