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Biomedical subjects

M Butler

Publications and source records attributed to M Butler.

At least 55 records · Page 3Linked to original sources
PubMed1994

A rat brain Sec1 homologue related to Rop and UNC18 interacts with syntaxin.

Sec1 is a hydrophilic protein that plays an essential role in exocytosis from the yeast Saccharomyces cerevisiae. Two high copy suppressors of mutations in the Sec1 gene, SSO1 and SSO2, were recently identified that encode proteins of the syntaxin family. Syntaxin (a T-SNARE), together with SNAP-25 and synaptobrevin/VAMP (a T- and a V-SNARE, respectively), is thought to form the core of the docking-fusion complex in synaptic vesicle exocytosis. Proteins that exhibit similarity to Sec1 were identified in the nervous system of Drosophila melanogaster (Rop) and Caenorhabditis elegans (UNC18). Based on the amino acid sequence alignment of Sec1, Rop, and UNC18, we have used a PCR-based approach to isolate a rat brain cDNA encoding a Sec1 homologue. The cDNA hybridizes to a 3.5-kb brain-specific mRNA by Northern blot analysis and encodes a protein of 593 amino acids (rbSec1). Antibodies raised against a central portion of rbSec1 recognize a 67.5-kDa protein in total homogenates of rat brain but not of nonneuronal tissues. When incubated with a Triton X-100 brain extract, rbSec1-glutathione S-transferase (GST) fusion protein, but not GST protein alone, specifically interacts with syntaxin but not with SNAP-25 or synaptobrevin/VAMP. We conclude that the function of proteins of the Sec1 family in membrane fusion involves an interaction with a T-SNARE.

Amino Acid Sequence↗
PubMed1994

Listeria monocytogenes and severe newborn respiratory failure supported with extracorporeal membrane oxygenation.

OBJECTIVE: To determine the efficacy of extracorporeal membrane oxygenation (ECMO) in newborn infants with early-onset Listeria monocytogenes infection, necrotizing pneumonia, and severe respiratory failure. DESIGN: Patient series. SETTING: ECMO referral centers. PARTICIPANTS: The Extracorporeal Life Support Organization Registry database of patients supported with ECMO between 1975 and 1991. INTERVENTION: ECMO. MEASUREMENTS AND RESULTS: Nine neonates were identified who were supported with ECMO for severe respiratory failure associated with L monocytogenes infection. Microbiologic studies demonstrated L monocytogenes organisms in the blood of all infants, and pneumonia was diagnosed by roentgenogram and/or isolation of L monocytogenes organisms in tracheobronchial secretions. All infants experienced progressive respiratory deterioration by age 36 hours and were placed on venoarterial bypass by 96 hours, having met institution-based criteria predictive of 80% to 90% mortality. The duration of ECMO for patients with Listeria infection (median, 210 hours; range, 137 to 454 hours) was prolonged compared with the duration of ECMO for neonates in all other registry diagnostic categories (median, 114 hours; range, 1 to 744 hours; N = 5146, P = .035). Six of the nine infants recovered completely. CONCLUSIONS: These data suggest that ECMO is efficacious in patients with severe respiratory failure secondary to Listeria sepsis. Prolonged time on bypass should be expected when Listeria sepsis is associated with severe necrotizing pneumonia.

Bacteremia↗
PubMed1994

Profile of energy metabolism in a murine hybridoma: glucose and glutamine utilization.

The antibody-secreting murine hybridoma, CC9C10, was grown in batch culture in a medium containing 20 mM glucose and 2 mM glutamine. After 2 days of exponential growth, the glutamine content of the medium was completely depleted, whereas the glucose content was reduced to 60% of the original concentration. The glucose and glutamine metabolism was analyzed at midexponential phase by use of radioactively labelled substrates. Glycolysis accounted for the metabolism of most of the glucose utilized (> 96%) with flux through the pentose phosphate pathway (3.6%) and the TCA cycle (0.6%) accounting for the remainder. Glutamine was partially oxidised via glutaminolysis to alanine (55%), aspartate (3%), glutamate (4%), lactate (9%), and CO2 (22%). Calculation of the theoretical ATP production from these pathways indicated that glucose could provide 59% and glutamine 41% of the energy requirement of the cells.

Adenosine Triphosphate↗
PubMed1994

Adaptation of mammalian cells to non-ammoniagenic media.

