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Biomedical subjects

P Clark

Publications and source records attributed to P Clark.

At least 127 records · Page 7Linked to original sources
PubMed1995

Expression of protein kinase C isoenzymes in colorectal cancer tissue and their differential activation by different bile acids.

Expression of protein kinase C (PKC) isoenzymes was determined in paired samples of normal mucosa and colorectal cancer tissue from 13 patients. Total PKC activity in cancer tissue was significantly decreased compared to that in normal mucosa. Western blotting, using PKC isoenzyme-specific antibodies, showed that two PKC isoenzymes, PKC beta and PKC epsilon, were significantly decreased in cancer tissue. The level of PKC delta was increased in cancer tissue and the expression of PKC alpha and zeta was not altered significantly. Primary bile acids--cholic acid (CA) and chenodeoxycholic acid (CDCA)--and the principal secondary bile acids--deoxycholic acid (DCA), lithocholic acid (LCA) and ursodeoxycholic acid (UDCA)--were found to be potent and selective activators of partially purified PKC isoenzymes. PKC beta 1 was the isoenzyme most effectively activated by secondary bile acids. Our data provide a model for the involvement of secondary bile acids in colorectal carcinogenesis through specific PKC isoenzyme modulation in colorectal mucosa.

Bile Acids and Salts↗
PubMed1995

Actin cytoskeletal isoforms in human endothelial cells in vitro: alteration with cell passage.

The microfilamentous actin component of the cytoskeleton is crucial to endothelial angiogenesis and vascular permeability. Differences in actin cytoskeletal profiles in cultured human endothelial cells were explored: when first isolated, both primary human umbilical vein endothelial cells (HUVEC) and primary human placental microvascular endothelial cells (HPMEC) expressed F-actin, but not beta-actin or alpha-smooth muscle actin. A similar endothelial actin profile was observed in cryo-sections of freshly delivered term umbilical cord and placenta. In subsequent cell culture, although the actin cytoskeleton of HUVEC remained unchanged, the actin profiles of HPMEC altered after the second passage with the induction of alpha-smooth muscle actin expression, which was intercellularly heterogeneous and increased to 20% at P4. This behaviour occurred in HPMEC monolayers cultured on a variety of extracellular matrices. Comparisons with a spontaneously immortalized human microvascular cell-line, HGTEN 21, revealed that in prolonged passage, both alpha-smooth muscle actin and beta-actin were expressed, whereas HPMEC at P4 showed a lower level of beta-actin expression. Therefore, in comparison with large vessels, microvascular cells are more likely to dedifferentiate. This may reflect the ability of microvascular cells to remodel according to changing requirements for new vessel formation. In conclusion, passage of human microvascular endothelial cells, but not of larger vessel endothelial cells, alters the expression of actin isoforms. This may be important in relation to comparisons of in vitro and in vivo vascular permeability; higher passage microvascular endothelial cells should thus be used with caution in such studies.

Actins↗
PubMed1995

Inhibition of neutrophil oxidative burst and phagocytosis by meconium.

