Myristoylation of hippocalcin is linked to its calcium-dependent membrane association properties

Hippocalcin, a recently identified Ca(2+)-binding protein of the recoverin family exclusively expressed in the hippocampus, has a primary structure containing three putative Ca(2+)-binding sites (EF-hands) and a possible NH2-terminal myristoylation site. 45Ca blots demonstrated that every three EF-hand domains, expressed as fusion proteins in Escherichia coli, bind Ca2+, indicating that hippocalcin binds 3 mol of Ca2+/mol of protein. To determine whether hippocalcin is myristoylated, hippocalcin mRNA was translated in vitro in the presence of [3H]myristic acid. 3H label was resistant to hydroxylamine treatment, and replacement of NH2-terminal glycine with alanine prevented 3H label incorporation, indicating that in vitro translated hippocalcin covalently bound [3H]myristic acid at the NH2-terminal glycine. In vitro translated hippocalcin is quantitatively myristoylated, as evidenced by an electrophoretic mobility shift of [35S]methionine-labeled protein on two-dimensional gels. Native hippocalcin comigrated precisely with the in vitro translated hippocalcin on two-dimensional gels, suggesting that native hippocalcin is myristoylated. Native and in vitro translated hippocalcins, but not non-myristoylated mutagenic (Gly1-Ala1) hippocalcin, displayed Ca(2+)-dependent membrane association, indicating that myristoylation participates in its Ca(2+)-dependent membrane association properties. In vitro translated hippocalcin bound to phospholipid vesicles somewhat, however, phospholipid association was insufficient for its membrane association properties, suggesting that the NH2-terminal myristoyl moiety on hippocalcin interacts with lipid bilayers and facilitates interaction with other membrane proteins.     

Mlh1 is unique among mismatch repair proteins in its ability to promote crossing-over during meiosis

In eukaryotes, homologs of the bacterial MutS and MutL proteins function in DNA mismatch repair and recombination pathways. The mutL homolog MLH1 is required for nuclear mismatch repair. Previously, cytological analysis of MLH1-deficient mice has implied a role for Mlh1 in crossing-over during meiosis. Here we demonstrate that Saccharomyces cerevisiae diploids containing a deletion of MLH1 have reduced crossing-over in addition to a deficiency in the repair of mismatched DNA during meiosis. Absence of either of the meiosis-specific mutS homologs Msh4 or Msh5 results in a similar reduction in crossing-over. Analysis of an mlh1 msh4 double mutant suggests that both genes act in the same pathway to promote crossing-over. All genetic markers analyzed in mlh1 mutants display elevated frequencies of non-Mendelian segregation. Most of these events are postmeiotic segregations that represent unrepaired heteroduplex. These data suggest that either restorational repair is frequent or heteroduplex tracts are shorter in wild-type cells. Comparison of mlh1 segregation data with that of pms1, msh2, msh3, and msh6 mutants show that the ability to promote crossing-over is unique to MLH1. Taken together these observations indicate that both crossing-over and gene conversion require MutS and MutL functions and that Mlh1 represents an overlap between these two pathways. Models of Mlh1 function are discussed.     

The physiological position of the liver in the circulation is not a major determinant of its functional capacity

The zonal patterns of gene expression in the liver of the rat are not affected by alteration of the afferent hepatic blood source. We investigated whether afferent hepatic blood source or flow rate affects the metabolic capacity of the liver. Using microsurgical techniques, we changed the afferent hepatic blood source to solely arterial blood, solely portal blood or solely caval blood. The transhepatic flow rate was four times higher in arterialized than in cavalized livers. Liver function was tested 2 wk after surgery. Three liver functions were tested (elimination of hepatic iminodiacetic acid from the liver and elimination of galactose and ammoniumbicarbonate from the circulation). Our results show that the afferent hepatic blood flow rate rather than the source of the afferent hepatic blood affects the elimination of the substrates tested. We found that at the physiological flow rate of approximately 15 ml/min and beyond, metabolic function does not depend on the flow of the afferent hepatic blood but that at lower flow rates the flow becomes a major determinant of the metabolic function of the liver. We conclude that the position of the liver within the circulation (i.e. between the gastrointestinal tract and the systemic circulation) is apparently not a prerequisite for adequate metabolic activity, at least for the substrates tested, provided that the liver is sufficiently perfused with blood.     

