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Maximum heart rates (HR) of three soricine shrews and six other small mammals were measured in response to a single supramaximal dose of isoproterenol (Iso) under urethan anesthesia. The highest HR, 1,043 +/- 66 (SD) beats/min (n = 3), was in least shrew (Sorex minutus, mean body mass 3.02 +/- 0.81 g). Maximum HRs of common shrew (Sorex araneus, 7.16 +/- 1.54 g) and water shrew (Neomys fodiens, 12.80 +/- 1.54 g) were 938 +/- 29 (n = 7) and 887 +/- 21 (n = 6), respectively. In general, maximum HRs of soricine shrews and other small wild mammals followed the common mammalian pattern, fHmax/Iso = 443 x Mb-0.14, determined by body size. The exponent for this equation is smaller than that of resting HR (-0.25) (Stahl, J. Appl. Physiol. 22: 453-460, 1967), predicting crossover at approximately 3 g body mass. However, resting HRs of small mammals were clearly lower than expected on the basis of body mass. Lowering resting HR below the common mammalian level, with concomitant increase in stroke volume, seems to be a prerequisite for small mammals to regulate cardiac output against the ceiling of maximum HR. Electrophoretic analysis showed that the myosin of shrew ventricles is different from those of rodent species. In native conditions, shrew myosin, designated V1', migrated faster than the V3 and V1 forms of rat heart. On SDS gradient gel the single heavy chain of shrew myosin migrated slower than the alpha- or beta-chains of rat ventricle. Differences in the molecular weight of light chains were also noted between small mammals. Despite the notable differences in myosin composition, myosin-ATPase activity of the shrew hearts was similar to that of mouse and rat heart. Because duration of isometric contraction was inversely related to resting and maximum HRs, it was concluded that in the small mammals rate and duration of contraction are determined mainly by the release and uptake rate of myoplasmic Ca2+ and less by myosin-ATPase activity.
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PMID:Maximum heart rate of soricine shrews: correlation with contractile properties and myosin composition. 153 6

Hypertrophy of the urinary bladder was produced in rabbit by partial ligation of the urethra. Electrophoresis of the bladder smooth muscle myosin on highly porous (3.5-7% gradient) SDS-polyacrylamide gel revealed two heavy chain isoforms, SM-1 and SM-2 with approximate molecular weights of 204,000 and 200,000, respectively. The ratio of the SM-2 to SM-1 heavy chain is 3:1 for myosin isolated from normal bladder smooth muscle, and this ratio changes to about 1:1 in hypertrophied bladder. Despite a change in the ratio of SM-2 to SM-1, the myosin ATPase and the actin-activated ATPase activities are not altered in response to hypertrophy.
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PMID:Smooth muscle myosin isoform distribution and myosin ATPase in hypertrophied urinary bladder. 153 95

Inhibition of neurotransmitter release by tetanus toxin and botulinum neurotoxin A can be mimicked by intracellular application of the corresponding toxin light chains. The aim of this study was to determine whether the two-chain toxins are reduced by brain preparations to yield free light chains which would represent the ultimate toxins. The interchain disulfide of two-chain tetanus toxin was cleaved by rat cortex homogenate fortified with NADPH. Reduction was promoted further by addition of thioredoxin. Thioredoxin reductase was demonstrated in and purified from porcine brain cortex. The thioredoxin system which consisted of purified enzyme, thioredoxin and NADPH reduced both toxins. The resulting light chains appeared homogeneous in SDS gel electrophoresis. The complementary heavy chain of tetanus but not of botulinum toxin migrated in two bands, the faster one with the velocity of heavy chain obtained by chemical reduction. The major, slower form was converted into the faster by chemical but not by enzymatic reduction. Tetanus toxin, whether in its single-chain or two-chain version also occurred in two forms which differed by their electrophoretic mobility. The two forms of single-chain toxin were interconverted by chemical reduction or oxidation but not by the thioredoxin system. It is concluded that a) a thioredoxin system in brain tissue reduces the interchain disulfide of two-chain tetanus toxin and botulinum neurotoxin A, b) tetanus toxin but not botulinum neurotoxin A consists of two electrophoretically distinct forms which differ by the thiol-disulfide status of their heavy chains, c) the disulfide loop within the heavy chain of tetanus toxin is resistant to the thioredoxin system.
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PMID:Reductive cleavage of tetanus toxin and botulinum neurotoxin A by the thioredoxin system from brain. Evidence for two redox isomers of tetanus toxin. 157 25

