Biomedical subjects

R B Kirkpatrick

Publications and source records attributed to R B Kirkpatrick.

At least 19 recordsLinked to original sources

PubMed2001

Steady-state kinetic characterization of substrates and metal-ion specificities of the full-length and N-terminally truncated recombinant human methionine aminopeptidases (type 2).

The steady-state kinetics of a full-length and truncated form of the type 2 human methionine aminopeptidase (hMetAP2) were analyzed by continuous monitoring of the amide bond cleavage of various peptide substrates and methionyl analogues of 7-amido-4-methylcoumarin (AMC) and p-nitroaniline (pNA), utilizing new fluorescence-based and absorbance-based assay substrates and a novel coupled-enzyme assay method. The most efficient substrates for hMetAP2 appeared to be peptides of three or more amino acids for which the values of k(cat)/K(m) were approximately 5 x 10(5) M(-1) min(-1). It was found that while the nature of the P1' residue of peptide substrates dictates the substrate specificity in the active site of hMetAP2, the P2' residue appears to play a key role in the kinetics of peptidolysis. The catalytic efficiency of dipeptide substrates was found to be at least 250-fold lower than those of the tripeptides. This substantially diminished catalytic efficiency of hMetAP2 observed with the alternative substrates MetAMC and MetpNA is almost entirely due to the reduction in the turnover rate (k(cat)), suggesting that cleavage of the amide bond is at least partially rate-limiting. The 107 N-terminal residues of hMetAP2 were not required for either the peptidolytic activity of the enzyme or its stability. Steady-state kinetic comparison and thermodynamic analyses of an N-terminally truncated form and full-length enzyme yielded essentially identical kinetic behavior and physical properties. Addition of exogenous Co(II) cation was found to significantly activate the full-length hMetAP2, while Zn(II) cation, on the other hand, was unable to activate hMetAP2 under any concentration that was tested.

Amino Acid Substitution↗

PubMed2000

A modified metal-ion affinity chromatography procedure for the purification of histidine-tagged recombinant proteins expressed in Drosophila S2 cells.

We have developed a modified method of immobilized metal-ion affinity chromatography (IMAC) that can be used for the purification of histidine-tagged proteins from conditioned medium containing free copper ions. Classical methods of IMAC purification, using resins such as Ni-NTA, have proven inefficient for this type of purification and require multiple steps due to the interference of divalent copper ions with the binding of His-tagged protein to the charged resin. In contrast, this modified IMAC procedure, using chelating Sepharose instead of Ni-NTA, enables efficient purification from copper-containing medium in a single step. This method appears to rely upon a preferential interaction of protein-copper complexes with immobilized chelating resin. We have utilized this method to purify active, His-tagged murine interleukin 12 from the conditioned medium of Drosophila S2 cells coexpressing recombinant p40 and His-tagged p35 subunits and for the purification of the extracellular domain of the erythropoietin receptor. This method should be applicable to the purification of a wide variety of His-tagged fusion proteins expressed in Drosophila cells and in other systems where free metal ions are present.

Animals↗

PubMed2000

Human cartilage glycoprotein 39 (HC gp-39) mRNA expression in adult and fetal chondrocytes, osteoblasts and osteocytes by in-situ hybridization.

