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Biomedical subjects

A Grubb

Publications and source records attributed to A Grubb.

At least 127 records · Page 7Linked to original sources
PubMed1990

The amino terminal portion of cerebrospinal fluid cystatin C in hereditary cystatin C amyloid angiopathy is not truncated: direct sequence analysis from agarose gel electropherograms.

The isolated amyloid substance in hereditary cystatin C amyloid angiopathy (HCCAA) is mainly composed of a cystatin C variant devoid of the 10 amino terminal amino acid residues of extracellular cystatin C from healthy individuals. We have developed a procedure for protein sequencing directly from agarose gel electropherograms and used this in conjunction with isoelectric focusing to investigate the amino terminal sequence of cerebrospinal fluid (CSF) cystatin C in HCCAA patients. The amino-terminal sequence determined for cystatin C from a HCCAA patient CSF sample, Xaa-Ser-Pro-Gly-Lys-Pro-Pro-Xaa-Leu-Val-Gly-Gly-Pro-Met-Xaa-Ala-Xaa-Val, showed that the protein was not amino-terminally truncated. CSF cystatin C from all nine HCCAA patients investigated was found to have an isoelectric point identical to that of native cystatin C, and the truncated form of cystatin C isolated from amyloid deposits was shown to contribute to less than 1% of the total amount of cystatin C in CSF. The total cysteine proteinase inhibitory capacity of CSF from HCCAA patients was lower than that of CSF from other patients. This decreased CSF inhibitory capacity in HCCAA patients was caused by decreased levels of cystatin C, since the levels of the other two cysteine proteinase inhibitors found in CSF, alpha 2-macroglobulin and kininogen, were significantly higher than in CSF from non-HCCAA patients.

Amino Acid Sequence↗
PubMed1990

HC-IgA antibodies of different specificities are normally present in serum: quantitation by ELISA and relationship to the major Ig classes.

Enzyme-linked immunosorbent assays (ELISA) were developed for direct measurement of protein HC-IgA complexes (HC-IgA) in serum with antibody specificity for rabbit IgG (rheumatoid factor (RF) activity), lipopolysaccharide from Yersinia enterocolitica serotype O:3 (Y3) and tetanus toxoid (TT). About 80% of patients with rheumatoid arthritis had increased concentrations of HC-IgA-RF. The values were correlated with the concentrations of IgA-RF and IgM-RF. HC-IgA anti-Y3 was measured in 45 sera with anti-Y3 antibodies of IgM, IgG and IgA class. The HC-IgA anti-Y3 levels were correlated with those of anti-Y3 of IgG and IgM class, but not of IgA class. For HC-IgA anti-Y3, the closest correlation was that with the specific IgM antibody concentration, rs = 0.63 (p less than 0.001). In 25 normal sera, significant correlations were observed between HC-IgA anti-TT and specific antibodies of IgG and IgA class, but not of IgM class. In 107 sera containing IgA M-components, the total concentration of HC-IgA correlated poorly with both protein HC and with IgA concentrations. It was concluded that specific HC-IgA antibodies are normal constituents of serum, and that their concentrations are not directly related to the serum content of specific IgA antibodies.

Alpha-Globulins↗
PubMed1990

Cystatin C, a human proteinase inhibitor, blocks replication of herpes simplex virus.

Cystatin C is a human cysteine proteinase inhibitor present in extracellular fluids. Cystatin C and a tripeptide derivative (Z-LVG-CHN2) that mimics its proteinase-binding center, were tested for possible antiviral activity against herpes simplex virus type 1 (HSV) and poliovirus type 1. Both recombinant cystatin C and Z-LVG-CHN2 displayed strong inhibitory effects on HSV replication, whereas no significant effect on poliovirus replication was seen. The molar concentration of cystatin C that gave total inhibition of HSV replication was lower than that of either Z-LVG-CHN2 or of acyclovir, the drug currently most used against HSV infections. These results suggest that cysteine proteinase inhibitors might play a physiological role as inhibitors of viral replication and that such proteinase inhibitors, or peptide derivatives that mimic their proteinase-binding centers, might be used as antiviral agents.

