Influence of para-substituents on the oxidative metabolism of o-nitrophenols by Pseudomonas putida B2.

Pseudomonas putida B2 is able to grow on o-nitrophenol (ONP) as the sole source of carbon and nitrogen. ONP was converted by a nitrophenol oxygenase to nitrite and catechol. Catechol was then attacked by a catechol 1,2-dioxygenase and further degraded through an ortho-cleavage pathway. ONP derivatives which were para-substituted with a methyl-, chloro-, carboxy-, formyl- or nitro-group failed to support growth of strain B2. Relevant catabolic enzymes were characterized to analyze why these derivatives were not mineralized. Nitrophenol oxygenase of strain B2 is a soluble, NADPH-dependent enzyme that is stimulated by magnesium, manganese, and calcium ions. It is active toward ONP, 4-methyl-, 4-chloro-, and to a lesser extent, 4-formyl-ONP but not toward 4-carboxy- or 4-nitro-ONP. In addition, 4-formyl-, 4-carboxy-, and 4-nitro-ONP failed to induce the formation of nitrophenol oxygenase. Catechol 1,2-dioxygenase of strain B2 is active toward catechol and 4-methyl-catechol but only poorly active toward chlorinated catechols. 4-Methyl-catechol is likely to be degraded to methyl-lactones, which are often dead-end metabolites in bacteria. Thus, of the compounds tested, only unsubstituted ONP acts as an inducer and substrate for all of the enzymes of a productive catabolic pathway.     

Structural similarities between corresponding heat-shock proteins from different eucaryotic cells

Effects of heat treatments on chick embryo fibroblasts, Drosophila embryonic cells, and human lymphoblastoid cells have been compared. Cells from all three species synthesize large heat-shock proteins (hsps) with Mr = 70,000 and 84,000-85,000. Different small hsps with Mr between 22,000 and 27,000 are made at high rates in heat-treated chicken and Drosophila cells but could not be observed in human cells. The structural features of the large hsps from cells of the different organisms were compared by three methods of peptide mapping, namely the examination of tryptic digests by two-dimensional thin layer chromatography or by high pressure liquid chromatography and of incomplete V8 digests by polyacrylamide gel electrophoresis. The Mr = 84,000-85,000 polypeptides from all three organisms are closely related, the chicken and human polypeptides having many peptides in common. The relationship between the Mr = 70,000 polypeptides of the different organisms appears to be less close; possible explanations for this latter result are discussed. Rates of synthesis of total as well as poly(A)+ RNA are much lower in heat-treated than in untreated cells of all three organisms. Heat treatments induce dramatic changes in the shape of chick embryo fibroblasts as seen by microscopic examination. Human lymphoblastoid cells do not show changes in shape.     

Modulation of actin mRNAs in cultured vascular cells by matrix components and TGF-β1

Summary Alpha-smooth muscle actin is currently considered a marker of smooth muscle cell differentiation. However, during various physiologic and pathologic conditions, it can be expressed, sometimes only transiently, in a variety of other cell types, such as cardiac and skeletal muscle cells, as well as in nonmuscle cells. In this report, the expression of actin mRNAs in cultured rat capillary endothelial cells (RFCs) and aortic smooth muscle cells (SMCs) has been studied by Northern hybridization in two-dimensional cultures seeded on individual extracellular matrix proteins and in three-dimensional type I collagen gels. In two-dimensional cultures, in addition to cytoplasmic actin mRNAs which are normally found in endothelial cell populations, RFCs expressed α-smooth muscle (SM) actin mRNA at low levels. α-SM actin mRNA expression is dramatically enhanced by TGF-β₁. In addition, double immunofluorescence staining with anti-vWF and anti-α-SM-1 (a monoclonal antibody to α-SM actin) shows that RFCs co-express the two proteins. In three dimensional cultures, RFCs still expressed vWF, but lost staining for α-SM actin, whereas α-SM actin mRNA became barely detectable. In contrast to two-dimensional cultures, the addition of TGF-β₁ to the culture media did not enhance α-SM actin mRNA in three-dimensional cultures, whereas it induced rapid capillary tube formation. Actin mRNA expression was modulated in SMCs by extracellular matrix components and TGF-β₁ with a pattern very different from that of RFCs. Namely, the comparison of RFCs with other cell types such as bovine aortic endothelial cells shows that co-expression of endothelial and smooth muscle cell markers is very unique to RFCs and occurs only in particular culture conditions. This could be related to the capacity of these microvascular endothelial cells to modulate their phenotype in physiologic and pathologic conditions, particularly during angiogenesis, and could reflect different embryologic origins for endothelial cell populations. Supported by a Post-Doctoral Fellowship from the Swiss National Science Foundation (OK) and grant HL-RO1-28373 (JAM) from the Department of Human Services, Public Health Service, Washington, D.C.     

Sequence variation in the outer-surface-protein genes of Borrelia burgdorferi

Borrelia burgdorferi is a spirochete pathogen transmitted among warm- blooded hosts by ixodid ticks. Frequency-dependent selection for variant outer-surface proteins might be expected to arise in this species, since rare variants are more likely to avoid immune surveillance in previously infected hosts. We sequenced the OspA and OspB genes of nine North American strains and compared them with nine strains previously described. For each gene, the mean number of synonymous substitutions per synonymous site and the mean number of nonsynonymous substitutions per nonsynonymous site show only a twofold excess of silent mutations. Synonymous rates vary widely along the OspB protein. Some regions show a significant excess of silent substitutions, while divergence in other regions is constrained by biased base composition or selection. The presence, in antigenically important regions of the protein, of significant variation among strains, as well as evidence for recombination among strains, should be considered in attempts to develop vaccines against this disease.      