Although glutamine is used as a major substrate for the growth of mammalian cells in culture, it suffers from some disadvantages. Glutamine is deaminated through storage or by cellular metabolism, leading to the formation of ammonia which can result in growth inhibition. Non-ammoniagenic alternatives to glutamine have been investigated in an attempt to develop strategies for obtaining improved cell yields for ammonia sensitive cell lines. Glutamate is a suitable substitute for glutamine in some culture systems. A period of adaptation to glutamate is required during which the activity of glutamine synthetase and the rate of transport of glutamate both increase. The cell yield increases when the ammonia accumulation is decreased following culture supplementation with glutamate rather than glutamine. However some cell lines fail to adapt to growth in glutamate and this may be due to a low efficiency transport system. The glutamine-based dipeptides, ala-gln and gly-gln can substitute for glutamine in cultures of antibody-secreting hybridomas. The accumulation of ammonia in these cultures is less and cell yields in dipeptide-based media may be improved compared to glutamine-based controls. In murine hybridomas, a higher concentration of gly-gln is required to obtain comparable cell growth to ala-gln or gln-based cultures. This is attributed to a requirement for dipeptide hydrolysis catalyzed by an enzyme with higher affinity for ala-gln than gly-gln.

Adaptation, Physiological↗
PubMed1994

Modifying quiet room design enhances calming of children and adolescents.

OBJECTIVE: To determine whether altering design of a quiet room (QR) produced more rapid calming of agitated or aggressive hospitalized children. METHOD: One of five similar QRs was modified by painting the white walls tea rose, carpeting the vinyl floor, and painting a picturesque mural on one wall. The effects of these modifications were assessed in 19 patients (mean age = 9.6 years), using a within-subjects, repeated-measures design. Overt Aggression ratings were made at the time of placement, and at 5-minute intervals thereafter, until the child was dismissed. Children were blind to the fact that a study was being conducted; raters and staff were not. RESULTS: Total aggression ratings were 45% lower in the modified QR than in the standard QR (p < .03), and initial aggression scores fell by 50% during 5 minutes of placement in the modified QR, but only after 20 minutes of placement in the standard QR (p < .0001). Motor excitement and verbal aggression were the two component factors most strongly influenced by QR design. CONCLUSION: This preliminary report suggests that it may be possible to modify QRs to facilitate calming of aggressive, agitated children and provides preliminary support for redesign of QRs.

Acting Out↗
PubMed1994

Chromosomal localization of the human renal sodium phosphate transporter to chromosome 5: implications for X-linked hypophosphatemia.

Hypophosphatemic vitamin D-resistant rickets, an X-linked dominant disorder, is the most common form of vitamin D-resistant rickets in humans (McKusick number 307800). Biochemically, these patients exhibit hypophosphatemia due to a defect in the renal tubular reabsorption of phosphate. The human cDNA encoding for the renal phosphate transporter has been recently cloned using the expression system in the Xenopus laevis oocytes. Because hypophosphatemic vitamin D-resistant rickets has an X-linked mode of transmission, we hypothesized that the gene encoding the renal phosphate transporter might map to the X chromosome. In this report, we determined the chromosomal localization of the human renal phosphate transporter using three independent methods. First, DNA from somatic cell hybrid panels was examined by Southern blotting for the phosphate transporter. Second, the polymerase chain reaction was used to amplify DNA from somatic cell hybrids. Third, fluorescent in situ hybridization was used to sublocalize the renal phosphate transporter. All three methods localized the renal phosphate transporter to chromosome 5q13. Our results indicate that either derangement of a gene other than the phosphate transporter gene that is encoded on chromosome 5 is responsible for X-linked hypophosphatemic rickets or, alternatively, a gene encoded on the X chromosome has an epistatic effect on the expression of the renal phosphate transporter on chromosome 5.

Base Sequence↗
PubMed1993

Limited tryptic digestion near the amino terminus of bovine liver rhodanese produces active electrophoretic variants with altered refolding.