Amino Acid Sequence↗
PubMed1990

Identification of cystatin C, a cysteine proteinase inhibitor, as a major secretory product of human alveolar macrophages in vitro.

The major inhibitor of the cysteine class of proteinases found in human body fluids, such as spinal fluid, milk, and seminal plasma, is cystatin C. In this study we show that human bronchoalveolar fluid also contains cystatin C and examine cystatin C expression by alveolar macrophages in vitro. Immunoprecipitation of extracts of metabolically labeled cells and immunoblotting of cellular extracts and culture media show that cystatin C is synthesized as a 14 (+/- 0.5) kilodalton (kD) protein and that greater than 90% of the protein is released as the 14 kD product into the culture supernatant (26.5 +/- 6.8 ng per 10(6) cells per 24 h). Cystatin C is not one of the most abundant proteins secreted during the first 24 h in vitro, representing approximately 10 to 12% of the total protein released by normal nonsmoker macrophages. Alveolar macrophages obtained from cigarette smokers or nonsmoker macrophages exposed to zymosan in vitro released 10 to 55% less cystatin C than nonsmoker macrophages. We also assayed culture supernatants from macrophages of smokers and nonsmokers for functional cystatin C. Supernatants of nonsmoker macrophages inhibited cathepsin B-like amidolytic activity in a fluorometric assay at pH 5.5. The inhibition was blocked by adsorption with Sepharose-coupled cystatin C antibodies and the inhibitor subsequently recovered from the Sepharose beads. In contrast, supernatants from smoker macrophages had obvious cathepsin B-like activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Bronchoalveolar Lavage Fluid↗
PubMed1990

Assignment of allotypes G1m(a+) and G1m(a-) at the genomic level by polymerase chain reaction analysis.

A method of assignment of the human immunoglobulin allotypes G1m(a+) and G1m(a-) without the use of serological reagents is described. It is based upon oligonucleotide-directed enzymatic amplification of genomic segments encoding CH3 of gamma chains, followed by dot-blot hybridization of radioactively labelled oligonucleotides to the amplified DNA. The method was used to classify the immunoglobulin allotypes of 11 persons, six G1m(a+) and five G1m(a-), and the resultant classification agreed completely with that of classical serological typing.

Amino Acid Sequence↗
PubMed1990

AA-amyloidosis. Tissue component-specific association of various protein AA subspecies and evidence of a fourth SAA gene product.

Protein AA, the major repetitive protein subunit present in fibrils deposited in AA-amyloidosis, is an N-terminal cleavage product of a 104-amino acid precursor, serum amyloid A (SAA). Protein AA subspecies varying between 45 and 94 amino acids in length have been described. In this study it is shown that the different protein AA subspecies are not evenly distributed in amyloid deposits and that in single patients, certain subspecies of protein AA are deposited in specific tissue component sites. Thus larger protein AA subspecies occur in lower concentration in amyloid in the glomeruli compared to other sites and are especially found in amyloid in vessel walls. Three different SAA forms have been predicted from genomic and complementary DNA studies. The existence of a fourth type has been suspected from amino acid sequence studies of purified SAA. Protein AA derived from this fourth type of SAA is now shown to be present in amyloid fibrils in one of the patients studied in this paper.

Amino Acid Sequence↗
PubMed1990

Synthesis of cysteine proteinase inhibitors structurally based on the proteinase interacting N-terminal region of human cystatin C.