OBJECTIVE: Meconium in amniotic fluid has been associated with an increased prevalence of chorioamnionitis. In an effort to delineate the mechanism of this association, we determined the effect of meconium on the neutrophil's capacity for phagocytosis and microbial killing by oxidative burst in vitro. STUDY DESIGN: Sterile meconium samples were obtained from four fetuses at the time of breech delivery and were then pooled and lyophilized. Neutrophils were purified from whole blood of each of 13 pregnant nonlaboring patients. Phagocytosis and the oxidative burst of neutrophils in the presence and absence of meconium were assessed by single-cell analysis with flow cytometry. Phagocytosis was measured as the mean fluorescence intensity produced after 30 minutes of incubation with fluorescein-labeled Escherichia coli. Oxidative burst was measured as the mean fluorescence intensity resulting from the oxidation of internalized reduced dichlorodihydrofluorescein after 15 minutes of stimulation with phorbol myristate acetate. Oxidative burst was expressed as the neutrophil oxidative index and the net fluorescence intensity. Neutrophil oxidative index was equivalent to the quotient of the mean fluorescence intensity for phorbol myristate acetate-stimulated and unstimulated cells. Net fluorescence intensity was equivalent to the absolute difference between stimulated and unstimulated cells. RESULTS: Exposure of neutrophils to light and very light meconium each resulted in significantly lower mean neutrophil oxidative index compared with unexposed controls (3.2 +/- 4.9 and 4.2 +/- 5.9 vs 16.2 +/- 7.5, p = 0.00002 and p = 0.0007, respectively) and significantly lower mean net fluorescence intensity than that of control cells (112 +/- 220 and 188 +/- 294 vs 613 +/- 328, p = 0.0001 and p = 0.005, respectively). Phagocytosis was significantly impaired in the presence of moderate meconium compared with control cells (2239 +/- 393 vs 4645 +/- 2071, p = 0.0001). Light meconium did not significantly affect phagocytosis. CONCLUSION: Meconium has significant effects on neutrophil function in vitro. Both light and very light meconium inhibit the oxidative burst. Moderate meconium inhibits phagocytosis.

Chorioamnionitis↗
PubMed1995

Performance of a new DNA probe for the detection of group B streptococcal colonization of the genital tract.

OBJECTIVE: To test the performance characteristics of a new DNA probe designed for the rapid identification of heavy colonization of the genital tract with group B streptococci. METHODS: Vaginal and combined vaginal-perianal samples were collected from 193 pregnant women and cultured on colistin-nalidixic acid agar plates. Bacterial growth was classified semiquantitatively. Specimens were also tested by a new DNA probe in two formats: a direct assay performed on the swabs soon after collection and an assay performed after the swabs were incubated for 24 hours in an enriched culture medium. RESULTS: The agar cultures were positive in 36 of 193 patients (18.6%, 95% confidence interval 13.2-24). Nineteen women were lightly colonized, and 17 were heavily colonized (at least 10(4) colonies/mL). The combined vaginal-perianal swabs yielded positive results more often than the vaginal swabs alone (26 versus 16, chi 2 = 24, P < .01). In its direct form, the assay had only 8.3% sensitivity in identifying colonized women. In heavily colonized women, the sensitivity of the assay increased slightly to 12%. After a 16-24-hour incubation, the sensitivity of the assay was 81%. CONCLUSION: The direct assay is insufficiently sensitive for clinical use. The delayed assay offers no advantage over standard cultures.

DNA Probes↗
PubMed1995

A simple probe for DNA accessibility in chromatin.

When DNA is treated with Cu(II) and then heated, the melting temperature (Tm) of the DNA is dramatically decreased (8). The Cu(II) binds to the DNA in such a way as to destabilize the double helix and help to break the hydrogen bonds between the bases. When soluble chromatin is similarly treated with Cu(II) and heated, the Tm is unaffected. Apparently the Cu(II) cannot penetrate the chromatin structure and thus cannot initiate the DNA destabilization process. However, when H-1 histone is removed from the chromatin by affinity chromatography, subsequent treatment with Cu(II) does lead to a lowered Tm when the chromatin is heated. This Tm lowering is also achieved by two less drastic techniques that do not remove histone H-1, but decrease the affinity of the H-1 to the DNA: (1) a mild acetylation procedure that specifically modifies either 2 or 4 epsilon-amino groups of lysines on the H-1 histone, and (2) reaction with phosphate-binding divalent metal ions, e.g., Mg(II), Mn(II), or Co(II). Apparently, removal of H-1 or decreased affinity of H-1 for DNA increases the accessibility of the DNA to the Cu(II). This phenomenon suggests a very simple qualitative probe for the degree of structural change in chromatin produced by a change in the stability of the DNA-H-1 interaction.

Acetylation↗
PubMed1995

Major trauma in Australia: a regional analysis.

