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Transformation in bacteria is the uptake and incorporation of exogenous DNA into a cell's genome. Several species transform naturally during a regulated state defined as competence. Genetic elements in Streptococcus pneumoniae induced during transformation were identified by combining a genetic screen with genomic analysis. Six loci were discovered that composed a competence-induced regulon. These loci shared a consensus promoter sequence and encoded proteins, some of which were similar to proteins involved in DNA processing during transformation in other bacteria. Each locus was induced during competence and essential for genetic transformation.

The surface of Streptococcus pneumoniae is decorated with a family of choline-binding proteins (CBPs) that are non-covalently bound to the phosphorylcholine of the teichoic acid. Two examples (PspA, a protective antigen, and LytA, the major autolysin) have been well characterized. We identified additional CPBs and characterized a new CBP, CbpA, as an adhesin and a determinant of virulence. Using choline immobilized on a solid matrix, a mixture of proteins from a pspA-deficient strain of pneumococcus was eluted in a choline-dependent fashion. Antisera to these proteins passively protected mice challenged in the peritoneum with a lethal dose of pneumococci. The predominant component of this mixture, CbpA, is a 75-kDa surface-exposed protein that reacts with human convalescent antisera. The deduced sequence from the corresponding gene showed a chimeric architecture with a unique N-terminal region and a C-terminal domain consisting of 10 repeated choline-binding domains nearly identical to PspA. A cbpA-deficient mutant showed a >50% reduction in adherence to cytokine-activated human cells and failed to bind to immobilized sialic acid or lacto-N-neotetraose, known pneumococcal ligands on eukaryotic cells. Carriage of this mutant in an animal model of nasopharyngeal colonization was reduced 100-fold. There was no difference between the parent strain and this mutant in an intraperitoneal model of sepsis. These data for CbpA extend the important functions of the CBP family to bacterial adherence and identify a pneumococcal vaccine candidate.

Pathogenic bacteria rely on adhesins to bind to host tissues. Therefore, the maintenance of the functional properties of these extracellular macromolecules is essential for the pathogenicity of these microorganisms. We report that peptide methionine sulfoxide reductase (MsrA), a repair enzyme, contributes to the maintenance of adhesins in Streptococcus pneumoniae, Neisseria gonorrhoeae, and Escherichia coli. A screen of a library of pneumococcal mutants for loss of adherence uncovered a MsrA mutant with 75% reduced binding to GalNAcbeta1-4Gal containing eukaryotic cell receptors that are present on type II lung cells and vascular endothelial cells. Subsequently, it was shown that an E. coli msrA mutant displayed decreased type I fimbriae-mediated, mannose-dependent, agglutination of erythrocytes. Previous work [Taha, M. K., So, M., Seifert, H. S., Billyard, E. & Marchal, C. (1988) EMBO J. 7, 4367-4378] has shown that mutants with defects in the pilA-pilB locus from N. gonorrhoeae were altered in their production of type IV pili. We show that pneumococcal MsrA and gonococcal PilB expressed in E. coli have MsrA activity. Together these data suggest that MsrA is required for the proper expression or maintenance of functional adhesins on the surfaces of these three major pathogenic bacteria.

Pneumococcus has been shown to bind to epithelial cells of the nasopharynx and lung, and to endothelial cells of the peripheral vasculature. To characterize bacterial elements required for attachment to these cell types, a library of genetically altered pneumococci with defects in exported proteins was screened for the loss of attachment to glycoconjugates representative of the nasopharyngeal cell receptor, type II lung cells (LC) and human endothelial cells (EC). A mutant was identified which showed a greater than 70% loss in the ability to attach to all cell types. This mutant also showed decreased adherence to the glycoconjugates containing the terminal sugar residues GalNAcbeta1-3Gal, GalNAcbeta1-4Gal and the carbohydrate GlcNAc, which are proposed components of the pneumococcal receptors specific to the surfaces of LC and EC. Analysis of the locus altered in this mutant revealed a gene, spxB, that encodes a member of the family of bacterial pyruvate oxidases which decarboxylates pyruvate to acetyl phosphate plus H2O2 and CO2. This mutant produced decreased concentrations of H2O2 and failed to grow aerobically in a chemically defined medium, unless supplemented with acetate which presumably restores acetyl phosphate levels by the action of acetate kinase, further suggesting that spxB encodes a pyruvate oxidase. The addition of acetate to the growth medium restored the adherence properties of the mutant indicating a link between the enzyme and the expression of bacterial adhesins. A defect in spxB corresponded to impaired virulence of the mutant in vivo. Compared to the parent strain, an spxB mutant showed reduced virulence in animal models for nasopharyngeal colonization, pneumonia, and sepsis. We propose that a mutation in spxB leads to down-regulation of the multiple adhesive properties of pneumococcus which, in turn, may correlate to diminished virulence in vivo.

The study of Streptococcus pneumoniae has led to many insights into the pathogenesis of bacterial infections. The importance of the polysaccharide capsule of the organism in determining its virulence was indicated by studies of the protective role of anticapsular antibodies.1–3 Of comparable importance was the observation that noncapsulated pneumococci caused progressive disease in rabbits with agranulocytosis, demonstrating thereby the pathogenic properties of the pneumococcal soma.4 Investigation of the pathologic events in pneumococcal infection led to the discovery of DNA,2,3 the development of the first bacterial polysaccharide vaccine,5 and insights into the mechanism of action of penicillin,6 antibiotic tolerance, . . .

