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Biomedical subjects

L J Davis

Publications and source records attributed to L J Davis.

At least 73 records · Page 4Linked to original sources
PubMed1990

Characterization of an active single polypeptide form of the human immunodeficiency virus type 1 protease.

The pepsin-like aspartyl proteases consist of a single polypeptide chain with topologically similar amino- and carboxyl-terminal domains, each of which contributes 1 aspartic acid residue to the active site. This structure has been proposed to have evolved by gene duplication and fusion from a dimeric enzyme composed of two identical polypeptide chains, such as the aspartyl protease (PRT) of human immunodeficiency virus type 1 (HIV-1). To determine if a single polypeptide form of the HIV-1 protease would be enzymatically active, two protease coding regions were linked to form a dimeric gene (pFGGP). Expression of this gene in Escherichia coli yielded a protein with the expected molecular mass of 22 kDa. The in vitro kinetic parameters of PRT and FGGP (where FGGP is the single polypeptide form of the HIV-1 protease with 2 glycine residues connecting the two subunits) for three peptide substrates are similar. Construction and analysis of a CheY-GAG-FGGP fusion protein demonstrated that FGGP is capable of precursor processing in vivo. Mutation of one or both of the active site aspartates to either asparagine or glutamate rendered the enzyme inactive, demonstrating that both active site aspartate residues are required for enzymatic activity.

Amino Acid Sequence↗
PubMed1990

Derangement of non-specific immunity in Down syndrome subjects: low leukocyte chemiluminescence activity after phagocytic activation.

Metabolic activation of peripheral blood leukocytes (chemiluminescence) from 27 children with Down syndrome (DS) and 23 age and sex-matched control children after phagocytic stimulation by opsonized zymosan particles was investigated through a chemiluminescence assay. Using autologous plasma or serum as opsonizing media, phagocytic activity of circulating leukocytes was significantly decreased in DS subjects. A further decrease of phagocytic activity was found in neutrophils from DS children, when normal heterologous plasma or sera were used. On the other hand, sera or plasma from DS subjects significantly increased phagocytic activation of leukocytes from normal donors. In DS subjects opsonizing agents such as serum immunoglobulins and complement fractions were in the normal ranges of concentration. Thus, the impaired chemiluminescence of neutrophils was mainly due to a metabolic impairment at the cellular level. A decreased production of radicals derived from the oxygen metabolism in neutrophils may be an important step of immune derangement leading to the increased incidence of infectious diseases frequently associated with DS.

Adolescent↗
PubMed1989

Purification and characterization of human immunodeficiency virus (HIV) core precursor (p55) expressed in Saccharomyces cerevisiae.

The core structure of retroviruses, including the human immunodeficiency virus (HIV), consists of proteins that are initially synthesized as polyprotein precursors and then processed by a virally encoded protease yielding the mature core polypeptides. To obtain sufficient quantities of the purified HIV core precursor p55 for detailed studies, a segment of HIV DNA encoding the full length core precursor polyprotein p55 was expressed in Saccharomyces cerevisiae using a plasmid containing a constitutive galactose promoter. The expression of this DNA produced a protein with an estimated molecular size of 55,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE); this protein was immunoreactive to anti-HIV p24 antisera. Following cell lysis, freezing, and thawing, the expressed protein was an insoluble aggregate that served as the starting material for the purification process. Solubilization of the insoluble p55 with guanidine HCl followed by phenyl-Sepharose column chromatography and high performance liquid chromatography resulted in a preparation of p55 that was greater than 95% pure by SDS-PAGE, immunoreactive to anti-HIV core protein antibodies, and completely soluble in aqueous solution. The expressed p55 appeared to be myristoylated as evidenced by the incorporation of radiolabel following incubation of recombinant yeast cells with [3H]myristic acid; in addition the amino terminus of the final purified protein was blocked. Proteolytic digestion of purified p55 with synthetic HIV protease yielded the predicted amino- and carboxyl-terminal products; these were confirmed by amino acid sequence analysis. In contrast, digestion of purified p55 by the protease derived from the avian myeloblastosis virus resulted in fragments that were different in size from those produced by the HIV protease. The availability of the purified, full length water-soluble HIV core precursor will be useful in identifying agents that inhibit its processing by the HIV protease.

Amino Acid Sequence↗
PubMed1989

Kinetics of the glycation of bovine serum albumin by mannose and fucose in vitro.

Glycation of bovine serum albumin was measured for mannose and fucose at 37 degrees C. Mannose as well as fucose demonstrated an initial rapid increase in rate of formation of total adducts followed by a slower secondary reaction. The equilibrium constant for Schiff base formation was almost two times larger for mannose than fucose, although the Schiff base formed by fucose rearranged 1.5 times faster than that for mannose. Both sugars showed parallel lines for the formation of total and acid stable products after three hours. Discussion integrates new mechanistic data with previously suggested mechanisms.

