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Hb J-Anatolia [alpha 61(E10)Lys----Thr]: structural characterization and gene localization of a new alpha chain variant.

We report the characterization of a new hemoglobin variant having a single amino acid substitution (Lys----Thr) at position 61 of the alpha chain. In addition to the structural analysis, we also describe the strategy used for the identification of the base substitution and the localization of the defect at the gene level using polymerase chain reaction and hybridization with allele-specific oligonucleotides.

Alleles↗

MAP 1A and MAP 1B are structurally related microtubule associated proteins with distinct developmental patterns in the CNS.

Five high-molecular-weight microtubule-associated proteins (MAPs) were identified in brain tissue in previous work from this laboratory (Bloom et al., 1984). These proteins were termed MAP 1A, 1B, 1C, 2A, and 2B. The MAP 1's differed from the MAP 2's, and showed little evidence of interrelationship on the basis of immunological and biochemical comparison. We report here that MAP 1A and MAP 1B are, in fact, related at the level of subunit composition. Immunoprecipitation of the individual MAPs showed that both contained low-molecular-weight subunits of Mr 30,000 and Mr 19,000 (light chains 1 and 3). An additional subunit, light chain 2 (Mr 28,000), was primarily found in preparations of MAP 1A. The light chains co-sedimented with microtubules after chymotryptic digestion of the MAPs. This suggested an association of the light chains with the microtubule binding domains of the MAPs, which are identified here as distinct fragments of Mr 60,000 for MAP 1A and 120,000 for MAP 1B. A panel of monoclonal anti-MAP 1A and anti-MAP 1B antibodies, including one that reacts with a common phosphorylated epitope, was used to examine the distribution of these proteins in the developing rat brain and spinal cord. MAP 1B was found to be abundant in the newborn brain and to decrease with development, in contrast to MAP 1A which increased with development. By immunohistochemistry MAP 1B was found to be highly concentrated in developing axonal processes in the cerebellar molecular layer, the corticospinal tract, the mossy fibers in the hippocampus, and the olfactory nerve. Of particular interest, the mossy fiber and olfactory nerve staining persisted in the adult, indicating continued outgrowth of the mossy fibers as well as olfactory nerve axons. MAP 1A staining was, in contrast, weak or absent in developing axonal fibers but moderate in mature axons and intense in developing and mature dendritic processes. Our results indicate that MAP 1A and MAP 1B are structurally related components of the neuronal cytoskeleton with complementary patterns of expression.

Animals↗

Untranslated immunoglobulin kappa light chain mRNA in a lambda light chain-producing mouse myeloma, MOPC104E.

Fourteen clones were isolated in culture from a mouse myeloma, MOPC104E. All clones had kappa and lambda types of light chain mRNAs in approximately equimolar quantity as assayed by hybridization with specific complementary DNA (cDNA). However, the myeloma produces and secretes only lambda-type light chain protein. Both kappa- and lambda-type mRNAs in these clones were indistinguishable from kappa- and lambda-type mRNAs of other myelomas with respect to (a) adsorption to oligo-(dT) cellulose, (b) molecular size (12.6 S), and (c) thermal stability of the hybrids formed with corresponding cDNA. The kappa chain mRNA of MOPC104E cells, however, was translated very inefficiently both in vivo and in vitro, whereas the lambda chain mRNA was translated efficiently. These results indicate that each cell of MOPC104E myeloma synthesizes a crippled kappa chain mRNA in addition to a normal lambda chain mRNA.

Animals↗

C/A dynein isolated from sea urchin sperm flagellar axonemes. Enzymatic properties and interaction with microtubules.

C/A dynein is a novel dynein isolated from sea urchin sperm flagellar axonemes. It is composed of C and A heavy chains and some additional lower molecular mass polypeptide chains. The characterization of ATPase activity and the interaction of this dynein with microtubules polymerized from calf brain tubulin were investigated in this study. The ATPase activity of C/A dynein (0.3-0.4 mumol Pi/min per mg) was about one half that of outer arm 21 S dynein (0.6-0.8 mumol Pi/min per mg) at 25 degrees C. Vanadate inhibited the ATPase activity with a half-maximal inhibition at 1 microM. C/A dynein absorbed to the glass surface was able to translocate the microtubules towards its plus end. The velocity of the microtubule movement in the presence of 1 mM ATP was 4.0 to 4.5 microns/s at 22 degrees C. C/A dynein binds to and bundles the microtubules even in the presence of ATP. Cross-bridges were found between adjacent microtubules in the bundle with an axial periodicity of about 24 nm. The ATPase activity of C/A dynein was enhanced up to several-fold by the microtubules at concentration as low as 1 mg/ml. On the other hand, 21 S dynein bound to the microtubules with 24 nm axial periodicity only in the absence of ATP. Its ATPase activity was not activated by the microtubules. From these results, it is concluded that the manner of interaction with microtubules of C/A dynein is different from that of the outer arm dynein.

