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The addition of ethylene to 1,3-dienes and 1-vinylcycloalkenes, catalyzed by two ruthenium complexes, proceeds in a regioselective fashion to afford 3-methyl-1,4-dienes as products. For a steroidal-based 1-vinylcycloalkene, the addition is stereospecific, giving a product with a 20(S) configuration. [reaction: see text]
A combined enzyme-histochemical (ATPase reactivity) and immunohistochemical study has been performed on sections of rabbit masseter muscle. The majority of the fibres previously designated as type IIC and/or type I according to their ATPase activity were found to contain 'cardiac' alpha-myosin heavy chain in addition to other myosin heavy chains. All alpha-myosin heavy chain-containing fibres reveal ATPase activity after pre-incubation at pH 4.2-4.6 similar to that of the classical type I fibres, while, in that pH range, limb type IIC fibres show intermediate ATPase activity. One group of these fibres reveal ATPase activity after pre-incubation at pH 10.1-10.3 as well, but not at pH 10.4-10.5. These fibres contain exclusively either alpha- or alpha- and I-myosin heavy chains but do not contain the IIA-myosin heavy chain. The second part of the fibres reveals ATPase activity after treatment within the whole alkaline pre-incubation range (pH 10.1-10.5) and these fibres contain alpha-myosin and IIA-myosin but no I-myosin heavy chain. It is concluded that the classical IIC fibre type is not present in the rabbit masseter muscle. Furthermore, ATPase reactivity does not allow us to distinguish fibres on their myosin heavy chain content in rabbit masseter muscle.
The up-regulation of the keratan sulfate proteoglycan (ABAKAN) was examined using indirect immunohistochemical methods. Previous studies indicate that the keratan sulfate proteoglycan is associated with astrocytes in the optic nerve and in the developing rat brain. In model culture systems, this proteoglycan is capable of inhibiting the growth of neurites over laminin. To determine whether the proteoglycan is up-regulated specifically during reactive gliosis, stab wounds were made in the cerebral cortex of early postnatal rats, and the up-regulation of the proteoglycan was related to the developmentally regulated gliotic response to injury. Following a stab wound in the cortex of the late postnatal rat, reactive gliosis was consistently observed along with an up-regulation of ABAKAN. When the cortex was injured on postnatal day 2, there was a variable gliotic response and considerable variation in the regulation of proteoglycan expression. Biochemical analysis revealed that ABAKAN is a large proteoglycan with multiple keratan sulfate side-chains, at least one chondroitin sulfate side-chain and at least one additional carbohydrate chain with a terminal 3-sulfoglucuronic acid. Taken together, these data demonstrate that the boundary proteoglycan ABAKAN is also associated with reactive gliosis during early postnatal development.
Model melanoidins from monomeric, oligomeric, and polymeric carbohydrates, and amino acids formed under aqueous as well as water-free reaction conditions, were submitted to acidic catalyzed hydrolysis. Their degradation products were detected qualitatively and quantitatively by HPTLC and HPLC-DAD. A considerable amount of monomer carbohydrates from hydrolysis of model melanoidins formed under water-free reaction conditions was detected. It can be seen clearly that the amount of carbohydrates released increased with increasing degree of polymerization of the carbohydrates used as starting material. In comparison, the hydrolysis of melanoidins formed in aqueous condition resulted in only a small glucose release. It seems that in the Maillard reaction under water-free conditions, a significant amount of di- and oligomer carbohydrates were incorporated into the melanoidin skeleton as complete oligomer with intact glycosidic bond, forming side chains at the melanoidin skeleton. Additional side chains could be formed by transglycosylation reactions. With increasing water content, hydrothermolytic as well as retro-aldol reactions of the starting carbonyl components became significant, and therefore the possibility of forming side chains decreased. The results are consistent with the postulated melanoidin structure being built up mainly from sugar degradation products, probably branched via amino compounds.
A full-length cDNA clone specifying sandbar shark (Carcharhinus plumbeus) immunoglobulin light chain has been isolated and sequenced. By alignment with human lambda chains, the leader, framework, complementarity-determining, joining, and constant regions are clearly identified in the shark light chain. Approximately 40-50% identity is shared between the human and shark sequences in the variable and constant regions. We have performed sequence comparisons of the individual segments and constructed phylogenetic trees for the variable region. These studies identify the shark protein as a lambda chain. In addition, the sandbar shark light chain is only distantly related to that of horned shark (Heterodontus francisci) [Shamblott, M. J. & Litman, G. W. (1989) Proc. Natl. Acad. Sci. USA 86, 4684-4688], demonstrating that the long evolutionary time of divergence among shark species has led to the generation of substantial differences in sequence. The positions of the variable, joining, and constant gene segments in 14 genomic clones have been mapped. The segments are linked in individual clusters (variable, joining, constant) occupying 3-7 kilobases. Cluster arrangement can be grouped into two patterns based upon spacing between the genes in the individual clones. This arrangement is fundamentally different from that observed in higher vertebrates.