Fucose↗
PubMed1989

Human immunodeficiency virus protease. Bacterial expression and characterization of the purified aspartic protease.

The protease of human immunodeficiency virus has been expressed in Escherichia coli and purified to apparent homogeneity. Immunoreactivity toward anti-protease peptide sera copurified with an activity that cleaved the structural polyprotein gag p55 and the peptide corresponding to the sequence gag 128-135. The enzyme expressed as a nonfusion protein exhibits proteolytic activity with a pH optimum of 5.5 and is inhibited by the aspartic protease inhibitor pepstatin with a Ki of 1.1 microM. Replacement of the conserved residue Asp-25 with an Asn residue eliminates proteolytic activity. Analysis of the minimal peptide substrate size indicates that 7 amino acids are required for efficient peptide cleavage. Size exclusion chromatography is consistent with a dimeric enzyme and circular dichroism spectra of the purified enzyme are consistent with a proposed structure of the protease (Pearl, L.H., and Taylor, W.R. (1987) Nature 329, 351-354). These data support the classification of the human immunodeficiency virus protease as an aspartic protease, likely to be structurally homologous with the well characterized family that includes pepsin and renin.

Aspartic Acid Endopeptidases↗
PubMed1989

The acute effect on levels of catecholamines and metabolites in brain, of a single dose of MPTP in 8 strains of mice.

The neurotoxic agent MPTP produces profound and sustained changes in the biochemistry of the brain in mice after a single dose of 20 mg/kg. Acute neurochemical changes have been examined in detail in 8 strains of mice. Coloured mice were more intoxicated than white mice when treated with MPTP: white mice were little affected behaviourally by a dose that could kill a sensitive coloured strain. Although changes in levels of dopamine were a good indication of the effect of treatment--particularly in the dopaminergic areas, the best discriminator between strains was changes in the levels of adrenaline and noradrenaline. Changes in levels of dopamine, which generally decreased in coloured mice, were not good indicators of toxicity. The changes in levels of adrenaline and noradrenaline discriminated better between strains: they were usually decreased in coloured mice but could substantially increase in white strains, particularly in certain areas of the brain. This study showed that there is a very considerable genetic component to the acute toxicity of MPTP within a single species. Even very closely related mice reacted quite differently to the effect. Also, it showed that white mice, in particular those from the commonly used BALB/c strain, are a poor model for any study of the toxicity of MPTP.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1989

Clinical studies in psychogenic stuttering of adult onset.

Twelve patients (6 men and 6 women), ages 21-79 years, who, as far as it could be determined from their case histories, began to stutter for the first time in adulthood were classified by examining physicians and speech-language pathologists as having psychogenic stuttering. The case histories of these patients were examined to determine the details of their speech and allied complaints, the course and duration of stuttering, and if any psychologically stressful events surrounded the onset and development of the disorder. Minnesota Multiphasic Personality Inventory (MMPI) data on 10 of the 12 patients were analyzed independently. The main findings were (a) stuttering was the chief complaint of 11 patients; (b) all 12 patients had additional neurologic-like complaints that turned out to be nonorganic; (c) duration of stuttering from onset to examination ranged from 4 hr to 1.5 years; (d) the stuttering of most patients was variable in severity and returned to normal either spontaneously or as a result of speech therapy or psychotherapy; (e) psychologic stress was associated with the onset in 10 patients: the stress was chronic in 4 patients and acute in 6; (f) MMPI profiles of 9 of 10 patients were abnormal, with four profiles reflective of a conversion reaction; (g) differentiating neurologic from psychogenic stuttering and the need for interdisciplinary collaboration were essential in all 12 cases; and (h) speech therapy and psychotherapy are justified when psychogenic stuttering is diagnosed.

Adult↗
PubMed1989

Self-administered alcoholism screening test (SAAST): comparison of classificatory accuracy in two cultures.

Results of stepwise discriminant analyses of the Self-Administered Alcoholism Screening Test (SAAST) administered to 181 alcoholics and 123 nonalcoholics in Mexico City were compared with results of a similar analysis of a sample in the United States (Rochester, MN). With sensitivities of 90% and 95%, specificities were 91.1% to 99.2% in the Mexican sample for total score and a nine-item version derived from the Mexican sample gave specificities between 95.1% and 99.2% at 90% and 95% sensitivities, as with the Rochester sample. The two items with greatest predictive power were the same in both cultures.

Adult↗
PubMed1988

High affinity phlorizin binding to the LLC-PK1 cells exhibits a sodium:phlorizin stoichiometry of 2:1.

