Cloning and Sequencing of the Gene Encoding an Aldehyde Dehydrogenase That Is Induced by Growing Alteromonas sp. Strain KE10 in a Low Concentration of Organic Nutrients

The protein composition of Alteromonas sp. strain KE10 cultured at two different organic-nutrient concentrations was determined by using two-dimensional polyacrylamide gel electrophoresis. The cellular levels of three proteins, OlgA, -B, and -C, were considerably higher in cells grown in a low concentration of organic nutrient medium (LON medium; 0.2 mg of carbon per liter) than cells grown in a high concentration of organic nutrient medium (HON; 200 mg of C liter⁻¹) or cells starved for organic nutrients. In the LON medium, the cellular levels of the Olg proteins were higher at the exponential growth phase than at the stationary growth phase. A sequence of the gene for OlgA revealed that the amino acid sequence had a high degree of similarity to the NAD⁺-dependent aldehyde dehydrogenases of several bacteria. OlgA, expressed in Escherichia coli, catalyzed the dehydrogenation of acetaldehyde, propionaldehyde, and butyraldehyde. The aldehyde dehydrogenase activity of KE10 was higher in cells growing exponentially in LON medium than in HON. OlgA may be involved in the growth under low-nutrient conditions. The physiological role of OlgA is discussed here.     

ENDOMEMBRANE STRUCTURE AND THE CHLOROPLAST PROTEIN TARGETING PATHWAY IN HETEROSIGMA AKASHIWO (RAPHIDOPHYCEAE, CHROMISTA)

Chloroplasts in heterokont algae are surrounded by four membranes and probably originated from a red algal endosymbiont that was engulfed and retained by eukaryotic host. Understanding how nuclear-encoded chloroplast proteins are translocated from the cytoplasm into the chloroplast across these membranes could give us some insights about how the endosymbiont was integrated into the host cell in the process of secondary symbiogenesis. In multiplastid heterokont algae such as raphidophytes, it has been unclear if the outermost of the four membranes surrounding the chloroplast (the chloroplast endoplasmic reticulum [CER] membrane) is continuous with the nuclear envelope and rough endoplasmic reticulum (ER). Here, we report detailed ultrastructural observations of the raphidophyte Heterosigma akashiwo (Hada) Hada ex Y. Hara et Chihara that show that the CER membranes were continuous with ER membranes that had attached ribosomes, implying that the chloroplast with three envelope membranes is located within the ER lumen, that is, topologically the same structure as that of monoplastid heterokont algae. However, the CER membrane of H. akashiwo had very few, if any, ribosomes attached, unlike the CER membranes in other heterokont algae. To verify that proteins are first targeted to the ER, we assayed protein import into canine microsomes using a precursor for a nuclear-encoded chloroplast protein, the fucoxanthin-chlorophyll a / c protein of H. akashiwo. This demonstrated that the precursor has a functional signal sequence for ER targeting and is cotranslationally translocated into the ER, where a signal sequence of about 17 amino acids is removed. Based on these data, we hypothesize that in H. akashiwo , nuclear-encoded chloroplast protein precursors that have been cotranslationally transported into the ER lumen are sorted in the ER and transported to the chloroplasts through the ER lumen.     

ENDOMEMBRANE ULTRASTRUCTURE AND POSSIBLE CHLOROPLAST PROTEIN IMPORT PATHWAY IN HETEROSIGMA AKASHIWO (RAPHIDOPHYCEAE)