Structure and evolution of teleost mitochondrial control regions

We amplified and sequenced the mitochondrial control region from 23 species representing six families of teleost fish. The length of this segment is highly variable among even closely related species due to the presence of tandemly repeated sequences and large insertions. The position of the repetitive sequences suggests that they arise during replication both near the origin of replication and at the site of termination of the D-loop strand. Many of the conserved sequence blocks (CSBs) observed in mammals are also found among fish. In particular, the mammalian CSB-D is present in all of the fish species studied. Study of potential secondary structures of RNAs from the conserved regions provides little insight into the functional constraints on these regions. The variable structure of these control regions suggests that particular care should be taken to identify the most appropriate segment for studies of intraspecific variation. Correspondence to: T.D. Kocher     

Recombinant human [Cys281]insulin-like growth factor-binding protein 2 inhibits both basal and insulin-like growth factor I-stimulated proliferation and collagen synthesis in fetal rat calvariae.

It is recognized that insulin-like growth factors (IGFs) are bound to specific high-affinity insulin-like growth factor-binding proteins (IGFBPs). The role of IGFBPs in bone metabolism is not well established. The effect of recombinant human [Cys281]IGFBP-2 ([Cys281]rhIGFBP-2) on bone formation in 21-day-old fetal rat calvariae was investigated. [Cys281]rhIGFBP-2 was expressed in and purified from conditioned medium of a clonal Chinese hamster ovary cell line. IGF-I-stimulated cell proliferation was inhibited dose dependently by [Cys281]rhIGFBP-2, with half-maximal inhibition observed at 2 x 10(-8) M. Suppression of the IGF-I-stimulated DNA synthesis was observed at an apparent dose ratio of 1:10. [Cys281]rhIGFBP-2 (10(-6) M) also inhibited the basal incorporation of [3H]thymidine into DNA by up to 45%. Insulin-stimulated cell proliferation was not affected in the presence of the binding protein. In addition, [Cys281]rhIGFBP-2 inhibited bone collagen synthesis under basal and IGF-I-stimulated conditions. In contrast, [Cys281]rhIGFBP-2 did not alter the parathyroid hormone-stimulated bone cell proliferation rate. In conclusion, binding of hIGF-I to rhIGFBP-2 results in an inhibition of the actions of free IGF-I on bone cell replication and matrix synthesis. Parathyroid hormone-stimulated cell proliferation is not mediated by an increase in free IGFs.     

Biosynthesis and structure of membrane and secretory immunoglobulins

Summary Almost all of the body's extracellular immunoglobulin (Ig) is derived from Ig-secreting plasma cells of lymphoid tissues. The secreted material is a heterogeneous mixture of different classes and specificities. Lymphoid tissues also contain a large number of essentially non-secretory cells — B lymphocytes — which bear Ig firmly associated with their plasma membranes. Ig molecules thus exist in two functionally different forms, as membrane-bound antigen receptors on the surface of B lymphocytes on the one hand, and as humoral secreted Ig antibodies on the other. On B cells, membrane-bound heavy chains have an apparent mol. wt. slightly larger than that of secreted heavy chains from plasma cells. Membrane-bound but not secreted heavy chains bind detergents, thus suggesting the presence of a hydrophobic region in membrane-bound heavy chains, which is absent in secreted heavy chains. Most investigations have dealt with immunoglobulin M. The two types of IgM heavy chains differ at their carboxy termini. Recent investigations at the nucleic acid level demonstrate that membrane-associated µ chains contain a 41-residue hydrophobic tail adjacent to the last constant domain, whereas secretory µ chains contain a 20-residue hydrophilic tail. At the present time, evidence is accumulating that all membrane-bound Ig heavy chain classes may contain similar hydrophobic structures necessary for anchorage of the molecules into the lipid bilayer.     

A deleterious effect associated with UNH159 is attenuated in twin embryos of an inbred line of blue tilapia Oreochromis aureus

Offspring of a highly inbred gynogenetic line of Oreochromis aureus displayed 12‐fold increase in twinning rate compared to the outbred population. Asymmetric conjoined twins, which consist of a normal embryo attached to a malformed‐atrophic twin, were frequently encountered in both gynogenetic (90·7%) and outbred (38·2%) embryos. The monozygotic origin of these twins was determined using five microsatellite markers. Progeny of heterozygous parents for the microsatellite UNH159 were separated into sub‐sets of twins and normal full‐sibs. Consistent with previous reports, the normal embryo sub‐set exhibited elimination of both types of homozygotes for the UNH159 genetic marker at 2–8 days after fertilization. Unexpectedly, this elimination was less frequent in twins. The UNH159 marker as well as RNA‐binding motif protein, X‐linked (rbmx), SRY‐box containing gene 3 (sox3) and alpha‐thalassemia/mental retardation syndrome X‐linked (atrx) genes were mapped to linkage group 2. These gene orthologues are all located on the mammalian X chromosome and atrx is necessary for the X‐chromosome inactivation.