Caldesmon content of mammalian smooth muscles

Summary In the present study we have used a quantitative immunoblotting method to measure the caldesmon content of a variety of smooth muscles with distinctly different contractile phenotypes. Two tonic vascular smooth muscles and several phasic smooth muscles were examined. The caldesmon, actin and myosin contents of each muscle type were measured. Smooth muscle from large arteries (i.e. bovine aorta and porcine carotid artery) had the lowest caldesmon content and phasic muscles (e.g. rat uterus and guinea pig taenia coli) had the highest. The molar ratio of monomeric caldesmon to monomeric actin was 1205 for the aorta and carotid arteryversus 122–28 for the taenia coli and uterus. The molar ratio of caldesmon to monomeric myosin heavy chain was 19 for the aorta and carotidversus 12 for the uterus and taenia coli. The caldesmon contents of canine trachealis and rabbit ileum were intermediate between these extremes. Evidence was found for the presence of both tissue- and species-specific caldesmon isoforms. The relatively high caldesmon content in rat uterus and guinea pig taenia coli suggests the possibility that the contractile phenotype associated with phasic smooth muscles may be dependent on the presence of caldesmon.     

Induction of interferon and host resistance in vivo by double-stranded complexes of copolyribonucleotide of inosinic and guanylic acids with polyribocytidylic acid

Summary Several double-stranded complexes of copolyribonucleotide of inosinic and guanylic acids with polyribocytidylic acid (poly IGC) were found to possess interferon inducing activity stronger than poly ICin vivo, Their activity increased in parallel with increase in the ratio of guanine base to hypoxanthine base in these copolymers as far as double-strand formation was observed with polyribocytidylic acid. Many other combinations of copolyribonucleotide with homopolyribonucleotide were also investigated, and several of them were found to induce interferon. However, the interferon inducing effects of these combinations including complementary base-pairings of hypoxanthine and cytosine increased in parallel with the length of the base-pairings, thus approaching to that of poly IC. It is, therefore, supposed that the activity of poly IG C is somewhat different from poly IC and that those of other combinations owe to the essential structure of poly IC. Furthermore, kinetics of interferon induction, cross tolerance to reinduction, and antiviral effectsin vivo of poly IGC and poly IC were studied.     

Determination of biogenic stimulators by the yeast nephelometric test

Summary Optimal condition for the A. F. Sysoev's yeast nephelometric test have been established. They are: age of yeasts 1–3 days. Incubation temperature 27–28°; suspension of animal tissue 150 to 1100 (live weight of tissue). The method thus modified may be used for standardization of tissue preparations.Presented by Academian V. P. Filatov     

Zinc uptake by proximal cells isolated from rabbit kidney: Effects of cysteine and histidine

The aim of this study was to characterize the mechanisms of zinc transport in proximal cells isolated from rabbit kidney cortex. Uptakes of ⁶⁵Zn were assessed under initial rate conditions, after 0.5 min of incubation. The kinetic parameters obtained at 20°C were a K _m of 15.0±1.5 M, a J _max of 208.0±8.4 pmol min^–1 (mg protein)^–1, and an unsaturable constant of 0.259±0.104 (n=8). Cadmium competitively inhibited the zinc uptake, with a K _i value of 13.0±2.8 M, while zinc competitively inhibited ¹⁰⁹Cd uptake by isolated cells. Cysteine and histidine stimulated zinc transport at an amino acidzinc molar ratio ranging from 11 to 81. This stimulation was not observed in the absence of a sodium gradient. At a molar ratio greater than 161 (i.e., 400 M cysteine or histidine and 25 M Zn), there was evidence of inhibition. These data suggest that zinc enters renal proximal cells (a) as a free ion via a saturable carrier-mediated process or an unsaturable pathway and (b) complexed with cysteine or histidine, by means of a sodium/amino acid cotransport mechanism.     

