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Biomedical subjects

S Howell

Publications and source records attributed to S Howell.

At least 19 recordsLinked to original sources
PubMed1992

A survey of membrane peptidases in two human colonic cell lines, Caco-2 and HT-29.

The expression of cell-surface peptidases was examined in two human colon carcinoma cell lines, Caco-2 and HT-29. Enzymic assays revealed the presence of eight cell-surface peptidases on a Caco-2 cell line (passage number 82-88), namely aminopeptidase N, dipeptidyl peptidase IV, peptidyl dipeptidase A (angiotension-converting enzyme), aminopeptidase P, aminopeptidase W, endopeptidase-24.11, gamma-glutamyl transpeptidase and membrane dipeptidase. The presence of dipeptidyl peptidase IV and endopeptidase-24.11 was also confirmed immunochemically. After 15 days culture, the activities of aminopeptidase P, peptidyl dipeptidase A and alkaline phosphatase activities on Caco-2 cells reached a plateau, and that of membrane dipeptidase began to decline. In contrast, aminopeptidase N, dipeptidyl peptidase IV and endopeptidase-24.11 activities were still rising after 26 days in culture. Caco-2 cells of passage number 181-183 were found to lack endopeptidase-24.11, but maintained dipeptidyl peptidase IV expression. Two populations of HT-29 cells were surveyed. Both the standard, undifferentiated population and a differentiated population expressed only three peptidases: dipeptidyl peptidase IV, aminopeptidase W and carboxypeptidase M. In the differentiated HT-29 cells the activity of dipeptidyl peptidase IV after 14-21 days was beginning to plateau whereas aminopeptidase W activity was still rising and that of carboxypeptidase M had begun to decline. These differences in activity profiles observed among this group of cell-surface peptidases indicate that these cell lines, especially Caco-2, are useful models to study the regulation of their expression.

Aminopeptidases↗
PubMed1992

The complete sequences of trout (Salmo gairdneri) thymosin beta 11 and its homologue thymosin beta 12.

Two forms of beta-thymosins, designated thymosin beta 11 and thymosin beta 12, were isolated from trout (Salmo gairdneri) spleen. This suggests that the presence of two beta-thymosins, previously thought to be a property of mammalian tissues only, is a more general phenomenon in vertebrate species. Both trout beta-thymosins were found to be N-terminally blocked by a group identified as acetyl by m.s. Automated protein sequencing of tryptic, thermolytic and Staphylococcus aureus in 41-residue V8 proteinase fragments revealed that one of the two beta-thymosins corresponds to the previously reported 41-residue-long sequence of thymosin beta 11 with two substitutions at positions 5 and 7, i.e. Asn instead of Asp, and Glu instead of Gln, whereas the other beta-thymosin, designated thymosin beta 12, was found to be a 42-residue polypeptide closely similar in sequence to thymosin beta 11, with five substitutions (i.e. at positions 5, 7, 10, 11 and 41, with Asp, Ala, Ser, Asn and Thr instead of Asn, Glu, Ala, Ser and Ser respectively) and one addition at position 42 (Ala). Comparison of the known six sequences of beta-thymosins together with the sequences reported here showed that the sequence similarity of the two beta-thymosins in trout (86%) is greater than that of the two beta-thymosins in mammalian species (74%) and that residues at 28 positions are identical in all beta-thymosins, the longer conserved segments located at positions 16-26 and 31-38.

Amino Acid Sequence↗
PubMed1991

A highly sensitive E.L.I.S.A. for endopeptidase-24.11, the common acute-lymphoblastic-leukaemia antigen (CALLA, CD-10), applicable to material of porcine and human origin.