OBJECTIVE: To examine the expression pattern of human cartilage glycoprotein 39 (HC gp-39) mRNA in human cartilage and bone. DESIGN: In-situ hybridization analysis was used to examine the expression pattern of human cartilage glycoprotein 39 (HC gp-39) mRNA in adult human osteoarthritic articular cartilage from various stages of disease, as well as in human osteophytic tissue and in human fetal bone. RESULTS: In cartilage from patients with mild osteoarthritic cartilage degeneration, HC gp-39 was expressed at moderate to high levels only in chondrocytes of the superficial zone. In advanced OA cartilage, cloning chondrocytes of the superficial zone expressed high levels of HC gp-39 and chondrocytes of the mid- and deep zones were also positive. HC gp-39 was undetectable in the chondrocytes of normal articular cartilage. In osteophytic tissue, the expression of HC gp-39 mRNA was intense in flattened, end-stage osteoblasts and in primary osteocytes in both endochondral and intramembranous bone formation. Proliferating osteoblasts expressed low to moderate levels. Notably, mature osteocytes were negative for HC gp-39 expression. Chondrocytes in the secondary ossification center of developing fetal cartilage demonstrated high expression while growth plate and mineralized cartilage chondrocytes had lower expression. Osteoblasts at sites of endochondral and intramembranous bone formation were positive for expression of HC gp-39. CONCLUSIONS: The stage-specific expression of HC gp-39 in fetal development and adult remodelling bone and cartilage provides evidence for a specific functional or structural role for HC gp-39 in bone and cartilage tissue. HC gp-39 is expressed in diseased human osteoarthritic cartilage and osteophyte, but not in non-diseased tissue, and its distribution within the tissue changes as disease progresses. OA is characterized not only by cartilage degeneration, but by increased subchondral bone formation and osteophytosis. The results from this study indicate that the increased HC gp-39 expression in OA serum and synovial fluid may reflect not only cartilage degeneration but increased osteogenesis.

Adipokines↗

PubMed1999

cDNA sequence and characterization of the gene that encodes human myotrophin/V-1 protein, a mediator of cardiac hypertrophy.

The predominant response of the heart to sustained increased work load is development of ventricular hypertrophy, principally as a result of hypertrophy of cardiomyocytes. The molecular mechanisms and factors involved in cardiomyocyte hypertrophy are poorly understood. Myotrophin is a novel 12-kilodalton protein recently implicated as a factor associated with and able to induce cardiac hypertrophy. Cloning of rat myotrophin revealed that this protein is identical to the functionally undefined rat, murine and chicken V-1 proteins. Although human myotrophin has been purified to homogeneity, its gene has not been characterized. In this report we describe the cloning, expression, purification and characterization of the human homolog of myotrophin/V-1 protein. Sequence analysis indicators high homology (>90%) between all species at both the nucleotide and amino acid levels, and Southern blot analysis of genomic DNA from diverse species verifies that myotrophin/V-1 is a highly conserved gene. Northern analysis indicates wide-spread expression of a single human transcript, and examination of mRNA distribution in 50 human tissues by dot blot analysis indicates ubiquitous expression with relatively high expressioon in adult and fetal heart. We verify that recombinant human myotrophin produces cardiomyocyte hypertrophy, and we demonstrate for the first time that elevated levels of myotrophin/V-1 protein mRNA are expressed in human dilated cardiomyopathic hearts. We report the novel findings that myotrophin expression is elevated in ischemic hearts, and that myotrophin expression correlates positively with ventricular mass in a hypoxic rat model of induced right ventricular hypertrophy.

Amino Acid Sequence↗

PubMed1998

Genetic characterization of the murine Ym1 gene and identification of a cluster of highly homologous genes.

The murine Ym1 gene belongs to a family of mammalian genes that are homologous to the chitinases from lower organisms, such as insects, nematodes, bacteria, and plants. All of these homologous mammalian proteins (except one member) have no demonstrable chitinase activity and therefore cannot be considered chitinases. The biological functions of these proteins remain unknown. However, these proteins may function through binding to carbohydrate polymers. In an effort to characterize the murine Ym1 gene better, we have delineated its genomic structure. We mapped the chromosomal location of the Ym1 gene to a central region of mouse chromosome 3 (in the region syntenic to human chromosome 1p13) and that of another murine chitinase-like gene, Brp39, to a central region of mouse chromosome 1 (in the region syntenic to human chromosome 1q31). In addition, we identified several genes highly homologous to Ym1 that cluster at the Ym1 locus. We show that these genes are expressed selectively in different mouse tissues.

Amino Acid Sequence↗

PubMed1998

Pulmonary infections in hospitalized patients with cirrhosis.