Peptides spanning the entire, or part of, the Gly4-Glu21 segment of the human cysteine proteinase inhibitor cystatin C have been synthesized. Peptides containing residues on the N-terminal side of Gly11 were rapidly cleaved by papain at the bond Gly11-Gly12 whereas a peptide starting at residue Gly11 was not, thus demonstrating 1. that the N-terminal segment of cystatin C has an amino acid sequence that would allow rapid interaction between this segment and the substrate pocket of papain and, if this interaction takes place, that 2. the cystatin C residue Gly11 would be in the P1 position, and 3. the major interaction would be between residues Arg8-Val10 and the papain substrate pocket subsites S4, S3 and S2, respectively. Several modified peptide derivatives containing either diazomethane groups or peptide bond isosters were synthesized based on the structure of the Leu9-Gly11 segment of cystatin C and tested for their cysteine proteinase inhibiting capacity. The peptidyl derivatives, t-butyloxycarbonyl-valyl-glycyl-diazomethane and benzyloxycarbonyl-leucyl-valyl-glycyl-diazomethane irreversible inhibited the cysteine proteinases papain, bovine cathepsin B and streptococcal proteinase, but did not influence the activity of serine, aspartic or metallo-proteinases.

Amino Acid Sequence↗
PubMed1989

Ig-binding bacterial proteins also bind proteinase inhibitors.

Protein G is a streptococcal cell wall protein with separate binding sites for IgG and human serum albumin (HSA). In the present work it was demonstrated that alpha 2-macroglobulin (alpha 2M) and kininogen, two proteinase inhibitors of human plasma, bound to protein G, whereas 23 other human proteins showed no affinity. alpha 2M was found to interact with the IgG-binding domains of protein G, and in excess alpha 2M inhibited IgG binding and vice versa. A synthetic peptide, corresponding to one of the homologous IgG-binding domains of protein G, blocked binding of protein G to alpha 2M. Protein G showed affinity for both native and proteinase complexed alpha 2M but did not bind to the reduced form of alpha 2M, or to the C-terminal domain of the protein known to interact with alpha 2M receptors on macrophages. Binding of protein G to alpha 2M and kininogen did not interfere with their inhibitory activity on proteinases, and the interaction between protein G and the two proteinase inhibitors was not due to proteolytic activity of protein G. The finding that protein G has affinity for proteinase inhibitors was generalized to comprise also other Ig binding bacterial proteins. Thus, alpha 2M and kininogen, were shown to bind both protein A of Staphylococcus aureus and protein L of Peptococcus magnus. The results described above suggest that Ig-binding proteins are involved in proteolytic events, which adds a new and perhaps functional aspect to these molecules.

Bacterial Proteins↗
PubMed1989

High-level expression of active human cystatin C in Escherichia coli.

Expression of the human cysteine proteinase inhibitor, cystatin C (CysC) in the cytoplasm of Escherichia coli was studied using a cDNA fragment encoding the cysteine proteinase inhibitor controlled by the phage lambda pR/cI857 system. The yield of CysC was low, probably due to proteolytic degradation. By fusing the cysC cDNA to a DNA fragment encoding the signal peptide of the E. coli outer membrane protein A, it was possible to produce a substantial amount of CysC in the periplasm. The processing of the signal peptide was shown to be quantitative and to result in CysC with the correct N-terminal amino acid. Yields higher than 1000 micrograms CysC/ml can be obtained by initiating the product formation at a moderate temperature (40 degrees C) late in an optimized fermentation process. A method that gives selective extraction of the periplasmic proteins and at the same time stabilizes CysC has been used.

Bacteriophage lambda↗
PubMed1989

Bacterial growth blocked by a synthetic peptide based on the structure of a human proteinase inhibitor.

