Evidence that the serum inhibitor of hyaluronidase may be a member of the inter-alpha-inhibitor family.
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A study of the uncharacterized serum inhibitors of hyaluronidase, first described half a century ago, was undertaken. Activity was measured against bovine testicular hyaluronidase using a microtiter-based assay and reverse hyaluronan substrate gel zymography. The predominant inhibitory activity was magnesium-dependent and could be eliminated by protease or chondroitinase digestion and by heat treatment. Kinetics of inhibition were similar against hyaluronidases from testis and snake and bee venoms. The inhibitor had no effect on Streptomyces hyaluronidase, indicating that inhibition was not through protection of the hyaluronan substrate. Inhibition levels in serum were increased in mice following carbon tetrachloride or interleukin-1 injection, inducers of the acute-phase response. Reverse zymography identified a predominant band of 120-kDa relative molecular size, with two bands of greater and one of smaller size. The predominant protein was tentatively identified as a member of the inter-alpha-inhibitor family. Inhibition was also observed using either purified inter-alpha-inhibitor or an inter-alpha-inhibitor-related 120-kDa complex. Inter-alpha-inhibitor, found in the hyaluronan-rich cumulus mass surrounding mammalian ova and the coat of fibroblasts and mesothelial cells, may function to stabilize such matrices by protecting against hyaluronidase degradation. Turnover of circulating hyaluronan is extraordinarily rapid, with a half-life of 2-5 min. Prompt increases in levels of serum hyaluronan occur in patients with shock, septicemia, or massive burns, increases that can be attributed, in part, to suppression of degradation by these acute-phase reactants, the inhibitors of hyaluronidase. (+info)
Nucleotide sequence determination of bacteriophage T2 and T6 species I ribonucleic acids.
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The nucleotide sequences of species I RNA coded for by bacteriophages T2 and T6 have been analyzed using 32-P-labeled material from T2 and T6-infected cultures of Escherichia coli. The T1 and pancreatic ribonuclease digestion products were partially analyzed and the results were compared with nucleotide sequences from T4 species I RNA to obtain a minimum estimate of the number of nucleotide sequence differences among the three species I RNAs. Analysis of fragments obtained by digestion with epsilon-carboxymethyl-lysine-41-pancreatic ribonuclease and with E. coli Q13 S30 crude extract was also performed to provide some additional confirmation for the nucleotide sequences that were derived for the T2 and T6 species I RNAs. T2 species I RNA was found to be different at three positions in the nucleotide sequence, and unlike T4 species I RNA, contained in addition the modified nucleotide, psi, in a region where the proposed secondary structure is identical to the TpsiC-loop of a tRNA. T6 species I RNA was found to contain nucleotide differences from the T4 species I RNA sequence at four positions. The U at position 119 in the sequence appears to be modified to psi only to a small extent. While a biological function for species I RNA is unknown, the fact that there is over 97% homology in the sequences suggests strong evolutionary pressures to retain the nucleotide sequence in the T-even genomes. (+info)
Mediation systems in bacterial lipopolysaccharide-induced hypotension and disseminated intravascular coagulation. I. The role of complement.