Serum creatine kinase activity following forearm flexion isometric exercise

Summary The purpose of this study was to 1) compare serum creatine kinase (CK) activity following two forearm flexion isometric exercise regimens differing in work to rest ratio, and 2) examine the CK response to a repeated bout of isometric exercise. Eleven males were tested on two sessions (bouts) spaced 1 week apart. For bout 1, five subjects (group A) performed a forearm flexion isometric exercise consisting of 40 10-s maximal contractions with 20-s inter-trial rests (1020), while six (group B) performed 40 maximal 10-s contractions with 5-s inter-trial rests (105). The increase in serum CK activity following the 1020 exercise (143%) was significantly greater than that following the 105 exercise (52%). The 1020 exercise was also associated with greater tension generation over trials. One week later, both groups performed a bout of 1020 exercise. A substantial reduction in the serum CK response was found following this second bout. The data suggest that for bout 1 the isometric exercise associated with the greater overall tension levels resulted in the greater CK response. However, when the 1020 exercise was repeated 1 week later, a substantial reduction in the CK response was found which was unrelated to the tension generated.This study was supported by a University Faculty Research Grant No. 2-03021     

Activation of myosin ATPase by actin isolated from cultured BHK cells and the effect of gelsolin

Summary Activation of skeletal muscle myosin and myosin subfragment-1 (S1) by actin purified from the cytoplasm of cultured BHK cells was studied using the fluorescence of pyrene-labelled BHK F-actin and its quenching by S1 and by an enzyme-linked ATPase assay. At non-saturating concentrations, both muscle and BHK actin activated skeletal muscle myosin to the same degree: at 30° C and an ionic strength of 108mm, 1 m actin approximately doubled the ATPase of myosin or of S1. The association between BHK actin and S1 was also followed in a fluorescence stop flow: the rate of ATP binding monitored by the loss of light scattering upon dissociation of actin was again the same for BHK and muscle actin. The similarity of activation of myosin ATPase by BHK and muscle actin at low actin concentrations (i.e. the similarity of V_max/K_m) suggests that bothV _max andK _m are similar for the two types of actin. The effect of varying filament length on actin activation of myosin ATPase was examined using pig plasma or BHK gelsolin to regulate the length. For both types of actin, maximum enhancement of the actomyosin ATPase activity was observed at an actin/gelsolin ratio of about 301, whereas inhibition was observed at lower ratios. Both activation and inhibition of actomyosin ATPase were apparent in the absence or presence of calcium; differences were observed only in the extent and the time course of the effect.     

Nonselective cationic currents elicited by extracellular ATP in human B-lymphocytes

Adenosine 5-triphosphate-(ATP)-induced whole-cell currents were studied in human B-lymphocytes, transformed by the Epstein-Barr virus, by means of the tight-seal voltage-clamp technique. During bath application of ATP, the membrane conductance was increased. The change of membrane conductance occurred within milliseconds. The dose response relationship for the ATP^4–-elicited membrane current (I _p) was fitted by the Hill function with a Hill coefficient of 1 and a K _D value of 0.2 mmol/l. Adenosine, as well as the Mg²⁺-bound form of ATP, did not effect the membrane conductance. I _p did not desensitize within 1 min and could be evoked repeatedly up to 100 times in 1 cell in the presence of the G-protein blocker Guanosine 5-o-(2-thiodiphosphate) (GDP [S]). Therefore, it seems that ion channels in form of P _2Z purinoceptors are involved in the observed effects. The permeability (P) sequence for cations carrying I _p was P _Ca:P _K:P _Cs:P _Na:P _TRIS= 3521.210.1. The reversal potential of I _p was not changed by substitution of intracellular Cl^– for aspartate, indicating that anions are not involved in the purinoceptor-dependent conductance. A single-channel conductance of P _2Z-receptor-dependent ion channels of about 3 pS was determined by noise analysis of I _p.     