The phlorizin binding properties of luminal membrane vesicles isolated from the LLC-PK1 cells, a continuous epithelial cell line derived from pig kidney, are studied. Scatchard analysis of this binding indicates the existence of a single high affinity sodium-dependent site with KD = 0.4 microM at 266 mM sodium. The specificity properties of this site indicate that it represents the binding of phlorizin to the hexose binding site of the sodium-dependent D-glucose transporter previously identified in this cell line. Both phlorizin equilibrium binding and the rate of phlorizin binding were found to be sigmoidal functions of sodium concentration. A Hill analysis of these data was consistent with a sodium:phlorizin stoichiometry of 2:1 in good agreement with the sodium:glucose stoichiometry already established in these cells. Phlorizin dissociation was also found to be sodium-dependent. On the basis of the phlorizin binding data presented here, a number of models of the binding of phlorizin and sodium to the transporter can be excluded. An analysis of a random binding model consistent with the data is presented. The significance of the LLC-PK1 sodium-dependent D-glucose transporter as a model system for related renal and intestinal transporters is discussed.

Animals↗
PubMed1988

Cell surface antigens of human melanocytes and melanoma. Expression of adenosine deaminase binding protein is extinguished with melanocyte transformation.

It has been proposed that the pathogenesis of melanoma proceeds through multiple stages, ranging from benign proliferation of melanocytic cells to acquisition of the capacity to invade tissues and metastasize. During investigations of cell surface antigens expressed by melanocytes and melanoma, we identified an antigen system that was expressed by cultured normal melanocytes but not by melanoma cell lines. mAbs against this antigen detected a 120-kD cell surface glycoprotein on melanocytes. This molecule had been identified previously as the binding protein for adenosine deaminase (ADAbp). ADAbp was expressed by 51 melanocyte cell lines derived from normal fetal, newborn, and adult skin and adult choroid, but not by 102 melanoma cell lines derived from primary and metastatic lesions. Studies with radiolabeled bovine adenosine deaminase, confirmed that melanocytes expressed binding sites for adenosine deaminase, but no binding sites were detected on cultured melanoma cells. Further studies showed that ADAbp+ melanocytes became ADAbp- upon malignant transformation in vitro. Immunohistochemical studies on a panel of frozen tissues demonstrated reactivity of anti-ADAbp mAbs with epidermal melanocytes and benign junctional nevi, but not with potentially premalignant dysplastic nevi or primary/metastatic melanoma lesions. These studies demonstrate that ADAbp expression is lost with malignant transformation of melanocytes, presumably at an early stage in the transformation process.

Adenosine Deaminase↗
PubMed1988

Resurrecting the ICAS: a competitor for the MAC in screening medical patients for alcoholism?

Although the MacAndrew scale (MAC) is the most widely used of the MMPI alcoholism screening scales, evidence to support its continued use is equivocal, and recent data raise serious questions about its efficacy in the screening of medical patients. In comparison, the Institutionalized Chronic Alcoholic Scale (ICAS), an 8-item scale, has an equivalent correct classification rate among male alcoholics and is significantly better than the MAC scale at identifying female alcoholics. Furthermore, misclassification rates among medical patients and normal persons are substantially lower for the ICAS than for the MAC scale, although the ICAS overidentifies potential alcoholism among normal women and female medical patients. However, reliability coefficients across the criterion and contrast samples are disappointingly low, and the use of the ICAS cannot be defended on that basis.

Adolescent↗
PubMed1988

Screening medical patients for alcoholism with the MMPI: a comparison of seven scales.

Seven alcoholism scales for the MMPI were evaluated for effectiveness in screening medical patients. Sensitivity and specificity were assessed by use of five contrast samples: (1) 736 inpatients from an alcohol and drug dependence unit; (2) 4,923 unselected medical outpatients; (3) 7,044 selected medical outpatients; (4) 214 psychiatric inpatients; and (5) 1,408 contemporary normal persons. None of these scales can be recommended for use in screening medical patients. However, endorsement of MMPI item 215 should lead to further evaluation because classification was more accurate with this single item than with any of the scales evaluated.

Adolescent↗
PubMed1988

Alcoholism in elderly persons: medical aspects and prognosis of 216 inpatients.

We describe 216 elderly patients (65 years of age or older) who were treated for alcoholism in an inpatient treatment program. Emphasis is placed on the demographics, medical characteristics, results of laboratory tests, outcome of treatment, and various comparisons of early- and late-onset alcoholism. The frequency of serious medical disorders among these patients was higher than would be expected for the overall population of a similar age. Elderly alcoholics have more abnormal results of commonly used laboratory tests than do younger alcoholics. Our data show that the elderly alcoholic can be successfully treated in a medically oriented inpatient treatment program. The concept of less-intensive treatment for the elderly alcoholic is generally not supported. More-intensive treatment may be necessary for some of these patients because of the high frequency of accompanying major medical and psychiatric problems. Early-onset alcoholism predominated, but we found no major differences between the two groups.

