Identification of arg-30 as the essential residue for the enzymatic activity of Taiwan cobra phospholipase A2

Three cohorts of patients with the factor V Leiden mutation were recruited independently (heterozygotes, homozygotes and combined thrombophilia). The antithrombotic efficacy of oral anticoagulation and the predictive value for recurrence of an idiopathic as opposed to a precipitated first event were determined. Idiopathic first events occurred at an older age than precipitated events (43 v 26 years, LR = 23.31, P < 0.001). None of the patients had a recurrent event while on warfarin but the median time to recurrence after stopping warfarin was 9 years (95% CI 0.7-17.3 years). The time to recurrence was shorter when the first event was idiopathic as opposed to precipitated (3.5 v 13 years, LR = 4.76, P = 0.029). A calculation of benefit to risk of oral anticoagulation with a target INR of 2.5 does not support the use of long-term therapy in all patients with the factor V Leiden mutation following a first thrombotic event.     

Characterization of PepB, a group B streptococcal oligopeptidase

Group B streptococci were recently reported to possess a cell-associated collagenase. Although the enzyme hydrolyzed the synthetic collagen-like substrate N-(3-[2-furyl]acryloyl)-Leu-Gly-Pro-Ala, we found that neither the highly purified enzyme nor crude group B streptococcal cell lysate solubilized a film of reconstituted rat tail collagen, an activity regarded as obligatory for a true collagenase. We cloned and sequenced the gene for the enzyme (pepB). The deduced amino acid sequence showed 66.4% identity to the PepF oligopeptidase from Lactococcus lactis, a member of the M3 or thimet family of zinc metallopeptidases. The group B streptococcal enzyme also showed oligopeptidase activity and degraded a variety of small bioactive peptides, including bradykinin, neurotensin, and peptide fragments of substance P and adrenocorticotropin.     

Prevention of neonatal group B streptococcal infection

Neonatal group B streptococcal infection is the primary cause of neonatal morbidity related to infection. It can often be prevented by identifying and treating pregnant women who carry group B streptococci or who are at highest risk of transmitting the bacteria to newborns. Increasing evidence and expert opinion support intrapartum treatment of women at relatively high risk of delivering an infant with group B streptococcal infection. Such women can be identified through the use of an anogenital culture for group B streptococci obtained at 35 to 37 weeks of gestation and by the presence of at least one of many risk factors associated with neonatal infection. These risk factors include preterm labor or rupture of the membranes at less than 37 weeks of gestation, previous delivery of an infant with invasive group B streptococcal disease, group B streptococcal bacteriuria during the present pregnancy, maternal intrapartum fever of 38 degrees C (100.4 degrees F) or higher and rupture of the fetal membranes for 18 hours or more. The recommended agent for intrapartum chemoprophylaxis is intravenous penicillin G; clindamycin is used in penicillin-allergic women. The use of risk markers alone to guide the administration of intrapartum antibiotics is much more cost-effective than other preventive strategies, but it exposes more women and infants to antibiotic-associated risks. Management of the infants of treated mothers is empiric and is currently guided by expert opinion.     

Alternative algorithms for prevention of perinatal group B streptococcal infections

The well-known association of hypertension and diabetes mellitus and the lack of suitable animal models to study diabetic hypertension prompted us to transfer 4 chromosomal regions with quantitative trait loci (QTLs) for blood pressure of the spontaneously hypertensive SHR rat onto the genetic background of the diabetes-prone and normotensive BB/OK rat. Four congenic strains developed are named as BB. Sa (Chr.1), BB.Bp2 (Chr.18), BB.1K (Chr.20) and BB.Xs (Chr.X). Because the systolic blood pressure is significantly elevated in all congenics, renal related traits were investigated in serum and urine. Comparing BB/OK and their congenic derivatives, significant differences were found in all serum and in 7 out of 8 urine constituents studied. Most significant differences were found between BB/OK and BB.Bp2 rats. Significant differences were also found between the different congenic strains indicating that each congenic strain has its own phenotype and that each chromosomal region contains most probably further QTLs for some of the traits studied.     