SummaryThe pharmacokinetics and tolerability of factor XIII (FXIII) from plasma were compared with those of FXIII from placenta in a randomised, double-blind, crossover study involving 13 patients with congenital FXIII deficiency. Both FXIII activity and FXIII antigen were monitored. No difference was seen in the mean half-lives of the two preparations (9.3 days and 9.1 days for plasma and placenta FXIII activity, respectively). Response was similar for both preparations, but was slightly greater for FXIII from plasma.Similar results were found for recovery (65% vs 60%). The area under the data completed by extrapolation was significantly higher for FXIII from plasma. No differences between preparations in terms of efficacy or tolerability were observed. It can be concluded that treatment with FXIII concentrate from plasma is as efficient as with FXIII concentrate from placenta in terms of recovery and half-life. Both preparations were equivalent in terms of safety during the observation period. With the administration of monthly injections of approximately 30 U/kg serious bleeding events were prevented and no other serious adverse events occurred.

To identify elements participating in the process of transformation, a bank of genetically altered mutants of Streptococcus pneumoniae with defects in exported proteins was assessed for a decrease in transformation efficiency. One mutant consistently transformed 10-fold less than the parent strain. Sequence analysis and reconstitution of the altered locus revealed a gene, plpA (permease-like protein), which encodes a putative substrate-binding protein belonging to the family of bacterial permeases responsible for peptide transport. The derived amino acid sequence for this gene was 80% similar to AmiA, a peptide-binding protein homologue from pneumococcus, and 50% similar over 230 amino acids to Spo0KA which is a regulatory element in the process of transformation and sporulation in Bacillus subtilis. PlpA fusions to alkaline phosphatase (PhoA) were shown to be membrane associated and labelled with [3H]-palmitic acid, which probably serves as a membrane anchor. Experiments designed to define the roles of the plpA and ami determinants in the process of transformation showed that: (i) mutants with defects in plpA were > 90% transformation deficient while ami mutants exhibited up to a fourfold increase in transformation efficiency; (ii) compared to the parental strain, the onset of competence in an ami mutant occurred earlier in logarithmic growth, whereas the onset was delayed in a plpA mutant; and (iii) the plpA mutation decreases the expression of a competence-regulated locus. Since the permease mutants would fail to bind specific ligands, it seems likely that the substrate-permease interaction modulates the process of transformation.

When colonies of encapsulated isolates of Streptococcus pneumoniae are viewed with oblique, transmitted light on a transparent surface, they are heterogeneous in appearance because of variation in opacity. There is spontaneous phase variation among at least three discernible phenotypes at frequencies from 10(-3) to 10(-6). The ability to detect differences in opacity varies according to serotype, but variation is independent of capsule expression. Electron microscopy shows no difference in chain length but suggests that autolysis occurs earlier in the growth of the transparent variant. There was no identifiable difference in membrane protein profiles of opaque and transparent variants of the same strain. In an infant rat model of nasopharyngeal carriage, there was no significant colonization by opaque variants. Efficient and stable colonization by the transparent variants was observed, suggesting a selective advantage for this phenotype in the nasopharynx. In contrast, there was no difference in the incidence of bacteremia or in the 50% lethal dose among the variants following their intraperitoneal inoculation. These results suggest that phase variation which is marked by differences in colonial morphology may provide insight into the interaction of the pneumococcus with its host.

A strategy was developed to mutate and genetically identify exported proteins in Streptococcus pneumoniae. Vectors were created and used to screen pneumococcal DNA in Escherichia coli and S. pneumoniae for translational gene fusions to alkaline phosphatase (PhoA). Twenty five PhoA+ pneumococcal mutants were isolated and the loci from eight of these mutants showed similarity to known exported or membrane-associated proteins. Homologues were found to: (i) protein-dependent peptide permeases, (ii) penicillin-binding proteins, (iii) Clp proteases, (iv) two-component sensor regulators, (v) the phosphoenolpyruvate: carbohydrate phosphotransferases permeases, (vi) membrane-associated dehydrogenases, (vii) P-type (E1E2-type) cation transport ATPases, (viii) ABC transporters responsible for the translocation of the RTX class of bacterial toxins. Unexpectedly one PhoA+ mutant contained a fusion to a member of the DEAD protein family of ATP-dependent RNA helicases suggesting export of these proteins.