Cysteine proteinases are important not only in the intracellular catabolism of peptides and proteins and in the processing of prohormones and proenzymes, but also in the penetration of normal human tissue by malignant cells and possibly microorganisms, including viruses. Cystatin C is a human cysteine proteinase inhibitor present in extracellular fluids. We have synthesized peptide derivatives mimicking the proposed proteinase-binding centre of cystatin C and find that they irreversibly inhibit cysteine proteinases. Several bacteria produce proteinases, so we tested a tripeptide derivative (Z-LVG-CHN2) for in vitro anti-bacterial activity against a large number of bacterial strains belonging to thirteen different species. It was found to inhibit specifically the growth of all strains of group A streptococci. The susceptibility of these human pathogens to the peptide was compared with that to well-established anti-streptococcal antibiotics such as tetracycline and bacitracin. Moreover, the peptide was active in vivo against group A streptococci: mice injected with lethal doses of these bacteria were cured by a single injection of Z-LVG-CHN2. The cysteine proteinase produced by group A streptococci was isolated and found to be inhibited by Z-LVG-CHN2; moreover, excess proteinase relieved the growth inhibition caused by the peptide derivative, suggesting that the antibacterial activity of Z-LVG-CHN2 is due to inhibition of this cysteine proteinase. This strategy of blocking proteinases with peptide derivatives that mimic naturally occurring inhibitors could be useful in the construction of new agents against other microorganisms, including viruses.

Amino Acid Sequence↗
PubMed1989

Isolation and characterization of a tumor necrosis factor binding protein from urine.

Tumor necrosis factor (TNF)/cachectin can produce both beneficial and harmful manifestations. Mechanisms may operate to counteract potentially harmful effects such as shock and cachexia. The TNF binding protein (TNF-BP), which is found at increased levels in serum and urine of patients with chronic renal failure, may play such a role. TNF-BP was purified 1,000,000-fold to homogeneity from urine of patients with chronic renal failure by use of ion exchange chromatography, affinity chromatography on TNF-Sepharose and reverse phase chromatography. The purified protein contained only one chain with an apparent Mr on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 30,000. The aminoterminal amino acid sequence D-S-V-X-P-Q-G-K-Y-I-H-P-Q-V-N-S-I-X-K-T revealed no significant homologies with previously described protein sequences. TNF-BP may act as a regulator of the bioactivities of TNF/cachectin.

Amino Acid Sequence↗
PubMed1989

Mutation in the cystatin C gene causes hereditary brain hemorrhage.

Hereditary cystatin C amyloid angiopathy (HCCAA) is an autosomal dominant disorder leading to massive brain hemorrhage and death in young adults (Jensson et al., 1987). A variant of a potent inhibitor of cysteine proteinases, cystatin C (Barrett et al., 1984), is deposited as amyloid fibrils in the cerebral arteries of the patients (Ghiso et al., 1986). We have used the full length cystatin C cDNA probe (Abrahamson et al., 1987) to demonstrate a mutation in the codon for leucine at position 68, which abolishes an Alu I restriction site in cystatin C gene of the HCCAA patients. The Alu I marker has been used to show that this mutation is transmitted only in the affected members in all eight families investigated, proving that the mutated cystatin C gene causes HCCAA. This DNA marker will be useful for the diagnosis of HCCAA in patients, asymptomatic affected individuals and also for pre-natal diagnosis. HCCAA is the first human disorder known to be caused by an abnormal gene for a cysteine proteinase inhibitor.

Amyloidosis↗
PubMed1988

Identification of retinol as one of the protein HC chromophores.

Protein HC (alias alpha 1-microglobulin) contains so far unidentified yellow-brown fluorescent chromophores. Several preparations of human protein HC were extracted with hexane. Most of the extracts contained a substance which, upon reversed-phase HPLC, co-eluted with all-trans- retinol and had an absorption spectrum identical to that of retinol. The substance was also, like retinol, destroyed by exposure to ultraviolet light and acid pH. These observations strongly support the proposal that protein HC is a member of the newly defined lipocalin protein superfamily. The highest retinol-protein HC molar ratio of the investigated protein HC preparations was 1.6 x 10(-3) indicating that retinol is not the only ligand bound to protein HC. This was confirmed by comparing the absorption spectrum of protein HC before and after hexane extraction.

