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Biomedical subjects

K Titani

Publications and source records attributed to K Titani.

At least 145 records · Page 8Linked to original sources
PubMed1991

The growth inhibitory factor that is deficient in the Alzheimer's disease brain is a 68 amino acid metallothionein-like protein.

We have purified and characterized the growth inhibitory factor (GIF) that is abundant in the normal human brain, but greatly reduced in the Alzheimer's disease (AD) brain. GIF inhibited survival and neurite formation of cortical neurons in vitro. Purified GIF is a 68 amino acid small protein, and its amino acid sequence is 70% identical to that of human metallothionein II with a 1 amino acid insert and a unique 6 amino acid insert in the NH2-terminal and the COOH-terminal portions, respectively. The antibodies to the unique sequence of GIF revealed a distinct subset of astrocytes in the gray matter that appears to be closely associated with neuronal perikarya and dendrites. In the AD cortex, the number of GIF-positive astrocytes was drastically reduced, suggesting that GIF is down-regulated in the subset of astrocytes during AD.

Aged↗
PubMed1991

Neural cell adhesion molecule mediates contact-dependent inhibition of growth of near-diploid mouse fibroblast cell line m5S/1M.

A near-diploid mouse fibroblast cell line m5S/1M used in this study shows a high sensitivity to contact-dependent inhibition of growth, and the addition of EGF causes both morphological change and loss of contact-dependent inhibition of growth. The m5S/1M cell and its transformants obtained by x-ray irradiation have been used to search for the cell surface glycoproteins that are responsible for the growth regulation via cell-cell interactions. Lectin blotting analyses showed that the expression of the cell surface glycoprotein of 140 kD (140KGP) is highly sensitive to the transformation induced either by x-ray irradiation or by the EGF stimulation. We purified the 140KGP and found that it was composed of two glycoproteins. The major component of 140KGP was identified as neural cell adhesion molecule (NCAM) by amino acid sequence analyses of the peptide fragments and by the cross-reactivity with anti-NCAM mAb, clone H28.1.2.3. Monoclonal antibody against 140KGP (clone LN-10) recognizes all three isoforms of NCAM expressed on m5S/1M cell and showed that the expression of NCAM was highly sensitive to the transformation. Furthermore, the immobilized LN-10 strongly inhibited the growth of actively proliferating m5S/1M cells and the LN-10 in a soluble form showed a significant growth-stimulating effect on the confluent quiescent cultures of m5S/1M cells. The results show that NCAM plays a major role in the contact-dependent inhibition of growth of m5S/1M, and that NCAM might be involved in the regulation of cell growth during embryogenesis and formation of nervous systems.

Amino Acid Sequence↗
PubMed1991

Production and characterization of functional domains of human fibronectin expressed in Escherichia coli.

An efficient expression system was constructed in Escherichia coli that produced a 33-kDa fragment, C-274, of human fibronectin with a strong cell-adhesive activity. The entire sequence of the heparin-binding domain with 271 amino acids, H-271, was also expressed. Deletion analysis of the type III repeats showed that the heparin-binding site was at type III-13. The cell-adhesive activity of a fusion protein, CH-271, containing the cell- and the heparin-binding domains was twice that of C-274 when BHK but not B16-F10 melanoma cells were tested; H-271 alone was inactive. Recombinant proteins containing the CS1 sequence of the IIICS region were more active than C-274 and CH-271 with B16-F10. However, H-296, which contained both H-271 and CS1, was almost inactive with BHK. CH-296, which contained CS1 at the C-terminus of CH-271, was more active with B16-F10 than H-296 and C-CS1, which was produced by the deletion of H-271 from CH-296. Thus, the cell-binding domain was active with both kinds of cells. The heparin-binding domain promoted the adhesion of both kinds of cells only when linked to the cell-binding domain or CS1. CS1 was specific for the adhesion of B16-F10 but was not essential.

Animals↗
PubMed1991

Artificial cell adhesive proteins engineered by grafting the Arg-Gly-Asp cell recognition signal: factors modulating the cell adhesive activity of the grafted signal.