In studying the molecular defects responsible for cross-reacting material-positive hemophilia A, we have identified two patients in whom the nonfunctional factor VIII-like protein has abnormal, slower-moving heavy or light chains on SDS/PAGE. Both patients have severe hemophilia A (less than 1% of normal factor VIII activity) with a normal plasma level of factor VIII antigen. The molecular defects were identified by denaturing gradient gel electrophoresis screening of PCR-amplified products of the factor VIII-coding DNA sequence followed by nucleotide sequencing of the abnormal PCR products. In patient ARC-21, a methionine-to-threonine substitution at position 1772 in the factor VIII light chain creates a potential new N-glycosylation site at asparagine-1770. In patient ARC-22, an isoleucine-to-threonine substitution at position 566 creates a potential new N-glycosylation site at asparagine-564 in the A2 domain of the factor VIII heavy chain. The mobility of these chains on SDS/PAGE was normal after N-Glycanase digestion and procoagulant activity was generated--to a maximum of 23% and 45% of control normal plasma. Abnormal N-glycosylation, blocking factor VIII procoagulant activity, represents a newly recognized mechanism for the pathogenesis of severe hemophilia A.
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PMID:Hemophilia A due to mutations that create new N-glycosylation sites. 159 97

The essential role of Factor VIII:C (FVIII:C, anti-hemophilia factor A) as a cofactor for Factor IXa-dependent activation of Factor X has been established. In this paper, we describe that capillary endothelial cells from bovine adrenal medulla express active FVIII:C gene. Accumulation of FVIII:C in conditioned media from an 8-day-old culture is approximately twice as high as that stored in the cell when immunoprecipitated FVIII:C was analyzed for its ability to convert Factor X to Factor Xa. Analysis of [35S]methionine-labeled and immunoprecipitated FVIII:C from cells or conditioned media on SDS-PAGE under fully denatured conditions indicated that the newly synthesized FVIII:C consists of heavy chain of M(r) 200,000 and light chain of M(r) 46,000. The secreted FVIII:C in the non-reduced condition however, has a molecular weight of 270,000 which suggests that in native protein, the heavy and light chains are held together by S-S bonds. Furthermore, susceptibility of the immunoprecipitated FVIII:C to N-glycanase digestion establishes that the endothelial cells derived FVIII:C contains asparagine-linked carbohydrate side chains.
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PMID:Expression of blood clotting factor VIII:C gene in capillary endothelial cells. 162 40

A humanized rat monoclonal antibody (Campath 1H) has been expressed in HeLa cells using recombinant vaccinia viruses. Heavy and light chain recombinant viruses were constructed separately and when grown independently produced proteins of the expected molecular weights. Expressed heavy chain was entirely intracellular but light chain was mainly excreted and processed. When cells were infected at high multiplicity with both heavy and light chain recombinants a proportion of the heavy chain was then found in the extracellular medium. This secreted heavy chain was shown to be associated with light chain as judged by co-electrophoresis in non-reducing SDS polyacrylamide gels and by co-purification on protein-A sepharose. The secreted heavy and light chain complexes were functionally active as an antibody, with activity comparable to authentic Campath 1H antibody as assessed by ELISA, T-cell binding and antigen binding assays. Production of antibody in this system was achieved in the absence of serum, which is an important consideration in the production of monoclonal antibodies (MAbs). The amount of antibody produced was 0.2-0.4 micrograms/10(6) cells without optimization of expression levels. The wide host cell range of vaccinia virus together with the recently developed methods for increasing expression levels make this an attractive candidate as a flexible general vehicle for producing MAbs.
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PMID:Synthesis and secretion of a functional antibody in a vaccinia virus expression system. 163 59