We have further characterized pulmonary infections by bronchoalveolar lavage in hospitalized patients with cirrhosis. Sixty-seven consecutive patients admitted to the Ohio State University Medical Center from 1992 to 1995 with liver disease who underwent bronchoscopy with bronchoalveolar lavage were identified. Twenty-one patients with cirrhosis and pneumonia were further analyzed. During the same period, we consecutively identified 23 patients without liver disease or immunosuppression, 19 patients with HIV infections, and 30 patients with cancer or pharmacologic immunosuppression who had bronchoscopy with bronchoalveolar lavage for pneumonia. These groups were included in these analyses as a control and immunosuppressed controls, respectively. Bronchoscopy isolated respiratory pathogens in 16 patients (76.2%) with cirrhosis and 6 patients (26.1%) in the control group (p = 0.002). Fungal organisms were most commonly found in patients with cirrhosis although several patients had more than one organism identified. The control group had mostly bacterial pathogens; the immunosuppressed controls were commonly infected with opportunistic organisms. Six (85.7%) of 7 patients with cirrhosis and fungal pneumonia died of their respiratory illness. Hospitalized patients with cirrhosis commonly have opportunistic pulmonary infections; diagnostic bronchoscopy and empiric antifungal therapy should be considered in those who do not respond to antibiotics.

Adult↗

PubMed1997

Induction and expression of human cartilage glycoprotein 39 in rheumatoid inflammatory and peripheral blood monocyte-derived macrophages.

Human cartilage glycoprotein 39 (HC gp-39) has been described as a major secreted product of cultured articular chondrocytes, synovial fibroblasts, and the osteosarcoma line MG63. However, its expression in these cells types has not been directly linked to corresponding cell types in vivo. In this report, expression of HC gp-39 is demonstrated from peripheral blood-derived macrophages in association with their differentiation from monocytes to macrophages. Consistent with macrophage specificity, HC gp-39 expression is also induced upon selective stimulation of the pluripotent promyelocytic leukemia cell line HL-60 toward the monocyte/macrophage lineage with vitamin D3 or phorbol 12-myristate 13-acetate (PMA), while treatments stimulating granulocyte and eosinophilic pathways do not induce expression. Furthermore, HC gp-39 expression levels correlate with the degree of morphological differentiation induced by PMA and vitamin D3 treatments. PMA-induced mRNA expression occurs by 36 h and is a secondary transcriptional response since its synthesis is inhibited by cycloheximide. Apparently, HC gp-39 expression is tied to later events in the differentiation of monocytes into macrophages. The in vivo significance of these results is validated by the in situ detection of HC gp-39 mRNA in inflammatory macrophages associated with rheumatoid synovium. Thus, macrophages appear to be an important source of HC gp-39, which has been shown to be present at elevated levels in the blood and synovium of rheumatoid arthritis patients. The implications of this extend well beyond the previously restricted observations in cell types associated with the joint and suggest a potential involvement of macrophage-derived HC gp-39 in other aspects of inflammation, tissue remodeling, and host defense.

Adipokines↗

PubMed1996

Isoforms of alkaline phosphatase determined by isoelectric focusing in patients with chronic liver disorders.

Fractionation of alkaline phosphatase isoenzymes and isoforms by isoelectric focusing is a simple procedure that resolves up to 17 fractions having alkaline phosphatase activity. The fractions are stable at 4 degrees C, and undergo only slight changes during repeated freeze-thaw cycles. Pretreatment with phospholipase-C or sialidase changes the isoelectric focusing patterns of alkaline phosphatase in serum; we recommend they not be used owing to the loss of information. We found that the alkaline phosphatase fractions provide diagnostic information in addition to that given by the common liver-function tests in patients with chronic liver diseases. Primary biliary cirrhosis and primary sclerosing cholangitis showed similar biochemical changes, but they are very different from alcoholic cirrhosis based on the common liver-function tests and the alkaline phosphatase isoform patterns obtained by isoelectric focusing. Analysis of the laboratory data using neural networks has some limited use in distinguishing chronic and chronic-active hepatitis of any cause. We have confirmed the tissue assignments made by Griffiths (Prog Clin Biochem 1989; 8:63-74) for the alkaline phosphatase fractions in liver as obtained by isoelectric focusing: Fractions 1a and 1b show a strong correlation with biliary diseases, and fractions 2, 3, and 4 show consistent increases in patients with primary disorders of hepatocytes; these fractions have good sensitivity for hepatocyte injury, but their specificity is limited. Fraction 10 may be a marker of activated T-lymphocytes; it was abnormal in most of our patients suggesting that it is a sensitive but non-specific test. Analysis of alkaline phosphatase by isoelectric focusing deserves further evaluation, because it may facilitate the diagnosis of certain chronic liver disorders and could be a supplement to the biopsy.