Endopeptidase-24.11 is a widely distributed cell-surface enzyme with a key role in the metabolism of neuropeptides. It is now known to be identical with CD-10, the common acute-lymphoblastic-leukaemia antigen (CALLA). An e.l.i.s.a. is described which utilizes two antibodies, one monoclonal, the other polyclonal, generated to pig endopeptidase-24.11. These antibodies cross-reacted with human endopeptidase-24.11, thus making the assay applicable to both species. By using optimum conditions for the e.l.i.s.a., as little as 25 pg of pure pig endopeptidase-24.11 could be quantified at 95% confidence limits. E.l.i.s.a. of tissue homogenates from a variety of pig tissues and of human kidney correlated well with enzymic assays. However, the use of detergents to solubilize the antigen greatly decreased the sensitivity of the e.l.i.s.a. The e.l.i.s.a. is 1000-fold more sensitive than the immunoradiometric assay and has advantages in specificity over enzymic assays. Daudi cells, some leukaemic cells shown to be CALLA-positive, and Caco-2 cells, could also be assayed, but N2 cavitation was necessary to fragment the cells, and only part of the total endopeptidase-24.11 activity in Daudi cells was recognized by the e.l.i.s.a.

Animals↗
PubMed1991

Clinical evaluation of intraperitoneal Pseudomonas exotoxin immunoconjugate OVB3-PE in patients with ovarian cancer.

OVB3-PE is an immunotoxin composed of a murine monoclonal antibody reactive with human ovarian cancer and conjugated to Pseudomonas exotoxin (PE). Twenty-three patients with refractory ovarian cancer were treated intraperitoneally (IP) with escalating doses of OVB3-PE to study toxicity, pharmacokinetics, antiimmunotoxin antibody formation, and antitumor response. Dose-limiting CNS toxicity occurred after repeated doses at 5 and 10 micrograms/kg. Other non-dose-limiting toxicities included transient elevation of liver enzymes, fever, and gastrointestinal toxicity. Pharmacokinetics of IP and serum OVB3-PE were determined in 16 patients. Peak peritoneal fluid levels exceeded the in vitro median effective dose at all doses tested. At doses of 1 to 2 micrograms/kg, the immunotoxin concentration in the peritoneal fluid remained constant for up to 8 hours and dropped to negligible levels after 12 hours. At the 5 and 10 micrograms/kg doses, levels remained high for up to 24 hours (greater than 100 ng/mL) and then gradually decreased and became undetectable (less than 4 ng/mL) after 72 hours. Serum levels of OVB3-PE were also analyzed in 16 patients. At doses of 1 micrograms/kg and 2 micrograms/kg, serum levels were not detectable (less than 5 ng/mL). However, after doses of 5 or 10 micrograms/kg, peak serum level occurred at 24 hours after each dose and dropped to negligible levels by 72 hours. Sera from 12 patients were analyzed for anti-PE antibodies and antibodies to mouse immunoglobulin (HAMA). All patients developed antibodies against PE within 14 days of therapy. Domain II of PE appeared to be the most immunogenic portion of the PE molecule. HAMA was detected on day 14 of therapy in nine patients, on day 21 in two, and on day 28 in one patient. No clinical antitumor responses were observed. We conclude that IP OVB3-PE at dose levels of 5 micrograms/kg (x 3) and 10 micrograms/kg (x 2) is accompanied by dose-limiting toxic encephalopathy. Neurologic toxicity is likely to be due to crossreactivity of OVB3 to normal human brain tissue, which was not appreciated during preclinical screening.

ADP Ribose Transferases↗
PubMed1990

Limited proteolysis and 'in vitro' mutagenesis of bovine brain inositol monophosphatase identifies an N-terminal region important for activity.

