Changes in the fatty-acid profile of cyanide-utilizing Yersinia species

A cyanide-utilizing Yersinia species was isolated from the cyanide-bearing gold-plating industrial wastewater. Analysis of the fatty acid composition of the organism revealed that it contains large amounts of saturated fatty acids. The unsaturated hydroxy- and cyclopropyl-ring-bearing fatty acids are present in low concentrations. A comparison of the fatty acid composition with other Yersinia species shows that the genus Yersinia appears homogeneous, and that fatty-acid data of Yersiniae do not reflect the distance between Yersiniae species.     

Diacylglycerol-induced membrane targeting and activation of protein kinase Cepsilon: mechanistic differences between protein kinases Cdelta and Cepsilon

Two novel protein kinases C (PKC), PKCdelta and PKCepsilon, have been reported to have opposing functions in some mammalian cells. To understand the basis of their distinct cellular functions and regulation, we investigated the mechanism of in vitro and cellular sn-1,2-diacylglycerol (DAG)-mediated membrane binding of PKCepsilon and compared it with that of PKCdelta. The regulatory domains of novel PKC contain a C2 domain and a tandem repeat of C1 domains (C1A and C1B), which have been identified as the interaction site for DAG and phorbol ester. Isothermal titration calorimetry and surface plasmon resonance measurements showed that isolated C1A and C1B domains of PKCepsilon have comparably high affinities for DAG and phorbol ester. Furthermore, in vitro activity and membrane binding analyses of PKCepsilon mutants showed that both the C1A and C1B domains play a role in the DAG-induced membrane binding and activation of PKCepsilon. The C1 domains of PKCepsilon are not conformationally restricted and readily accessible for DAG binding unlike those of PKCdelta. Consequently, phosphatidylserine-dependent unleashing of C1 domains seen with PKCdelta was not necessary for PKCepsilon. Cell studies with fluorescent protein-tagged PKCs showed that, due to the lack of lipid headgroup selectivity, PKCepsilon translocated to both the plasma membrane and the nuclear membrane, whereas PKCdelta migrates specifically to the plasma membrane under the conditions in which DAG is evenly distributed among intracellular membranes of HEK293 cells. Also, PKCepsilon translocated much faster than PKCdelta due to conformational flexibility of its C1 domains. Collectively, these results provide new insight into the differential activation mechanisms of PKCdelta and PKCepsilon based on different structural and functional properties of their C1 domains.     

Expression and spectroscopic characterization of a large fragment of the mu-opioid receptor

We report here a procedure for the production in Escherichia coli and subsequent purification and characterization of an 80-residue fragment of the human mu-opioid receptor. The fragment ('TM2-3'), which comprises the second and third transmembrane segments as well as the first extracellular loop of the receptor, was expressed as a fusion with glutathione-S-transferase. The fusion protein, which accumulated in insoluble inclusion bodies, was solubilized with N-lauroylsarcosine, and TM2-3 was obtained by thrombin cleavage of the fusion protein followed by reversed-phase HPLC purification. CD spectroscopy of TM2-3 in lysophosphatidylcholine micelles showed that TM2-3 adopts approximately 50% alpha-helical structure in this environment, with the remainder consisting of disordered and/or beta-structure. This is consistent with the assumption of an alpha-helical structure by the two membrane-spanning regions and a nonhelical structure in the loop region of TM2-3. Fluorescence spectroscopy and fluorescence quenching experiments suggested that the extracellular loop lies near the surface of the lysophosphatidylcholine micelle. Our work shows that the study of large receptor fragments is a technically accessible approach to the study of the structural properties of the mu-opioid receptor and, possibly, other G-protein-coupled receptors as well.     

Mechanism of diacylglycerol-induced membrane targeting and activation of protein kinase Cdelta

The regulatory domains of novel protein kinases C (PKC) contain two C1 domains (C1A and C1B), which have been identified as the interaction site for sn-1,2-diacylglycerol (DAG) and phorbol ester, and a C2 domain that may be involved in interaction with lipids and/or proteins. Although recent reports have indicated that C1A and C1B domains of conventional PKCs play different roles in their DAG-mediated membrane binding and activation, the individual roles of C1A and C1B domains in the DAG-mediated activation of novel PKCs have not been fully understood. In this study, we determined the roles of C1A and C1B domains of PKCdelta by means of in vitro lipid binding analyses and cellular protein translocation measurements. Isothermal titration calorimetry and surface plasmon resonance measurements showed that isolated C1A and C1B domains of PKCdelta have opposite affinities for DAG and phorbol ester; i.e. the C1A domain with high affinity for DAG and the C1B domain with high affinity for phorbol ester. Furthermore, in vitro activity and membrane binding analyses of PKCdelta mutants showed that the C1A domain is critical for the DAG-induced membrane binding and activation of PKCdelta. The studies also indicated that an anionic residue, Glu(177), in the C1A domain plays a key role in controlling the DAG accessibility of the conformationally restricted C1A domain in a phosphatidylserine-dependent manner. Cell studies with enhanced green fluorescent protein-tagged PKCdelta and mutants showed that because of its phosphatidylserine specificity PKCdelta preferentially translocated to the plasma membrane under the conditions in which DAG is randomly distributed among intracellular membranes of HEK293 cells. Collectively, these results provide new insight into the differential roles of C1 domains in the DAG-induced membrane activation of PKCdelta and the origin of its specific subcellular localization in response to DAG.     