When the enzyme rhodanese was partially digested by immobilized trypsin, it retained greater than 50% of its original activity although less than 10% of the undigested enzyme remained. The predominant daughter species were two 31-kDa polypeptides whose amino termini corresponded to either residue 44 or 45 of the enzyme's sequence. Following digestion, charged species were isolated by ion exchange chromatography. Denaturing electrophoresis revealed that a 4-kDa peptide remained associated with the 31-kDa fragment. This 4-kDa peptide appears to correspond to the amino-terminal 45 residues of rhodanese. Further proteolysis gave a 2.5-kDa peptide that dissociated under non-denaturing conditions without apparent change in migration of the 31-kDa fragment on SDS gels. Refolding of undigested, urea-denatured rhodanese restored much of its activity. Similar treatment of rhodanese following limited tryptic digestion resulted in no regain of activity. Refolding of a mixture of intact and digested rhodanese resulted in regain of activity appropriate for the amount of intact rhodanese in the sample, indicating that clipped rhodanese does not inhibit refolding of intact rhodanese. It is concluded that portions of the amino terminus of rhodanese are important in the enzyme's folding, but are not essential for the enzyme's sulfurtransferase activity.

Animals↗
PubMed1993

Association of GAD-65, but not of GAD-67, with the Golgi complex of transfected Chinese hamster ovary cells mediated by the N-terminal region.

Glutamic acid decarboxylase (GAD) is the enzyme responsible for synthesis of the neurotransmitter gamma-aminobutyric acid in neurons and pancreatic beta cells. It is represented by two isoforms, GAD-65 and GAD-67, which are the products of two different genes and differ substantially only at their N-terminal regions. GAD-65 is a dominant autoantigen in stiff-man syndrome and insulin-dependent diabetes mellitus. In neurons and beta cells, GAD is concentrated around synaptic vesicles and synaptic-like microvesicles, respectively, as well as in the area of the Golgi complex. The mechanisms responsible for specific targeting of GAD to these organelles are not yet understood. The elucidation of the mechanism of subcellular targeting of GAD may be relevant to understanding its role as an autoantigen. In this study, the cloned genes for GAD-65 and GAD-67 were expressed separately in Chinese hamster ovary (CHO) cells and COS cells. While GAD-67 had a diffuse cytoplasmic localization, GAD-65 had a punctate distribution, with most of the immunoreactivity being concentrated in the area of the Golgi complex. A chimeric protein in which the 88 N-terminal amino acids of GAD-67 were replaced by the 83 N-terminal amino acids of GAD-65 was targeted to the Golgi complex, indicating that the N-terminal region of GAD-65 contains a targeting signal sufficient for directing the remaining portion of the molecule, highly similar in GAD-65 and GAD-67, to the Golgi complex-associated structures.

Amino Acid Sequence↗
PubMed1993

Interactive intermediates are formed during the urea unfolding of rhodanese.

Structural transitions have been studied on the pathway for urea denaturation of rhodanese. Unlike guanidinium hydrochloride, urea gives no visible precipitation. Increasing urea concentrations cause a transition in which the enzyme activity is completely lost by 4.5 M urea, and there is a shift of the intrinsic fluorescence maximum from 335 nm for the native enzyme to 350 nm. There is a maximum exposure of organized hydrophobic surfaces at 4.5 M urea as reported by the fluorescence of 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid. Above 4.5 M urea, this probe reports the progressive loss of organized hydrophobic surfaces. The polarization of the intrinsic fluorescence falls with increasing urea concentrations in a complex transition showing that rhodanese flexibility increases in at least two phases. Rhodanese becomes increasingly susceptible to digestion by subtilisin between 3.5 and 4.5 M urea, giving rise to large fragments. At urea concentrations > 5 M, rhodanese is completely digested. There is a small increase in the rate of sulfhydryl accessibility between 3.5 and 4.5 M urea, but there is a large increase in the sulfhydryl accessibility above 4.5 M urea. Dimethyl suberimidate cross-linking shows the presence of associated species in 3-5 M urea, but there are few cross-linkable species at lower or higher urea concentrations. These results are consistent with a model in which urea unfolding of rhodanese is associated with the initial production of a species having organized regions of structure with exposed hydrophobic surfaces separated by flexible elements.

Animals↗
PubMed1993

Glucose and glutamine metabolism of a murine B-lymphocyte hybridoma grown in batch culture.