Animals↗

Regional differences in the expression of myosin light chains and tropomyosin subunits during development of chicken breast muscle.

Types of myosin light chains and tropomyosins present in various regions and at different developmental stages of embryonic and posthatched chicken breast muscle (pectoralis major) have been characterized by two-dimensional gel electrophoresis. In the embryonic muscle all areas appear to accumulate both slow and fast forms of myosin light chains in addition to alpha and beta forms of tropomyosin. During development regional differences in myosin and tropomyosin expression become apparent. Slow myosin subunits become gradually restricted to areas of the anterior region of the muscle and finally become localized to a small red strip found on its anterior deep surface. This red region is characterized by the presence of slow and fast myosin light chains, alpha-fast, alpha-slow, and beta-tropomyosin. In all other areas of the muscle examined only fast myosin light chains, beta-tropomyosin and the alpha-fast form of tropomyosin, are found. In addition, beta-tropomyosin also gradually becomes lost in the posterior regions of the developing breast muscle. In the adult, the red strip area represents less than 1% of the total pectoralis major mass and of the myosin extracted from this area approximately 15% was present as an isozyme that comigrated on nondenaturing gels with myosin from a slow muscle (anterior latissimus dorsi). The red region accumulates therefore fast as well as slow muscle myosin. Thus while the adult chicken pectoralis major is over 99% fast white muscle, the embryonic muscle displays a significant and changing capacity to accumulate both fast and slow muscle peptides.

Animals↗

Studies on the mechanism of hemorrhage production by five proteolytic hemorrhagic toxins from Crotalus atrox venom.

The sites and relative rates of peptide bond hydrolysis of the oxidized B chain of bovine insulin by two hemorrhagic proteinases, Ht-a and Ht-b, isolated from the venom of the Western Diamondback Rattlesnake, Crotalus atrox, were investigated. The results were compared with previous results on the digestion of the same substrate by the C. atrox hemorrhagic proteinases Ht-c, d, and e. Both toxins, Ht-a and Ht-b, were found to cleave the His5-Leu6, His10-Leu11, Ala14-Leu15, and Tyr16-Leu17 bonds of the oxidized insulin B chain. In addition, Ht-a cleaves the Asn3-Gln4 bond, whereas Ht-b cleaves the Gly23-Phe24 bond. The cleavage specificity of Ht-b on the insulin B chain is identical to that of the weakly hemorrhagic isoenzymes Ht-c and Ht-d. Hemorrhagic proteinase Ht-a cleaves the Ala14-Leu15 bond most rapidly, having a turnover number of 12.6 min-1 which was approximately twice as fast as the Tyr16-Leu17 bond turnover of cleavage. Hemorrhagic proteinase Ht-b, on the other hand, cleaves the Tyr16-Leu17 bond fastest, having a turnover number of 61.3 min-1. The Ala14-Leu15 bond is the peptide bond cleaved the second fastest by Ht-b, with a turnover number of 48.2 min-1. The five hemorrhagic toxins from Crotalus atrox venom were also examined for their capability to hydrolyse basement membrane preparations, and the identities of the digested proteins comprising the basement membrane were determined. From the results it is concluded that all five hemorrhagic toxins cleave laminin and the component of the basement membrane referred to as band a.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

Relative locations of the beta and delta chains of the acetylcholine receptor determined by electron microscopy of isolated receptor trimer.

The monomeric unit of the acetylcholine receptor of electric tissue of Torpedo californica has previously been shown to have a subunit composition of alpha 2 beta gamma delta. Receptor in membrane isolated from Torpedo electric tissue occurs as both monomer and dimer. In the dimer which is the predominant form, the monomeric units are cross-linked via a disulfide bond between delta chains. The addition of diamide to receptor-rich membrane causes the formation of trimer and higher oligomers in which the monomeric units are linked by disulfide bonds alternately between pairs of delta chains and between pairs of beta chains. We have isolated receptor trimer and determined the relative locations of the monomeric units by scanning transmission electron microscopy of negatively stained preparations. In face view, the trimer appears as three approximately 90 A disks, each with a central, densely staining pit. From the angles of the triangle formed by the lines connecting the centers of the monomers in the trimer, we infer that the beta-beta disulfide bond is separated from the delta-delta disulfide bond by an angle in the range of 50-80 degrees.

