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Biomedical subjects

M C Young

Publications and source records attributed to M C Young.

At least 19 recordsLinked to original sources
PubMed2001

Dissection of the ATP-driven reaction cycle of the bacteriophage T4 DNA replication processivity clamp loading system.

Processive DNA replication requires the loading of a multisubunit ring-shaped protein complex, known as a sliding or processivity clamp, onto the primer-template (p/t) DNA. This clamp then binds to the replication polymerase to form a processive polymerase holoenzyme. The processivity of the holoenzyme derives from the topological properties of the clamp, which encircles the DNA without actually binding to it. Multisubunit complexes known as clamp-loaders utilize ATP to drive the placement of this ring around the DNA. To further understand the role of ATP binding and hydrolysis in driving clamp-loading in the DNA replication system of bacteriophage T4, we report the results of a series of presteady-state and steady-state kinetic ATPase experiments involving the various components of the reconstituted system. The results obtained are consistent with a mechanism in which a slow step, which involves the binary ATP-bound clamp-clamp loader complex, activates this complex and permits p/t DNA to bind and stimulate ATP hydrolysis. ATP hydrolysis itself, as well as the subsequent (after clamp-loading) dissociation of the clamp-loader and the slippage of the loaded clamp from the p/t DNA construct, are shown to be fast steps. A second slow step occurs after ATP hydrolysis. This step involves the dissociated clamp loader complex and may reflect ADP release. Only one molecule of ATP is hydrolyzed per clamp-loading event. Rate constants for each step, and an overall reaction mechanism for the T4 clamp-loading system, are derived from these data and from other results in the literature. The principles that emerge fit into a general framework that can apply to many biological processes involving ATP-driven reaction cycles.

Adenosine Triphosphatases↗
PubMed2001

Antifungal polysulphides from Petiveria alliacea L.

Bioactivity-directed fractionation of the CH(2)Cl(2)/MeOH (2:1, v/v) extract of the roots of Petiveria alliacea, using mutant yeast strains of Saccharomyces cerevisiae and fungi Cladosporium cladosporioides and C. sphaerospermum led to the isolation of dipropyl disulphide (1), dibenzyl sulphide (2), dibenzyl disulphide (3), dibenzyl trisulphide (4), dibenzyl tetrasulphide (5), benzylhydroxymethyl sulphide (6) and di(benzyltrithio) methane (7). Of these, 5-7 are new compounds and this is the first report of the natural occurrence of 2 and 3.

Antifungal Agents↗
PubMed2000

nor-Lignans from the leaves of Styrax ferrugineus (Styracaceae) with antibacterial and antifungal activity.

Chemical examination of the leaves of Styrax ferrugineus yielded 5-[3''-(beta-D-glucopyranosyloxy)propyl]-7-methoxy-2-(3',4'-dimethoxyphenyl) benzofuran, along with the known nor-lignans 5-(3''-hydroxypropyl)-7-methoxy-2-(3',4'-methylenedioxyphenyl) benzofuran, 5-(3''-hydroxypropyl)-7-methoxy-2-(3',4'-dimethoxyphenyl)benzofuran, 5-[3''-(beta-D-glucopyranosyloxy)propyl]-7-methoxy-2-(3',4'-methylenedioxyphenyl)benzofuran and the lignan, dihydrodehydrodiconiferyl alcohol. All arylpropanoids isolated showed antibacterial and antifungal activities. The structures of the isolates were established by spectroscopic analysis.

Anti-Bacterial Agents↗
PubMed2000

Antifungal amides from Piper hispidum and Piper tuberculatum.

Piper hispidum and Piper tuberculatum accumulate amides bearing isobutyl, pyrrolidine, dihydropyridone and piperidine moieties. The isolation and characterization of several representatives including two hitherto unreported amides were performed by chromatographic techniques and by analysis of spectroscopic data. The antifungal activity of each amide was determined by direct bioautography against Cladosporium sphaerospermum.

Amides↗
PubMed1999

Sudden death due to ischaemic heart disease in young aboriginal sportsmen in the Northern Territory, 1982-1996.

OBJECTIVE: To estimate the incidence of sport-related sudden cardiac death due to ischaemic heart disease (IHD) in competitive young Aboriginal sportsmen. SETTING: Northern Territory (NT), 1982-1996. DESIGN: Retrospective case series with cases identified from Australian Bureau of Statistics cause-of-death listings and NT coronial autopsy records. MAIN OUTCOME MEASURES: Circumstances and incidence of sport-related sudden cardiac deaths due to IHD; autopsy findings. RESULTS: Between 1982 and 1996, there were eight sudden cardiac deaths due to IHD and related to sporting activity among Aboriginal sportsmen aged 15-37 years in the NT. Six were associated with games of Australian (rules) football. All occurred in the Top End of the NT in the wet season, and all occurred after the first half, or within an hour of, a game. Four of the players had macrosopic myocardial abnormalities (hypertrophy or previous infarcts) on autopsy. The estimated incidence of IHD-related sudden cardiac death among Aboriginal Australian football players in the NT was 19-24 per 100,000 player-years, compared with 0.54 per 100,000 player-years among Australian rules footballers of similar ages in Victoria. CONCLUSIONS: Incidence of sudden cardiac death attributable to underlying IHD was extremely high among young NT Aboriginal Australian footballers. Prevention will best be achieved by funding culturally appropriate long-term strategies to reduce the incidence of IHD. However, in the short-term, community-controlled programs with education of athletes, heat-stress reduction strategies, and cardiovascular screening should reduce the incidence of sudden cardiac death in sport.

Adult↗
PubMed1999

Iridoid and seco-iridoid glucosides from Chioccoca alba (Rubiaceae).