Characterization of histidine residues essential for receptor binding and activity of nerve growth factor

The role of the four histidine residues in receptor binding and activity of mouse nerve growth factor (NGF) was investigated using both site-directed mutagenesis and chemical modification with diethyl pyrocarbonate. Replacement of His-75 or His-84 with alanine resulted in decreased biological activity and decreased affinity for p140(trkA); however, with H75A only, a 5-fold increased affinity toward p75(LANR) was observed. The effect of simultaneous replacement of both His-75 and His-84 was neither additive nor synergistic. Slight perturbations in circular dichroism spectra and weakened self-association of the mutants indicated that His-75 and His-84 may be involved in stability, dimerization, and/or folding of NGF. Diethyl pyrocarbonate modification of His-4 and His-8 in the H75A/H84Q double mutant abolished neuritogenesis, binding to both receptors, and phosphorylation of p140(trkA) in PC12 cells. These chemical and mutational results confirm and clarify previous evidence for the involvement of His-75 and His-84 (Dunbar, J. C., Tregear, G. W., and Bradshaw, R. A. (1984) J. Protein Chem. 3, 349-356) or His-4 and His-8 (Shih, A., Laramee, G. R., Schmelzer, C. H., Burton, L. E., and Winslow, J. W. (1994) J. Biol. Chem. 269, 27679-27686) in receptor binding of NGF. At least three and possibly all four histidines, which are located in three spatially distinct regions, contribute to maintenance of functional sites that are essential for receptor binding and activity of NGF.     

Risk of preterm delivery in pregnant women with group B streptococcal urinary infections or urinary antibodies to group B streptococcal and E. coli antigens

OBJECTIVE: To establish whether there is an association between preterm delivery and either group B streptococcal urinary infection or the presence of urinary antibodies to group B streptococcal or E. coli antigens. DESIGN: A prospective study with urine culture and antibody measurement performed at the first antenatal visit and at 28 weeks gestation. SETTING: Ninewells Hospital, Dundee. SUBJECTS: Two thousand and forty-three women registering consecutively at an antenatal clinic. MAIN OUTCOME MEASURE: Delivery at less than 37 weeks gestation. RESULTS: No increase in preterm delivery was observed in women with positive urine cultures for group B streptococci either at booking or at 28 weeks, even when confirmed by positive repeat cultures. Preterm delivery was more common in women with elevated urinary antibodies to E. coli antigens at booking (relative risk 1.81, 95% CI 1.22-2.68, P = 0.005) and at 28 weeks (relative risk 2.36, 95% CI 1.60-3.48, P < 0.0001) and to group B streptococcal antigens at 28 weeks (relative risk 2.24, 95% CI 1.46-3.43, P = 0.0003). CONCLUSIONS: These data do not support previous reports that positive urine cultures for group B streptococci are associated with an increased risk of preterm delivery. Our report of an association between elevated levels of urinary antibodies and preterm delivery is a new finding consistent with the possibility that a local inflammatory response to uro-genital infection may be important in stimulating the onset of preterm labour. The results suggest that screening for urinary antibodies at 28 weeks gestation might help to identify a group of women at increased risk of prematurity.     