An artificial cell adhesive protein could be engineered by grafting the RGDS tetrapeptide, the core sequence of the major cell adhesive site of fibronectin, to a truncated form of Staphylococcal protein A (tSPA) via cassette mutagenesis of the tSPA expression vector pRIT2T [T. Maeda et al. (1989) J. Biol. Chem. 264, 15165-15168]. We synthesized a panel of tSPA derivatives grafted with various RGDS-containing oligopeptides to address the problem of how the cell adhesive activity of the resulting tSPA derivatives was affected by the length and amino acid sequence of the grafted oligopeptides and by the sites on tSPA where the extra oligopeptides were inserted. The results showed that (i) the amino acid residues flanking the RGDS core sequence played a key role in modulating the cell adhesive activity of the grafted RGDS signal; (ii) at least two sites on tSPA, each corresponding to on e of the two HindIII sites of pRIT2T, were competent in sustaining the cell adhesive activity of the grafted signal; and (iii) the divalent tSPA containing the RGDS signal at both sites was more active than monovalent derivatives containing only one signal at either site. These results provide a strategic basis for engineering of artificial cell adhesive proteins by grafting the RGDS signal.

Amino Acid Sequence↗
PubMed1991

Differential expression of beta amyloid protein precursor (APP) and tau mRNA in the aged human brain: individual variability and correlation between APP-751 and four-repeat tau.

We investigated the relationship between the differential expression of beta amyloid protein precursor (APP) and tau mRNA, and the extent of beta and tau deposition in three regions from each of the 38 aged brains obtained from consecutive autopsied cases. Remarkable variabilities were noted in the ratios of APP-770/-751/-695 and four-repeat tau among elderly individuals. There was no consistent alteration in the APP differential expression among beta plaque (-), (+), and (++(-) ) groups. Also, no differences in the four-repeat tau ratios were noted among tangle (-), (+), and (++) groups. Despite these great individual variabilities, APP-751 was found to be well-correlated with four-repeat tau. It is possible that APP-751 and four-repeat tau are increasing during aging, while APP-695 and three-repeat tau are decreasing.

Aged↗
PubMed1991

Activin-binding protein is present in pituitary.

A binding protein for activin was purified from bovine pituitary by affinity chromatography on dextran sulfate-Sepharose CL-4B and activin-Affi-Gel 10. A 52,700-fold purification over the starting crude homogenate was achieved. The purified preparation showed two bands of 36 and 33 kilodalton in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. The ability of each form of the protein to specifically bind activin was determined by ligand blot analysis and binding competition study. Each protein was found to have the same NH2-terminus and its sequence was identical to that of follistatin, which is a specific inhibitor of identical to that of follistatin, which is a specific inhibitor of FSH release. Moreover, the binding protein was shown to inhibit the spontaneous FSH release from cultured pituitary cells as does follistatin. These properties are the same as activin-binding protein that we have obtained from rat ovary. These results support a conclusion that activin-binding protein/follistatin exists also in the pituitary. Activin-binding protein has an ability to inhibit the activin-induced augmentation of FSH release from cultured pituitary cells as does inhibin. However, the inhibitory pattern by the binding protein was quite distinct from that of inhibin, suggesting that there may be different mechanism(s) for their antagonistic actions. Stoichiometric inhibition as shown by gel filtration analysis indicates that activin-binding protein binds activin to form an inactive equimolar complex having neither stimulatory nor inhibitory activity for FSH secretion by the pituitary. These findings suggest that activin is actually involved in FSH secretion in the pituitary and that the activin action in the pituitary is regulated by activin-binding protein/follistatin.

Activins↗
PubMed1991

Characterization of antisera directed against follistatin/activin-binding protein peptides.

An attempt was made to develop immunologic probes directed against follistatin/activin-binding protein (ABP), for use in investigating the distribution of ABP in various tissues. Five oligopeptides corresponding to different regions of the predicted ABP amino acid sequence (peptides 1-12, 28-43, 123-134, 270-281 and 300-315) were synthesized chemically, and coupled to Limulus polyphemus hemolymph hemocyanin. The peptide-hemocyanin conjugates were then used to immunize rabbits, and the immunoresponses were monitored by enzyme-linked immunosorbent assay. Reactivity of the antipeptide antisera with ABP was determined by SDS-polyacrylamide gel electrophoresis and immunoblotting analysis. All of the peptides produced immune responses. The antiserum to peptide 123-134 recognized all forms of ABP, whereas the antiserum to peptide 300-315 reacted specifically and sensitively with the long forms of ABP. These two antisera exhibited only a limited cross-reaction with other proteins or none at all. Therefore, they will be useful for studying the distribution of ABP in various tissues.