The adherence of Bordetella pertussis to ciliated cells and macrophages is critical to colonization and infection of the respiratory tract. Adherence to both types of cells involves the recognition of eukaryotic carbohydrates by the bacterial adhesin filamentous hemagglutinin (Fha). The carbohydrate recognition domain (CRD) of Fha is considered an important antigen for subcomponent vaccines to maximize the generation of antiadherence antibodies capable of protecting against colonization. For identification of the CRD of Fha, a bank of eight monoclonal antibodies (MAbs) that mapped to four contiguous regions were tested for their ability to block Fha binding to lactosylceramide or to block bacterial binding to ciliated cells. Only MAb 12.5A9, which maps to amino acid residues 1141 to 1279, blocked both Fha binding to lactosylceramide and bacterial binding to ciliated cells. An 18-kDa polypeptide corresponding to this region was expressed in Escherichia coli. Cell lysates containing this protein bound to lactosylceramide in a manner identical to that of native Fha. Mutant strains of B. pertussis that contained an in-frame deletion of the coding sequence for this region produced a truncated Fha that showed negligible cross-reactivity with MAb 12.5A9. In an adherence assay, these mutant strains failed to bind efficiently to either ciliated cells or macrophages. The numbers of adherent bacteria for these strains were reduced to the number obtained with a nonadherent strain. We conclude that the region defined by residues 1141 to 1279 of Fha constitutes a CRD critical for bacterial adherence and represents a potential candidate for a subcomponent vaccine.

During the course of human infection, Bordetella pertussis colonizes sequential niches in the respiratory tract that include intracellular and extracellular environments. In vitro the expression of virulence factors such as the adenylate cyclase toxin is coordinately regulated by the bvg locus, which is an example of a two-component sensory transduction system. With this toxin as a reporter, enzyme activities were compared between a wild-type and an altered strain to determine whether bacterial entry into human macrophages affected gene expression. BPRU140, a strain containing an inducible expression vector, produced enzyme activity independent of bvg. Samples of the parent, the induced, and the uninduced BPRU140 were incubated individually with macrophages for 30 min. Extracellular bacteria were then killed by gentamicin. The number of viable intracellular bacteria and the internalized bacterial enzyme activity were measured over time. By 2.5 hr all samples reached a steady-state concentration of 10(5) bacteria per 10(6) macrophages. Following an initial peak of enzyme activity, adenylate cyclase values for the parent and the uninduced BPRU140 decreased to a basal level, while the values for the induced strain remained at least 3-fold greater. Therefore, compared with the persistence of enzyme in the induced strain BPRU140, the decrease in enzyme production by the parent and the uninduced BPRU140 upon entry into macrophages indicates in vivo down-modulation of gene expression. These observations support the hypothesis that sensory transduction contributes to adaptations for bacterial survival in the infected host.

Laboratory investigation of the haemostatic system deserves particular procedures in the quality control of analytical variables as well as preanalytical variables. This paper reviews the precautions that have to be taken in the blood prelevement, the transport of the tubes and the performance of the laboratory tests aimed to investigate the haemostatic system in order to obtain reliable results.

Bordetella pertussis is bound to glycoconjugates on human cilia and macrophages by multiple adhesins, including pertussis toxin. The cellular recognition properties of the B oligomer of pertussis toxin were characterized and the location and structural requirements of the recognition domains were identified by site-directed mutagenesis of recombinant pertussis toxin subunits. Differential recognition of cilia and macrophages, respectively, was localized to subunits S2 and S3 of the B oligomer. Despite greater than 80% sequence homology between these subunits, ciliary lactosylceramide exclusively recognized S2 and leukocytic gangliosides bound only S3. Substitution at residue 44, 45, 50, or 51 in S2 resulted in a shift of carbohydrate recognition from lactosylceramide to gangliosides. Mutational exchange of amino acid residues 37-52 between S2 and S3 interchanged their carbohydrate and target cell specificity. Comparison of these carbohydrate recognition sequences to those of plant and animal lectins revealed that regions essential for function of the prokaryotic lectins were strongly related to a subset of eukaryotic carbohydrate recognition domains of the C type.

Genetic exchange by natural transformation in Streptococcus pneumoniae occurs in a cell-density dependent process and is initiated by a small extracellular signalling molecule, the competence-stimulating peptide (CSP). comC, the gene for this peptide, has previously been identified and encodes a 44 amino acid pre-peptide that is apparently processed to an active molecule that consists of the C-terminal 17 amino acids. We have sequenced the region adjacent to comC and shown that it is the first gene of an operon, com, consisting of two downstream elements, comD and comE, which encode members of the two-component family of sensor regulators. Null mutants with defects in either comC or comD were transformation deficient and failed to respond to exogenous CSP. A comC mutant did not exhibit any detectable CSP activity, while a comD mutant that contained an intact comC produced minimal CSP activity. In mixed-culture experiments consisting of isogenic pairs of pneumococci (Csp+ and Csp-), we showed that induction of competence by quorum sensing was independent of CSP. Northern analysis showed that com was transcribed as a single polycistronic message, while analysis of strains with transcriptional fusions showed that com was constitutively expressed under conditions that both promoted or repressed the development of competence. Finally, we showed genetically and biochemically a CSP-dependent transcription of rec, a competence-induced locus, and that ComD and ComE are required for this CSP-dependent expression.

New insight has been gained into the mechanisms underlying the tissue tropism and inflammation of pneumococcal infection. Virulence has been linked to a transparent colonial morphology. Adherence has been characterized at the molecular level, and the importance of receptors arising upon activation of eukaryotic cells in promoting the progression to disease has been established. The contribution of peptidoglycan and teichoic acid to the generation of inflammation has suggested the need to couple anti-inflammatory therapy with antibiotic treatment in order to improve the outcome of invasive disease. Elucidation of the pathogenesis of pneumococcal infection, including the identification of virulence determinants by recently developed genetic strategies, can provide a paradigm for new mechanisms that are active in gram-positive bacterial infections and that are clearly distinct from the familiar pathways triggered by endotoxin.