Identification of a cysteine residue essential for activity of protein farnesyltransferase. Cys299 is exposed only upon removal of zinc from the enzyme

Protein farnesyltransferase (FTase) is a zinc metalloenzyme that performs a post-translational modification on many proteins that is critical for their function. The importance of cysteine residues in FTase activity was investigated using cysteine-specific reagents. Zinc-depleted FTase (apo-FTase), but not the holoenzyme, was completely inactivated by treatment with N-ethylmaleimide (NEM). Similar effects were detected after treatment of the enzyme with iodoacetamide. The addition of zinc to apo-FTase protects it from inactivation by NEM. These findings indicated the presence of specific cysteine residue(s), potentially located at the zinc binding site, that are required for FTase activity. We performed a selective labeling strategy whereby the cysteine residues exposed upon removal of zinc from the enzyme were modified with [3H]NEM. The enzyme so modified was digested with trypsin, and four labeled peptides were identified and sequenced, one peptide being the major site of labeling and the remaining three labeled to lesser extents. The major labeled peptide contained a radiolabeled cysteine residue, Cys299, that is in the beta subunit of FTase and is conserved in all known protein prenyltransferases. This cysteine residue was changed to both alanine and serine by site-directed mutagenesis, and the mutant proteins were produced in Escherichia coli and purified. While both mutant proteins retained the ability to bind farnesyl diphosphate, they were found to have lost essentially all catalytic activity and ability to bind zinc. These results indicate that the Cys299 in the beta subunit of FTase plays a critical role in catalysis by the enzyme and is likely to be one of the residues that directly coordinate the zinc atom in this enzyme.     

Identification of histidine residues in Wolinella succinogenes hydrogenase that are essential for menaquinone reduction by H2

This study aimed to demonstrate that sufficient Ph-negative blood progenitors could be collected following administration of glycosylated rhG-CSF (lenograstim) to patients with Philadelphia chromosome (Ph)-positive chronic myeloid leukaemia (CML) who responded to recombinant alpha-interferon (alpha-IFN) (Ph-positive marrow metaphases < 35%). 23 patients received lenograstim (150 microg/m2) once daily for a median of 9 d. Peak circulating numbers of white blood cells (36.4 x 10(9)/l), CD34+ cells (24/ microl) and colony-forming unit-granulocyte-macrophage (CFU-GM; 1346.5/ml) occurred at a median of day 8, day 8 and day 7, respectively. Two to six (median three) leukaphereses (LK) were performed from days 5 to 12. The median number of mononuclear cells (MNC), CD34+ cells and CFU-GM collected per patient was 7.35 x 10(8/)kg, 2.72 x 10(6)/kg and 10.23 x 10(4)/kg, respectively. 22/23 patients had LK which contained either 10(4) CFU-GM/kg and/or 10(6) CD34+ cells/kg; 47LK (from 20/22 patients) were evaluable for cytogenetics. The median percentage of Ph-positive cells was 0, and 43/47 LK (91%) contained <35% Ph-positive cells; 25 (53%) were entirely negative. Sixteen of 20 evaluable patients had one or more LK with <35% Ph-positive cells, and 12 had at least one 100% Ph-negative LK. Mobilization and collection of Ph-negative cells were not influenced by the dose or duration of alpha-IFN administration before (or during) lenograstim administration or by the quality of cytogenetic response (complete v major) during lenograstim administration. No significant side-effects were observed. Thus, lenograstim administration can result in satisfactory Ph-negative blood progenitor cell collection. Autologous transplantation of such cells may be used when indicated.     

The calcium-binding sites of heparinase I from Flavobacterium heparinum are essential for enzymatic activity