Cutting edge: CD4 is the receptor for the tick saliva immunosuppressor, Salp15

Salp15 is an Ixodes scapularis salivary protein that inhibits CD4+ T cell activation through the repression of TCR ligation-triggered calcium fluxes and IL-2 production. We show in this study that Salp15 binds specifically to the CD4 coreceptor on mammalian host T cells. Salp15 specifically associates through its C-terminal residues with the outermost two extracellular domains of CD4. Upon binding to CD4, Salp15 inhibits the subsequent TCR ligation-induced T cell signaling at the earliest steps including tyrosine phosphorylation of the Src kinase Lck, downstream effector proteins, and lipid raft reorganization. These results provide a molecular basis to understanding the immunosuppressive activity of Salp15 and its specificity for CD4+ T cells.     

Metabolic response of Bacillus sphaericus 1593M for dual-substrate limitation in continuous and total-cell-retention cultures

A chemically defined medium has been developed to support the growth and the production of mosquito larvicidal factor(s) (MLF) of Bacillus sphaericus 1593M. On the basis of the data of steady-state continuous cultures, it has been understood that acetate can serve as a sole carbon and energy source for B. sphaericus 1593M. Utilization of acetate by B. sphaer-icus 1593M and the production of MLF are further enhanced by the addition of glutamate at low concentrations, both in steady-state continuous as well as in total-cell-retention cultures (TCRC). A two-step TCRC procedure resulted in better biomass and MLF production by B. sphaericus 1593M. It was also found that glutamate can serve as a carbon source as well as a growth factor in the presence of acetate and hence is a partially substitutable carbon source. Received: 3 January 1997 / Accepted: 31 January 1997     

Conformation of a double-membrane-spanning fragment of a G protein-coupled receptor: effects of hydrophobic environment and pH

Overcoming the problems associated with the expression, purification and in vitro handling of membrane proteins requires an understanding of the factors governing the folding and stability of such proteins in detergent solutions. As a sequel to our earlier report (Biochim. Biophys. Acta 1747(2005), 133-140), we describe an improved purification procedure and a detailed structural analysis of a fragment of the mu-opioid receptor ('TM2-3') that comprises the second and third transmembrane segments and the extracellular loop that connects them. Circular dichroism (CD) spectroscopy of TM2-3 in 2,2,2-trifluoroethanol gave a helical content similar to that predicted by published homology models, while spectra acquired in several detergents showed significantly lower helical contents. This indicates that this part of the mu-opioid receptor has an intrinsic propensity to be highly helical in membrane-like environments, but that in detergent solutions, this helical structure is not fully formed. Proteolysis of TM2-3 with trypsin showed that the helical portions of TM2 and TM3 are both shorter than their predicted lengths, indicating that helix-helix interactions in the full-length receptor are apparently important for stabilizing their conformation. Lengthening the alkyl chain of the detergent led to a small but significant increase in the helicity of TM2-3, suggesting that hydrophobic mismatch could play an important role in the stabilization of transmembrane helices by detergents. Protonation of aspartic acid residues in detergent-solubilized TM2-3 also caused a significant increase in helicity. Our results thus suggest that detergent alkyl chain-length and pH may influence membrane protein stability by modulating the stability of individual transmembrane segments.     

Purpose-driven biomaterials research in liver-tissue engineering

Bottom-up engineering of microscale tissue ("microtissue") constructs to recapitulate partially the complex structure-function relationships of liver parenchyma has been realized through the development of sophisticated biomaterial scaffolds, liver-cell sources, and in vitro culture techniques. With regard to in vivo applications, the long-lived stem/progenitor cell constructs can improve cell engraftment, whereas the short-lived, but highly functional hepatocyte constructs stimulate host liver regeneration. With regard to in vitro applications, microtissue constructs are being adapted or custom-engineered into cell-based assays for testing acute, chronic and idiosyncratic toxicities of drugs or pathogens. Systems-level methods and computational models that represent quantitative relationships between biomaterial scaffolds, cells and microtissue constructs will further enable their rational design for optimal integration into specific biomedical applications.     

Fura-2 used as a probe to show elevated intracellular free calcium in platelets of Dahl-sensitive rats fed a high salt diet

Elevated intracellular free calcium concentration [Ca2+]i in vascular smooth muscle cells has been implicated in the pathophysiology of hypertension. Platelet [Ca2+]i was measured using the fluorescent indicator, Fura-2, in Dahl sensitive (DS) and resistant (DR) rats given high (8% NaCl) and low (0.4% NaCl) salt diets, as well as in the spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats. The aim of this study was to show whether [Ca2+]i is elevated in salt induced hypertension. Platelet [Ca2+]i and systolic blood pressure (SBP) were higher (p less than 0.001) in DS rats given a high than low salt diets. In contrast, no changes in platelet [Ca2+]i and SBP were observed in DR rats. In SHR, platelet [Ca2+]i and SBP were higher (p less than 0.001) than in the WKY rats. Platelet [Ca2+]i correlated with SBP in all groups of rats (r = 0.929; p less than 0.001, n = 38). The parallel increase in SBP and [Ca2+]i in the DS high salt rats and the SHR suggests that an increased [Ca2+]i is involved in the pathophysiology of hypertension in the two models which differ with respect to the pathogenesis of their hypertension. This increase in [Ca2+]i therefore seems to reflect an abnormality in [Ca2+]i handling in hypertension regardless of its cause.