To gain access to tissues within the human host, Streptococcus pneumoniae initially colonizes the nasopharynx and then interacts with glycoconjugates on the surfaces of target cells at various sites of infection. Although pneumococcal adhesins are currently unknown, exported proteins on the bacterial surface are potential candidates. To identify bacterial elements involved in this process, mutants of S. pneumoniae with defects in exported proteins were screened for the inability to adhere to cells representative of three in vivo niches: (i) agglutination of bovine erythrocytes, which reflects adherence to cells which reside in the nasopharynx; (ii) human type II pneumocytes (lung cells [LC]), representing the alveolar site of infection; and (iii) human vascular endothelial cells (EC), representing the endovascular site. The capacity of the mutants to adhere during the course of pneumococcal disease was also assessed by using cytokine-activated LC and EC. All of the 30 mutants analyzed produced hemagglutination values comparable with those of the parent strain. Four independent mutants demonstrated a greater than 50% decrease in adherence to both LC and EC. Sequence analysis of the altered alleles from these strains showed that mutations had occurred in two previously identified loci, plpA and ami, which belong to the family of genes encoding protein-dependent peptide permeases. Mutations in the ami locus resulted in an inability to recognize the GalNAc beta 1-4Gal glycoconjugate receptor present on resting LC and EC, whereas mutations in plpA resulted in a failure to recognize a GalNAc beta 1-3Gal glycoconjugate receptor also present on resting cells. Mutations in neither allele affected recognition of GlcNAc receptors present on cytokine-activated LC and EC. These results suggest that peptide permeases modulate pneumococcal adherence to epithelial and endothelial cells either by acting directly as adhesins or by modulating the expression of adhesins on the pneumococcal surface during the initial stages of colonization of the lung or the vascular endothelium.

To study competence and the process of transformation (TFN) in pneumococci, we developed a method for isolating TFN- mutants using insertional inactivation coupled with fusions to the gene for alkaline phosphatase (phoA). One TFN- mutant transformed 2 log units less efficiently than the parent strain. Reconstitution of the mutated region revealed a locus, rec, that contains two polycistronic genes, exp10 and the previously identified recA (B. Martin, J. M. Ruellan, J. F. Angulo, R. Devoret, and J. P. Claverys, Nucleic Acids Res. 20:6412, 1992). Exp10 is likely to be a membrane-associated protein, as it has a prokaryotic signal sequence and an Exp10-PhoA fusion localized with cell membranes. On the basis of sequence similarity, pneumococcal RecA is a member of bacterial RecA proteins responsible for homologous recombination of DNA. DNA-RNA hybridization analysis showed that this locus is transcribed as a polycistronic message, with increased transcription occurring during competence. With an Exp10-PhoA chimera used as a reporter, there was a 10-fold increase in the expression of the rec locus during competence while there was only minimal expression under growth conditions that repressed competence. The TFN- mutant containing the exp10-phoA fusion produced activator, a small extracellular polypeptide that induces competence, and the expression of rec was induced in response to activator. Therefore, the rec locus is directly required for genetic transformation and is regulated by the cell signaling mechanism that induces competence.

An invasive form of the CaM-sensitive adenylyl cyclase from Bordetella pertussis can be isolated from bacterial culture supernatants. This isolation is achieved through the use of QAE-Sephadex anion-exchange chromatography. It has been demonstrated that the addition of exogenous Ca2+ to the anion-exchange gradient buffers will affect elution from the column and will thereby affect the isolation of invasive adenylyl cyclase. This is probably due to a Ca2(+)-dependent interaction of the catalytic subunit with another component in the culture supernatant. Two peaks of adenylyl cyclase activity are obtained. The Pk1 adenylyl cyclase preparation is able to cause significant increases in intracellular cAMP levels in animal cells. This increase occurs rapidly and in a dose-dependent manner in both N1E-115 mouse neuroblastoma cells and human erythrocytes. The Pk2 adenylyl cyclase has catalytic activity but is not cell invasive. This material can serve, therefore, as a control to ensure that the cAMP which is measured is, indeed, intracellular. A second control is to add exogenous CaM to the Pk1 adenylyl cyclase preparation. The 45-kDa catalytic subunit-CaM complex is not cell invasive. Although the mechanism for membrane translocation of the adenylyl cyclase is unknown, there is evidence that the adenylyl cyclase enters animal cells by a mechanism distinct from receptor-mediated endocytosis. Calmodulin-sensitive adenylyl cyclase activity can be removed from preparations of the adenylyl cyclase that have been subjected to SDS-polyacrylamide gel electrophoresis. This property of the enzyme has enabled purification of the catalytic subunit to apparent homogeneity. The purified catalytic subunit from culture supernatants has a predicted molecular weight of 45,000. This polypeptide interacts directly with Ca2+ and this interaction may be important for its invasion into animal cells. Finally, the technique for purifying the catalytic subunit by SDS-polyacrylamide gel electrophoresis may prove useful in studying the interaction of the adenylyl cyclase with other components produced by the bacteria, as well as the interaction of the enzyme with eukaryotic target cells.