Amino Acid Sequence↗
PubMed1991

Immunohistochemical localization of follistatin in rat tissues.

We have used immunohistochemistry to localize follistatin/activin-binding protein in adult male and female rats. A polyclonal antibody directed against a follistatin peptide (residues 123-134) was used as a specific immunologic probe. Intense and specific follistatin immunoreactivity was evident in spermatogenic cells of seminiferous tubules in the testis. The predominant staining was in nuclei of spermatocytes and spermatids, but no immune reaction was observed in spermatogonia or spermatozoa. Moderate immunoreactivity was detected in Leydig cells. Sertoli cells were follistatin-negative. Significant immunoreactivity was evident in ovarian granulosa cells. The intensity of the staining changed with follicle development: no immunoreactivity was observed in granulosa cells of primordial to primary follicles, but the cells of secondary to Graafian follicles displayed moderate to strong staining and finally luteal cells of the corpus luteum became negative. The epithelial lining of the oviduct and the smooth muscle of the myometrium of the uterus were intensely immunoreactive. Immunoreactive follistatin staining was present in the pituitary: a group of round-shaped cells were specifically stained. Immunostainable follistatin was visible in the epithelial layers of renal tubules with moderate to strong staining reactivity. Hepatic cells in the liver demonstrated homogeneous immunoreactivity from moderate to strong. The cortex of the adrenal gland, white pulp of the spleen and the brain cortex were also stained weakly but distinctly with the antiserum. In conclusion, immunoreactive follistatin is widespread in rat tissues, suggesting that follistatin/activin-binding protein is a ubiquitous protein, regulating a wide variety of activin actions.

Adrenal Glands↗
PubMed1991

Urinary fibronectin fragments (a potential tumor marker) measured by immunoenzymometric assay with domain-specific monoclonal antibodies.

We found that urinary fibronectin (UFN) in cancer patients was almost all fragmented and consisted mainly of the cell-binding domain. We developed a two-site immunoenzymometric assay for UFN, using two monoclonal antibodies that both recognize this domain of fibronectin. The amount of UFN was expressed as arbitrary units per milligram of creatinine. Some 2% of the 623 healthy subjects had UFN above the clinical cutoff point (200 arb. units/mg creatinine), as did 14% of the 271 patients with nonmalignant diseases. In contrast, concentrations of UFN exceeded the cutoff point in 59% of the 589 patients with cancer. In 37 patients with gastrointestinal cancer tested for UFN and for one or more of three established serum tumor markers, UFN was found in 25, significantly more often than the other markers. These results indicated that UFN is a marker that may be helpful in evaluating many kinds of cancer.

Adult↗
PubMed1991

[Domain structure of human von Willebrand factor, and its modulators involved in the platelet adhesion process in vitro].

von Willebrand factor (vWF), a macromolecular glycoprotein synthesized in endothelial cells and megakaryocytes circulates in blood as heterogenous multimers and plays a critical role in the formation of platelet plugs. vWF is composed of an identical subunit with a molecular weight (MW) of 270 kDa, which is held together by disulfide bonds. Functional domains of vWF for factor VIII, heparin, platelet glycoprotein (GP) Ib, collagen, and GP IIb/IIIa locate in this order from the N-to C-terminus of subunit. The GP Ib binding domain is cryptic in normal circulation and only becomes expressed when vWF is associated with the subendothelial matrix or with fibrin. In vitro, an antibiotic ristocetin or the snake venom botrocetin isolated from Bothrops jararaca mimics the active component of subendothelial matrix and causes the binding of vWF to GP Ib. To elucidate the mechanism of in vitro process of vWF-GPIb binding, we describe here the isolation and characterization of two distinct forms of botrocetin. Since ristocetin is a heavily glycosylated proteoid, it is hard to characterize the structure on the basis of amino acid analysis. The apparent MW of the one-chain botrocetin was 28 kDa before and 32 kDa after reduction of disulfide bonds, while that of the two-chain botrocetin was 27 kDa before and 15/14.5 kDa after reduction. Amino acid composition of the two species revealed a similar high content of potentially acidic residues (greater that 60 Asx and Glx residues/molecules) but significant differences in the content of Cys and Phe residues. The N-terminal sequence of the one-chain was Ile-Ile/Val-Ser-Pro-Pro-Val-Cys-Gly-Asn-Glu-.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

The complete amino acid sequence of the mature form of rat sepiapterin reductase.

