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Biomedical subjects

H Kim

Publications and source records attributed to H Kim.

At least 649 records · Page 36Linked to original sources
PubMed1995

Proteolytic fragmentation with high specificity of mouse immunoglobulin G. Mapping of proteolytic cleavage sites in the hinge region.

We report the results of fragmentation of mouse IgG by clostripain, lysyl endopeptidase, metalloendopeptidase, and V8 protease that have a narrower substrate specificity than papain and pepsin. A panel of mouse monoclonal switch variant antibodies with IgG1, IgG2a, and IgG2b subclasses were examined. Cleavage sites by these proteases were mapped on the hinge region of each of the IgG subclasses. It was demonstrated that lysyl endopeptidase can cleave the core hinge portion of IgG2a and IgG2b without perturbing the inter-chain disulfide bridges. Digestion products were successfully isolated by a combined use of protein A affinity chromatography and HPLC techniques. This is a first successful attempt of obtaining the F(ab')2 fragment of IgG2b by proteolytic digestion.

Alkylation↗
PubMed1995

Model for the complex between protein G and an antibody Fc fragment in solution.

BACKGROUND: Streptococcal protein G and staphylococcal protein A are bacterial antibody-binding proteins, widely used as immunological tools, whose antibody-binding domains are structurally quite different. The binding of protein G to Fc fragments is competitive with respect to protein A, suggesting that the binding sites for protein A and protein G on Fc overlap, notwithstanding the fact that they lack sequence or structural similarity. RESULTS: To resolve this issue, the residues involved in the interaction between an IgG-binding domain of protein G (domain II) and the Fc fragment of mouse IgG2a have been identified by use of 13C and 15N NMR. Binding of protein G domain II selectively perturbed resonances from residues between the CH2 and CH3 domains of Fc, whereas in domain II the residues affected are primarily those on the alpha-helix and the third strand of the beta-sheet. This information was used, together with the structures of the two uncomplexed proteins, to construct a model of the complex, using Monte Carlo minimization techniques. In this model, the alpha-helix of protein G lies in the same position as helix 1 of protein A in the crystal structure of the protein A:Fc complex, but its orientation differs from the latter by 180 degrees. CONCLUSIONS: The interactions of the bacterial antibody-binding proteins with their 'target' immunoglobulins involve a very versatile set of protein-protein interactions. First, the IgG-binding domains of protein A and protein G have quite different three-dimensional structures, but bind to sites on the Fc fragment that overlap extensively. Secondly, protein G employs two quite different regions of its surface to bind to the Fab and Fc regions of IgG.

Antigens, Bacterial↗
PubMed1995

Regulation of neurofibromin expression in rat sciatic nerve and cultured Schwann cells.

Loss of function mutations at the NF1 locus may act intrinsically in Schwann cells to cause the formation of benign Schwann cell tumors (neurofibromas) in patients with type 1 neurofibromatosis. To identify contexts in Schwann cells in which such mutations may play an important role, we measured the levels of NF1 mRNA and neurofibromin in rat sciatic nerve during development, after axotomy, and in cultured rat Schwann cells. NF1 mRNA was present in developing sciatic nerve throughout the period of active Schwann cell proliferation and myelination. After nerve transection, no alteration in NF1 message level was detected, but neurofibromin levels increased, as assessed by immunohistochemistry and Western blotting, suggesting that, in vivo, neurofibromin expression in Schwann cells is post-transcriptionally induced during Wallerian degeneration. Cultured rat Schwann cells constitutively expressed NF1 mRNA and neurofibromin. Schwann cell proliferation induced by exposure to serum and forskolin was not associated with changes in NF1 mRNA or neurofibromin expression, whereas Schwann cell proliferation induced by extracts of embryonic brain membranes was associated with increased NF1 message and neurofibromin expression. Thus, Schwann cells, both in vivo and in vitro, express NF1 mRNA constitutively; the expression of NF1 mRNA and neurofibromin is modulated by only some mitogenic stimuli in Schwann cells.

Animals↗
PubMed1995

Overexpression and characterization of human tetrameric pyruvate dehydrogenase and its individual subunits.