The extracellular calmodulin-sensitive adenylate cyclase produced by Bordetella pertussis is synthesized as a 215-kDa precursor. This polypeptide is transported to the outer membrane of the bacteria where it is proteolytically processed to a 45-kDa catalytic subunit which is released into the culture supernatant [Masure, H.R., & Storm, D.R. (1989) biochemistry 28, 438-442]. The gene encoding this enzyme, cyaA, is part of the cya operon that also includes the genes cyaB, cyaD, and cyaE. A comparison of the predicted amino acid sequences encoded by cyaA, cyaB, and cyaD with the amino acid sequences encoded by hlyA, hlyB, and hlyD genes from the hemolysin (hly) operon from Escherichia coli shows a large degree of sequence similarity [Glaser, P., Sakamoto, H., Bellalou, J., Ullmann, A., & Danchin, A. (1988) EMBO J. 7, 3997-4004]. Complementation studies have shown that HlyB and HlyD are responsible for the secretion of HlyA (hemolysin) from E. coli. The signal sequence responsible for secretion of hemolysin has been shown to reside in its C-terminal 27 amino acids. Similarly, CyaB, CyaD, and CyaE are required for the secretion of CyaA from Bordetella pertussis. We placed the cyaA gene and a truncated cyaA gene that lacks the nucleotides that code for a putative C-terminal secretory signal sequence under the control of the lac promoter in the plasmid pUC-19. These plasmids were transformed into strains of E. coli which contained the hly operon. The truncated cyaA gene product, lacking the putative signal sequence, was not secreted but accumulated inside the cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Bordetella pertussis, the etiologic agent of whooping cough, produces a calmodulin-sensitive adenylate cyclase which elevates intracellular cAMP in a variety of eucaryotic cells. Exogenous calmodulin added to the partially purified adenylate cyclase has been shown to inhibit invasion of animal cells by this enzyme (Shattuck, R. L., and Storm, D. R. (1985) Biochemistry 24, 6323-6328). In this study, several properties of the calmodulin-sensitive adenylate cyclase are shown to be influenced by Ca2+ in the absence of calmodulin. The presence or absence of Ca2+ during QAE-Sephadex ion exchange chromatography produced two distinct chromatographic patterns of adenylate cyclase activity. Two different forms of the enzyme (Pk1 and Pk2EGTA) were isolated by this procedure. Pk1 adenylate cyclase readily elevated intracellular cAMP levels in mouse neuroblastoma cells (N1E-115) while Pk2EGTA adenylate cyclase had no effect on cAMP levels in these cells. Gel exclusion chromatography of Pk1 adenylate cyclase gave apparent Stokes radii (RS) of 43.5 A (+/- 1.3) in the presence of 2 mM CaCl2 and 33.8 A (+/- 0.94) in the presence of 2 mM EGTA [( ethylenebis (oxyethylenenitrilo)]tetraacetic acid). These Stokes radii are consistent with molecular weights of 104,000 (+/- 6,400) and 61,000 (+/- 3,600), respectively. Pk2EGTA adenylate cyclase had an apparent RS of 33.0 (+/- 1.2) (Mr = 60,600 (+/- 2,800] in the presence of Ca2+ or excess EGTA. At 60 degrees C, Pk1 adenylate cyclase exhibited a Ca2+-dependent heat stability with a half-life for loss of enzyme activity of 10.3 min in 5 mM CaCl2 and a half-life of 2.8 min in the presence of 0.1 microM CaCl2. The stability of Pk2EGTA adenylate cyclase was not affected by changes in free Ca2+. The adenylate cyclase preparations described above were submitted to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and enzyme activity was recovered from gel slices by extraction with detergent containing buffers. The catalytic subunit isolated from SDS-polyacrylamide gels was activated 7-fold in the presence of Ca2+ with maximum activity observed at 1 microM free Ca2+. With both preparations, the apparent molecular weight of the catalytic subunit on SDS gels was 51,000 in the presence of 2 mM CaCl2 and 45,000 in the presence of 2 mM EGTA. The catalytic subunit of the enzyme was purified to apparent homogeneity by preparative SDS-polyacrylamide gel electrophoresis and resubmitted to SDS gel electrophoresis in the presence or absence of free Ca2+. The purified catalytic subunit also exhibited a Ca2+-dependent shift in its mobility on SDS gels.(ABSTRACT TRUNCATED AT 400 WORDS)

A method is described for the rapid purification of both S-100 protein and calmodulin from crude bovine brain extracts by the use of a fluphenazine-Sepharose affinity column eluted stepwise with decreasing concentrations of free Ca2+. Protein containing only alpha-subunit was purified from preparations of S-100 protein by anion-exchange chromatography. This protein co-migrated with the alpha-subunit of S-100 protein on sodium dodecyl sulphate/urea/polyacrylamide-gel electrophoresis and had an amino acid composition identical with that previously reported for this subunit. The results of u.v.-absorption and fluorescence-emission spectroscopy indicate that the tryptophan residue of the purified alpha-subunit of S-100 protein undergoes a Ca2+-induced change in environment. Measurements of changes in tryptophan fluorescence with increasing Ca2+ concentrations suggest an apparent dissociation constant of the alpha-subunit for Ca2+ of 7 × 10(-5)M in the absence of K+. In the presence of 90mM-K+ this value is increased to 3.4 × 10(-4)M.