Animals↗

Role of the N-terminal region of the A chain in beta 1-bungarotoxin from the venom of Bungarus multicinctus (Taiwan-banded krait).

The N-terminal alpha-amino groups of beta 1-bungarotoxin (beta 1-Bgt) from Bungarus multicinctus venom were modified with trinitrobenzene sulfonic acid and the modified derivative was separated by high performance liquid chromatography. The trinitrophenylated (TNP) derivative contained two TNP groups at the alpha-amino groups of A chain and B chain and showed a marked decrease in enzymatic activity. Methionine residues at positions 6 and 8 of the A chain were oxidized with chloramine T or cleaved with cyanogen bromide to remove the N-terminal octapeptide. Oxidation of methionine residues and removal of the N-terminal octapeptide caused a precipitous decrease in enzymatic activity, whereas antigenicity remained unchanged. The presence of dihexanoyllecithin influenced the interaction between beta 1-Bgt and 8-anilinonaphthalene sulfonate (ANS) and revealed that beta 1-Bgt consists of two types of ANS-binding sites, one at the substrate binding site of the A chain and the other might be at the B chain. The modified derivatives still retained their affinity for Ca2+ and ANS, indicating that the N-terminal region is not involved in Ca2+ and substrate binding. A fluorescence study revealed that the alpha-amino group of the A chain was in the vicinity of substrate binding site and that the TNP alpha-amino groups were in proximity to Trp-19 of the A chain. In addition, the study showed that the N-terminal region is important for stabilizing the architectural environment of Trp-19. The results, together with the proposal that Trp-19 of the A chain is involved in substrate binding, suggest that the N-terminal region of the A chain plays a crucial role in maintaining a functional active site for beta 1-Bgt.

Amino Acids↗

Synthesis of copolymers containing an active ester of methacrylic acid by RAFT: controlled molecular weight scaffolds for biofunctionalization.

We report the controlled radical copolymerization of N-(2-hydroxypropyl)methacrylamide (HPMA) with a monomer containing an active ester, N-methacryloyloxysuccinimide (NMS), by reversible addition fragmentation chain transfer (RAFT). The large difference in the reactivity ratios of HPMA and NMS resulted in significant variations in copolymer composition with increasing conversion during batch copolymerization. The use of a semi-batch copolymerization method, involving the gradual addition of the more reactive NMS, allowed uniformity of copolymer composition to be maintained during the polymerization. We synthesized polymers in a wide range of molecular weights (M(n) = 3000-50,000 Da) with low polydispersities (1.1-1.3). The effect of the ratio of monomer to chain transfer agent (CTA) on the molecular weight of the polymer was investigated. Given the numerous applications of poly(HPMA)-based conjugates in designing polymeric therapeutics, these controlled molecular weight activated polymers represent attractive scaffolds for biofunctionalization. As a demonstration, we attached a peptide to the activated polymer backbone to synthesize a potent controlled molecular weight polyvalent inhibitor of anthrax toxin.

Biopolymers↗

Inhibition of inducible nitric oxide synthase gene expression and enzyme activity by epigallocatechin gallate, a natural product from green tea.

Chronic inflammation has been implicated as the underlying factor in the pathogenesis of many disorders. In the past decade, inflammation-related endogenous production of reactive nitrogen species, similar to oxygen free radicals, has also been suggested as a risk factor for cancer, in addition to the well-studied exogenous nitroso compounds. Epidemiological, in vitro, and animal model studies have implicated green tea to be protective against nitroso compound-induced and inflammation-related cancer. Therefore, we investigated the effect of epigallocatechin-3-gallate (EGCG), one of the known biologically active catechins contained in green tea, on the production of nitric oxide (NO.). We have shown previously that EGCG reduces NO. production as measured by nitrite accumulation in the culture medium. Expanding on this finding, in this report we show that EGCG may do so by two mechanisms: reduction of inducible nitric oxide synthase (iNOS) gene expression and inhibition of enzyme activity. Addition of 1-10 microM EGCG to lipopolysaccharide- and interferon-gamma-activated mouse peritoneal cells reduced iNOS mRNA expression concentration dependently, to 82-14%, as measured by relative reverse transcription-polymerase chain reaction. Addition of 50-750 microM EGCG, in a concentration-dependent manner, inhibited the enzyme activity of iNOS, to 85-14%, and neuronal nitric oxide synthase (nNOS), to 93-56%, as measured by citrulline formation. EGCG competitively inhibited binding of arginine and tetrahydrobiopterin, and the gallate structure is important for this action.