Phototactic migration of Dictyostelium cells is linked to a new type of gelsolin-related protein

The molecular and functional characterization of a 125-kDa Ca2+-extractable protein of the Triton X-100-insoluble fraction of Dictyostelium cells identified a new type of a gelsolin-related molecule. In addition to its five gelsolin segments, this gelsolin-related protein of 125 kDa (GRP125) reveals a number of unique domains, two of which are predicted to form coiled-coil regions. Another distinct attribute of GRP125 concerns the lack of sequence elements known to be essential for characteristic activities of gelsolin-like proteins, i.e. the severing, capping, or nucleation of actin filaments. The subcellular distribution of GRP125 to vesicular compartments suggests an activity of GRP125 different from actin-binding, gelsolin-related proteins. GRP125 expression is tightly regulated and peaks at the transition to the multicellular pseudoplasmodial stage of Dictyostelium development. GRP125 was found indispensable for slug phototaxis, because slugs fail to correctly readjust their orientation in the absence of GRP125. Analysis of the GRP125-deficient mutant showed that GRP125 is required for coupling photodetection to the locomotory machinery of slugs. We propose that GRP125 is essential in the natural environment for the propagation of Dictyostelium spores. We also present evidence for further representatives of the GRP125 type in Dictyostelium, as well as in heterologous cells from lower to higher eukaryotes.     

CTL epitope generation is tightly linked to cellular proteolysis of a Listeria monocytogenes antigen

Listeria monocytogenes is a pathogenic intracellular bacterium that secretes proteins into the cytosol of host cells. A major secreted protein, p60, is processed by the host cell into the nonamer peptides p60 217-225 and p60 449-457, which are presented to CTL by H-2Kd MHC class I molecules. Herein, we use two membrane permeable peptide aldehyde protease inhibitors, LLnL and Z-LLF, to inhibit cytosolic proteolysis in L. monocytogenes-infected cells. These inhibitors, which have been shown to inhibit proteasomes, completely abrogate cytosolic p60 degradation. The effect of LLnL and Z-LLF on p60 epitope generation was determined by acid-eluting, HPLC-purifying, and quantifying p60 217-225 and p60 449-457 from infected cells. We show a direct linkage between p60 degradation and epitope generation. However, the two inhibitors have quantitatively different effects on the generation of the two epitopes. Our findings implicate proteasomes in the earliest stages of Ag degradation and suggest that different CTL epitopes can be generated by distinct proteolytic processes.     

Leukocyte infection by the granulocytic ehrlichiosis agent is linked to expression of a selectin ligand

Human granulocytic ehrlichiosis (HGE) is an emerging tickborne illness caused by an intracellular bacterium that infects neutrophils. Cells susceptible to HGE express sialylated Lewis x (CD15s), a ligand for cell selectins. We demonstrate that adhesion of HGE to both HL60 cells and normal bone marrow cells directly correlates with their CD15s expression. HGE infection of HL60 cells, bone marrow progenitors, granulocytes, and monocytes was blocked by monoclonal antibodies against CD15s. However, these antibodies did not inhibit HGE binding, and anti-CD15s was capable of inhibiting the growth of HGE after its entry into the target cell. In contrast, neuraminidase treatment of HL60 cells prevented both HGE binding and infection. A cloned cell line (HL60-A2), derived from HL60 cells and resistant to HGE, was deficient in the expression of alpha-(1, 3)fucosyltransferase (Fuc-TVII), an enzyme known to be required for CD15s biosynthesis. Less than 1% of HL60-A2 cells expressed CD15s, and only these rare CD15s-expressing cells bound HGE and became infected. After transfection with Fuc-TVII, cells regained CD15s expression, as well as their ability to bind HGE and become infected. Thus, CD15s expression is highly correlated with susceptibility to HGE, and it, and/or a closely related sialylated and alpha-(1,3) fucosylated molecule, plays a key role in HGE infection, an observation that may help explain the organism's tropism for leukocytes.     