Adult↗
PubMed1988

Alcoholism in elderly persons: a study of the psychiatric and psychosocial features of 216 inpatients.

The medical records of 216 elderly persons, admitted to the hospital for treatment of alcoholism, were reviewed. Concern of family and friends was the most common factor motivating patients for admission. Patients with late-onset alcoholism reported an association between a life event and problem drinking more frequently than did the early-onset alcoholics. The most common associated psychiatric disorders were tobacco dependence (67%), organic brain syndrome (25%), atypical or mixed organic brain syndrome (19%), and affective disorder (12%). Fourteen percent of patients also had a drug abuse or dependence problem, all using legally prescribed drugs. Psychiatric diagnoses and results of psychologic testing did not differ between early-onset and late-onset alcoholism groups. In a 60-patient cohort studied for correlation of outcome of treatment for alcoholism with major psychiatric diagnoses, no associations were found.

Affective Disorders, Psychotic↗
PubMed1988

Active human immunodeficiency virus protease is required for viral infectivity.

Retroviral proteins are synthesized as polyprotein precursors that undergo proteolytic cleavages to yield the mature viral proteins. The role of the human immunodeficiency virus (HIV) protease in the viral replication cycle was examined by use of a site-directed mutation in the protease gene. The HIV protease gene product was expressed in Escherichia coli and observed to cleave HIV gag p55 to gag p24 and gag p17 in vitro. Substitution of aspartic acid residue 25 (Asp-25) of this protein with an asparagine residue did not affect the expression of the protein, but it eliminated detectable in vitro proteolytic activity against HIV gag p55. A mutant HIV provirus was constructed that contained the Asn-25 mutation within the protease gene. SW480 human colon carcinoma cells transfected with the Asn-25 mutant proviral DNA produced virions that contained gag p55 but not gag p24, whereas virions from cells transfected with the wild-type DNA contained both gag p55 and gag p24. The mutant virions were not able to infect MT-4 lymphoid cells. In contrast, these cells were highly sensitive to infection by the wild-type virions. These results demonstrate that the HIV protease is an essential viral enzyme and, consequently, an attractive target for anti-HIV drugs.

Asparagine↗
PubMed1988

Analysis of two 'home remedy' contact lens cleaners.

In this study, we compared the cleaning efficacy of two "home remedy" contact lens cleaners, baking soda and popcorn salt, to the FDA approved "soft" abrasive cleaner, Opticlean. Ten deposited soft contact lenses were cut into four equal sections. The first section was left uncleaned and served as a control. The other three sections were cleaned with Opticlean, a baking soda-distilled water paste, and a mixture of Pliagel and popcorn salt, respectively. A quantitative analysis indicated that the area of the lens surface covered by deposits was not significantly different for the three experimental sections, but all three were significantly cleaner than the control. In addition, there were numerous deep scratches on the lens surfaces cleaned with baking soda and popcorn salt.

Bicarbonates↗
PubMed1988

Cardiovascular effects of fentanyl reversal by naloxone at varying arterial carbon dioxide tensions in dogs.

Clinical reports, as well as animal studies, have described cardiovascular and sympathetic stimulation after the administration of naloxone (NX) to reverse opioid-induced respiratory depression. This investigation examines the effect of PaCO2 on hemodynamic and adrenergic responses to NX, by means of 24 experiments carried out in six dogs. Each dog underwent NX reversal of fentanyl (FEN) at three different PaCO2 levels: 20, 35, and 60 mm Hg. In a final series of six experiments, the dogs were exposed to increasing PaCO2 after autonomic block by total spinal anesthesia and vagotomy. During enflurane anesthesia, 50 micrograms/kg FEN decreased mean arterial blood pressure (MAP), heart rate (HR), and plasma concentrations of norepinephrine (NE) and epinephrine (EPI) significantly. NX 0.4 mg promptly returned HR and MAP to baseline or above in all experiments; catecholamine (CA) levels increased only in hypercapnic dogs. Increases in HR were the same in all series. MAP, EPI, and NE levels were significantly greater than pre-FEN baseline values only in hypercapnic dogs 1 minute after NX and were also significantly higher in hypercapnic than in hypocapnic dogs at this time. NE levels were greater in hypercapnic dogs at all time periods after NX. In blocked dogs, neither F nor NX had any effects on hemodynamic functions or plasma CA levels; the institution of hypercapnia caused significant decreases in HR, MAP, and systemic vascular resistance. This direct circulatory depressant action of an elevated PCO2 may have attenuated the indirectly mediated excitatory hemodynamic effects of NX in intact dogs, thus explaining the relatively greater effect of hypercapnia on adrenergic than on hemodynamic responses to reversal.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