The energy metabolism of a mammalian cell line grown in vitro was analyzed by substrate consumption rates and metabolic flux measurements. The data allowed the determination of the relative importance of the pathways of glucose and glutamine metabolism to the energy requirements of the cell. Changes in the substrate concentrations during culture contributed to the changing catalytic activities of key enzymes, which were determined. 1. A murine B-lymphocyte hybridoma (PQXB1/2) was grown in batch culture to a maximum cell density of 1-2 x 10(6) cells/mL in 3-4 d. The intracellular protein content showed a maximum value during the exponential growth phase of 0.55 mg/10(6) cells. Glutamine was completely depleted, but glucose only partially depleted to 50% of its original concentration when the cells reached a stationary phase following exponential growth. 2. The specific rates of glutamine and glucose utilization varied during culture and showed maximal values at the midexponential phase of 2.4 nmol/min/10(6) cells and 4.3 nmol/min/10(6) cells, respectively. 3. A high proportion of glucose (96%) was metabolized by glycolysis, but only limited amounts by the pentose phosphate pathway (3.3%) and TCA cycle (0.21%). 4. The maximum catalytic activity of hexokinase approximates to the measured flux of glycolysis and is suggested as a rate-limiting step. In the stationary phase, the hexokinase activity reduced to 11% of its original value and may explain the reduced glucose utilization at this stage. 5. The maximal activities of two TCA cycle enzymes were well above the measured metabolic flux and are unlikely to pose regulatory barriers. However, the activity of pyruvate dehydrogenase was undetectable by spectrophotometric assay and explains the low level of flux of glycolytic metabolites into the TCA cycle. 6. A significant proportion of the glutamine (36%) utilized by the cells was completely oxidized to CO2. 7. The measured rate of glutamine transport into the cells approximated to the metabolic flux and is suggested as a rate-limiting step. 8. Glutamine metabolism is likely to occur via glutaminase and amino transaminase, which have significantly higher activities than glutamate dehydrogenase. 9. The calculated potential ATP production suggests that, overall, glutamine is the major contributor of cellular energy. However, at the midexponential phase, the energy contribution from the catabolism of the two substrates was finely balanced--glutamine (55%) and glucose (45%).

Adenosine Triphosphate↗
PubMed1993

Haemospermia: how to proceed?

Haemospermia is an alarming symptom but does it signify serious disease and how should it be investigated? A retrospective review of 44 men showed no evidence of malignancy and infection as the commonest cause. Standard investigation with midstream specimen of urine, intravenous pyelogram and cystoscopy is unhelpful. Microscopy and culture of a first stream specimen of urine or expressed prostatic secretions is the investigation of choice. Cystoscopy should be reserved for patients with recurrent haemospermia.

Adult↗
PubMed1993

Altered diaphragm function modifies neonatal lung growth: biologic morphometric assessment.

Thoracic volume relationships supported by the diaphragm are important mechanical components of neonatal lung growth. Alterations modify regional lung growth. We used stereologic morphometry to study effects of altered diaphragm function on alveolar growth in neonatal pigs. Nine piglets (1 mo) were divided into three groups: unilateral phrenectomy, noncompliant patch replacement of diaphragm, and sham. Seven days later lungs were fixed in situ with 2.5% glutaraldehyde by airway installation at 20 cm H2O. Five 1-mm cubes were cut from lung corresponding to apex/RUQ(A), base/RLQ(B), apex/LUQ(C), and base/LLQ(D) and prepared for light microscopy (n = 25/quad/animal). Stereologic morphometry involved point counting for air volume density and point intersect to determine the surface area density of the alveolar spaces. Results were analyzed for variance and by Tukey range testing. Variance of air volume % between groups-quadrants B, C, D had decreased air volume % in phrenectomy group compared with patch and sham group (P < .05). Variance of air volume % within groups, for phrenectomy group A (81.5%), is different from all others (B = 70.6%, C = 75.5%, D = 66.7%); C is different from D (P < .05). For patch and sham group, D (P = 74.8%, S = 80.7%) is different from A (P = 84.1%, S = 86.6%) and C (P = 86.2%, S = 84.4%). Variance of surface area density between groups--quadrant D had increased surface area density % in phrenectomy group compared to sham group (P < .05). Variance of surface area density % within groups, for phrenectomy group, A (37.9 mm-1) is different from D (55.7 mm-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed1993

Uptake of glutamate, not glutamine synthetase, regulates adaptation of mammalian cells to glutamine-free medium.