The influence of Poly I∶C on the course of infection in mice inoculated with West Nile Virus

Summary In mice infected intraperitoneally with a hundred per cent lethal dose of West Nile virus a significant reduction in mortality was found if treatment with the complex of synthetic polyriboinosinic and polycytidylic acids (Poly IC) was given four hours prior to or twenty hours after virus challenge.Treatment induced large amounts of circulating interferon a few hours after inoculation. Only a slight difference in maximum viraemia in the various groups was found, but viraemia developed later in the mice given Poly IC a few hours before virus injection. Infection of the brain developed later in the groups treated with Poly IC.Using various doses of West Nile virus almost the same mortality was found in the group given a lethal virus dose but treated with Poly IC and the group receiving sublethal virus dose and no Poly IC treatment. Maximum of viraemia was high in the former group, while in the latter group it was significantly lower. Therefore it is supposed that Poly IC in these experiments did not protect through an interferon mediated suppression of the viraemia but rather through an effect of the interferon exerted directly upon the target organ. A reduction of circulating HI antibodies was found in the group on which Poly IC had the most pronounced effect.     

Effects of sonication on physicochemical and biological properties of synthetic double-stranded polyribonucleotides as interferon inducer

Summary Two kinds of synthetic double-stranded polyribonucleotides, poly IGC¹ and poly I:C and their constituent single-stranded polymers were subjected to sonication. Sonication of both poly IGC and poly IC resulted in decreases in viscosity, molecular size and heterogeneity in the size distribution. In poly IGC, whose average sedimentation constant was larger than or around 11 S, these changes were accompanied with enhancements of interferon inducing activity in rabbits and mice and antiviral activity in mice, and moreover with a decrease in the systemic toxicity in mice. In poly IC, however, such an enhancement in the interferon inducing activity was observed only when its molecular size corresponded to that of poly IGC. Previous sonication of poly C of the relatively large molecular size (> 10 S) has also been shown effective, to a certain extent, in obtaining double-stranded RNA of smaller size distribution with increased interferon inducing activity and lowered toxicity. It has been shown that these changes induced by sonication were based on the breakage of phosphodiester bonds of both double and single-stranded polyribonucleotides. On the basis of the analyses of the correlations between molecular sizes and the biological activities, it has been suggested that, while toxicity decreases always when the molecular size becomes smaller, the optimal size of the double-stranded polyribonucleotide complexes for interferon production ranges roughly from 10 S (9.1×10⁵ daltons) to 5 S (1.2 ×10⁵ daltons).     

Increasing incidence of meningococcal disease in Spain associated with a new variant of serogroup C

Serogroup B has been the main cause of meningococcal disease in Spain since at least 1979, but in recent years an increase in the prevalence of infection due to serogroup C meningococci has been detected. In 1996, for the first time, most cases of meningococcal disease were caused by serogroup C strains. The sero/subtype of all serogroup C meningococci received from 1993 to June 1996 was determined, and the results showed that C2bP1.2,5, the most common phenotype in 1995 and 1996 (63% and 65%, respectively), represented only 4.8% of strains in 1993. The C2bP1.2,5 epidemic strains appear to be responsible for the high prevalence of serogroup C in Spain. One hundred fifty-one randomly selected serogroup C strains were analyzed by multilocus enzyme electrophoresis, ribotyping, and pulsedfield gel electrophoresis. Pulsed-field gel electrophoresis provided the most accurate information: more than 80% of the C2bP1.2,5 and C2bP1.2 isolates exhibited one of two very closely related profiles, while most of the C:2b:NST and C2bP1.5 strains had a pattern located at a genetic distance of 0.24 from those two profiles. The results show that C2bP1.2,5 strains represent a subclone or a genetic variant of the previously identified Spanish epidemic clone C2bnon-subtypable strains.     

Characterization of the cellular response of spleen cells in BALB/c mice inoculated with Mycoplasma pneumoniae or the P1 protein

BALB/c mice were intranasally infected or intraperitoneally inoculated with Mycoplasma pneumoniae whole cells or were immunized with the isolated adhesin (P1 protein). Spleen cells were isolated and tested in vitro for proliferation activity after stimulation with the P1 protein and sonicated M. pneumoniae whole antigen preparations. In frequency analysis experiments the P1 protein-specific proliferative response of spleen lymphocytes increased from 1/11494 in mice immunized once to 1/3246 in eightfold-inoculated mice, demonstrating that the P1 protein is a prominent immunogen of M. pneumoniae cells. Depletion experiments showed that T and B cells are activated in a 21 relation. Fluorescence-activated cells sorting analysis revealed a shift of the CD4/CD8 ratio from 21 in control mice up to 31 in M. pneumoniae-, and to 3.41 in P1 protein-immunized mice, as well as an increase in interleukin 2 receptor-bearing cells and macrophage cell populations. The results indicate that this animal model is appropriate to study host-M. pneumoniae interactions and vaccination schedules.     