Animals↗

Chemical modification of oleanene type triterpenes and their inhibitory activity against HIV-1 protease dimerization.

Oleanolic acid derivatives with different lengths of 3-O-acidic acyl chains were synthesized and evaluated for their inhibitory activity against HIV-1 protease. The lengths of the acidic chains were optimized to 6 and 8 carbons. Changing a 3-ester bond to an amide bond or dimerization of the triterpenes retained their inhibitory activity against HIV-1 protease. Introduction of an additional acidic chain to C-28 of oleanolic acid increased the inhibitory activity appreciably, though a derivative with only one acidic chain linked at C-28 also showed potent activity against HIV-1 protease. The inhibitory mechanism was proved directly by size exclusion chromatography to be inhibition of dimerization of the enzyme polypeptides. The ester bonds of the triterpene derivatives were found to be stable to lipase under mild alkaline conditions.

Chromatography, Gel↗

Electrophoretic analyses of atrial and ventricular cardiac myosins from foetal and adult rabbits.

1. Rabbit cardiac myosins from atrium and ventricle were found to differ in their native form in pyrophosphate gel electrophoresis as well as in their light chain pattern in one- and two-dimensional electrophoretic systems. 2. The myosin light chain pattern in sodium dodecylsulfate gel electrophoresis differs between atrial and ventricular tissues in the mammalians (rabbit, dog, human, pig, sheep and rat) but not in the chicken. 3. In foetal rabbit ventricle an additional light chain is observed which, in two-dimensional electrophoresis with adult cardiac light chains, was found to be the same as atrial light chain-1.

Animals↗

Attempted isolation of a heparin proteoglycan from bovine liver capsule.

(1) Polysaccharides were isolated from bovine liver capsule by extraction with 2m-potassium chloride followed by precipitation from 0.8m-potassium chloride with cetylpyridinium chloride. Chondroitin sulphate was eliminated by digestion with hyaluronidase. The yield of heparin was approx. 40% of that obtained after extraction of the papain-digested tissue. (2) The macromolecular properties of the hyaluronidase-digested polysaccharide were studied by gel chromatography on Sephadex G-200 of the intact, as well as of the alkali-degraded, material. The results suggested the presence of single heparin chains in addition to a dermatan sulphate proteoglycan. (3) A purified heparin preparation was analysed for amino acids and neutral sugars. Xylose, galactose and serine were found in amounts corresponding to 0.1, 0.2, and 0.4 residue/polysaccharide chain (mol.wt. 7400), respectively. It is suggested that the isolated material had been degraded by a polysaccharidase with endo-enzyme properties.

Animals↗

Molecular mechanisms of anaphylaxis: lessons from studies with murine models.

Studies with murine models demonstrate 2 pathways of systemic anaphylaxis: one mediated by IgE, Fc epsilonRI, mast cells, histamine, and platelet-activating factor (PAF), and the other mediated by IgG, Fc gammaRIII, macrophages, and PAF. The former pathway requires much less antibody and antigen than the latter. As a result, IgG antibody can block IgE-mediated anaphylaxis induced by small quantities of antigen without mediating Fc gammaRIII-dependent anaphylaxis. The IgE pathway is most likely responsible for most human anaphylaxis, which generally involves small amounts of antibody and antigen; similarities in the murine and human immune systems suggest that the IgG pathway might mediate disease in persons repeatedly exposed to large quantities of antigen. Mice, like human subjects, can experience IgE/Fc epsilonRI/mast cell-mediated gastrointestinal and systemic anaphylaxis in response to ingested antigen. Gastrointestinal symptoms depend on serotonin and PAF; mediator dependence of systemic symptoms has not been determined. Both local and systemic anaphylaxis induced by ingested antigens might be blocked by IgA and IgG antibodies. IL-4 and IL-13 signaling through the IL-4 receptor alpha chain, in addition to promoting the mastocytosis and IgE antibody production that mediate most human anaphylaxis, exacerbates the effector phase of anaphylaxis by increasing target cell responsiveness to vasoactive mediators. As a result, IL-4 receptor alpha chain antagonists might be particularly effective suppressors of anaphylaxis.

Anaphylaxis↗

Interior segment regrowth configurational-bias algorithm for the efficient sampling and fast relaxation of coarse-grained polyethylene and polyoxyethylene melts on a high coordination lattice.