Bovine brain inositol monophosphatase is rapidly cleaved by endoprotease lys-C at a single site in the absence of SDS. Further sites are revealed only after prolonged incubation with high concentrations of protease. The initial cleavage occurs near one end of the enzyme, generating an N-terminally-derived 36-residue peptide, which is blocked, and a large 28 kDa fragment bearing a free N-terminus. The start sequence of this fragment was found to be Xaa-Ser-Pro-Ala-Asp-Leu-Val, consistent with the cDNA sequence, and Lys-36-Ser-37 was identified as the cleavage site. The activity of the cleaved enzyme was markedly decreased to 3% of that of the native enzyme, although its dimeric structure was preserved. The 36-residue peptide was not covalently associated with the large fragment after proteolytic cleavage, although the possibility of non-covalent association could not be excluded. Finally, the epitope for the inhibitory monoclonal antibody G-2A4 [Gee, Howell, Ryan & Ragan (1989) Biochem J. 264. 793-798] was found to lie proximal to the endoprotease lys-C cleavage site. In vitro mutagenesis further mapped the epitope for monoclonal antibody G-2A4 to residues around Cys-8 of the enzyme. These results suggest that the N-terminal region of the enzyme is important for activity.

Amino Acid Sequence↗
PubMed1990

Effect of sulfhydryl-deficient diets on hepatic metallothionein, glutathione, and adenosine 3'-phosphate 5'-phosphosulfate (PAPS) levels in rats.

Low dietary concentrations of methionine and cysteine are known to decrease hepatic glutathione content. However, it is not known if restricting the dietary content of these sulfur containing amino acids also affects hepatic levels of adenosine 3'-phosphate 5'-phosphosulfate (PAPS), the cofactor for sulfation, or metallothionein, a protein rich in sulfhydryl groups. Rats were fed diets lacking cysteine and containing various concentrations of methionine (0.15, 0.3, or 0.6%) for 8 days. Control diet contained 0.3% each of methionine and cysteine. Hepatic glutathione levels were decreased approximately 75% in rats fed diets containing 0.15 or 0.3% methionine. In contrast, PAPS and hepatic metallothionein concentrations were not decreased by the low sulfhydryl diets. Additionally, rats on the various diets were challenged by the administration of ZnCl2 (3 mmol/kg. sc). In both control rats and rats maintained on sulfhydryl-deficient diets, ZnCl2 increased hepatic metallothionein to the same level. However, significantly lower levels of PAPS were observed after ZnCl2 in rats receiving sulfhydryl-deficient diets than in controls. In summary, restriction of dietary sulfhydryl markedly decreases the hepatic content of glutathione and has a minor effect on PAPS concentration, but does not decrease the basal hepatic concentration of metallothionein or its induction by ZnCl2.

Adenine Nucleotides↗
PubMed1990

An electron microscopic evaluation of the enamel surface subsequent to various debonding procedures.

In this study, enamel surface roughness was investigated following the use of various combinations of bonding agents, burrs and polishing procedures. Orthodontic brackets were bonded to 135 premolar teeth extracted from adolescents. Subsequent to debonding and finishing, the enamel surface was gold coated and examined in the scanning electron microscope. Each 200x photomicrograph was graded according to surface roughness and assigned to one of four selected standard grades. The use of different composites and different burs showed no significant effect on the finish of the enamel surface using Chi-square tests. Only two finishing procedures had a significant effect on surface roughness. The use of a Soflex disc followed by pumice slurry resulted in the roughest enamel surface and the use of pumice alone produced the smoothest enamel surface.

Adolescent↗
PubMed1989

A monoclonal antibody to bovine brain inositol monophosphatase. Immunoaffinity purification of the brain and kidney enzymes and evidence for their structural identity.

A monoclonal IgG2b(K) antibody, G-2A4, has been generated against bovine brain myo-inositol monophosphatase (EC 3.1.3.25). The identity of the antigen recognized by the antibody was established by using e.l.i.s.a. and Western blotting procedures, and by immunoprecipitation of enzyme activity from crude brain supernatant. In addition, the hydrolysis of Ins1P by crude brain extract was inhibited by up to 83% by the pure antibody. Under identical conditions, the hydrolysis of Ins(1,4)P2 was unaffected. An immunoadsorbent column containing monoclonal antibody G-2A4 covalently attached to CNBr-activated Sepharose 4B has been used for rapid purification of the brain enzyme. Elution conditions have been optimized to allow isolation of the enzyme in high yield (54%) with full retention of column-binding capacity. The enzyme was electrophoretically homogeneous, Mr 30,000 and of higher specific activity than that purified conventionally. Chromatography of the pure enzyme on high resolution ion-exchange columns revealed some charge heterogeneity, possibly indicative of some type of post-translational modification. The immunoadsorbent column has also been used to purify the bovine kidney cortex enzyme to homogeneity. Partial proteolytic fragmentation patterns of the brain and kidney enzymes using endoprotease glu-C were identical, suggesting that they are almost certainly products of the same gene.