Apolipoprotein E genotype is a determinant of low-density lipoprotein cholesterol and of its response to a low-cholesterol diet in Type 1 diabetic patients with elevated urinary albumin excretion

BACKGROUND: Immunoablative high-dose cyclophosphamide without stem-cell rescue induces durable, complete remission in most patients with aplastic anemia. OBJECTIVE: To determine the efficacy of high-dose cyclophosphamide in various refractory, severe autoimmune diseases. DESIGN: Prospective phase II study. SETTING: Johns Hopkins University (Baltimore, Maryland) and Hahnemann University (Philadelphia, Pennsylvania). PATIENTS: Eight patients with refractory, severe autoimmune disease. Intervention: Immunoablative high-dose cyclophosphamide (50 mg/kg of body weight per day) for 4 consecutive days. MEASUREMENTS: Clinical and laboratory variables of autoimmune disease. RESULTS: Seven patients improved markedly: Five achieved complete remission and two achieved partial remission. Four patients have remained in continuous complete remission for 3 to 21 months, and two patients in partial remission continue to improve after 14 and 19 months of follow-up. High-dose cyclophosphamide was well tolerated; median times to a neutrophil count of 0.5 x 10(9) cells/L and platelet transfusion independence were 17 and 16 days, respectively. CONCLUSIONS: Immunoablative high-dose cyclophosphamide without stem-cell rescue can induce complete remission in patients with refractory, severe autoimmune disease. Reemergence of marrow function is similar to that seen after autologous transplantation and does not carry the risk for reinfusion of autoaggressive lymphocytes with the autograft.     

A unique variant of a homeobox gene related to Drosophila cut is expressed in mouse testis

The wild-type gene encoding granule-bound starch synthase (GBSS) is capable of both complementing the amylose-free (amf) potato mutant and inhibiting the endogenous GBSS gene expression in wild-type potato. Co-suppression of the endogenous GBSS gene, easily visualised by staining the starch with iodine, occurred when the full-size GBSS sequence (genomic), GBSS cDNA or even the mutant amf allele were introduced into the wild-type potato. Conversely, introduction of the GBSS promoter sequence alone, did not result in co-suppression in the 80 analysed transformants. Neither the orientation of the GBSS gene with respect to kanamycin resistance nor the presence of an enhancer influenced the frequency of plants showing a co-suppression phenotype. After crossing a partially complemented amf mutant with a homozygous wild-type plant, the F1 offspring segregated into plant phenotypes with normal and decreased expression of the GBSS gene. This decreased expression correlated with the presence of a linked block of five T-DNA inserts which was previously shown to be correlated with partial complementation of the amf mutant. This crossing experiment indicates that co-suppression can cause inhibition of gene expression of both inserted and endogenous wild-type GBSS genes. The frequency of partially complemented amf plants was equal to the frequency of co-suppressed wild types when a construct, with an enhancer in front of the GBSS promoter, was used (pWAM 101E). This might suggest that partial complementation of the amf genotype caused by unstable expression of the transgene can be overcome by inserting an enhancer in front of the GBSS promoter.     