Chloroplasts in heterokont algae probably originated from a red algal endosymbiont which was engulfed and retained by a eukaryotic host, and are surrounded by four envelope membranes. The outermost of these membranes is called chloroplast ER (CER) and usually connects with the nuclear envelope. This information, however, is based mainly on studies on single‐plastid heterokont algae. In multi‐plastid heterokont algae, it is still unclear whether CER is continuous with the nuclear envelope. Since nuclear‐encoded chloroplast proteins are synthesized by ribosomes on the ER membrane, clarifying the ER‐CER structure in the heterokont algae is important in order to know the targeting pathway of those proteins. We did a detailed ultrastructural observation of endomembrane systems in a multi‐plastid heterokont alga: Heterosigma akashiwo, and confirmed that the CER membrane was continuous with the ER membrane. However, unlike the CER membranes in other heterokont algae, it seemed to have very few ribosome attached. We also performed experiments for protein targeting into canine microsomes using a precursor for a nuclear‐encoded chloroplast protein, a fucoxanthin‐chlorophyll protein (FCP), of H. akashiwo, to see if the protein is targeted to the ER. It demonstrated that the precursor has a functional signal sequence for ER targeting, and is co‐translationally translocated into the microsomes. Based on these data, we propose a hypothesis that, in H. akashiwo, nuclear‐encoded chloroplast protein precursors that have been co‐translationally inserted into the ER lumen are sorted in the ER and transported to the chloroplasts through the ER.     

The human SDF1 gene polymorphism is located on a mutational hot spot that was identified by the hominoid genome study.

Nucleotide sequences of a part of the stromal cell-derived factor-1 (SDF-1) gene 3' untranslated region were studied among hominoids (chimpanzees, gorillas, orangutans and gibbons). An identical sequence to the human SDF1-3'G allele was found in chimpanzees and gibbons, whereas that to the 3'A allele was found in gorillas. Based on the sequence data and the hominoid phylogenetic relation, it was suggested that an adenine nucleotide at nucleotide position (np) 801 in humans and gorillas was independently introduced into each lineage after the specific divergence and an ancestral hominoid sequence of this site (np 799-802) was deduced as CCGG. The present data showing a mutational hot spot on this site suggest the possible presence of multiple origins of the worldwide distribution of the SDF1-3'A allele in humans.     

Conformational comparison of mu-selective endomorphin-2 with its C-terminal free acid in DMSO solution, by 1H NMR spectroscopy and molecular modeling calculation.

In order to make clear the structural role of the C-terminal amide group of endomorphin-2 (EM2, H-Tyr-Pro-Phe-Phe-NH2), an endogenous mu-receptor ligand, in the biological function, the solution conformations of endomorphin-2 and its C-terminal free acid (EM2OH, H-Tyr-Pro-Phe-Phe-OH), studied using two-dimensional 1H NMR measurements and molecular modeling calculations, were compared. Both peptides were in equilibrium between the cis and trans isomers around the Tyr-Pro omega bond in a population ratio of approximately/= 1:2. The lack of significant temperature and concentration dependence of NH protons suggested that the NMR spectra reflected the conformational features of the respective molecules themselves. Fifty possible 3D structures for the each isomer were generated by the dynamical simulated annealing method under the proton-proton distance constraints derived from the ROE cross-peaks. These energy-minimized conformers, which were all in the phi torsion angles estimated from J(NHCalphaH) coupling constants within +/- 30 degrees, were then classified in groups one or two according to the folding backbone structures. All trans and cis EM2 conformers adopt an open conformation in which their extended backbone structures are twisted at the Pro2-Phe3 moiety. In contrast, the trans and cis conformers of EM2OH show conformational variation between the 'bow'-shaped extended and folded backbone structures, although the cis conformers of its zwitterionic form are refined into the folded structure of the close disposition of C- and N-terminal groups. These results indicate clearly that the substitution of carboxyl group for C-terminal amide group makes the peptide flexible. The conformational requirement for mu-receptor activation has been discussed based on the active form proposed for endomorphin-1 and by comparing conformational features of EM2 and EM2OH.     