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We have studied the role of complement in lipopolysaccharide (LPS)-induced hypotension and disseminated intravascular coagulation (DIC) by comparing the effects of injection of three preparations of LPS from E. Coli 0111:B4, S. minnesota Re595, and S. marcescens. Injections of nonlethal doses of these LPS preparations into normal rabbits produced decreases in mean arterial blood pressure during a 5-h period. When rabbits treated with cobra venom factor (CoF) to deplete C3 were injected with the various LPS preparations, mean arterial pressures fell at a rate and extent essentially identical to that observed in normal rabbits. Rabbits genetically deficient in C6 also demonstrated LPS-induced hypotensive changes. Only minimal, or no changes in plasma C3 levels or serum CH50 values were detected in normal rabbits after LPS injection. Hypotensive changes were also induced in rabbits when complement was rapidly activated by intravenous injection of CoF. In contrast to the hypotension induced by LPS, the fall in arterial pressure associated with the consumption of complement was short lived and required the rapid consumption of considerable amounts of C3. The occurrence of DIC noted in normal rabbits injected with each preparation of LPS was not inhibited in either rabbits treated with cobra factor or in C6-deficient rabbits. The DIC was most pronounced after injection of Re595 and S. marcescens LPS. Injection of the various LPS preparations produced a rapid disappearance of circulating neutrophils and mononuclear cells, which occurred with the same kinetics and to the same extent in normal, CoF-treated, and C6-deficient rabbits. Injection of either Re595 LPS or S. marcescens LPS produced a biphasic disappearance of circulating 51Cr-platelets. In contrast, injection of 0111:B4 LPS affected only slightly the rate of disappearance of 51Cr-platelets. Depletion of C3 by cobra factor treatment had no effect on the disappearance of platelets in animals injected with 0111:B4. In marked contrast cobra factor treatment greatly reduced the initial rapid disappearance of platelets in rabbits injected with either Re595 or S. marcescens LPS, but had no effect in the secondary disappearance phase. (+info)
Characterization of the internal motions of a chimeric protein by 13C NMR highlights the important dynamic consequences of the engineering on a millisecond time scale.
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By transferring the central curaremimetic beta hairpin of the snake toxin alpha into the scaffold of the scorpion charybdotoxin, a chimeric protein was constructed that reproduced the three-dimensional structure and partially reproduced the function of the parent beta hairpin, without perturbing the three-dimensional structure of the scaffold [1]. Picosecond to hour time scale motions of charybdotoxin and the engineered protein were observed, in order to evaluate the dynamic consequences of the six deletions and eight mutations differentiating the two molecules. The chimeric protein dynamics were also compared to that of toxin alpha, in order to examine the beta hairpin motions in both structural contexts. Thus, 13C R1, R1rho and 1H-->13C nOe were measured for all the CalphaHalpha and threonine CbetaHbeta vectors. As the proteins were not labeled, accordion techniques combined to coherence selection by pulsed field gradients and preservation of magnetization following equivalent pathways were used to considerably reduce the spectrometer time needed. On one hand, we observed that the chimeric protein and charybdotoxin are subjected to similar picosecond to nanosecond time scale motions except around the modified beta sheet region. The chimeric protein also exhibits an additional millisecond time scale motion on its whole sequence, and its beta structure is less stable on a minute to hour time scale. On the other hand, when the beta hairpin dynamics is compared in two different structural contexts, i.e. in the chimeric protein and the curaremimetic toxin alpha, the picosecond to nanosecond time scale motions are fairly conserved. However, the microsecond to millisecond time scale motions are different on most of the beta hairpin sequence, and the beta sheet seems more stable in toxin alpha than in the chimera. The slower microsecond to hour time scale motions seem to be extremely sensitive to the structural context, and thus poorly transferred from one protein to another. (+info)
Envenomation by the neotropical colubrid Boiruna maculata (Boulenger, 1896): a case report.
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This is a case report of a Boiruna maculata snake bite in a child admitted to the Hospital Municipal de Pronto Socorro de Porto Alegre, Porto Alegre, RS, Brazil. The patient was bitten on the lower left limb, and exhibited pronounced local manifestations of envenomation. She was treated with Bothrops antivenom and was discharged from the hospital five days later with marked improvement of envenomation. (+info)
Guinea-pig nephrotoxic nephritis. I. The role of complement and polymorphonuclear leucocytes and the effect of antibody subclass and fragments in the heterologous phase.