The reflex effect of certain drugs acting on the receptors of the mesenteric veins in the cat

Summary By employing the perfusion method of the humorally isolated protion of the anterior mesenteric vein in cat the author studied the reflex effect of solutions of adrenalin (110⁶), caffeine sodium benzoate (110³), strophanthin(12·10⁶), glyceryl trinitrate (12·10⁵) and cardiazol (12·10⁶) on the heart rate, arterial pressure, and respiration. Adrenalin provoked a rise of arterial pressure and stimulation of respiration by reflex action, while other substances mostly reduced the arterial pressure and depressed the respiration. Caffeine frequently decreased the rate heart. The reflex origin of these reactions was proved by control experiments with 2% procaine solution and labeled phosphorus P³².Presented by Active Member AMN SSSR V. V. Parin     

Amino acids 519–524 of Dictyostelium myosin II form a surface loop that aids actin binding by facilitating a conformational change

Residues 519–524 of Dictyostelium myosin II form a small surface loop on the actin binding face, and have been suggested to bind directly to actin through high affinity hydrophobic interactions. To test this hypothesis, we have characterized mutant myosins that lack this loop in vivo and in vitro. A mutant myosin in which this loop was replaced by an Ala residue (519–524/+A) was non-functional in vivo. Replacement with a single Gly residue instead of Ala yielded partial function, suggesting that structural flexibility, rather than hydrophobicity, is the key feature of the loop. The in vivo phenotype of the mutant enabled us to identify a number of additional amino acid changes that restore function to the 519–524/+A mutation. Intriguingly, many of these, including L596S, were located at some distances away from the 519–524 loop. We have also isolated suppressors for the L596S mutant myosin, which was not functional in vivo. The suppressors for 519–524/+A and those for L596S showed complementary charge patterns. In ATPase assays, 519–524/+A S1 showed very low activity and little enhancement by actin, whereas L596S S1 was hyper active and displayed enhanced affinity for actin. In motility assays, 519–524/+A myosin released actin filaments upon addition of ATP and was unable to support movements. L596S myosin was also inactive, but in this case actin filaments stayed immobile even after the addition of ATP. Transient kinetic measurements demonstrated that 519–524/+A S1 is not only slower than wild type to bind actin filaments, but also slower to dissociate from actin filaments. Based on these results, we concluded that the 519–524 loop is not a major actin binding site but aids actin binding by facilitating a critical conformational change.     

Biochemical characteristics,phage patterns,and O1 factor analysis ofEscherichia coli O1∶K1∶H7∶F11 and O1∶K1∶H−∶F9 strains isolated from patients with urinary tract infections

Biochemical characteristics, O1 antigen factors and phage patterns were examined in 35 urinary O1K1 isolates ofEscherichia coli different in H and F antigen. Fermentation of dulcitol, decarboxylation of ornithine, requirement for nicotinamide, and determination of O1 factor d allowed maximum differentiation. On the basis of these tests the strains could be divided into two major groups which are obviously of different clonal origin. Members of clone 1 represented by serotypes O1K1H7F11 (12 strains) and O1K1H^–F11 (5 strains) were characterized by positive biochemical reactions and absence of O1 antigen factor d. Negative biochemical tests and presence of O1 antigen factor d were shown by strains of clone 2 which were of serotypes O1K1H^–F9 (14 strains) and O1K1H^–F^– (3 strains). Phage patterns are less well correlated with clonal assignment. However, strains of clone 2 were not susceptible to K1-specific phage D and were non-typable with another set of 13 phages.     