Mammalian oocytes are surrounded by an extracellular glycocalyx, the zona pellucida (ZP). In the mouse, the ZP is composed of three glycoproteins, designated mZP1, mZP2, and mZP3. Extensive studies in this species have resulted in the identification of primary (mZP3) and secondary (mZP2) receptors for spermatozoa. In this paper we present evidence for the occurrence of poly-N-acetyllactosaminyl glycans and an O-linked trisaccharide on mZP2 and mZP3. When exhaustively digested with endo-beta-galactosidase, an enzyme known to cleave repeating units of acetyllactosamine (3Gal beta 1, 4GlcNAc beta 1), mZP2 and mZP3 showed an apparent reduction in size by 23 kDa and 16 kDa, respectively. Experimental evidence included in this report indicates that polylactosaminyl glycans are present on N-linked sugar chains. In addition, O-linked sugar chains of mZP3 have been characterized. First, treatment of de-N-glycosylated mZP3 with O-glycanase in the presence of exo-glycosidases (sialidase, alpha-L-fucosidase, and N-acetylglucosaminidase) caused an apparent reduction in its size by 2-3 kDa as determined by SDS-PAGE. Second, treatment of the de-N-glycosylated mZP3 with mild alkali in the presence of 1 M NaB3H4 released radiolabeled oligosaccharide (OS) that eluted from a high-resolution Bio-Gel P-4 column at the position of a trisaccharide. The radiolabeled OS had the following structure: GlcNAc-->Gal beta 1,3GalNAcol. The structure was established by sizing on the Bio-Gel P-4 column, followed by examination of the susceptibility of the OS to exo-glycosidases and by its absorbability to immobilized lectin (PNA). Potential roles of N-linked and O-linked sugar chains in sperm-egg interaction are herein discussed.
A 5-year-old child heterozygous for beta thalassaemia has the clinical picture of thalassaemia intermedia. Restriction endonuclease mapping shows that the child is homozygous for a triplicated alpha gene complex. The greater degree of globin chain imbalance resulting from two additional alpha chain genes is the likely mechanism for this unusually severe clinical phenotype.
The concentration of immunoglobulins in paired serum and cerebrospinal fluid (CSF) specimens from 15 patients with systemic lupus erythematosus (SLE) and central nervous system (CNS) involvement (CNS-SLE) were determined based on their light chain properties. Eight patients with SLE without CNS involvement and 20 patients with noninflammatory neurological diseases were also studied for comparison. We found that both of the CSF Ig-k (immunoglobulin kappa) and Ig-1 (immunoglobulin lambda) indices were significantly elevated, indicating the increased intrathecal immunoglobulin synthesis of both kappa and lambda types in patients with CNS-SLE. Moreover, the CSF Ig-k and Ig-1 indices correlated well with each other, and both indices were found to decrease remarkably when the CNS manifestations subsided after successful treatment. In 4 of 15 patients, intrathecal synthesis of free light chains in addition to Ig bound light chains seemed to be increased. These observations provide substantial evidence for polyclonal B lymphocyte activation within the CNS.
We have constructed and expressed a series of mutant influenza virus hemagglutinins, each containing a new consensus site for glycosylation in addition to the seven sites found on the wild-type protein. Oligosaccharide side chains were added with high efficiency at four of the five novel sites, located on areas of the protein's surface that are not normally shielded by carbohydrate. Investigations of the structure, intracellular transport, and biological activities of the mutant hemagglutinin molecules indicated that (a) supernumerary carbohydrate side chains can be used to shield or disrupt functional epitopes on the surface of hemagglutinin, and (b) the presence of an additional oligosaccharide may cause temperature-dependent defects in the transport of the glycoprotein. We discuss the addition of supernumerary oligosaccharides as a general tool for shielding chosen areas of the surface of proteins that enter or traverse the secretory pathway.
PTPzeta/RPTPbeta, a receptor-type protein tyrosine phosphatase synthesized as a chondroitin sulfate (CS) proteoglycan, uses a heparin-binding growth factor pleiotrophin (PTN) as a ligand, in which the CS portion plays an essential role in ligand binding. Using an organotypic slice culture system, we tested the hypothesis that PTN-PTPzeta signaling is involved in the morphogenesis of Purkinje cell dendrites. An aberrant morphology of Purkinje cell dendrites such as multiple and disoriented primary dendrites was induced in slice cultures by (1) addition of a polyclonal antibody against the extracellular domain of PTPzeta, (2) inhibition of protein tyrosine phosphatase activity, (3) enzymatic removal of the CS chains, (4) addition of exogenous CS chains, and (5) addition of exogenous PTN, all of which disturb PTN-PTPzeta signaling. These treatments also reduced the immunoreactivity to GLAST, a glial glutamate transporter, on Bergmann glial processes. Furthermore, a glutamate transporter inhibitor also induced the abnormal morphogenesis of Purkinje cell dendrites. Altogether, these findings suggest that PTN-PTPzeta signaling regulates the morphogenesis of Purkinje cell dendrites and that the mechanisms underlying that regulation involve the GLAST activity in Bergmann glial processes.