This study was undertaken to evaluate the frequency, distribution, cause, pattern, and outcome of patients suffering from major trauma in the State of Victoria over a 1-year period. No previous study in Australia has attempted a comprehensive regional analysis of major trauma. All major trauma admissions resulting from blunt, penetrating, and burns injury were identified, and data collected from emergency departments and intensive care log books at 25 major metropolitan and rural hospitals from the January 3, 1992 to February 28, 1993 by onsite data collectors. The total number of patients admitted into the study was 2,944. There were 1,076 major trauma cases with an Injury Severity Score greater than 15 in a population of 4.2 million people. The type of injury was predominantly blunt (87.5%), with only a small percentage of penetrating injuries (6.4%) and burns (6%). Major trauma in pediatric cases is less common (132 cases). The most common causes of injury were road transport (56%) and falls (22%). The overall outcome of the group was favorable when compared with the Major Trauma Outcome Study group (Z = 1.4, M = 0.93, W = 0.52). There was an unexpectedly low number of patients suffering from major trauma. Outcome using Trauma and Injury Severity Score methodology was favorable when compared with North America.

Adolescent↗
PubMed1995

Coronary artery bypass surgery in patients with inherited antithrombin deficiency.

Antithrombin (AT) replacement in coronary artery bypass grafting procedures in three individuals with inherited antithrombin deficiency is described. All three had a significant personal or family history of thrombotic disease. All patients achieved satisfactory AT levels throughout bypass and in the postoperative period. All received heparin prophylaxis in the postoperative period. None suffered thrombotic or bleeding complications.

Aged↗
PubMed1995

Patterns of injury from major trauma in Victoria.

Basic demographic and injury data were collected on all major trauma patients (ISS > 15) presenting to 25 Victorian hospitals over a 1 year period (March 1992-February 1993). A total of 1076 patients were identified with an Injury Severity Score (ISS) > 15. Of these, 957 resulted from blunt trauma, 68 from penetrating trauma and 51 from burns. Most serious blunt injury was transport-related (n = 652) but falls made up a significant proportion (n = 206). The pattern of injury in blunt trauma demonstrated in this study showed a preponderance of serious head, thoracic and limb injuries with less frequent occurrences of abdominal, spine and facial injuries. In major penetrating trauma, serious injuries of the thorax and abdomen were more frequent. Head injury is the most common cause of morbidity in major trauma patients. Motor vehicle accidents caused the majority of head injuries but, proportionately, head injury was more common in pedal cycle, pedestrian, motorcycle injuries and falls. The low frequency of major abdominal trauma has important implications for surgical training and resource allocation. In Victoria, various injury prevention interventions have been introduced such as compulsory wearing of bicycle helmets, a safer home environment and behavioral modifications through advertising. Injury prevention strategies must continue to target the populations at risk and assess the impact of interventions by accurate injury surveillance.

Accidents, Home↗
PubMed1995

Guidance of oligodendrocytes and their progenitors by substratum topography.

Oligodendrocyte progenitors arise in subventricular zones and migrate extensively during development before differentiating into mature oligodendrocytes, which myelinate nerve tracts in the central nervous system. We have used microfabricated substrata, containing periodic patterns of contours similar to those of central nervous system axons to assess the influence in vitro of substratum topography on oligodendrocytes isolated from 7 day rat optic nerve. Antiganglioside antibody A2B5 positive oligodendrocyte-type 2 astrocyte progenitors, and galactocerebroside positive and myelin basic protein positive oligodendrocytes, were highly aligned by surface contours as small as 100 nm depth and 260 nm repeat spacing. Rat optic nerve astrocytes also aligned on surface contours, but rat hippocampal and cerebellar neurons were unresponsive. Oligodendrocytes demonstrated enhanced parallel extension of their processes on narrow repeating topography in an arrangement similar to that found in the intact optic nerve. This is in marked contrast to the phenotype displayed by this cell type on planar substrata. Neither oligodendrocytes nor oligodendrocyte-type 2 astrocyte progenitors showed high-order F-actin cytoskeletal networks; thus their alignment on gratings is unlikely to result from deformation of actin cables and focal contacts. In contrast, aligned astrocytes showed striking arrangements of actin stress fibres. These results establish glial cells as potentially the most topographically sensitive cell types within the central nervous system. Furthermore, the topographical pattern inducing maximal alignment of oligodendrocyte lineage cells corresponds to the diameters of single axons within the 7 day optic nerve. Thus the migration of oligodendrocyte-type 2 astrocyte progenitors and axonal ensheathment by oligodendrocytes may be guided by axonal topography within the developing nerve.