In the accompanying paper (Shriver, Z., Liu, D., Hu, Y., and Sasisekharan, R. (1999) J. Biol. Chem. 274, 4082-4088), we have shown that calcium binds specifically to heparinase I and have identified two major calcium-binding sites (CB-1 and CB-2) that partly conform to the EF-hand calcium-binding motif. In this study, through systematic site-directed mutagenesis, we have confirmed the accompanying biochemical studies and have shown that both CB-1 and CB-2 are involved in calcium binding and enzymatic activity. More specifically, we identified critical residues (viz. Asp210, Asp212, Gly213, and Thr216 in CB-1 and Asn375, Tyr379, and Glu381 in CB-2) that are important for calcium binding and heparinase I enzymatic activity. Mutations in CB-1 resulted in a lower kcat, but did not change the product profile of heparinase I action on heparin; conversely, mutations in CB-2 not only altered the kcat for heparinase I, but also resulted in incomplete degradation, leading to longer saccharides. Fluorescence competition experiments along with heparin affinity chromatography suggested that mutations in CB-1 alter heparinase I activity primarily through decreasing the enzyme's affinity for its calcium cofactor without altering heparin binding to heparinase I. Compared with CB-1 mutations, mutations in CB-2 affected calcium binding to a lesser extent, but they had a more pronounced effect on heparinase I activity, suggesting a different role for CB-2 in the enzymatic action of heparinase I. These results, taken together with our accompanying study, led us to propose a model for calcium binding to heparinase I that includes both CB-1 and CB-2 providing critical interactions, albeit via a different mechanism. Through binding to CB-1 and/or CB-2, we propose that calcium may play a role in the catalytic mechanism and/or in the exolytic processive mechanism of heparin-like glycosaminoglycan depolymerization by heparinase I.     

Opportunities for prevention of perinatal group B streptococcal disease: a multistate surveillance analysis. The Neonatal Group B Streptococcal Disease Study Group

OBJECTIVE: To evaluate the potential impact of ACOG and Centers for Disease Control and Prevention (CDC) consensus strategies for the prevention of perinatal group B streptococcal disease. METHODS: We evaluated cases of early-onset group B streptococcal disease identified by active surveillance during 1995, in four areas in North America with an aggregate 186,000 births per year. We reviewed the hospital records of mothers and infants and any prenatal records available on site. Cases were determined to be preventable based on whether group B streptococcal screening could have been performed prenatally, sensitivity of screening, presence of obstetric complications, and opportunity to administer antibiotics. RESULTS: We reviewed records for 245 of 246 infants with early-onset group B streptococcal disease in the surveillance areas. Most of the 53 case-mothers who delivered preterm and 192 who delivered full-term had had at least one prenatal visit (83% and 99%, respectively). Few case-mothers had prenatal group B streptococcal screening cultures, although compliance was high for other prenatal screening tests. Fifty-four percent of case-mothers had a recognized obstetric risk factor for group B streptococcal disease: labor or rupture of membranes at less than 37 weeks, rupture of membranes for 18 hours on longer, or temperature 38C or greater. The estimated preventable portion of early-onset group B streptococcal cases was 78% for the screening-based approach (range 74% to 82% by area), compared with 41% for the risk-based approach (range 39% to 53% by area). CONCLUSION: Comprehensive implementation of either of the recommended prevention strategies could potentially prevent a substantial proportion of early-onset group B streptococcal disease.     

Safety and immunogenicity of capsular polysaccharide-tetanus toxoid conjugate vaccines for group B streptococcal types Ia and Ib

The translocation of spin-labeled analogues of phosphatidylcholine (4-doxylpentanoyl-PC, SL-PC), phosphatidylethanolamine (SL-PE), phosphatidylserine (SL-PS), and sphingomyelin (SL-SM) from the outer to the inner leaflet of the plasma membrane bilayer was investigated in dog kidney MDCK II and human colon Caco-2 cells. Disappearance from the outer leaflet was assayed using back-exchange to serum albumin. Experiments with cells in suspension as well as with polarized cells on filters were performed at reduced temperatures (10 and 20 degreesC) to suppress endocytosis and hydrolysis of spin-labeled lipids. For both epithelial cell lines, a fast ATP-dependent inward movement of the aminophospholipids SL-PS and SL-PE was found, while SL-SM was only slowly internalized without any effect of ATP depletion. The kinetics of redistribution of SL-PC were clearly different between the two cell lines. In MDCK II cells, SL-PC was rapidly internalized in an ATP-dependent and N-ethylmaleimide-sensitive manner and at a rate similar to that of the aminophospholipids. In contrast, in Caco-2 cells the inward movement of SL-PC was much slower than that of the aminophospholipids, did not depend on ATP, and was not N-ethylmaleimide-sensitive. Inhibitor studies indicated that the outward-translocating multidrug resistance P-glycoprotein present in these cells did not affect the kinetics of inward translocation. Internalization was always similar on the apical and basolateral cell surface, suggesting the presence of the same phospholipid translocator(s) on both surface domains of epithelial cells. We propose that Caco-2 cells contain the well-known aminophospholipid translocase, while MDCK II cells contain either two translocases, namely, the aminophospholipid translocase and a phosphatidylcholine-specific translocase, or one translocase of a new type, translocating aminophospholipids as well as phosphatidylcholine.     