The amino acid sequence was determined for bovine brain P-57, a neurospecific, membrane-associated, calmodulin-binding protein. It consists of a single 239-residue polypeptide chain blocked at its amino terminus and containing an unusually hydrophilic amino acid composition. Seventy percent of the molecule is composed of Glu/Gln, Ala, Lys, Asp/Asn, and Pro; there is only one aromatic residue. A lack of favorable cleavage sites required that a particularly wide variety of digests and subdigests be performed to obtain appropriate sets of overlapping peptides. This protein is clearly homologous with the cDNA-derived sequence of mouse brain P-57, although the bovine protein is 12 amino acid residues longer; the homology is less obvious in the middle sections of the two sequences. Bovine brain P-57 lacks homology with any other protein in an updated sequence database. A segment reported to interact with calmodulin (Arg-Gly-His-Ile-Thr-Arg-Lys-Lys-Leu) is placed at residues 43-51 within the only extended segment of P-57 that carries the net positive charge that would favor that interaction. There is no hydrophobic segment characteristic of many proteins that interact with membranes.

Urine obtained from normal pregnant women as well as from patients with hCG-secreting tumors frequently contains native hCG and free hCG subunits when separated on Sephadex G-100. In addition, a small amount of an immunoreactive, hCG-like, low molecular weight substance is usually observed in those chromatograms and represents less than 1% of the total immunoreactive hCG present. Two patients with widely metastatic hCG-secreting tumors excreted disproportionately large quantities of that low molecular weight substance, and that observation raised the possibility that this substance was a secretory and not a degradative product of the hCG molecule. The small immunoreactive hCG-like substance was subsequently characterized immunologically, biologically, and physically. The hCG fragment displayed a biphasic dose-response line in a homologous hCG RIA. The slope of the upper portion of the dose-response line was equal to that for native hCG, but the slope of the lower component of the dose-response line was significantly different from that for hCG. The immunoreactive hCG substance cross-reacted with hCG beta but not with either hCG alpha or hCG beta carboxyl-terminus. The small molecular size immunoreactive hCG-like substance bound to Concanavalin A-Sepharose 4B and eluted with 0.2 M alpha-D-methyl glucopyranoside, contained no significant intrinsic biological activity when tested in the in vitro Leydig cell bioassay and also failed to compete with labeled hCG for specific ovarian LH/hCG receptors. Consequently, that small urinary immunoreactive hCG substance behaved neither as a hCG agonist or antagonist. It exhibited a plasma half-life of 4.5 min when the appropriate Sephadex G-100 fractions were injected into immature female rats. The small molecular size immunoreactive hCG-like substance may be a secretory or breakdown product of hCG-secreting cells.

HNA was assayed using a substrate plasma, thrombin and various concentrations of heparin, results being expressed in units of heparin neutralized. Technical conditions of the assay were analysed. HNA values depended on the kind of substrate, the use of CaCl2, the thrombin concentration. A loss of HNA was observed after heating or adsorption of the media. With similar citrate and overall protein composition of both blank (substrate alone) and sample curves (substrate and tested medium), a good relation was obtained between amount of HNA measured in dilutions of different media. Values obtained for serum and plasma are relatively high and not comparable with the low values obtained for platelet extract.

The main haemostasis changes observed in a screening study performed in 40 patients who underwent an open heart surgery with extracorporeal circulation (ECC) are: a significant drop in platelet count from the onset of the ECC to the third postoperative day, a decrease of platelet retention and aggregation during ECC with an 8-day persistently increased heparin-neutralizing activity in plasma but not in serum, a moderate decrease of plasma factors I, II, VII-X, X and XIII and a more important drop in factor V which disappears 24 h after ECC, a transitory increase of fibrinolysis during ECC and the lack of FDP elevation in the serum. These disorders require a very good neutralization of the heparin used during ECC. The ratio protamine/heparin can be established by a titration clotting time test. Protamine chloride seems to be more efficacious and to act more quickly than protamine sulfate for the neutralization. An overload in protamine can enhance the hemostatic, biological and clinical disorders. The preventive administration of platelet concentrate immediately after the heparin neutralization contributes to reduce the bleeding disorders related to the quantitative and qualitative platelet defects.

The fundamental treatment of haemophilia consists in blood transfusion or transfusion of blood derivatives.Fresh blood, fresh plasma stored in the frozen state at minus 30°, or lyophilized in parti...

SummaryBleeding time, clotting time and capillary resistance test have been studied in 100 normal people.1° As far as our Belgian people are concerned and with the technical conditions described, positivity of the capillary resistance test has to be considered far above the values generally admitted as normal : 45 % of normal subjects have more than 10 petechial spots, 8 % of them more than 80. Differences in the size of the spots are more significative than differences in number.2° Results obtained in the determination of bleeding time are in the limits generally considered as normal (96 % of the results are between 1 and 3 minutes).3° Clotting time is appreciated by the moment at which the first fine thread of fibrin appears on a paraffined slide in the first drop of blood collected from the finger. If temperature is maintained between 18° and 20° this time does not exceed 16 minutes; in the second or third drop of blood this time is 14 minutes. On non paraffined slides these limits are reduced to 13 mi...