Animals↗
PubMed1994

Electron transfer in the nitric-oxide synthases. Characterization of L-arginine analogs that block heme iron reduction.

Heme iron reduction in the nitric-oxide synthases (NOSs) requires calmodulin binding and is associated with increased NO synthesis and NADPH oxidation (Abu-Soud, H. M., and Stuehr, D. J. (1993) Proc. Natl. Acad. Sci., U. S. A. 90, 10769-10772). Here, we examined how L-arginine and the analogs N omega-methyl-L-arginine (NMA), N omega-nitro-L-arginine methyl ester (NAME), and d-(thioureido)-L-norvaline (thiocitrulline) affect electron flux through neuronal and macrophage NOS. L-Arginine and NMA increased or decreased NOS NADPH consumption depending on the isoform, while thiocitrulline and NAME decreased NADPH oxidation in both NOS by 73-86% relative to their ligand-free rates. Kinetic studies showed that thiocitrulline and NAME inhibited NOS NADPH consumption through binding within the substrate binding site. Thiocitrulline and NAME did not affect the NADPH-dependent reduction of NOS flavins nor NOS cytochrome c reduction, indicating that they blocked electron flux at a point beyond the flavins in the electron transfer sequence. Thiocitrulline and NAME inhibited both NADPH-dependent and dithionite-mediated heme iron reduction in the NOS isoforms relative to the substrate-free NOS, whereas L-arginine and NMA did not. Thus, L-arginine and NMA increase or decrease electron flux through the NOS by coupling NADPH oxidation to NO synthesis (L-arginine), or by occupying the substrate binding site with minimal catalytic coupling (NMA). In contrast, thiocitrulline and NAME decrease electron flux through both NOS isoforms by decreasing the reduction potential of the heme iron. Inhibition of heme iron reduction by substrate analogs is unusual and represents a new means to modulate electron flow through the NOS.

Amino Acid Oxidoreductases↗
PubMed1994

Mutational analysis of the fingers and palm subdomains of human immunodeficiency virus type-1 (HIV-1) reverse transcriptase.

We have analyzed the human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT) polymerase domain between amino acids 91 and 157 by site-directed mutagenesis. We have constructed a series of amino acid substitutions using BspMI cassettes, and have assayed the RNA-dependent DNA polymerase, DNA-dependent DNA polymerase, and RNase H activities of the mutant HIV-1 RTs. The regions of HIV-1 RT between amino acids 91 and 119 and between amino acids 151 and 157 lie within the palm subdomain and include part of the polymerase active site. A number of amino acids within these regions have been identified as being directly or indirectly involved with polymerization, since amino acid substitutions at these residues decrease the polymerase activity without affecting RNase H activity. The region of HIV-1 RT between amino acids 120 and 150 lies within the fingers subdomain of the HIV-1 polymerase. We believe that the fingers subdomain plays a role in positioning the template. Many amino acid substitutions in this region decrease or abolish both the polymerase and the RNase H functions.

Amino Acids↗
PubMed1994

L-arginine and calmodulin regulation of the heme iron reactivity in neuronal nitric oxide synthase.