Limited proteolysis of preparations of mature rabbit cornea with pepsin allows the recovery of approximately 90% of the tissue collagen in soluble form. Using recently described selective precipitation techniques, the soluble cornea collagen can be resolved into two major fractions. The predominant fraction, which accounts for about 95% of the solubilized collagen, was identified by means of solubility properties, electrophoretic and chromatographic properties, as well as amino acid analyses of its constituent chains, as Type I collagen. The alternate fraction, which accounts for virtually all of the remainder of the solubilized cornea collagen, was identified by means of the same criteria as collagen comprised of the A and B chains. In addition, the stoichiometry of the latter chains in preparations of cornea collagen indicate that in the cornea these chains are likely to participate in the formation of only one type of collagen molecule with the chain composition, AB2. And finally, the demonstration that Type I and the AB2 collagens are the predominant forms of collagen recoverable from the mature cornea strongly suggests that molecules comprised of the A and B chains are not necessarily confined to basement membrane structures.
The C.C58 and C.AKR congeneic strains of mice differ from BALB/c at loci on chromosome 6 which govern kappa light chain variable region (V kappa) polymorphisms and the Lyt-2 and Lyt-3 alloantigens. Amino acid sequence analysis of light chains of myelomas induced in these strains revealed one light chain, C.C58 M75, that had an NH2-terminal serine and differed sufficiently from published V kappa sequences to define a new V kappa group, V kappa (Ser), apparently not expressed by BALB/c mice. Peptide map analysis indicated that the M75 light chain contained the IB-peptide marker, a V kappa polymorphism expressed by C.C58 but not BALB/c mice, which is determined by the IgK-Trpa allele present on chromosome 6. This same light chain was found by isoelectric focussing to correspond to IgK-Ef1a, another V kappa genetic marker of C.C58 and C.AKR. Isoelectric focussing of approximately 200 C.C58 and C.AKR myeloma light chains revealed three additional C.C58 and four C.AKR light chains that corresponded to IgK-Ef1a-specific light chains. All three additional C.C58 light chains belonged to the V kappa (Ser) group and contained the IB-peptide marker. Thus, the differences in V kappa repertoires represented by the IB-peptide and IgK-Ef1a markers and controlled by genes on chromosome 6 appear to reflect expression (or failure of expression) of a distinct group of V kappa regions.
In this study, myosin types in human skeletal muscle fibers were investigated with electrophoretic techniques. Single fibers were dissected out of lyophilized surgical biopsies and typed by staining for myofibrillar ATPase after preincubation in acid or alkaline buffers. After 14C-labelling of the fiber proteins in vitro by reductive methylation, the myosin light chain pattern was analysed on two-dimensional gels and the myosin heavy chains were investigated by one-dimensional peptide mapping. Surprisingly, human type I fibers, which contained only the slow heavy chain, were found to contain variable amounts of fast myosin light chains in addition to the two slow light chains LC1s and LC2s. The majority of the type I fibers in normal human muscle showed the pattern LC1s, LC2s and LC1f. Further evidence for the existence in human muscle of a hybrid myosin composed of a slow heavy chain with fast and slow light chains comes from the analysis of purified human myosin in the native state by pyrophosphate gel electrophoresis. With this method, a single band corresponding to slow myosin was obtained; this slow myosin had the light chain composition LC1s, LC2s and LC1f. Type IIA and IIB fibers, on the other hand, revealed identical light chain patterns consisting of only the fast light chains LC1f, LC2f and LC3f but were found to have different myosin havy chains. On the basis of the results presented, we suggest that the histochemical ATPase normally used for fibre typing is determined by the myosin heavy chain type (and not by the light chains). Thus, in normal human muscle a number of 'hybrid' myosins were found to occur, namely two extreme forms of fast myosins which have the same light chains but different heavy chains (IIA and IIB) and a continuum of slow forms consisting of the same heavy chain and slow light chains with a variable fast light chain composition. This is consistent with the different physiological roles these fibers are thought to have in muscle contraction.