Pyruvate dehydrogenase (E1), an alpha 2 beta 2 tetramer, is the first component of the pyruvate dehydrogenase complex which catalyzes a two-step oxidative decarboxylation of pyruvic acid. To overexpress human E1 and its subunits individually, cDNAs for the mature forms of human E1 alpha and E1 beta were subcloned either individually or together into a plasmid pQE-9 and expressed in Escherichia coli M15. A polyhistidine extension was added at the NH2-termini of the recombinant E1 alpha and E1 beta for the rapid purification of the proteins by Ni-nitrilotriacetic-agarose chromatography. The polyhistidine extension on either E1 alpha or E1 beta subunit did not affect the activity of the recombinant tetrameric E1. Highly purified recombinant human E1 catalyzed the partial reactions of the oxidative and nonoxidative conversion of pyruvic acid with the same efficiency as E1 purified from bovine kidney. Recombinant human E1 interacted with thiamin pyrophosphate by forming a charge transfer complex band at 330 nm that changed during the catalytic cycle. Recombinant human E1 was phosphorylated by E1-kinase (with concomitant inactivation) by incorporating nearly three phosphoryl groups per mole of E1. When expressed individually, E1 alpha and E1 beta subunits lacked any catalytic activity in the oxidative or nonoxidative reactions. Spectral studies demonstrated that there was no thiamin pyrophosphate binding to either recombinant E1 alpha or E1 beta subunit. The E1 alpha subunit retained the ability to be phosphorylated; however, the incorporation of phosphoryl groups into recombinant E1 alpha alone was only about 12% of that observed with the tetrameric E1. These findings show that both subunits are required for formation of the active center and catalysis.

Base Sequence↗
PubMed1995

Multimodality therapy for adenocarcinoma of the esophagus.

Few reports exist detailing results of multimodality treatment for adenocarcinoma of the esophagus. We have treated 28 such patients using a preoperative regimen consisting of two courses of cisplatin and 5-fluorouracil with radiation (either 3,000 or 3,600 cGy). There were 25 men and 3 women (mean age, 62.9 years; range, 35 to 86 years), and 16 patients were known to have Barrett's esophagus. Dysphagia was present for a mean of 2.7 months, and the average weight loss was 6.5 kg. Tumors ranged from 2 to 10 cm in length (mean, 5.2 +/- 1.8 cm) with American Joint Committee on Cancer clinical stage I in 2 patients, stage II in 19 patients, and stage III in 7. Dysphagia improved in 23 patients (82%), and in 8 (29%) no tumor was detected during radiologic and endoscopic staging after neoadjuvant therapy. Four patients refused operation. Esophagectomy via standard Ivor Lewis approach was accomplished in 20 of 24 patients (87%) undergoing operation. There were no operative deaths, and mean hospital stay was 15.5 +/- 11.6 days. Four patients (17%) were complete responders with no tumor in the resected specimen. Actuarial survival in the 28 patients at 1, 2, and 3 years is 71%, 28%, and 20% respectively. Of the 20 esophagectomy patients, 6 are alive with no evidence of disease at 10, 50, 54, 70, 77, and 84 months. Three of these were complete responders. Only 1 of the 8 patients no undergoing resection is alive at 16 months with no evidence of disease after further radiotherapy and chemotherapy.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenocarcinoma↗
PubMed1995

Relative contributions of pituitary-adrenal hormones to the ontogeny of behavioral inhibition in the rat.

Recent investigations revealed that adrenalectomized (ADX) rat pups exhibit deficits in behavioral inhibition. Furthermore, administration of exogenous corticosterone (CORT) restores behavioral inhibition in ADX pups. Although these studies suggest that CORT has an important role in the development of behavioral inhibition, the relative behavioral effects of elevated pituitary hormone secretion induced by ADX are not known. Therefore, experiments were conducted to assess the potential behavioral effects of elevated adrenocorticotropin (ACTH) secretion induced by ADX and to further evaluate the contribution of endogenous CORT to the development of behavioral inhibition. In Experiment 1., we verified that 10-day-old ADX rats exhibit high levels of plasma ACTH throughout the preweaning period associated with the development of behavioral inhibition. In Experiment 2, 10-day-old pups were hypophysectomized (HYPOX) and ADX and were compared behaviorally to sham-operated controls on day 14. When tested in the presence of an anesthetized unfamiliar adult male rat, HYPOX + ADX pups exhibited low levels of freezing accompanied by ultrasonic vocalizations. These pups also had reduced concentrations of plasma ACTH and CORT. In Experiment 3, 10-day-old pups were HYPOX and tested for behavioral inhibition on day 14. In comparison to sham-operated controls, HYPOX rats exhibited significantly lower levels of freezing and had reduced plasma concentrations of ACTH and CORT. Results demonstrate clearly that deficits in freezing occur even in the presence of low plasma ACTH concentrations. Therefore, elevated secretion of pituitary hormones is not a major factor that contributes to the ADX-induced deficits in behavioral inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Cortex Hormones↗
PubMed1995

Pyridine induction of Sprague-Dawley rat renal cytochrome P4502E1: immunohistochemical localization and quantitation.