Two cell lines (McCoy and MDCK) were studied in an attempt to understand the metabolic changes associated with adaptation to glutamine-free medium (GMEM + gmate). McCoy cells assumed normal growth rates after 2-3 passages in this medium whereas MDCK cells showed no growth in GMEM + gmate. The glutamine synthetase (GS) activity of both cell lines was elevated (up to x 9) as glutamine was depleted from normal media (GMEM + gmine). The high activity of GS was maintained during McCoy cell growth in GMEM + gmate. However, there was no apparent significant difference between the two cell lines in the pattern of changes of GS activity in response to glutamine. The cellular uptake rates of glutamine and glutamate from the medium differed significantly between the two cell lines. During the adaptation of McCoy cells to GMEM + gmate, the rate of glutamate uptake doubled to a value of 0.54 nmol/min per mg cell protein whereas the maximum value for MDCK cells was considerably lower (0.04 nmol/min per mg cell protein). We propose that the difference in intrinsic ability for glutamate transport accounts for the difference in growth response between the two cell lines in the glutamine-free medium.

Amino Acid Transport System X-AG↗
PubMed1992

Reducing sugars can induce the oxidative inactivation of rhodanese.

The enzyme rhodanese (thiosulfate sulfurtransferase, EC 2.8.1.1) is inactivated on incubation with reducing sugars such as glucose, mannose, or fructose, but is stable with non-reducing sugars or related polyhydroxy compounds. The enzyme is inactivated with (ES) or without (E) the transferable sulfur atom, although E is considerably more sensitive, and inactivation is accentuated by cyanide. Inactivation of E is accompanied by increased proteolytic susceptibility, a decreased sulfhydryl titer, a red-shift and quenching of the protein fluorescence, and the appearance of hydrophobic surfaces. Superoxide dismutase and/or catalase protect rhodanese. Inactive enzyme can be partially reactivated during assay and almost completely reactivated by incubation with thiosulfate, lauryl maltoside, and 2-mercaptoethanol. These results are similar to those observed when rhodanese is inactivated by hydrogen peroxide. These observations, as well as the cyanide-dependent, oxidative inactivation by phenylglyoxal, are explained by invoking the formation of reactive oxygen species such as superoxide or hydrogen peroxide from autooxidation of alpha-hydroxy carbonyl compounds, which can be facilitated by cyanide.

Fructose↗
PubMed1992

Neuropsychological battery choice and theoretical orientation: a multivariate analysis.

In order to investigate the tests selected by neuropsychologists to make up clinical batteries, a large survey of neuropsychological test usage was cluster analyzed. This provided groupings of tests that are endorsed in common. Theoretical orientation within neuropsychology also was included in the analysis to determine which tests and clusters of tests are more and less associated with the reported orientation of the neuropsychologist. Fifteen clusters of tests were found. Strong and appropriate associations with the eclectic, hypothesis testing, process approach, Halstead-Reitan, Luria, and Benton orientations were seen.

Attitude of Health Personnel↗
PubMed1992

Characterization of glutamine metabolism in two related murine hybridomas.

Aspects of glutamine metabolism were examined in two related hybridomas, a high-producing line (PQX B1/2) and a low-producing line (PQX B2/2). The growth and metabolic characteristics of both cell lines were identical or very similar. During batch growth glutamine was completely exhausted from the medium and an examination of the fate of [14C]glutamine highlighted the importance of this amino acid as an energy source. The relative enzyme activities and the amount of ammonia produced during growth indicated that glutamine is oxidized preferentially via the transamination pathway. The overall rate of glutamine utilization from the growth medium was similar to the rate of [14C]glutamine uptake which suggests that transport may regulate glutamine metabolism.

Animals↗
PubMed1992

Endomysial antibody: is it the best screening test for coeliac disease?

The sensitivities and specificities of the IgA and IgG antigliadin antibody and the IgA antireticulin antibody have been compared with the recently described endomysial antibody directed against the basement membrane of smooth muscle in monkey oesophagus. One hundred and seventeen patients with adult coeliac disease (21 untreated), 84 patients with inflammatory bowel disease, systemic lupus erythematosus and rheumatoid arthritis (comprising the disease control group), 47 normal controls and a miscellaneous group of 29 patients, who were selected because of a positive reticulin staining pattern, were investigated. These results were correlated with the degree of abnormality of the intestinal mucosa in patients with adult coeliac disease. Endomysial antibodies were found in all patients with untreated coeliac disease and subtotal villous atrophy and in 47% of patients on a non-strict gluten free diet. One patient on a strict gluten free diet was positive and had partial villous atrophy while all patients in disease control groups were negative. Results were variable with the antireticulin and antigliadin antibodies. Sensitivity and correlation with subtotal villous atrophy in the untreated patients was 100%. It is concluded that the endomysial antibody is superior to other current antibody tests and should be used in preference for the diagnosis of coeliac disease.

Adolescent↗