The partial amino acid sequence of rat sepiapterin reductase was determined using peptides generated by cleavage of the S-carboxyamidomethylated protein with Achromobacter protease I, cyanogen bromide, chymotrypsin or BNPS-skatole. The protein began with N-acetyl methionyl residue at the N-terminus and ended with isoleucyl residue at the C-terminus. The present results essentially coincided with the amino acid sequence predicted from the nucleotide sequence of the cDNA recently reported by Citron et al. (Proc. Natl. Acad. Sci. USA 87, 6436-6440 (1990)), clarified the processing event during the biosynthesis and provided the complete amino acid sequence of the mature form of the enzyme.

Alcohol Oxidoreductases↗
PubMed1990

Antibodies raised against different oligopeptide segments of human dopamine-beta-hydroxylase.

We raised antibodies against 3 oligopeptide segments of human dopamine-beta-hydroxylase (hDBH) corresponding to the N-terminal (hDBH-N), the intermediate (hDBH-I), and the C-terminal (hDBH-C) amino acid sequences (residues 26-43, 452-468, and 582-598), respectively. We characterized the antibodies in terms of specificity by means of Western blotting and immunohistochemistry. Anti-hDBH-N antiserum recognized DBH in the brain (noradrenergic neurons in the pons and medulla oblongata) and adrenal medulla, not only of human but also of mouse, rat and house shrew. In contrast, anti-hDBH-C antiserum recognized only human DBH. These observations suggest that the antibody raised against the hDBH-C terminal peptide may specifically recognize only human DBH.

Adrenal Glands↗
PubMed1990

Structures of the asparagine-linked oligosaccharides of guinea-pig factor B of the alternative complement pathway.

This paper describes the structures of the asparagine-linked oligosaccharides of two forms of guinea-pig Factor B of the alternative complement pathway with different Mr values. Oligosaccharides were quantitatively liberated from both glycoproteins by hydrazinolysis, fractionated by paper electrophoresis and Bio-Gel P-4 column chromatography, and their structures determined by sequential exoglycosidase digestions in conjunction with methylation analysis. Both glycoproteins were shown to have the same biantennary complex-type oligosaccharides but it is suggested that they contain different numbers of oligosaccharide chains.

Animals↗
PubMed1990

Structural changes in the oligosaccharide chains of IgG in autoimmune MRL/Mp-lpr/lpr mice.

The structures of the asparagine-linked oligosaccharide chains of IgG from autoimmune arthritic MRL/Mp-lpr/lpr (MRL-lpr/lpr) mice and control MRL/Mp(-)+/+ (MRL(-)+/+) mice were investigated. Two subpopulations of IgG, M1-I and M1-II, were obtained from serum of MRL-lpr/lpr mice by column chromatography on protein A-Sepharose CL-4B. Although M1-I did not bind to the column, its elution was retarded, whereas M1-II was bound and was eluted in acidic buffer. IgG (Mn) from MRL(-)+/+ mice showed the same chromatographic behavior as M1-II. The structures of oligosaccharide chains liberated quantitatively by hydrazinolysis from IgG samples Mn, M1-I, M1-II, and a pooled mixture (M1) of M1-I and M1-II were determined by sequential exoglycosidase digestion, lectin (RCA120) affinity HPLC, and by methylation analysis. Their oligosaccharide structures were the same and shown to be biantennary complex-type chains +/- Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6(+/- Gal beta 1----4GlcNAc beta 1----2Man alpha 1----3)Man beta 1----4GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAc. The proportion of each oligosaccharide in Mn and M1-II was the same but differed from that in M1-I where the degree of the galactosylation was significantly decreased which caused the change in the oligosaccharide pattern of total serum IgG (M1) of autoimmune MRL-lpr/lpr mice. This phenomenon, which is also found in total serum IgG of patients with rheumatoid arthritis, suggests that alteration of oligosaccharides in IgG may be a common feature in animals which develop arthritis with the production of rheumatoid factor regardless of species.

Animals↗
PubMed1990

Rapid purification and characterization of human platelet glycoprotein V: the amino acid sequence contains leucine-rich repetitive modules as in glycoprotein Ib.