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In guinea-pig nephrotoxic nephritis induced by a sheep antibody there was minimal glomerular capillary deposition of C3 and accumulation of polymorphonuclear leucocytes (PMN) in the heterologous phase. The C4-deficient strain developed the same injury as normal Duncan-Hartley animals. Complement depletion with cobra venom factor, polymorph depletion with nitrogen mustard or anti-PMN serum and treatment with antihistamines provided no protection. The relationship between the dose of nephrotoxic antibody and the proteinuria was similar for gamma1 and gamma2 subclasses and the F(ab')2 fragment of gamma1 antibody. However, the F(ab') and F(ab) antibody fragments, though fixing on the glomerular basement membrane, did not cause proteinuria. It is concluded that the development of proteinuria in this system: is largely independent of the complement-polymorph system; is due to the fixation of the F(ab')2 fragment of the antibody molecule; and does not depend on an intact Fc piece. (+info)
Homozygous human C3 deficiency. The role of C3 in antibody production, C-1s-induced vasopermeability, and cobra venom-induced passive hemolysis.
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Studies of the family of a patient with marked deficiency of the third component of complement (C3) demonstrated that the patient was homozygous for a blank allele at the C3 locus, C3-. Metabolic studies with purified radiolabeled C3 in the patient revealed a mildly elevated fractional catabolic rate and a markedly reduced synthesis rate, consistent with a lack of C3 synthesis as the patient's primary defect. There was also a mild increase in the rate of conversion of purified C3 added to her serum and incubated at 37 degrees C in vitro. Major blood group-compatible erythrocytes from a patient with paroxysmal nocturnal hemoglobinuria had the same shortened survival in the C3-deficient patient as in a normal control. Although no leukocytosis developed in the patient in spontaneous infection by pyogenic organisms, there was a normal leukocytosis in response to the injection of thyphoid vaccine. The intradermal injection of C-1s, which produces a marked increase in vasopermeability in the skin of normal subjects, produced no definite change in the patient, possibly implicating C3 or a protein in the alternative pathway as the normal mediator of this response. The patient's serum exhibited near-normal immune adherence activity, confirming the lack of requirement of C3 for this function. C5 inactivation and passive hemolysis of unsensitized guinea pig erythrocytes occurred normally in C3-deficient serum on incubation with cobra venom factor, indicating that C3 is not required for these reactions. The patient's humoral antibody response to both protein and carbohydrate antigens was entirely normal, making it unlikely that C3 is required for antigen processing. (+info)
Increasing molecular diversity of secreted phospholipases A(2) and their receptors and binding proteins.
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Secreted phospholipases A(2) (sPLA(2)s) form a large family of structurally related enzymes which are widespread in nature. Snake venoms are known for decades to contain a tremendous molecular diversity of sPLA(2)s which can exert a myriad of toxic and pharmacological effects. Recent studies indicate that mammalian cells also express a variety of sPLA(2)s with ten distinct members identified so far, in addition to the various other intracellular PLA(2)s. Furthermore, scanning of nucleic acid databases fueled by the different genome projects indicates that several sPLA(2)s are also present in invertebrate animals like Drosophila melanogaster as well as in plants. All of these sPLA(2)s catalyze the hydrolysis of glycerophospholipids at the sn-2 position to release free fatty acids and lysophospholipids, and thus could be important for the biosynthesis of biologically active lipid mediators. However, the recent identification of a variety of membrane and soluble proteins that bind to sPLA(2)s suggests that the sPLA(2) enzymes could also function as high affinity ligands. So far, most of the binding data have been accumulated with venom sPLA(2)s and group IB and IIA mammalian sPLA(2)s. Collectively, venom sPLA(2)s have been shown to bind to membrane and soluble mammalian proteins of the C-type lectin superfamily (M-type sPLA(2) receptor and lung surfactant proteins), to pentraxin and reticulocalbin proteins, to factor Xa and to N-type receptors. Venom sPLA(2)s also associate with three distinct types of sPLA(2) inhibitors purified from snake serum that belong to the C-type lectin superfamily, to the three-finger protein superfamily and to proteins containing leucine-rich repeats. On the other hand, mammalian group IB and IIA sPLA(2)s can bind to the M-type receptor, and group IIA sPLA(2)s can associate with lung surfactant proteins, factor Xa and proteoglycans including glypican and decorin, a mammalian protein containing a leucine-rich repeat. (+info)