Alpha-Globulins↗
PubMed1988

Mutation in cystatin C gene causes hereditary brain haemorrhage.

Hereditary cystatin C amyloid angiopathy (HCCAA) is an autosomal dominant disorder in which a cysteine proteinase inhibitor, cystatin C, is deposited as amyloid fibrils in the cerebral arteries of patients and leads to massive brain haemorrhage and death in young adults. A full length cystatin C cDNA probe revealed a mutation in the codon for leucine at position 68 which abolishes an Alu I restriction site in the cystatin C gene of HCCAA patients. The Alu I marker has been used to show that this mutation is transmitted only in affected members of all eight families investigated, and that the mutated cystatin C gene causes HCCAA.

Cerebral Hemorrhage↗
PubMed1988

Efficient production of native, biologically active human cystatin C by Escherichia coli.

A cDNA encoding the mature human cysteine proteinase inhibitor cystatin C was fused to the coding sequence for the Escherichia coli outer membrane protein A signal peptide, and the recombinant gene was expressed in E. coli under the control of the lambda PR promoter, an optimized Shine-Dalgarno sequence and the lambda cI 857 repressor. When induced at 42 degrees C, such cells expressed large amounts of recombinant cystatin C. The recombinant protein was isolated in high yield and characterized. All physicochemical properties investigated, including the positions of disulfide bonds, indicated that the E. coli derived cystatin C was identical to cystatin C isolated from human biological fluids, except that the proline residue in position three was not hydroxylated. The recombinant protein displayed full biological activity against papain, cathepsin B and dipeptidyl peptidase I.

Amino Acid Sequence↗
PubMed1988

Effects of plasmapheresis on the plasma concentration of proteins used to monitor the disease process in multiple myeloma.

We studied the influence of plasmapheresis on the plasma concentrations of proteins (IgG, IgA, beta 2-microglobulin) used for estimation of tumour cell mass in patients with multiple myeloma. Simultaneously, the effects of plasmapheresis on plasma concentrations of proteins (CRP, alpha 1-antitrypsin, haptoglobin) used for the assessment of inflammatory processes were studied. Also, changes in the plasma concentration of protein HC, a recently described protein occurring both as a free protein and as an IgA complex, were studied. The influence of plasmapheresis varied for the different proteins. The plasma levels of IgG, IgA, CRP, alpha 1-antitrypsin, and haptoglobin decreased during plasmapheresis. The simultaneous reduction of the level of protein HC was smaller than that of IgG, IgA and haptoglobin. The plasma concentration of beta 2-microglobulin did not decrease during plasmapheresis. The recovery rates of the proteins were found to be approximately inversely related to their molecular weights.

Alpha-Globulins↗
PubMed1988

Protein HC-IgA complexes carry antibody activities.

Polyclonal protein HC-IgA complexes (HC-IgA) were isolated from two different serum pools. Their hydrodynamic volumes were found to be slightly greater than that of monomeric IgA but less than that of dimeric IgA. Sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis of reduced and carboxymethylated complexes followed by immunoblotting showed that the complexes contained normal light and heavy Ig chains, and one polypeptide chain with Mr = 90,000, which carried both IgA alpha chain and protein HC epitopes. Enzyme-linked immunosorbent assays (ELISA) demonstrated that the isolated HC-IgA carried about 0.1 and 4%, respectively, of the antibody activities against one carbohydrate antigen (Yersinia enterocolitica serotype 0:3 lipopolysaccharide) and one protein antigen (rabbit IgG, i.e. antigen for rheumatoid factors) of the IgA populations of the two serum pools. HC-IgA with rheumatoid factor activity could also be demonstrated in the unfractionated serum pool. The binding of HC-IgA in the ELISA was not mediated through its protein HC part. The present observations show that HC-IgA carries antibody activities and constitutes a unique class of IgA complexes, since it does not dissociate under denaturating conditions after reduction. It may represent a further biological potential of the humoral immune system.

Alpha-Globulins↗