Acquisition of chemoresistance in a human ovarian carcinoma cell is linked to a defect in cell cycle control

Chemoresistance is a major concern in cancer erradication; it involves various mechanisms, including defects in the apoptosis program induced by anticancer drugs. In order to further explore the mechanisms underlying the development of chemoresistance in ovarian carcinoma after cisplatin treatment, we established an in vitro model, mimicking a clinical protocol of administration of cisplatin. Therefore, IGROV1 ovarian carcinoma cells were exposed for 2 hr to the drug and allowed to recover for several weeks; this way of exposure was reiterated with escalating doses. We followed changes in cytotoxicity of the drug, cell cycle kinetics and long-term survival of cells after cisplatin treatment, and found that resistance to cisplatin was not associated with altered apoptosis pathway, since both cisplatin sensitive and resistant cells underwent apoptosis in a similar way. Acquisition of resistance to cisplatin was associated with the ability of the treated cells to progress through the cell cycle beyond the G1/S checkpoint; although most cells died by apoptosis, a few surviving cells proliferated and recolonized the cultures. Compared to sensitive cells, the chemoresistant variants were able to override the G1/S checkpoint whatever the dose, and the recurrent cells recolonized the cultures much faster. Analysis of alterations in gene expression suggests that the defect in cell cycle regulation could take place at the level of the cdk inhibitor p21(CIP1/WAF1).     

Thermodynamic and immunological properties of a monoclonal antibody to human blood group A

A murine anti-A monoclonal antibody was obtained by the hybridoma technique. This antibody, of an IgM nature, is capable of agglutinating A1, A2 and A3 red blood cells. Thermodynamic study confirmed its monoclonal character; its association constant is 1,6 10(6) l/mole. The enthalpy change of the antigen/antibody reaction is nul which indicates the absence of the role of temperature on antibody fixation. This weak affinity makes it necessary to concentrate the supernatant so as to enable use of these reagents under the same conditions as those used at present.     

Selective interactions among the multiple connexin proteins expressed in the vertebrate lens: the second extracellular domain is a determinant of compatibility between connexins

Gap junctions are collections of intercellular channels composed of structural proteins called connexins (Cx). We have examined the functional interactions of the three rodent connexins present in the lens, Cx43, Cx46, and Cx50, by expressing them in paired Xenopus oocytes. Homotypic channels containing Cx43, Cx46, or Cx50 all developed high conductance. heterotypic channels composed of Cx46 paired with either Cx43 or Cx50 were also well coupled, whereas Cx50 did not form functional channels with Cx43. We also examined the functional response of homotypic and heterotypic channels to transjunctional voltage and cytoplasmic acidification. We show that all lens connexins exhibited sensitivity to cytoplasmic acidification as well as to voltage, and that voltage-dependent closure of heterotypic channels for a given connexin was dramatically influenced by its partner connexins in the adjacent cell. Based on the observation that Cx43 can discriminate between Cx46 and Cx50, we investigated the molecular determinants that specify compatibility by constructing chimeric connexins from portions of Cx46 and Cx50 and testing them for their ability to form channels with Cx43. When the second extracellular (E2) domain in Cx46 was replaced with the E2 of Cx50, the resulting chimera could no longer form heterotypic channels with Cx43. A reciprocal chimera, where the E2 of Cx46 was inserted into Cx50, acquired the ability to functionally interact with Cx43. Together, these results demonstrate that formation of intercellular channels is a selective process dependent on the identity of the connexins expressed in adjacent cells, and that the second extracellular domain is a determinant of heterotypic compatibility between connexins.     

The lunatic fringe gene is a target of the molecular clock linked to somite segmentation in avian embryos