Glycoprotein V (GPV) is a membrane-associated, 82 Kd platelet glycoprotein that is hydrolyzed during thrombin activation to yield 69 Kd fragment. We have developed a rapid and simple method for isolation of the protein from platelet extracts using a combination of gel permeation, anion-exchange, and lectin affinity chromatography. The partial amino acid sequence was determined by analysis of peptides generated by digestion of the S-carboxyamido-methylated protein with Achromobacter protease I or cyanogen bromide. The sequence shows a remarkable periodicity of leucine residues, which is homologous to the consensus sequence of a highly diversified protein super-family with a common repetitive module. Thrombin cleavage site was determined to be located at the C-terminal region of GPV by analysis of the products separated by sizing and reversed-phase high performance liquid chromatography. By lectin blot analysis, the existence of mucin-type carbohydrate chains was indicated, as well as the existence of asparagine-linked carbohydrate chains shown by the amino acid sequence analysis. From these data, we report a structural model of GPV that is analogous to glycoprotein Ib.

Amino Acid Sequence↗
PubMed1990

Diversity of oligosaccharide structures on the envelope glycoprotein gp 120 of human immunodeficiency virus 1 from the lymphoblastoid cell line H9. Presence of complex-type oligosaccharides with bisecting N-acetylglucosamine residues.

The N-linked oligosaccharide structures on the envelope glycoprotein gp120 of human immunodeficiency virus 1 derived from chronically infected lymphoblastoid (H9) cells have been investigated by enzymatic microsequencing after release from protein by hydrazinolysis, labeling with NaB3H4, and chromatography on adsorbent columns of Phaseolus vulgaris erythrophytohemagglutinin and Ricinus communis agglutinin (Mr 120,000) and on Bio-Gel P-4. A substantially greater diversity of oligosaccharide structures was detected than among those released by hydrazinolysis from recombinant gp120 produced in Chinese hamster ovary cells and investigated by similar procedures (Mizuochi, T., Spellman, M.W., Larkin, M., Solomon, J., Basa, L.J., and Feizi, T. (1988) Biochem J. 254, 599-603) and among those released by endoglycosidases from virus-derived gp120 isolated from infected H9 cells after metabolic labeling with D-[2-3H]mannose or D-[6-3H]glucosamine (Geyer, H., Holschbach, L., Hunsmann, G., and Schneider, J. (1988) J. Biol. Chem. 263, 11760-11767). In this study, 16% of the oligosaccharides were identified as complex-type bi-, tri-, and tetraantennary sialo-oligosaccharides with bisecting N-acetylglucosamine residues. Such structures were lacking on recombinant gp120 and could not be detected on the metabolically labeled, virus-derived glycoprotein. As in the earlier investigations, complex-type chains lacking bisecting N-acetylglucosamine residues, hybrid-type chains, and a series of high mannose-type structures with 5-9 mannose residues were identified. In addition, an array of complex-type chains having one or more outer chains with beta-galactosyl residues were detected in this study, but with additional substitutions that require further investigation. The number of potential N-glycosylation sites on gp120 is on the order of 20, but the oligosaccharide structures are far more numerous. Thus, the salient conclusion from this and earlier investigations is that alternative structures occur on at least some of the glycosylation sites and that numerous glycosylation variants of this glycoprotein are produced even within a single cell line. Since the glycosylation is the product of host cell glycosyltransferases, an even greater number of glycosylation variants of gp120 are predicted to arise from the heterogeneous cell populations harboring the virus in in vivo infection.

Borohydrides↗
PubMed1990

Purification and complete amino acid sequence of novel beta 2-microglobulin.

We have previously reported that novel beta 2-microglobulin (beta 2m) is a metabolite derived from beta 2m in ultrafiltrate of patients on long-term hemodialysis (LT-HD). Chromatofocusing showed the presence of at least two major novel beta 2m's with isoelectric points of 5.38 and 5.22. In the present study we purified one of major novel beta 2m's and determined the complete amino acid sequence. We demonstrate herein that the novel beta 2m has the same sequence as native beta 2m except for the 17th residue from the N-terminus which was identified as Asp instead of Asn in native beta 2m, suggesting a possible deamidation during LT-HD.

Amino Acid Sequence↗