Previous research has shown that i.p. injection of rats with pyridine results in a significant increase in immunoreactive renal cytochrome P4502E1 (alcohol-inducible form) in a dose- and time-dependent manner. However, the cellular location of renal P4502E1 in rats was not reported. Thus, it was not known whether the pyridine-induced increase in renal P4502E1 resulted from increased production of the enzyme in cells which normally express P4502E1 or from de novo expression in cells normally devoid of the protein. To address these questions, rats were injected i.p. with either 200 mg pyridine/kg body wt./day for 1, 2, 3, or 4 days (n = 2/group) or injected once with an equal volume of sterile, pyrogen-free saline (control group; n = 2). Kidney tissue samples from saline- and pyridine-exposed rats were processed by light microscopy and were immunochemically stained to detect rat cytochrome P4502E1. Most of the immunoreactive P4502E1 was located within renal cortical epithelial cells lining proximal and distal tubules of the cortex with lesser--but consistent--amounts present in tubular epithelial cells within the inner and outer medulla. Pyridine exposure resulted in a 2-3-fold increase in P4502E1 immunoreactivity in proximal cortical tubules surrounding glomeruli and cortical blood vessels. The results of this study demonstrate a cell-specific distribution of cytochrome P4502E1 within the rat kidney and indicate that pyridine exposure results in a selective induction of immunoreactive P4502E1 in tubule epithelial cells which constitutively express this enzyme. The results of this study provide a morphologic basis for interpreting cell-specific nephrotoxicity due to xenobiotics that are biotransformed to toxic metabolites by renal P4502E1.

Animals↗
PubMed1995

Structures of revertant signal sequences of Escherichia coli ribose binding protein.

Recently we reported (Yi et al., 1994) that the alpha-helical content of the signal peptide of Escherichia coli ribose binding protein, when determined by circular dichroism (CD) and two-dimensional NMR in trifluoroethanol/water solvent, is higher than that of its nonfunctional mutant signal peptide. In the present investigation, the structures of the signal peptides of two revertant ribose binding proteins in the same solvent were also determined with CD and two-dimensional 1H NMR spectroscopy. According to the CD results, both of these revertant signal peptides showed an intermediate helicity between those of wild-type and mutant signal peptides, the helical content of the revertant peptide with higher recovery of the translocation capability being higher. On the other hand, the alpha-helix regions of the wild-type and the revertant peptides as determined by NMR were shown to be the same. This discrepancy may be due to the difference in stability between identical alpha-helical stretches in wild-type and revertant peptides. A good correlation was observed between the helical content of these four ribose binding protein signal peptides in TFE/water as studied by CD and their in vivo translocation activities. It appears, therefore, that both the proper length of the helix and the stability are of functional significance.

Amino Acid Sequence↗
PubMed1995

A phylogenetic analysis of the genus Saccharomonospora conducted with 16S rRNA gene sequences.

Nearly complete sequences of 16S rRNA genes of representative strains of the genus Saccharomonospora were determined following the isolation and cloning of the amplified genes. The sequences were aligned with those of representatives of the family Pseudonocardiaceae, and a phylogenetic tree was inferred by the neighbor-joining method. The genus Saccharomonospora formed a distinct clade within the evolutionary radiation encompassed by the family Pseudonocardiaceae. The average nucleotide similarity value found between the type strains of the four validly described Saccharomonospora species was 97.5% +/- 1.0%. The most distant relationship was found between Saccharomonospora azurea and Saccharomonospora viridis K73 (96.3% similarity). In contrast, Saccharomonospora azurea K161 and "Saccharomonospora caesia" K163 had identical 16S rRNA gene sequences. The nucleotide sequence data suggest that the genus Saccharomonospora contains several new centers of variation.