Incidence and significance of candida antigen in low-risk and high-risk patient populations

A latex agglutination test which detects candida antigen in patients with disseminated infection was used to screen 328 patients. Of these patients 100 had renal failure but no signs or symptoms of candida infection, and 100 had high rheumatoid factor titers but also no signs or symptoms of candida infection. The remaining 128 patients were considered at high risk of developing systemic candida infection. Sequential titers were also determined in a number of these patients. The incidence of candida antigen titers 14 in the low-risk population was 3% as compared to 53% in the high-risk group. None of the patients with positive rheumatoid factor titers had candida antigen titers 12, however 13% had nonspecific agglutination at titers > 12. The incidence of nonspecific agglutination increased with increasing rheumatoid factor titers. In general the candida antigen titers correlated well with clinical findings and with the course of infection in those patients in whom sequential candida antigen titers were determined. This latex test thus appears to be a useful adjunct procedure for the diagnosis of serious candida infections.     

Characterization ofEscherichia coli serotype O12∶K1∶H7 isolates from an immunocompetent carrier with a history of spontaneous abortion and septicemia

Escherichia coli isolates from an immunocompetent woman with a history of repeated amnion infections and spontaneous abortion were characterized.Escherichia coli were isolated from stool, blood and cervix swab samples taken over a 21-month period after the last abortion which followed septicemia during pregnancy. All samples except the last cervix swab contained isolates of serotype O12K1H7, which produced adhesins, P fimbriae, type I fimbriae and the iron-chelator aerobactin. Pulsed-field gel electrophoresis revealed identical Xbal restriction patterns of the O12K1H7 isolates, suggesting that one particularEscherichia coli strain was responsible for the severe extraintestinal infections during pregnancy. The strain was able to persist in the intestine of the woman despite antibiotic therapy.     

Regional location of α1-antichymotrypsin and α1-antitrypsin genes on human chromosome 14

The human protease inhibitor genes ₁ antitrypsin (₁-PI) and ₁-antichymotrypsin (₁-ACT) are acute-phase proteins which are induced in response to inflammation. These inhibitors function to limit the activity of serine proteases in vivo. ₁-PI acts as an inhibitor of neutrophil elastase to protect the elastin fibers of the lung. Genetic deficiencies of ₁-PI result in development of chronic pulmonary emphysema. The physiologic role of ₁-ACT has not been clearly defined, but it also appears to function in the maintenance of protease-protease inhibitor equilibrium in the lung. Nucleic acid and protein sequence homologies detected between ₁-PI and ₁ t-ACT suggested an evolutionary relationship. Gene mapping experiments were performed to determine if these protease inhibitor genes reside at the same chromosomal locus in man. In situ hybridization data demonstrate that both ₁-PI and ₁-ACT map to the same region, q31–q32.3, on chromosome 14.     

Sequence specificity of mutations induced by benzo[a]pyrene-7,8-diol-9,10-epoxide at endogenousaprt gene in CHO cells

We have determined the spectrum of mutations induced, by±trans-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a] pyrene (BPDE) at the endogenous aprt locus in an hemizigous Chinese hamster ovary cell line exposed to 0.7 M BPDE. Southern analysis of 59 independent mutants revealed no major genomic alterations, indicating that gene inactivation was the result of a point mutation. This conclusion was confirmed by the cloning and sequencing of 21 of these mutants. The predominant mutation, the GCTTA transversion, comprised 62% of the spectrum, but other base pair substitutions and frameshifts were recovered. An examination of the target sequences for BPDE mutation revealed that mutations were localized within runs of GC base pairs. However, approximately half of these GC runs involved a particular sequence—a run of guanines flanked by adenine residues. Of seven such sites within the coding sequence ofaprt, mutations were clustered within five of them. This class of sequence occurs at codon 61 of the human C-Ha-ras1 protooncogene and may account for the selective activation of this codon by BPDE.     