Neuronal nitric oxide synthase (NOS) is a calmodulin-dependent, flavin-containing hemoprotein that forms NO from L-arginine, NADPH, and molecular oxygen. Calmodulin binding to NOS triggers reduction of its heme groups (Abu-Soud, H., and Stuehr, D.J. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10769-10762), leading to NADPH oxidation and NO synthesis. We have examined how L-arginine and calmodulin control the ligand binding and electron acceptor properties of the NOS heme iron. In the absence of bound calmodulin, ferric NOS exhibited a Kd of 0.6 microM for L-arginine, as determined by the substrate-dependent shift in heme spin equilibrium toward a high spin state. L-Arginine binding reduced the affinity of the ferric NOS heme for cyanide by 8-fold. Carbon monoxide binding to substrate-free ferrous NOS occurred at a rate of 2 x 10(5) M-1 S-1; this rate was decreased 12-fold when L-arginine was bound. In contrast, bound calmodulin did not significantly affect cyanide or carbon monoxide binding to the NOS heme, nor did it alter NOS binding affinity for L-arginine. Anaerobic titration of a calmodulin-bound, L-arginine-free NOS with NADPH led to incomplete reduction of the heme iron; full reduction was achieved only in the presence of added L-arginine. Thus, our data suggest that L-arginine controls NOS heme iron reactivity in at least two ways: 1) it slows ligand interactions by binding in the distal pocket very near the heme and 2) it also appears to increase the reduction potential of the iron. In contrast, bound calmodulin does not alter the NOS affinity for L-arginine or heme ligands and may function solely as a switch that enables electrons to pass from the flavin domain onto the heme iron.

Amino Acid Oxidoreductases↗
PubMed1994

A structural model for fidelity in transcription.

Distances between the metal ions bound to the product terminus i site and the substrate i + 1 site of Escherichia coli RNA polymerase range from 5.0 to 5.6 A when the substrate is complementary to a template base and from 6.5 to 7.0 A for a noncomplementary relationship. The metal bound to the substrate at the i + 1 site exhibits a constant distance to the three phosphates on the substrate regardless of complementarity, but the distance to base and ribose protons changes. The differences in these geometric parameters are explained by the ability of the enzyme to assume two conformations, one to place correct nucleotide substrates in optimal position for bond formation and the other to prevent incorrect nucleotides from assuming such a position. In this scheme a metal-triphosphate complex can move toward or away from the terminal 3' OH group of the growing RNA chain, to assure fidelity of transcription.

Base Composition↗
PubMed1994

Correlations between dynamic urethral profilometry and perivaginal pelvic muscle activity.

Of 208 ambulatory female subjects evaluated for complaints of urinary incontinence, complete history, physical findings, and urodynamic data were available on 163 patients allowing correlation of measures of perivaginal muscle activity to urethral profilometry measurement of sphincter strength. Perivaginal measures include pelvic digital exam score as well as vaginal electromyography with a modified perinometer. Urethral profilometry was performed at rest and during pelvic muscle contractions in both supine and standing positions. There was a moderate and significant correlation (r = 0.19 to 0.32) between profilometry measures of voluntary sphincter contractions and perivaginal EMG parameters of endurance peak and area, as well as to the digital test parameters of pressure and displacement. The correlation values between the vaginal EMG and the Digital Test scale of perivaginal strength were higher (r = 0.28 to 0.74). When the patients with pure stress incontinence were stratified by degrees of incontinence (mild, moderate, severe), urethral prolfilometry measures were a more accurate indicator of severity of incontinence than measures of perivaginal strength or the degree of bladder neck mobility as measured by the Q-tip test.

Adult↗
PubMed1994

Artificial ultraviolet whole-body radiation does not modify serum lipoprotein, plasma fibrinogen, plasminogen or antithrombin III concentrations in post-myocardial infarction patients.

The relationship of ischaemic heart disease (IHD) to seasonal and latitude variation has prompted speculation that exposure to the ultraviolet component of solar radiation may reduce IHD risk. This hypothesis was partially tested by exposing 14 post-myocardial infarction patients to a 6 week course of artificial whole-body ultraviolet radiation (UVR). Serum lipoprotein and plasma coagulation factor concentrations were measured before and after the course of UVR. Results were compared with similar measurements from a placebo-controlled group of 13 post-myocardial patients. Despite a more than two-fold rise in mean serum 25-OHD, serum lipoprotein and plasma fibrinogen, antithrombin III and plasminogen concentrations did not change significantly in the UVR group. Significant but minor change in prothrombin time and thrombin time in the placebo group appear unlikely to be of biological significance. Seasonal and latitude variation in these IHD risk factors appear unrelated to corresponding variation in solar UVR exposure.

Antithrombin III↗