SummaryAuthors have treated six cases of leukaemia and two cases of lymphomas with urethane as proposed by Paterson and al. 3 gr. of the drug were given a day.In the cases of leukaemia a decrease of the while cells count has been observed while the amount of red cells increased at the same time. The bone marrow showed a decrease of the immature granulocytes.The spleen decreased in size. The results were not so good in the cases of lymphoma.There is no doubt that this new therapy is useful.

An elderly woman had dysmyelopoietic syndrome with a rapidly fatal course. On initial marrow and unstimulated blood cell examination a structural anomaly of the X chromosome was observed with a 46,X,idic(X)(q13) karyotype with the exception of two cells showing a 47,XXX karyotype. Peripheral blood lymphocytes showed a normal 46,XX karyotype. The specificity of these findings is discussed.

SummaryAuthor has studied from a statistical point of view the results obtained in normal people when determining the number of blood platlets by a procedure described by Van Herwerden en Van Goids...

P-57 is a neurospecific calmodulin binding protein that was discovered by virtue of its unusual interactions with calmodulin-Sepharose [Andreasen, T. J., Luetje, C. W., Heideman, W., & Storm, D. R. (1983) Biochemistry 22, 4615-4618; Cimler, B. M., Andreasen, T. J., Andreasen, K. I., & Storm, D. R. (1985) J. Biol. Chem. 260, 10784-10788]. In contrast to other calmodulin binding proteins, P-57 has higher affinity for calmodulin-Sepharose in the absence of calcium compared to that in the presence of calcium. In this study, we report the chemical and physical properties of P-57 purified from detergent-solubilized bovine brain membranes. The amino acid composition of P-57 is distinctive in that it contains a single phenylalanine residue with no other aromatic amino acids and a relatively high percentage of proline and alanine. In the presence of 0.05% Lubrol PX, its predicted secondary structure from circular dichroism spectroscopy is 1% alpha-helix, 21% beta-sheet, and 78% random coil. The hydrodynamic characteristics of the protein-detergent complex and the molecular weight of the protein were determined by gel filtration and sucrose density gradient sedimentation in the presence and absence of calmodulin. The P-57-detergent complex has an apparent Stokes radius (Rs) of 4.58 nm and a sedimentation coefficient (S20,w) of 1.44 S while the Stokes radius and S20,w for the P-57-calmodulin-detergent complex are 5.33 nm and 2.32 S, respectively. Perrin analysis of a 5-[[[(iodoacetyl)amino]ethyl]amino]-1-naphthalenesulfonic acid (AEDANS) derivative of P-57 confirmed the Stokes radius determined by gel filtration.(ABSTRACT TRUNCATED AT 250 WORDS)

Medroxyprogesterone acetate (MPA), a potent progestagen, is used as a very efficient contraceptive agent. The method of administration is by a threemonthly or sixmonthly intramuscular injection. The method is particularly convenient when the desired number of children has been reached. Fertility is sometimes reestablished only slowly after stopping the injections. The main side effects are linked to endometrial atrophy (blood loss, amenorrhoea). Other side effects are infrequent and subjective. A controversy has risen over the product, by reference to its effects on beagle dogs. We have reviewed the literature. We also present the characteristics of 313 patients treated during (all together) 8,000 months. These patients belong to a rather poor sample of the population (amongst them many immigrant women), aged about 30, with above average pregnancy and parity rates. We also studied, in 31 patients, serum levels of glucose and lipids, and haemostasis. Side effects and reasons for discontinuing the drug are reported. The Pearl index was 0,75% women-years. There was no average effect on weight, blood tension, glycemia. Triglycerides increased slightly, and there was a discreet activation of coagulation with inhibition of the fibrinolytic activity, but without clinical consequences. We conclude that we can go on prescribing the drug, in view of our study and after reviewing the literature.

In an experimental study on the rabbit (80 animals) the haemostasis changes observed during the perfusion of dextrans of various molecular weights, gelatin and stable plasma protein solution (1.5 g/kg/h during 3 h) are compared using the normal animal and the animal with DIC induced by thrombin perfusion (100 U/kg/h during 3 h). With the normal animal, no significant disorders of haemostasis have been observed during perfusion of dextran 40 or SPPS. On the contrary, the perfusions with dextran 75 or gelatin are associated with modifications of platelet activities and of platelet and plasma plasmininhibiting activities similar to those found in thrombophilia states. Perfusion with dextran 200 produces haemostasis changes similar to those found during consumptive coagulopathies. In the animals with DIC, preventive, simultaneous or curative administration of dextrans 40, 75, 200 or gelatin significantly enhances the haemostasis disorders produced by perfusion with thrombin alone, this is opposed to SPPS which has no influence.