Alkaline Phosphatase↗

PubMed1996

Comparison of a rapid hepatitis B immunization schedule to the standard schedule for adults.

We report a prospective, randomized, single-blinded trial comparing immunogenicity of rapid (0, 1, and 2 months) versus standard schedule (0, 1, 6 months) hepatitis B vaccinations of healthy adults with recombinant hepatitis B vaccine (Engerix-B, 20 micrograms i.m.) (230 of 234) negative to hepatitis B were randomized and completed the study. Groups were similar in age, weight, race, and obesity rate, but the rapid schedule group had more women. Both groups reached > or = 100 mIU/mL at a similar rate, but a higher seroprotection rate at > or = 500 mIU/mL was reached by the standard schedule. No demographic variables influenced the effect of dose schedule on anti-hepatitis B titer. We conclude that rapid schedule vaccination gives a rate that is quicker than, and identical to, the rate of seroprotection of the standard schedule vaccination.

Adult↗

PubMed1995

Heavy chain dimers as well as complete antibodies are efficiently formed and secreted from Drosophila via a BiP-mediated pathway.

We have constructed a stable Drosophila cell line co-expressing heavy chain (HC) and light chain (LC) immunoglobulins of a humanized monoclonal antibody (mAb) that recognizes the F antigen of respiratory syncytial virus (Tempest, P. R., Bremmer, P., Lambert, M., Taylor, G., Furze, J. M., Carr, F. J., and Harris, W. J. (1991) Bio/Technology 9, 266-271. These cells efficiently secrete antibody with substrate binding activity indistinguishable from that produced from vertebrate cell lines. Significantly, the Drosophila homologue of the immunoglobulin binding chaperone protein (BiP), hsc72, was found to interact specifically with the immunoglobulin HC in an ATP-dependent fashion, similar to the BiP-HC interaction known to occur in vertebrate cells. This is, in fact, the first substrate ever shown to interact specifically with Drosophila hsc72. Most surprisingly, expression of heavy chains in the absence of LC led to the efficient secretion of heavy chain dimers. Moreover, this secretion occurred in association with hsc72. This dramatically contrasts with what is seen in vertebrate cells where in the absence of LC, HC remains sequestered inside the cell in stable association with BiP. Our results clearly suggest that Drosophila BiP can substitute for its mammalian counterpart and chaperone the secretion of active IgG. However, the finding that Drosophila BiP can also uniquely chaperone heavy chain dimers indicates mechanistic differences that may relate to the evolved need for retaining immature IgGs in vertebrates.

Animals↗

PubMed1995

An abundantly secreted glycoprotein from Drosophila melanogaster is related to mammalian secretory proteins produced in rheumatoid tissues and by activated macrophages.

An abundantly secreted 47-kDa glycoprotein, DS47, was purified from Drosophila melanogaster (Dm) Schneider line-2 cells, a line exhibiting macrophage-like properties. DS47 is also secreted from several Dm cell lines resembling S2 but not from lines that are morphologically distinct. A cDNA cline was isolated from an S2 cell cDNA library using oligodeoxyribonucleotide probes based on the DS47 amino acid (aa) sequence and found to encode a novel secretory glycoprotein of 452 aa. Analysis of DS47 protein production and mRNA expression during fly development indicates that both are present throughout the entire Dm life cycle, suggesting that DS47 may be important at all developmental stages. In larvae, the DS47 message is made in the fat body and by hemocytes, and secreted into the hemolymph. DS47 is related to a human cartilage glycoprotein, HC gp-39, that is secreted from cell types associated with the arthritic joint, such as synovial cells and activated macrophages. Interestingly, the HC gp-39 message is most readily detected in the human liver, an organ that is somewhat analogous to the Dm fat body. DS47 also shares homology to a mouse secretory glycoprotein, YM-1, identified in activated macrophages. These homologies extend to the chitinase gene family and include a conserved cysteine aa motif, as well as two blocks of aa within the enzymatic active site, although neither DS-47 nor HC gp-39 exhibit chitinase activity. Potential functions of this conserved protein family are discussed.