Cathepsin B was purified from normal human liver and several human tumour tissues and partially characterized. Three forms of cathepsin B, with molecular masses of 25 kDa, 26 kDa (the two appearing as a doublet) and 30 kDa, were detected in SDS/polyacrylamide gels. The 25-26 kDa doublet was associated with the fractions from tumours and normal liver containing the highest cathepsin B activity. Cathepsin B from both sources showed similar pH optima. Both normal liver and tumour cathepsin B exhibited similar kinetics against selected synthetic substrates. At neutral pH and 24 degrees C, cathepsin B from both normal liver and tumour exhibited a lower Km and a higher kcat./Km than at pH 6.0. Their inhibitory profiles against synthetic inhibitors were also similar. Immunological studies with a monospecific antibody against the mature double-chain form of human liver cathepsin B and an antibody against a cathepsin B-derived synthetic peptide established the immunological similarity of liver and tumour enzymes. The N-terminal sequences of the 25 kDa and 26 kDa forms were identical with that of the heavy chain of the mature double-chain form of human cathepsin B, whereas the N-terminal sequence of the 30 kDa species was identical with that of the single-chain form of human cathepsin B. Treatment of the double-chain form of cathepsin B from normal liver and tumours with the endoglycosidase peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase converted the 26 kDa form into 25 kDa in SDS/polyacrylamide gels, suggesting that cathepsin B may exist as both glycosylated and unglycosylated forms. Our results, in contrast with those reported earlier for mouse cathepsin B, indicate that human liver and tumour cathepsin B are similar.
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PMID:Human tumour cathepsin B. Comparison with normal liver cathepsin B. 163 35

Three mouse IgG1 monoclonal antibodies (MAbs), named FA1, FA2, and FA3, against cardiac myosin heavy chain (MHC) with high specificity have been obtained. The immunogen used to generate these MAbs was the high-salt- and detergent-insoluble fraction of adult rat myocardial tissue. Western blots showed that these MAbs reacted with a 200 kD protein band, which comigrated with the heavy chain of purified rat cardiac myosin in SDS-PAGE. Immunofluorescence microscopy revealed that the antigen recognized by these MAbs was localized at the A-band of isolated myofibrils. The tissue-, species-, and isoform-specificities of these MAbs were examined by Western blots on various muscle samples. FA2 recognized fish, frog, chicken, rabbit, bovine, mouse and rat cardiac MHC, as well as rabbit skeletal and rat aorta smooth muscle MHC. This antibody reacted equally well with both alpha- and beta-isoforms of MHC. FA1 did not crossreact with any MHC tested so far but with rat cardiac MHC. It appeared to react only with alpha-isoform of MHC. FA3 recognized only rat, bovine and rabbit cardiac MHC with the specificity to bovine and rabbit atrial MHC. Elisa competition assay revealed that different epitopes on the antigen molecules were recognized by these three MAbs, although there was a partial overlap between the epitopes for FA1 and FA2. These anti-MHC MAbs will be most useful in investigating the expression of MHC during myocardial development.
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PMID:Monoclonal antibodies against cardiac myosin heavy chain. 170 14

A 29-year-old women, who had been treated by hemodialysis for 5 years because of chronic renal failure, developed bleeding tendency in March 1989. Laboratory data showed prolonged activated partial thromboplastin time which was not corrected by addition of normal plasma; factor VIII activity was less than 1% and factor VIII inhibitor 70 Bethesda units/ml. The inhibitor was eluted in the second peak which corresponded to IgG when the plasma was subjected to Sephacryl S 200 column. The further purified IgG fraction by passing through protein A column showed a factor VIII inhibitor activity of 52 Bethesda units/ml. The factor VIII inhibitor epitopes were examined by western blotting technique using factor VIII purified by monoclonal antibody as the antigen. The factor VIII preparation used was composed of a doublet of light chain (Mr 80,000) and three heavy chains (Mr 160,000-200,000) when examined by immunoblotting using anti-factor VIII light and heavy chains monoclonal antibodies after SDS-PAGE. Factor VIII inhibitor that arose in a hemophilia A patient recognized the light chain, and the inhibitor in this case reacted to the heavy chain of factor VIII.
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PMID:[Characterization of the factor VIII inhibitor in a patient with chronic renal failure]. 170 30

A fusion protein consisting of the humanised Fab fragment of the anti CEA MAb BW 431 and the human beta-glucuronidase was expressed in BHK cells. Functional testing revealed that the specificity and avidity of the humanised V region was similar to the original murine MAb BW 431. Furthermore, the enzymatic activity, pH sensitivity and stability of the human beta-glucuronidase in the fusion protein was comparable to the activity of recombinant human beta-glucuronidase. Using anti-idiotype affinity chromatography, two molecules of a molecular weight of 125 kDa or 250 kDa could be visualized under nonreducing conditions in SDS-PAGE. Reducing conditions revealed a 25 kDa light and 100 kDa heavy chain. Due to its suitable biological characteristics this fusion protein might be an appropriate molecule allowing a site specific antibody directed enzyme prodrug therapy (ADEPT) in vivo.
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PMID:Molecular and functional characterisation of a fusion protein suited for tumour specific prodrug activation. 173 23


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