Comparison of NNA agar culture and selective broth culture for detection of group B streptococcal colonization in women

In 1996, the Centers for Disease Control and Prevention recommended the use of a selective broth culture for the improved detection of genital tract or anorectal carriage of group B streptococci (GBS) in pregnant women. In order to verify this recommendation in our laboratory, we compared the sensitivity of Todd-Hewitt medium with gentamicin and nalidixic acid (SBM) with our current method of direct plating on blood agar medium containing neomycin and nalidixic acid (NNA). Five hundred consecutive cervicovaginal and anorectal specimens submitted for GBS culture were included in the study. Swabs were plated onto NNA and the swabs were immersed in SBM, followed by overnight incubation at 35 degrees C. On the following day, the NNA plates were examined for colonies typical of GBS and the organisms were identified by the CAMP test or by latex agglutination. SBM cultures were subcultured onto blood agar and CNA agar plates, and the plates were reincubated for 24 h. Negative specimens from either medium were incubated for an additional 24 h and were examined again before finalization of the results. GBS were recovered from 78 specimens by both methods; from SBM only for 17 specimens (sensitivity, 86%) and from NNA only for 16 specimens (sensitivity, 85%). A moderate to heavy growth of Enterococcus faecalis was observed on plates containing NNA-positive, SBM-negative specimens. Competitive growth studies suggested that E. faecalis suppressed the growth potential of GBS in SBM. Our study suggests that direct plating on NNA, as a single method, is equivalent in sensitivity to SBM for the recovery of GBS, and the results are often available 24 h sooner. However, it appears that both direct plating and selective broth amplification techniques are required for the maximum level of identification of colonization with GBS in pregnant women.     

Identification of the essential cysteinyl residue located in the active site of human phenylethanolamine N-methyltransferase

Rabbit (Oryctolagus cuniculus) red cell and tissue acid phosphatases were studied by means of horizontal starch gel electrophoresis and isoelectric focusing followed by enzyme blotting. Red cell acid phosphatase 1 (ACP1) is monomorphic while tissue acid phosphatase 2 (ACP2) is polymorphic in a wild rabbit population, with two alleles: ACP2*1 (0.96) and ACP2*2 (0.04). A third locus homologous of human acid phosphatase 3 (ACP3) is characterized by the presence of three alleles (ACP3*1, ACP3*2 and ACP3*3). ACP3*1 is the most common allele and was detected in all populations, ACP3*2 was found in domestic breeds and in a wild population from Southern France, whereas ACP3*3 is typical of Portuguese wild rabbits. The geographical distribution of ACP3*2 and ACP3*3 is in agreement with the subspecific level of differentiation of the rabbit species in O. cuniculus cuniculus and O. c. algirus. The comparative study of the acid phosphatase activity in red cells of several mammalian species, including humans, suggests that ACP3 activity in erythrocytes exists only in rabbit.     