Heparin cofactor activity is measured with the amidolytic method according to 0degard (1975) during in vitro coagulation and during bleeding.Heparin cofactor activity decreases differently during coagulation process of PRP or PFP. It is suggested this is due to heparin neutralization by PF4 and/or to thrombin formation. Antithrombin III level is not modified.During bleeding, the same phenomenon is observed when the amount of heparin in the buffer is low enough. Decrease in heparin cofactor activity is already observed on a sample collected less than 90 seconds after the puncture, before any detectable thrombin formation.Decrease in heparin cofactor activity during bleeding is studied in pathological conditions. It is more marked in some hypercoagulable states.

SummaryA collaborative study of the first Belgian Standard for Factor VIII assays has been carried out. It involved participants working in five University Hospitals, who were asked: 1) to calibrate the Standard in VIII: C International Units; 2) to measure the VIII: C and VIII R: Ag levels in numerous samples of fresh normal plasma, against the Belgian Standard; and 3) to define whether the Belgian Standard could be used for VIII R: RCF measurements.One VIII R: Ag Unit was defined as the amount of antigen present in that volume of average fresh normal plasma containing 1 VIII: C International Unit.The calibration of VIII: C in International Units disclosed important differences between the five laboratories, probably due to the difficulties encountered in the use of the International Standard. When tested against the 2nd International Standard, the Belgian Standard was calculated to contain 0.99 VIII: C I.U. and 1.08 VIII R: Ag/vial; when tested against average fresh normal plasma taken as 100%, the Belg...

Antiheparin activity is usually achieved by a heparin-thrombin time assay. Platelet free substrate plasma is adsorbed or not, calcium chloride used or not. Those technical conditions are analysed.Results are expressed by the amount of heparin units neutralized in the assay. With non adsorbed plasma substrate, calcium chloride generates thrombin, increasing antiheparin like activity. Lower antiheparin like activity is also observed with BaSO4 plasma substrate and with thrombin free of factor Xa. The amount of heparin units neutralized varies from 2.9 to 1.2 according to the system used.Tested without calcium, with Xa free thrombin and BaSO4 adsorbed substrate plasma, there exists a good relationship between the amount of heparin neutralized and various dilutions of serum, platelet rich plasma and platelet extract.An assay based on anti-Xa effect of heparin, set up according to the heparin assay of Yin, is compared to the method described above.

Bordetella pertussis, the pathogen responsible for whooping cough, releases a soluble calmodulin-sensitive adenylate cyclase into its culture medium. Several investigators have shown that the partially purified adenylate cyclase is capable of entering animal cells and elevating intracellular cAMP levels [Confer, D. L., & Eaton, J. W. (1982) Science 217, 948-950; Shattuck, R. L., & Storm, D. R. (1985) Biochemistry 24,6323-6328]. However, the mechanism for entry of the catalytic subunit of the adenylate cyclase into animal cells is unknown. Recently, it was determined that the purified catalytic subunit of the enzyme is unable to enter animal cells [Masure, H. R., Oldenburg, D. J., Donovan, M. G., Shattuck, R. L., & Storm, D. R. (1988) J. Biol. Chem. 263, 6933-6940]. On the basis of these data and other observations, we hypothesized that the culture medium of B. pertussis contains one or more additional polypeptides which facilitate entry of the adenylate cyclase catalytic subunit into animal cells. In this study, we report that a cell-invasive preparation of B. pertussis adenylate cyclase was rendered noninvasive after passage through a wheat germ lectin-agarose column. A fraction was eluted from the wheat germ lectin-agarose column with N-acetyl-D-glucosamine. This fraction, when combined with the noninvasive adenylate cyclase, was able to restore the ability of the adenylate cyclase preparation to enter neuroblastoma cells and increase intracellular cAMP levels. Furthermore, the fraction eluted from the wheat germ lectin-agarose column was found to be trypsin and chymotrypsin sensitive, suggesting that this material was proteinaceous.(ABSTRACT TRUNCATED AT 250 WORDS)

In an experimental study in the rabbit, the modifications of some haemostasis parameters (platelet count, platelet retention and aggregation, platelet factors 3 and 4, platelet and plasma plasmin inhibiting activities, fibrinogen and other plasma factor levels, FDP), and histological findings are compared in both the normal animal and the animal with disseminated intravascular coagulation (DIC) induced by thrombin perfusion after administration of fibrinolytic inhibitors (plasminogen antiactivators and proteinase inhibitors). In the normal animal, the administration of fibrinolytic inhibitors is followed by haemostatic changes similar to those found in thrombophilic states. The modifications are more pronounced with plasminogen antiactivators than with proteinase inhibitors. In the animal with DIC, the administration of fibrinolytic inhibitors enhances the haemostatic and the biological disorders produced by thrombin perfusion. The effect of the plasminogen antiactivators is even more evident. The preventive administration of heparin reduces or abolishes the biological and histological disorders induced by thrombin; its beneficial effect is considerably reduced when thrombin is combined with fibrinolytic inhibitors. The administration of acetylsalicylic acid appears to be ineffective for the prevention of haemostatic and histological changes induced by thrombin perfusion.

A rational haemotherapy, consists, in giving all the needed blood components in suitable amounts, avoiding degraded products and undesirable contaminants, avoiding too, the wastage of unneeded components, useful for other patients, considering the ethic aspect of human blood donation and the logistic problems of blood transfusion services.