Occurrence of unusual tubular invaginations of the plasma membrane in smooth muscle cells of the lamprey,Lampetra japonica

Numerous tubular structures were observed in the surface region of smooth muscle cells making up the vascular walls in the lamprey, Lampetra japonica; they were designated as surface tubules. The limiting membrane of the surface tubules was connected to the plasma membrane, allowing communication of the lumen of the tubule with the extracellular space. Tannic acid reacted with osmium, serving as an extracellular marker, penetrated into the tubules but not into the intracellular organelles, such as the endoplasmic reticulum and the Golgi complex. The surface tubules were grouped in longitudinal parallel rows, separated from each other by tubule-free areas where dense plaques were present. Each tubule was fairly cylindrical (approximately 60 nm in diameter) and often ramified into two or three branches with a blind end. Occasionally, these tubules were encircled by the sarcoplasmic reticulum which was located immediately beneath the plasma membrane. Similar tubules were also observed in the surface region of vascular endothelial cells and fibroblasts in the adventitial connective tissue. The possibility that the surface tubules in the present observations are analogous to the smooth muscle caveolae or the striated muscle T-tubule is discussed.     

Compartmentation of ATP synthesis and utilization in smooth muscle: roles of aerobic glycolysis and creatine kinase

The phosphocreatine content of smooth muscle is of similar magnitude to ATP. Thus the function of the creatine kinase system in this tissue cannot simply be regarded as an energy buffer. Thus an understanding of its role in smooth muscle behavior can point to CK function in other systems. From our perspective CK function in smooth muscle is one example of a more general phenomenon, that of the co-localization of ATP synthesis and utilization. In an interesting and analogous fashion distinct glycolytic cascades are also localized in regions of the cell with specialized energy requirements. Similar to CK, glycolytic enzymes are known to be localized on thin filaments, sarcoplasmic reticulum and plasma membrane. In this chapter we will describe the relations between glycolysis and smooth muscle function and compare and contrast to that of the CK system. Our goal is to more fully understand the significance of the compartmentation of distinct pathways for ATP synthesis with specific functions in smooth muscle. This organization of metabolism and function seen most clearly in smooth muscle is likely representative of many other cell types.     

Endothelin-1 production by normal human cultured keratinocytes and its regulation

The possibility that cultured keratinocytes produce endothelins were investigated. The results showed that cultured keratinocytes derived from normal human skin produce endothelin-1. Moreover, keratinocyte endothelin-1 production was completely inhibited by the presence of actinomycin D in the medium. As in the case of endothelial cells, recombinant interleukin-1beta was capable of promoting endothelin-1 production in keratinocytes, whereas herapin inhibited it. Thrombin also inhibited endothelin-1 production. These results indicate that the mechanism of endothelin-1 production in keratinocytes is slightly different from the mechanism in vascular endothelial cells.     

Rapid Free Radical Reduction in the Perfused Rat Liver

The reduction of nitroxide free radicals was investigated in detail by Electron Paramagnetic Resonance (EPR) spectroscopy in perfused liver. The nitroxide free radical was rapidly reduced to the corresponding hydroxylamine more efficiently at the lower flow rate of 8 [ml/min], while at higher flow rates, the amount of reduced nitroxide showed a significant decrease. Oxidation of hydroxylamine using hydrogen peroxide provided dynamic information concerning the reduction of the free radical within the liver. In addition, liver homogenates were also investigated to determine the level of nitroxide uptake. The results suggested that a portion of the infused nitroxide was taken up by the liver and cleared from the circulation.     

Estimation of the contribution of the bioluminescent reaction rate and quantum yield to the enhancement of firefly bioluminescence in the presence of cationic liposomes.

Cationic liposomes containing phosphatidylcholine, cholesterol and distearyldimethylammonium chloride (DSDAC) enhanced maximum light emission (BL intensity) and total light emission from the firefly bioluminescence (BL) reaction. The increase in BL intensity was interpreted on the basis of the increase in both BL reaction rate and BL quantum yield (PhiBL) of the BL reaction. The increase in BL reaction rate was due to the increase in the localized concentration of BL reactants on the surface of cationic liposomes by electrostatic interaction. On the other hand, the increase in PhiBL was due to the change of light-emitting species in the presence of cationic liposomes. Each contribution of BL reaction rate and PhiBL to the enhancement of the BL intensity was estimated by measuring the BL reaction rate and PhiBL in the presence of cationic liposomes containing various amounts of DSDAC. The contribution of the BL reaction rate to the increase in the BL intensity was found to be two-fold greater than that of PhiBL.