Actinomycetales↗
PubMed1995

Lipoprotein (a) and apolipoprotein A-1 and B in schoolchildren whose grandparents had coronary and cerebrovascular events: a preliminary study of 12-13 year old Japanese children.

The aim of this study was to evaluate the relationship between the serum levels of lipoprotein (a) [Lp (a)] and apolipoproteins (apo A-1 and apo B) in schoolchildren with a history of coronary and cerebrovascular events in their grandparents. We measured serum concentrations of Lp (a) and apoliproteins immunochemically in 289 schoolchildren aged 12-13 years and questioned parents about coronary and cerebrovascular events in the children's grandparents. In boys and girls, mean +/- s.d. levels of apo A-1, apo B and Lp (a) were 134 +/- 20.3 and 136 +/- 17.4 mg/dL, 61 +/- 16 and 66 +/- 15 mg/dL and 12.5 +/- 15.3 and 12.5 +/- 15.1 mg/dL, respectively. There were no significant sex differences in the levels of apo A-1, apo B, and Lp (a). The Lp (a) levels (mean +/- s.d., 12.5 +/- 15.2 mg/dL; median 7.5 mg/dL, n = 289) were not affected by other variables. The Lp (a) distribution was strongly positively skewed and 75% of schoolchildren had very low levels. In the total 289 schoolchildren, thirty-two grandparents who had had coronary vascular events (21 myocardial infarction, 11 angina pectoris) and twenty-three grandparents who had had cerebrovascular events were recorded. By the boxplot statistical analysis, no difference was found in Lp (a) levels in children whose grandparents had myocardial infarction compared with those whose grandparents had no such history, or compared with those whose grandparents had suffered cerebrovascular events.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1995

Changes in apolipoproteins during the acute phase of Kawasaki disease.

To determine disturbances of lipid metabolism in patients with Kawasaki disease, we investigated changes in the serum levels of apolipoproteins and serum lipids. Results were as follows. Total serum cholesterol (TC) and high-density lipoprotein cholesterol (HDL-C) decreased during the early stage of Kawasaki disease. The apo A-I and A-II levels were low until the 2nd week of illness. Apo B decreased during the 1st week of illness, but rose slightly during the 2nd and 3rd weeks. The apo B/apo A-I ratio and the apo B/TC ratio were high in the early stages of illness. The group of patients with coronary artery lesions showed low levels of TC and HDL-C, and low levels of apo A-I, A-II and B during the early stages, compared with the group without coronary artery lesions. The apo B/TC ratio was significantly higher in the patients with coronary artery lesions during the 2nd week of illness. Our findings suggest an association between changes in serum lipids and apolipoproteins and coronary artery involvement in Kawasaki disease. These abnormalities may indicate the presence of early coronary arteriosclerosis.

Acute Disease↗
PubMed1995

Acute febrile diarrhoea in horses: 86 cases (1986-1991).

Eighty-six horses presented for examination at the Rural Veterinary Centre between January 1986 to December 1991 with acute diarrhoea. The average age of affected horses was 3.2 +/- 0.2 years (mean +/- SE), with 69% three years or younger. Sixty-one horses were male (36 stallions, 25 geldings) and 83 horses were Thoroughbreds (47) or Standardbreds (36). Sixty-six horses were undergoing routine training at the time of onset of diarrhoea. Eight horses were afflicted with a non-specific illness within one to five days before the onset of diarrhoea, whereas eight horses developed diarrhoea during or within 48 h of discontinuation of antimicrobial therapy. Three horses developed the diarrhoea within 24 h of road transportation. Clinically, the disorder was characterised by a fever, sudden onset of profuse diarrhoea, clinical evidence of dehydration (estimated to be 5 to 12% of body weight at the time of admission) and shock. Degenerative leucopaenia, hyponatraemia, hypochloraemia, hyperkalaemia, hyperglycaemia and azotaemia were characteristic laboratory findings. Laminitis was a sequel in about 30% of cases. The cause of diarrhoea remained undetermined in most cases. Salmonellas were isolated from faecal or tissue samples in only two cases. Similarly, there was no evidence of seroconversion to Erhlichia risticii in 17 cases. Sixty-two of the horses survived the acute phase of the disease in response to supportive care. In horses that did not survive, necropsies were performed and revealed sanguineous or turbid peritoneal fluid. The colonic and caecal walls were oedematous and thickened with serosal congestion and discolouration of these organs evident grossly.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