The adaptation of enzymes to temperature: catalytic characterization of glucosephosphate isomerase homologues isolated from Mytilus edulis and Isognomon alatus, bivalve molluscs inhabiting different thermal environments

Homologues of glucosephosphate isomerase (GPI, EC 5.3.1.9) were purified to homogeneity and kinetically characterized from Mytilus edulis and Isognomon alatus, two bivalve molluscs experiencing contrasting thermal environments. The enzyme isolated from I. alatus functions at warmer temperatures (25-35 C) than GPI from M. edulis, a species that inhabits colder marine littoral habitats (5-20 C). The former exhibits apparent first-order (with respect to substrate) catalytic rate constants (Vmax/KM) in vitro that become progressively greater than the mussel enzyme as the assay temperature is raised. Apparent zero-order catalytic rate constants (Vmax) are relatively less differentiated. Catalytic efficiency, defined as the rate at which a catalytic event occurs in either reaction direction for reference standard states (substrate concentrations), is greater for the enzyme from the tropical species (I. alatus) at all realistic combinations of temperature and substrate concentration except for the lowest temperatures and highest substrate concentrations, where the GPI from the boreal/temperate M. edulis is more efficient. This pattern of catalytic divergence appears to be due primarily to differentiation in Vmax/KM. These results and other published data are reviewed and shown to be inconsistent with claims that adaptation of enzymes to higher cell temperatures requires a loss in catalytic efficiency.      

Apolipoprotein E is secreted by cultured lipocytes of the rat liver

Hepatic lipocytes, the retinoid-storing cells of the liver, share several characteristics with vascular smooth muscle cells. To determine whether they also share the characteristic of apolipoprotein E secretion, we have compared the relative mRNA expression and protein secretion of apolipoprotein E, apolipoprotein A-I, and apolipoprotein A-IV in early primary cultures of lipocytes, hepatocytes, and Kupffer cells. Expression of apolipoprotein mRNAs was detected using the polymerase chain reaction and oligonucleotide primers specific for apolipoprotein E, apolipoprotein A-I, and apolipoprotein A-IV. Cellular mRNA concentrations were compared by dot blot analysis, and apolipoprotein secretion was assessed by immunoblot analysis of culture media. Apolipoprotein E mRNA was found in all three cell types, whereas apolipoprotein A-I and A-IV mRNAs were detected only in hepatocytes. Hepatocyte, lipocyte, and Kupffer cell media all contained a Mr approximately 36,000 protein identified by an antibody specific for rat apolipoprotein E. The relative concentration of apolipoprotein E mRNA per microgram of total cellular RNA in lipocytes, hepatocytes, and Kupffer cells was 1.0, 3.0, and 1.6, respectively. The relative secretion of apolipoprotein E per cell was also lowest in lipocytes, being twofold greater in hepatocytes and 1.4-fold greater in Kupffer cells. The secretion of apolipoprotein E by lipocytes is not only an additional smooth muscle cell-like characteristic of the hepatic lipocyte, but also raises the possibility of retinol mobilization upon apolipoprotein secretion.     

Accumulation of apolipoproteins in the regenerating and remyelinating mammalian peripheral nerve. Identification of apolipoprotein D, apolipoprotein A-IV, apolipoprotein E, and apolipoprotein A-I

In this report, we have identified two apolipoproteins (apo), apoD and apoA-IV, that, together with the previously identified apoA-I and apoE, accumulate in the regenerating peripheral nerve. These four apolipoproteins were identified in regenerating rat sciatic nerves by their molecular weights, their isoelectric points, and their recognition by specific antibodies. Antibodies were also used to document the changing concentrations of these apolipoproteins in homogenates of regenerating sciatic nerves collected 1 day to 6 weeks after a denervating crush injury. By 3 weeks after injury, at their peak accumulation, apoA-IV and apoA-I had increased 14- and 26-fold, respectively, relative to their concentrations in the normal nerve. Apolipoproteins D and E, in contrast, increased over 500- and 250-fold, respectively, by 3 weeks. These same apolipoproteins also accumulated in the regenerating sciatic nerves of two other species, the rabbit and the marmoset monkey. Immunocytochemistry showed that apoD was produced by astrocytes and oligodendrocytes in the normal central nervous system, and by neurolemmal or fibroblastic cells in the normal peripheral nervous system. Metabolic labeling of both apoD and apoE by [35S]methionine during an in vitro incubation of regenerating rat sciatic nerve segments confirmed that these apolipoproteins are synthesized by the nerve. Neither apoA-IV nor apoA-I was metabolically labeled, however, suggesting that they enter the nerve from the plasma. The results from this study provide evidence that several different apolipoproteins from various sources may play a role in lipid transport within neural tissues.     

脱辅基脂蛋白E(ApoE)及低密度脂蛋白(LDL)受体在外周神经中的双向运输

本文用免疫细胞化学法研究正常和再生的大白鼠外周神经中脱辅基脂蛋白E(ApoE)和低密度脂蛋白(LDL)受体的运输特性,发现在正常的外周神经中,ApoE及LDL受体在轴浆中均可沿轴突正行和逆行运输;再生的神经可产生ApoE,ApoE可逆行向细胞体转运。ApoE及LDL受体运输特性的研究为ApoE及LDL受体参与神经损伤后的修复和再生提供了进一步的证据。     

Micro-Manipulation of Cells in Mammals

An arrangement of apparatus for applying micro-manipulative procedures to cells in living mammals is described. It was found satisfactory for manipulation of pericapillary cells and capillary endothelium in the greater omentum of the cat.

An animal board, a Bausch and Lomb triple purpose micro-projector and a double Fitz micro-manipulator were mounted on a spring platform. The micro-needles were drawn from Pyrex rods by hand. The stage of the microscope was modified to protect the condenser from fluids. Warm saline solution was carried to the exposed omentum thru flexible rubber tubing.

The use of a micro-dissection chamber which immobilized the part of the omentum under manipulation is explained. The construction of this chamber is shown by diagrams. A camera support was bolted to the base of the micro-manipulator.

The arrangement of apparatus is shown by a photograph.     

Comparative cytotoxicity of fumonisin B1 in two cell lines derived from normal human bronchial epithelial cells using four distinct bioassay techniques

This study focuses on the cytotoxic effects of fumonisin B₁ (FB₁) on both immortalised and immortalised and subsequently transfected normal human bronchial epithelial (NHBE) cells of human origin using four bioassays. While the MTT, Neutral Red and hexosaminidase colorimetric assays showed little difference between the toxic effects on the two related cell lines, the clonogenic assay, measuring cell survival and proliferation, indicated that FB₁ had a more toxic effect on the nontransfected cells. This kind ofin vitro approach using cells which retain many characteristics of normal cell growth and differentiation can go some way to developing evaluation models for food safety in the case of mycotoxin contamination without resorting totally to whole animal testing. Nevertheless, one or two cytotoxicity tests may be inadequate for a complete appraisal of toxic potential: rather, as wide a range of methodologies as feasible should be employed initially before meaningful conclusions may be drawn.