Mutagenesis of endopolygalacturonase from Fusarium moniliforme: histidine residue 234 is critical for enzymatic and macerating activities and not for binding to polygalacturonase-inhibiting protein (PGIP)

The sequence encoding the endopolygalacturonase (PG) of Fusarium moniliforme was cloned into the E. coli/yeast shuttle vector Yepsec1 for secretion in yeast. The recombinant plasmid (pCC6) was used to transform Saccharomyces cerevisiae strain S150-2B; transformed yeast cells were able to secrete PG activity into the culture medium. The enzyme (wtY-PG) was purified, characterized, and shown to possess biochemical properties similar to those of the PG purified from F. moniliforme. The wtY-PG was able to macerate potato medullary tissue disks and was inhibited by the polygalacturonase-inhibiting protein (PGIP) purified from Phaseolus vulgaris. The sequence encoding PG in pCC6 was subjected to site-directed mutagenesis. Three residues in a region highly conserved in all the sequences known to encode PGs were separately mutated: His 234 was mutated into Lys (H 234-->K), and Ser 237 and Ser 240 into Gly (S 237-->G and S 240-->G). Each of the mutated sequences was used to transform S. cerevisiae and the mutated enzymes were purified and characterized. Replacement of His 234 with Lys abolished the enzymatic activity, confirming the biochemical evidence that a His residue is critical for enzyme activity. Replacement of either Ser 237 or Ser 240 with Gly reduced the enzymatic activity to 48% and 6%, respectively, of the wtY-PG. When applied to potato medullary tissue, F. moniliforme PG and wtY-PG caused comparable maceration, while the variant PGs exhibited a limited (S 234-->G and S 240-->G) or null (H 234-->K) macerating activity. The interaction between the variant enzymes and the P. vulgaris PGIP was investigated using a biosensor based on surface plasmon resonance (BIAlite). The three variant enzymes were still able to interact and bind to PGIP with association constants comparable to that of the wild type enzyme.     

Characterization of group B streptococcal invasion of human chorion and amnion epithelial cells In vitro

Group B streptococci (GBS) have been cultured from the chorioamnionic membrane of pregnant women, usually in association with chorioamnionitis and premature labor (K. A. Boggess, D. H. Watts, S. L. Hillier, M. A. Krohn, T. J. Benedetti, and D. A. Eschenbach, Obstet. Gynecol. 87:779-784, 1996). Colonization and infection of placental membranes can be a prelude to neonatal GBS infections even in the presence of intact membranes (R. L. Naeye and E. C. Peters, Pediatrics 61:171-177, 1978), suggesting that GBS cause chorioamnionitis or establish amniotic fluid infections by partial or complete penetration of the placental membranes. We have isolated and grown cultures of primary chorion and amnion cells from human cesarean-section placentas. This has provided a biologically relevant model for investigating GBS adherence to and invasion of the two epithelial barriers of the placental membrane. GBS adhered to chorion cell monolayers to a high degree. Pretreatment of GBS with trypsin reduced adherence up to 10-fold, which suggested that the bacterial ligand(s) was a protein. GBS invaded chorion cells at a high rate in vitro, and invasion was dependent on cellular actin polymerization. GBS could be seen within intracellular vacuoles of chorion cells by transmission electron microscopy. We also demonstrated that GBS were capable of transcytosing through intact chorion cell monolayers without disruption of intracellular junctions. GBS also adhered to amnion cells; in contrast, however, these bacteria failed to invade amnion cells under a variety of assay conditions. GBS interactions with the chorion epithelial cell layer shown here correlate well with epidemiological and pathological studies of GBS chorioamnionitis. Our data also suggest that the amnion cell layer may provide an effective barrier against infection of the amniotic fluid.     

Identification of glucuronan lyase from a mutant strain of Rhizobium meliloti

This review attempts to capture the history of research involved in the understanding of lipid metabolism via investigation of the sn-glycerol-3-phosphate acyltransferase (glycerol-P acyltransferase), the first step in the synthesis of lipids in E. coli. We will review the original identification of this enzymatic activity and its subsequent characterization. The biochemical and genetic regulation of this enzyme and gene are discussed, as well as the unique structural characterization of this integral membrane protein. Future perspectives regarding the regulatory and structural aspects of this key enzyme are discussed.