The membrane-bound glycoprotein (gp64) of the cellular slime mold Polysphondylium pallidum, is a putative cell-cell adhesion protein identified by adhesion-blocking antibody fragments. Since gp64 can be purified in a few days and in substantial yields, it is a good candidate for clarifying the structure of a cell-cell adhesion protein. This study reveals that gp64 possesses a glycolipid anchor which is sensitive to deamination but resistant to phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis. Although the anchor resistance to phosphatidylinositol-specific phospholipase C can be ascribed to the presence of an additional acyl chain on the inositol ring in the glycosyl phosphatidylinositol anchors, this was not the case. After a mild-base treatment that released an additional acyl chain on the inositol ring, only a trace amount of fatty acid was detected but, after strong acid hydrolysis, we detected both amide-linked fatty acids and a long-chain base. The long-chain base was further analysed by gas-chromatography/mass spectrometry and was found to be phytosphingosine. Both fatty acids and myo-inositol were also analysed by gas-chromatography/mass spectrometry. These data suggest that gp64 possesses a glycolipid anchor which contains ceramide and myo-inositol.
A cell-free protein-synthesizing system made from rabbit reticulocytes was used to incorporate (14)C-amino acids into hemoglobin. Electrophoretic analyses of the soluble products of this cell-free system revealed a fraction containing rabbit (14)C-alpha chains in addition to the rabbit (14)C-hemoglobin. The addition of isolated human hemoglobin beta chains to this system during active synthesis inhibited the release of newly synthesized rabbit (14)C-beta chains into solution from the ribosome fraction. This inhibition was possibly a result of hybrid hemoglobin formation between rabbit alpha and human beta chains. A model of hemoglobin construction in which soluble alpha chains are intermediates is suggested. These alpha chains may aid in the release of beta chains from the polyribosomes during the completion of the hemoglobin molecule.
During the initiation of intrinsic coagulation factors XI and XIa interact intimately with several other coagulation proteins (factor XIIa, high Mr kininogen, and factor IX) as well as with the platelet surface. To help elucidate these complex intramolecular interactions, we have prepared a collection of monoclonal antibodies directed against various epitopes in factor XI. We have utilized these reagents to isolate factor XI and the light chain of factor XIa on affinity columns, and to probe structure-function relationships involved in the interactions of factor XIa with factor IX. The isolated light chain of factor XIa retained greater than 90% of its amidolytic activity against the oligopeptide substrate pyro-Glu-Pro-Arg-pNA (S-2366), but only 3.8% of its clotting activity in a factor XIa assay and 1% of its factor IX activating activity in an activation peptide release assay. This suggests that regions of the heavy chain are required for development of coagulant activity and specifically for the interaction of factor XIa with factor IX. To test this hypothesis, the effects of three of the monoclonal antibodies (5F4, 1F1, and 3C1) on the function of factor XIa were examined. The results show that in a clotting assay the light chain-specific antibody (5F4) inhibits 100% of the factor XIa activity, whereas of the heavy chain-specific antibodies, one (3C1) inhibits 75% and another (1F1) only 17%. Similarly in the factor IX activation peptide release assay, antibody 5F4 inhibits 100% of the factor XIa activity, whereas 3C1 inhibits 75% and 1F1 inhibits 33%. We conclude that regions located in the heavy chain, in addition to those in the light chain, are involved in the interaction of factor XIa with factor IX and in the expression of the coagulant activity of factor XI.
Human hyaline cartilage contains three collagen chains in addition to the alpha 1(II) chain of Type II collagen. Two of these chains, tentatively designated the 1 alpha and 2 alpha chains, have been extensively characterized. Although similar in molecular weight to other collagen chains, by the combined criteria of solubility, electrophoretic mobilities, carboxymethylcellulose elution profiles, total amino acid compositions, carbohydrate contents, cyanogen bromide peptide profiles, limited V8 protease digestion profiles, and examination of selected cyanogen bromide peptides, the 1 alpha and 2 alpha chains are structurally unique collagen alpha chains. Because of similarities of the 1 alpha and 2 alpha chains to the alpha 1(V) and alpha 2(V) chains, respectively., of Type V collagen, specific comparisons between these collagen chains and their constituent peptides were made and yielded the conclusion that these chains are nonidentical, but share substantial similarities.
High frequency of hybridoma lines secreting large amounts of reaginic anti-DNP antibodies were generated by the fusion of splenic lymphocytes enriched by adoptive transfer of immune cells and the NSI plasmacytoma cell line. The murine IgE molecule binds DNP with high affinity and was specifically purified by immunoabsorption. The 190,000 dalton molecule is composed of a lambda-light chain and an epsilon-heavy chain of an apparent m.w. of about 90,000. Absorption studies with anti-class-specific antisera suggest that some of the hybrid clones secrete also a mixed molecule consisting of lambda- and kappa-light chains in addition to the epsilon-heavy chain. The monoclonal reaginic antibody binds to mast cells or rat basophilic leukemia cells and, upon binding of DNP-protein, triggers an anaphylactic reaction.