Phosphorylation of caldesmon by smooth-muscle casein kinase II

Summary A caldesmon kinase activity was partially purified from an extract of chicken gizzard smooth muscle by sequential chromatography on columns of DEAE-Sephacel, MonoQ and Superose 12. This kinase was identified as casein kinase II by Western blotting using peptide-directed antibodies raised against the , and subunits of human casein kinase II; the smooth muscle enzyme consisted of similar subunits of M_r 43 000 (), 39 000 (), and 27 000 (). Phosphorylation of caldesmon and casein by smooth muscle casein kinase II was optimal at 0.1 M NaCl, did not require second messengers, and was inhibited by heparin. The kinase utilized either GTP or ATP as a substrate. Caldesmon was phosphorylated to 1 mol P_i mol^-1 caldesmon by smooth muscle casein kinase II with a K_m for caldesmon of 4.9 M. Two-dimensional thin-layer electrophoresis indicated phosphate incorporation into both serine and threonine. All the incorporated phosphate was recovered in the N-terminal peptide (residues 1–152) generated by cleavage at cysteine 153 with 2-nitro-5-thiocyanobenzoic acid. Purification of tryptic phosphopeptides and N-terminal sequencing revealed two principal sites of phosphorylation: serine 73 and threonine 83. The following four synthetic peptides corresponding to this domain of caldesmon were examined as substrates of casein kinase II: A = RRREVNAQNSVAEEE; B = AQNSVAEEE; C = RSTDDEAA; D = SVAEEETKRSTDDE. Interestingly, only peptides C and D were phosphorylated and both only at threonine. Phosphorylation of intact caldesmon did not affect the pattern of chymotryptic digestion suggesting that it does not induce a significant conformational change in the protein substrate. Phosphorylation also had no effect on the binding of caldesmon to actin or on the caldesmon-mediated inhibition of actomyosin MgATPase activity. However, phosphorylation completely abolished the interaction of caldesmon with immobilized smooth muscle myosin. These results are consistent with the localization of the myosin-binding domain near the N-terminus of caldesmon and of the actin-binding domain near the opposite end of the elongated molecule. Casein kinase II may therefore play a role in regulating caldesmon-myosin interaction and the ability of caldesmon to cross-link actin and myosin filaments in smooth muscle.     

Myosin filament ATPase is enhanced by intramolecularly cross-linked actin

Summary Reaction of rabbit skeletal muscle F-actin with the lysine-directed photolabile cross-linker, N-5-azido-2-nitrobenzoyloxy succinimide was limited to Lysine-328 and Lysine-326, with Lysine-328 being labelled to a greater extent. Photolysis of the modified actin enhanced the actin-activated MgATPase activity of filamentous scallp myosin 3-4-fold more than unmodified actin, without affecting calcium sensitivity. Unphotolysed modified actin behaved as untreated actin, indicating that photolysis was essential for the effect. The actin-activated ATPase of filamentous rabbit myosin was similarly increased by photolysed N-5-azido-2-nitrobenzoyloxy succinimide-modified actin. After photolysis in either the monomeric (G-) or filamentous (F-) form, N-5-azido-2-nitrobenzoyloxy succinimide-modified actin moved as a monomeric (42 kDa) species on SDS gels, and depolymerized and polymerized readily, demonstrating that any cross-linking event produced by photolysis must be intramolecular. In contrast to the substantial increase in actin-activated ATPase activity observed when photolysed ANB-NOS-modified actin was added to filamentous myosin, the enhancement was not observed with the soluble HMM and S-1 fragments of myosin. Photolysed modified actin showed only poor movement on a rabbit HMM-coated surface in vitro motility assays. These results can be explained if the internally cross-linked G-actin subunits which comprise only a fraction of the actin population, either weaken the actin-actin contacts or have an increased affinity for myosin.Abbreviations ANB-NOS N-5-azido-2-nitrobenzoyloxy-succinimide - G-actin monomeric actin - F-actin filamentous actin - S-1 subfragment I - HMM heavy meromyosin - EGTA ethyleneglycol(bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid) - DTT dithiothreitol - DMSO dimethyl sulfoxide - NADodSO₄ sodium dodecyl sulphate - TEMED N,N,N,N-tetramethylethylenediamine - TB tris(hydroxmethy)aminomethane boric acid - Tris tris(hydroxymethy)aminomethane - PTH phenylthiohydantoin - TPCK N-tosyl-phenylalanine chloromethyl ketone - HPLC highperformance liquid chromatography - TFA trifluoroacetic acid