Animals↗
PubMed1989

Evidence for at least four different inositol bisphosphatases in bovine brain.

Bovine brain supernatant contains at least four enzymes capable of hydrolysing inositol bisphosphates. These activities may be distinguished on the basis of their metal, salt and pH dependence, sensitivity to Li+ ions and thiol-modification reagents, and on their molecular sizes. In addition to Li+-sensitive Ins(1,4)P2/Ins(1,3,4)P3 1-phosphatase [Gee et al. (1988) Biochem. J. 253, 777-782] which has an absolute requirement for Mg2+, two (Li+-insensitive and Mg2+-independent) phosphatases, capable of hydrolysing Ins(3,4)P2 and Ins(1,3)P2, respectively, have been identified. Both enzymes were inhibited by only moderate concentrations of salt, although for the former there was no obvious correlation between inhibitory potency and either the nature of the anion/cation or the ionic strength of the buffer. Ins(3,4)P2 phosphatase had a pH optimum of 7.6 and this activity could be resolved on gel-filtration columns into a two overlapping peaks of molecular mass 170 kDa and 450 kDa. Mg2+-independent Ins(1,3)P2 phosphatase had a pH optimum of 7.1 and displayed a single broad activity peak on gel-filtration columns. However, if assays were performed in the presence of Mg2+, a second Ins(1,3)P2 phosphatase was revealed (35 kDa), which had a pH optimum of 8.8 Ins(1,4)P2/Ins(1,3,4)P3 1-phosphatase, Ins(3,4)P2 phosphatase, Mg2+-independent Ins(1,3)P2 phosphatase and inositol monophosphatase were all inhibited by 5,5'-dithiobis(2-nitrobenzoic acid) with IC50 values of 34 microM, 65 microM and 560 microM and 1100 microM, respectively. The metabolism of Ins(1,3,4)P3 by brain supernatant was also examined. Product specificity was shown to be entirely dependent on the buffer conditions employed. In Mg2+-containing buffers, Ins(1,3,4)P3 was hydrolysed predominantly to Ins(3,4)P2, consistent with hydrolysis by Ins(1,4)P2/Ins(1,3,4)P3 1-phosphatase. In the presence of EDTA, Ins(1,3,4)P3 was degraded exclusively by a 4-phosphatase enzyme generating Ins(1,3)P2. Under these conditions, high concentrations of Ins(3,4)P2 blocked the hydrolysis of Ins(1,3,4)P3.

Animals↗
PubMed1989

Oral hygiene program for orthodontic patients.

The gingival and periodontal health of 62 adolescent orthodontic patients was assessed before and again after orthodontic treatment in a controlled clinical study. Before the commencement of orthodontic treatment, the experimental group received an oral hygiene program consisting of 4 weekly sessions of oral health education, instruction on plaque-control techniques and reviews in plaque removal performance. The control group did not receive this program. It was found that there was a general trend in the study population for improved gingival health as measured by the bleeding index, gingival index, plaque index and gingival crevicular fluid volume. Furthermore, the oral hygiene program induced in the experimental group significantly lower scores in the measured clinical indices compared to the control group. However, no significant difference was noted in the measured probing depths in all subjects before or after the study period.

Dental Plaque↗
PubMed1989

Relationship of diaphragmatic contractility to diaphragmatic blood flow in newborn lambs.