The most obvious segments of the vertebrate embryo are the trunk mesodermal somites which give rise to the segmented vertebral column and the skeletal muscles of the body. Mechanistic insights into vertebrate somitogenesis have recently been gained from observations of rhythmic expression of the avian hairy-related gene (c-hairy1) in chick presomitic mesoderm (PSM), suggesting the existence of a molecular clock linked to somite segmentation ([1]; reviewed in [2]). Here, we show that lunatic Fringe (IFng), a vertebrate homolog of the Drosophila Fringe gene, is also expressed rhythmically in PSM. The PSM expression of IFng was observed as coordinated pulses of mRNA resembling the expression of c-hairy1. We show that c-hairy1 and IFng expression in the PSM are coincident, indicating that both genes are responding to the same segmentation clock. The genes were found to differ in their regulation, however; in contrast to c-hairy1, IFng mRNA oscillations required continued protein synthesis, suggesting that IFng could be acting downstream of c-hairy1 in the clock mechanism. In Drosophila, Fringe has been shown to play a role in modulating Notch-Delta signalling [3,4], a pathway which in vertebrates has been implicated in defining somite boundaries [5-9]. These observations place the segmentation clock upstream of the Notch-Delta pathway during vertebrate somitogenesis.     

EDG1 is a functional sphingosine-1-phosphate receptor that is linked via a Gi/o to multiple signaling pathways, including phospholipase C activation, Ca2+ mobilization, Ras-mitogen-activated protein kinase activation, and adenylate cyclase inhibition

In Chinese hamster ovary (CHO) cells transiently transfected with an expression vector for EDG1, but not an empty vector, sphingosine-1-phosphate (SP) at a concentration as low as 10(-10) M caused an increase in the intracellular free Ca2+ concentration ([Ca2+]i) as a result of mobilization of Ca2+ from both intracellular and extracellular pools. In a CHO clone stably expressing EDG1 receptor (CHO-EDG1 cells), SP induced increases in the production of inositol phosphates and the [Ca2+]i and inhibited forskolin-induced increase in the cellular cAMP content, all in a manner sensitive to pertussis toxin. SP also activated mitogen-activated protein kinase in CHO-EDG1 cells in pertussis toxin-sensitive and Ras-dependent manners. To evaluate the spectrum of agonists for EDG1, we used human erythroleukemia (HEL) cells, which at naive state do not respond to SP or structurally related lipids with an increase in the [Ca2+]i. In HEL cells stably expressing EDG1 receptor (HEL-EDG1 cells), SP dose-dependently increased the [Ca2+]i with half-maximal and maximal concentration values of 10(-9) and 3 x 10(-7) M, respectively; sphingosylphosphorylcholine at exclusively high concentrations, but not sphingosine at all, also increased the [Ca2+]i. HEL-EDG1 cells bound 32P-labeled SP, which was displaced dose dependently by unlabeled SP. These results indicate that EDG1, a member of the EDG family G protein-coupled receptors, is a specific, high-affinity SP receptor.     

Cholesteryl ester transfer protein gene polymorphism is a determinant of HDL cholesterol and of the lipoprotein response to a lipid-lowering diet in type 1 diabetes

The TaqIB cholesteryl ester transfer protein (CETP) gene polymorphism (B1B2) is a determinant of HDL cholesterol in nondiabetic populations. Remarkably, this gene effect appears to be modified by environmental factors. We evaluated the effect of this polymorphism on HDL cholesterol levels and on the lipoprotein response to a linoleic acid-enriched, low-cholesterol diet in patients with type 1 diabetes. In 44 consecutive type 1 diabetic patients (35 men), CETP polymorphism, apolipoprotein (apo) E genotype, serum lipoproteins, serum CETP activity (measured with an exogenous substrate assay, n = 30), clinical variables, and a diet history were documented. The 1-year response to diet was assessed in 14 type 1 diabetic patients, including 6 B1B1 and 6 B1B2 individuals. HDL cholesterol was higher in 10 B2B2 than in 14 B1B1 homozygotes (1.63 +/- 0.38 vs. 1.24 +/- 0.23 mmol/l, P < 0.01). HDL cholesterol, adjusted for triglycerides and smoking, was 0.19 mmol/l higher for each B2 allele present. CETP activity levels were not significantly different between CETP genotypes. Multiple regression analysis showed that VLDL + LDL cholesterol was associated with dietary polyunsaturated:saturated fatty acids ratio (P < 0.02) and total fat intake (P < 0.05) in the B1B1 homozygotes only and tended to be related to the presence of the apo E4 allele (P < 0.10). In response to diet, VLDL + LDL cholesterol fell (P < 0.05) and HDL cholesterol remained unchanged in 6 B1B1 homozygotes. In contrast, VLDL + LDL cholesterol was unaltered and HDL cholesterol decreased (P < 0.05) in 6 B1B2 heterozygotes (P < 0.05 for difference in change in VLDL + LDL/HDL cholesterol ratio). This difference in response was unrelated to the apo E genotype. Thus, the TaqIB CETP gene polymorphism is a strong determinant of HDL cholesterol in type 1 diabetes. This gene effect is unlikely to be explained by a major influence on the serum level of CETP activity, as an indirect measure of CETP mass. Our preliminary data suggest that this polymorphism may be a marker of the lipoprotein response to dietary intervention.     