Adipokines↗

PubMed1995

Cirrhosis: a risk factor for cryptococcal peritonitis.

Cryptococcal peritonitis is usually associated with end-stage renal disease and peritoneal dialysis. Significant liver disease has not been well recognized as a risk factor for its development. We report two patients with cirrhosis who developed peritoneal infections with Cryptococcus neoformans. We also retrospectively review all cases of cryptococcal illness at the Ohio State University Medical Center from October 1990 to January 1994 and present a review of the literature regarding cryptococcal peritonitis associated with hepatic dysfunction. Cirrhotic patients with this entity present with subtle, nonspecific complaints resulting in delayed diagnoses, dissemination, and death. We suggest that clinicians maintain an increased awareness of this unusual but lethal entity in patients with liver impairment. Early and frequent abdominal paracenteses with bedside inoculations of fungal culture medium, India ink preparations, and serum cryptococcal antigen testing may hasten the diagnosis and institution of appropriate therapy.

Chronic Disease↗

PubMed1995

Fulminant hepatic failure associated with etodolac use.

Etodolac is a new pyranocarboxylic acid nonsteroidal anti-inflammatory agent with a unique chemical structure indicated for use in patients with painful musculoskeletal disorders and rheumatoid disease. Hepatotoxicity, in the form of reversible elevations in transaminases or bilirubin, occurs rarely. We present the first reported case of fulminant hepatic failure related to etodolac.

Aged↗

PubMed1994

Isolation of silencer-containing sequences causing a tissue-specific position effect on alcohol dehydrogenase expression in Drosophila melanogaster.

A transient expression assay has been used to investigate the cause of a tissue-specific position effect on Adh expression from a transgene insertion in Drosophila. A 15.4-kb genomic clone containing the 3.2-kb Adh insert along with flanking regions of genomic DNA is expressed in this assay in a tissue-specific pattern resembling the abnormal expression pattern of the position effect. The 3.2-kb Adh insert is expressed normally without the flanking sequences. A silencer element is located upstream of the Adh gene within a 2-kb fragment that acts in both orientations and at a distance of at least 6.5 kb from the larval Adh promoter to suppress ADH expression in a nontissue specific fashion. The DNA sequence of the 2-kb fragment indicates that it is a noncoding region. A 17-bp sequence is repeated within this region and may be associated with the silencer activity, since subclones from the 2-kb fragment, each containing one of the repeated regions, both retain full silencer activity. This silencer fails to suppress expression from an alpha 1-tubulin promoter-LacZ fusion construct or an hsp70 promoter-Adh fusion construct. In addition to the silencer, another element is located downstream of the Adh gene that produces a higher level of anterior than posterior midgut expression. These results suggest that the 5' silencer and the 3' element act together to create the tissue specific position effect characteristic of the GC-1 line.

Alcohol Dehydrogenase↗

PubMed1992

Tissue-specific position effects on alcohol dehydrogenase expression in Drosophila melanogaster.

Twenty transformed lines have been isolated as a result of the germ line insertion of a 3.2 kb alcohol dehydrogenase (Adh) gene fragment into an Adh negative strain of Drosophila melanogaster by P element-mediated transformation. More than half of these lines exhibited abnormal ADH expression. The level of ADH expression ranges from zero in some lines to near normal levels in others, and the pattern of ADH expression in the larval gut is also abnormal in many of these lines. Each of the abnormal tissue-specific patterns is stable and characterized by the absence or reduction of ADH expression in certain tissues. High levels of ectopic expression were not observed. In two of these lines, the pattern of ADH staining is highly restricted: it is limited to the medial midgut in line MM-50, and to the gastric caecae and the proventriculus in line GC-1. In heterozygotes between these two lines ADH is expressed in both of these tissues. To test the hypothesis that this abnormal expression is due to position effects, inserts were mobilized to new locations. The mobilized inserts exhibited new patterns of tissue-specific expression associated with new cytological insert locations, showing that the abnormal expression in lines MM-50 and GC-1 is due to tissue-specific position effects and not to mutations. The results are discussed in the context of chromatin structure as a possible cause of these position effects.