We demonstrate the application of a modified form of the configurational-bias algorithm for the simulation of chain molecules on the second-nearest-neighbor-diamond lattice. Using polyethylene and poly(ethylene-oxide) as model systems we show that the present configurational-bias algorithm can increase the speed of the equilibration by at least a factor of 2-3 or more as compared to the previous method of using a combination of single-bead and pivot moves along with the Metropolis sampling scheme [N. Metropolis, A. W. Rosenbluth, M. N. Rosenbluth, A. H. Teller, and E. Teller, J. Chem. Phys. 21, 1087 (1953)]. The increase in the speed of the equilibration is found to be dependent on the interactions (i.e., the polymer being simulated) and the molecular weight of the chains. In addition, other factors not considered, such as the density, would also have a significant effect. The algorithm is an extension of the conventional configurational-bias method adapted to the regrowth of interior segments of chain molecules. Appropriate biasing probabilities for the trial moves as outlined by Jain and de Pablo for the configurational-bias scheme of chain ends, suitably modified for the interior segments, are utilized [T. S. Jain and J. J. de Pablo, in Simulation Methods for Polymers, edited by M. Kotelyanskii and D. N. Theodorou (Marcel Dekker, New York, 2004), pp. 223-255]. The biasing scheme satisfies the condition of detailed balance and produces efficient sampling with the correct equilibrium probability distribution of states. The method of interior regrowth overcomes the limitations of the original configurational-bias scheme and allows for the simulation of polymers of higher molecular weight linear chains and ring polymers which lack chain ends.

Journal Article↗

Invariant chain targets HLA class II molecules to acidic endosomes containing internalized influenza virus.

The role of the HLA class II-associated invariant chain in the intracellular trafficking of HLA-DR molecules was examined in a transient expression system using HeLa cells. In the absence of alpha and beta polypeptides, invariant chain was retained in the endoplasmic reticulum (ER). In the absence of invariant chain, intracellular alpha beta heterodimers could be detected only in the ER and the Golgi apparatus. However, when alpha and beta subunits were coexpressed with invariant chain, HLA-DR molecules were detectable in peripheral cytoplasmic vesicles, which also contained invariant chain. In addition, an antibody directed to an acid-induced conformational determinant on the influenza hemagglutinin molecule detected internalized influenza virus in the HLA-DR-containing vesicles. These findings provide direct evidence that the invariant chain targets class II molecules to an acidic endosomal compartment and that this compartment, long suspected to be the site of antigen processing, is the site where class II molecules interact with natural antigen.

Antibodies↗

New polymerase chain reaction-based diagnostic techniques for bacterial respiratory infection.

Polymerase chain reaction protocols are now available for the diagnosis of all of the major bacterial respiratory tract pathogens. Molecular techniques are also being used to determine the susceptibility of Streptococcus pneumoniae and Mycobacterium tuberculosis. In addition, polymerase chain reaction-based diagnosis will find a place in the investigation of the epidemiology of some pathogens such as Bordetella pertussis and Chlamydia pneumoniae. The added sensitivity will allow the detection of mild or asymptomatic cases or carriers who may play a role in sustaining the pathogen in the community.

Journal Article↗

The effect of functional differences in the alpha and beta chains on the cooperativity of the osidation reduction reaction of hemoglobin.

Partially oxidized solutions of hemoglobin have been reacted with azide to determine the extent of oxidation, of the alpha and beta chains according to the method of McQuarrie and Gibson (J. Biol. Chem. (1971) 246, 517-522) In 2, 2'2'' nitriloethanol buffer the fraction of oxidized material represented by the beta chains decreases with decreasing extent of total oxidation, of the alpha chains. Upon addition of insitol hexaphosphate, the degree of perferntial oxidation in terms of a two-state model similar to the description of oxygenation by Edelstein (nature(1971) 230, 224-227) but with the incorporation of chain heterogeneity. The results indicate that the pH-dependent cooperativity of the oxidation-reduction reaction can be described in terms of a bell curbe of n versus log l, the allosteric somewhat lower and shifted slightly to the left, due in part to an affnity of beta chains for electrons approximately twince that of alpha chains. Because the curve is shifted to the left, oxidation-reduction equilibria at l values corresponding to pH 6 to lie on the right side of the bell curve where cooperativity the preferntial affity of beta chains for electrons rises to about 4 times that of alpha chains. As a consequence, the coreesponding bell curve is lowered with the Hill coeficient falling to unity or below in the range of l encountered. Thus the principal cause of decreased cooperativity is chain heterogeneity and not stabilization in the t state as suggested by Perutz; under these conditions the molecules of methemoglobin in the t state are only a fractional part of the population.

Binding Sites↗