We determined the relationship of diaphragmatic contraction rate to diaphragmatic blood flow (Qdi), metabolism, and contractility in nine open-chested mechanically ventilated newborn lambs. The diaphragm was paced for 15 min at slow (20/min) and fast (100/min) contraction rates each followed by a 30-min rest period. There was a mild reduction in transdiaphragmatic pressure (Pdi) during the slow contraction period accompanied by a shift to the right of the curve relating stimulation frequency (10-100 Hz) to Pdi. Pdi returned to control at the start of the fast contraction period, but then fell by 30% within 2 min with continued fast contraction rates. The frequency-Pdi curve was significantly shifted to the right. Qdi, O2 transport, and O2 consumption increased during slow contraction and to an even greater extent during fast contraction. Fractional O2 extraction reached an apparent maximum during slow contraction. Lactate efflux from the right phrenic vein during slow contraction remained unchanged from control. During fast contraction lactate efflux rose proportionately more than did O2 consumption. We conclude that the energy demands at fast rates of diaphragmatic contraction in newborn lambs cannot be met by aerobic metabolism alone despite increasing O2 transport to the diaphragm.

Animals↗
PubMed1989

Effects of high-frequency chest wall oscillation on respiratory control in humans.

We studied the spontaneous breathing patterns of 10 normal adult volunteers during high-frequency chest wall oscillation (HFCWO), accomplished by inflating and deflating a vest worn around each subject's thorax at 2.5 Hz. Tidal volumes generated by HFCWO averaged 100 ml. Mean vest pressure was maintained at approximately 35 cm H2O throughout each experiment, even when HFCWO was not applied. During HFCWO, subjects were instructed occasionally to exhale deeply to obtain end-tidal samples representative of PACO2. HFCWO increased the breath-to-breath variability of spontaneous respiration in all subjects, prolonging expiratory pauses and producing short apneas in some cases. PACO2 decreased significantly (p less than 0.05). The effects on minute ventilation, tidal volume, and inspiratory and expiratory durations remained variable across subjects, even when differences in PACO2 between control and HFCWO states were reduced through inhalation of a low CO2 mixture. None of the changes were statistically significant, although average expiratory duration increased by 29%. Ventilatory responses to CO2 with and without HFCWO were also measured. Normocapnic (PACO2 = 40 mm Hg) ventilatory drive increased significantly (p less than 0.05) in six subjects (Type 1 response) and decreased substantially in the others (Type 2 response); with hypercapnia, the changes in drive were attenuated in both groups. Consequently, CO2 sensitivity decreased in Type 1 subjects and increased in Type 2 subjects. A simple analysis based on this result shows that with HFCWO, Type 2 subjects breathing air will tend to have a lower spontaneous minute ventilation and become hypercapnic. Type 1 subjects will become hypocapnic, but minute ventilation may be higher or lower than control.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1989

Fulminant liver failure and pancreatitis associated with the use of sulfamethoxazole-trimethoprim.

We report the case of a 26-yr-old patient with fulminant liver failure and acute hemorrhagic pancreatitis secondary to the use of trimethoprim-sulfamethoxazole (Bactrim DS). Our patient presented with skin rash and decreased C3 and C4 levels, which we believed was due to a hypersensitivity reaction secondary to the sulfonamide component (sulfamethoxazole). To our knowledge, this is the first case reported in which sulfamethoxazole-trimethoprim has been implicated as a cause of fulminant liver failure and acute hemorrhagic pancreatitis simultaneously, and emphasizes the need of discontinuing this medication as soon as there is evidence of liver and pancreatic dysfunction.

Adult↗
PubMed1989

Fulminant idiopathic pseudomembranous colitis.

Pseudomembranous colitis is characterized by inflammatory plaques and pseudomembranes on the colonic mucosa. The disorder most commonly occurs after the use of antibiotics, which allow overgrowth of Clostridium difficile, a spore-forming, gram-positive rod that produces a toxin. Overgrowth of Staphylococcus aureus can also produce pseudomembranous colitis. In rare cases, pseudomembranous colitis is not associated with antibiotic use. When C. difficile is present, vancomycin or metronidazole usually produces a prompt response. In idiopathic cases, surgery may be required.

Aged↗