Alcohol Dehydrogenase↗

PubMed1991

Species-specific differences in tissue-specific expression of alcohol dehydrogenase are under the control of complex cis-acting loci: evidence from Drosophila hybrids.

Differences in the expression of alcohol dehydrogenase in the hindgut and testis of adult Drosophila virilis, D. texana, D. novamexicana and D. borealis flies were observed. These heritable differences do not arise due to chromosomal rearrangements, since the polytene chromosome banding patterns did not reveal any such gross chromosomal rearrangements near the Adh locus in any of the tested species. Analysis of the interspecific hybrids revealed that these differences are controlled by complex cis-acting genetic loci. Further, the cis-acting locus controlling the expression of ADH in testis was found to be separable by crossing-over.

Alcohol Dehydrogenase↗

PubMed1985

Strain differences in purified rat hepatic 3 alpha-hydroxysteroid UDP-glucuronosyltransferase.

Qualitative and quantitative differences of purified hepatic 3 alpha-hydroxysteroid UDP-glucuronosyltransferase were investigated in Wistar and Sprague-Dawley rats. Individual differences in the glucuronidation rate of androsterone and chenodeoxycholic acid were observed in hepatic microsomal fractions from Wistar but not Sprague-Dawley rats. No individual variation was observed in the glucuronidation of testosterone, p-nitrophenol or oestrone. The 3 alpha-hydroxysteroid UDP-glucuronosyltransferases from livers of Wistar and Sprague-Dawley rats were isolated and highly purified by using Chromatofocusing and affinity chromatography. The amount of 3 alpha-hydroxysteroid UDP-glucuronosyltransferase in the liver of Wistar rats exhibiting low rates for androsterone glucuronidation is about 10% or less than that found in hepatic microsomal fractions obtained from Wistar rats having high rates for androsterone glucuronidation. The apparent Km for androsterone with purified 3 alpha-hydroxysteroid UDP-glucuronosyltransferase from Wistar rats with high glucuronidation activity (6 microM) was not different from that observed for the enzyme purified from Sprague-Dawley animals, whereas that for the enzyme purified from Wistar rats with low glucuronidation activity was substantially higher (120 microM). Despite the differences in apparent Km values for androsterone, the apparent Km for UDP-glucuronic acid (0.3 mM) was not different in the different populations of rats.

Androsterone↗

PubMed1985

Androgens and estrogens affect hepatic bile acid sulfotransferase in male rats.

The effect of estrogens and androgens on hepatic glycolithocholate sulfotransferase activity was studied in male rats. Significant increases in specific activity were noted following treatment of rats for 21 days with 17 beta-estradiol, 17 alpha-ethynylestradiol, and the nonsteroidal estrogen agonists nafoxidine, tamoxifen, and diethylstilbestrol. Similar treatment of male rats with 5 alpha-dihydrotestosterone, hydrocortisone, norethindrone, and prolactin did not affect activity. To further assess the effect of androgens, male rats were castrated. Glycolithocholate sulfotransferase activity increased fivefold by 14 days after castration. Treatment of castrated rats with 5 alpha-dihydrotestosterone prevented the increase and maintained activity at the level of sham-operated animals. Castrated animals exhibited an additional increment in activity following treatment with 17 alpha-ethynylestradiol: specific activity in these animals rose to levels comparable with those measured in untreated female rats. These data suggest endogenous androgens maximally suppress hepatic glycolithocholate sulfotransferase activity in male rats. The data also indicate that activity is stimulated by estrogenic compounds of varied chemical structure and that stimulation is not solely due to suppression of androgen release by the testes as a consequence of estrogen treatment.

Androgens↗