Binding of insulin-like growth factor (IGF)-binding protein-5 to smooth-muscle cell extracellular matrix is a major determinant of the cellular response to IGF-I

Insulin-like growth factor-binding protein-5 (IGFBP-5) has been shown to bind to fibroblast extracellular matrix (ECM). Extracellular matrix binding of IGFBP-5 leads to a decrease in its affinity for insulin-like growth factor-I (IGF-I), which allows IGF-I to better equilibrate with IGF receptors. When the amount of IGFBP-5 that is bound to ECM is increased by exogenous addition, IGF-I's effect on fibroblast growth is enhanced. In this study we identified the specific basic residues in IGFBP-5 that mediate its binding to porcine smooth-muscle cell (pSMC) ECM. An IGFBP-5 mutant containing alterations of basic residues at positions 211, 214, 217, and 218 had the greatest reduction in ECM binding, although three other mutants, R214A, R207A/K211N, and K202A/R206N/R207A, also had major decreases. In contrast, three other mutants, R201A/K202N/R206N/R208A, and K217N/R218A and K211N, had only minimal reductions in ECM binding. This suggested that residues R207 and R214 were the most important for binding, whereas alterations in K211 and R218, which align near them, had minimal effects. To determine the effect of a reduction in ECM binding on the cellular replication response to IGF-I, pSMCs were transfected with the mutant cDNAs that encoded the forms of IGFBPs with the greatest changes in ECM binding. The ECM content of IGFBP-5 from cultures expressing the K211N, R214A, R217A/R218A, and K202A/R206N/R207A mutants was reduced by 79.6 and 71.7%, respectively, compared with cells expressing the wild-type protein. In contrast, abundance of the R201A/K202N/R206N/R208A mutant was reduced by only 14%. Cells expressing the two mutants with reduced ECM binding had decreased DNA synthesis responses to IGF-I, but the cells expressing the R201A/K202N/R206N/R208A mutant responded well to IGF-I. The findings suggest that specific basic amino acids at positions 207 and 214 mediate the binding of IGFBP-5 to pSMC/ECM. Smooth-muscle cells that constitutively express the mutants that bind weakly to ECM are less responsive to IGF-I, suggesting that ECM binding of IGFBP-5 is an important variable that determines cellular responsiveness.     

Development of a necklace microstructure during isothermal deformation and its properties relative to uniform microstructures

A necklace microstructure containing a controlled amount of fine (i.e., 4 to 6 μm) grains along the boundaries of larger (i.e., 30 to 40 ώm) warm-worked grains is intended to achieve a balance in mechanical properties for applications such as gas turbine disks. The development of this duplex grain microstructure critically depends on the starting microstructure, the strain and strain rate of the deformation process, and the subsequent heat-treatment conditions. These aspects of microstructural development were studied using isothermal compression tests on powder metallurgy (PM) ASTROLOY consolidated by “hipping” or extrusion. Tensile, creep, and stressrupture (S/R) properties of the necklace microstructure were also evaluated and compared with those of uniform grain microstructures. Formerly Senior Scientist, Research and Development Department, Cameron Forge Company