Phytochemical investigation of Chioccoca alba afforded three new iridoids, alboside I, alboside II and alboside III, and a new seco-iridoid alboside V. Alboside IV showed moderate activity towards the DNA repair-deficient mutant RS321 of Saccharomyces cerevisiae. The structural elucidation of the new compounds was performed by ES-MS and by 1D and 2D NMR spectroscopic methods.

Glucosides↗
PubMed1999

A chromene and prenylated benzoic acid from Piper aduncum.

In addition to nerolidol, 2',6'-dihydroxy-4'-methoxydihydrochalcone, methyl 2,2-dimethyl-8-(3'-methyl-2'-butenyl)-2H-1-chromene-6-carboxylate, methyl 2,2-dimethyl-2H-1-chromene-6-carboxylate and methyl 8-hydroxy-2,2-dimethyl-2H-1-chromene-6-carboxylate, two new natural products were isolated from the leaves of Piper aduncum, 2,2-dimethyl-2H-1-chromene-6-carboxylic acid and 3-(3',7'-dimethyl-2',6'-octadienyl)-4-methoxybenzoic acid. The structures of the isolates were established based on analysis of spectroscopic data, including ES-MS. The DNA-damaging activity of the isolated compounds was also investigated against mutant strains of Saccharomyces cerevisiae.

Benzoates↗
PubMed1999

Intra-aortic administration of epinephrine above aortic occlusion does not alter outcome of experimental cardiopulmonary resuscitation.

Intra-aortic balloon occlusion during experimental cardiopulmonary resuscitation (CPR) improves coronary perfusion pressure and resuscitability and provides unique access to the central circulation. It has been hypothesized that administration of epinephrine into the aortic arch in combination with aortic occlusion would further improve haemodynamics during CPR, resuscitability and 24 h survival. In 16 anaesthetised dogs intravascular catheters were placed for hemodynamic and blood gas monitoring. An aortic balloon catheter was placed by femoral artery insertion with its tip just distal to the left subclavian artery. Ventricular fibrillation for 7.5 min without CPR, 2.5 min of Basic Life Support with chest compressions and ventilation with 100% oxygen were followed by 30 min of Advanced Cardiac Life Support (ACLS) with systemic canine drug dosages. The intra-aortic balloon was inflated when ACLS started and gradually deflated shortly after restoration of spontaneous circulation (ROSC). Epinephrine, in 100 microg/kg boluses every 5 min until the heart was restarted or 30 min had elapsed was administered through the intra-aortic catheter in the experimental group (n = 8) and via a central venous catheter in the control group (n = 8). Coronary perfusion pressure increased during the ACLS period in both groups (P < 0.05) with no difference between the groups and there was no difference in the frequency of ROSC (experimental group 5/8, control group 4/8). Furthermore with respect to 24 h survival, there was no difference between the experimental group (2/8) and the control group (3/8). Severe macroscopic haemorrhagic necrosis of the myocardium in the dogs with ROSC was found in 4/5 in the experimental group compared to 1/4 in the control group. In conclusion, intra-aortic administration of 100 microg/kg epinephrine doses combined with aortic occlusion during experimental CPR did not alter outcome.

Analysis of Variance↗
PubMed1999

Search for antifungal and anticancer compounds from native plant species of Cerrado and Atlantic Forest.

Bioactivity-guided fractionation of several bioactive extracts obtained from Cerrado and Atlantic Forest plant species led to the isolation of potent DNA-damaging piperidine 1-5 and guanidine alkaloids 6-9 from Cassia leptophylla and Pterogyne nitens respectively, two common Leguminosae from Atlantic Forest. By means of biotechnological approach on Maytenus aquifolium, a species from Cerrado, moderate DNA-damaging sesquiterpene pyridine alkaloid 10-11 was isolated. Bioassay-guided fractionation on Casearia sylvestris, a medicinal plant species found in Cerrado and Atlantic Forest, led to the isolation of clerodane diterpenes 12-13 which showed effect on DNA. In addition, we have reported several interesting potent antifungal iridoids: 1 beta-hydroxy-dihydrocornin (14), 1 alpha-hydroxy-dihydrocornin (15), alpha-gardiol (16), beta-gardiol (17), plumericin (18), isoplumericin (19), 11-O-trans-caffeoylteucrein (20); ester derivative: 2-methyl-4-hydroxy-butyl-caffeoate (21), amide N-[7-(3',4'-methylenedioxyphenyl)-2Z, 4Z-heptadienoyl] pyrrolidine (22) and triterpene viburgenin (23).

Antifungal Agents↗
PubMed1998

Antifungal amide from leaves of Piper hispidum.

Bioactivity-guided fractionation of a CH2Cl2 extract from leaves of Piper hispidum (Piperaceae) yielded a new pyrrolidine amide, N-[7-(3',4'-methylenedioxyphenyl)-2(Z),4(Z)-heptadienoyl] pyrrolidine 1, in addition to two known amides N-[5-(3', 4'-methylenedioxyphenyl)-2(E)-pentadienoyl] pyrrolidine and N-[2-(3', 4'-methylenedioxy-6'-methoxyphenyl)-2(Z)-propenoyl]pyrrolidine. The structure of compound 1 was elucidated by interpretation of spectral data, including ES-MS. Compound 1 showed antifungal activity against Cladosporium sphaerospermum.

Antifungal Agents↗
PubMed1998

Triterpenes and saponins from Rudgea viburnioides.

A novel triterpene, viburgenin (1), has been isolated from an extract of the ripe fruit rinds of Rudgea viburnioides, together with the known saponins, arjunglucoside I and trachelosperosides B-1 and E-1, and the triterpenes trachelosperogenin B (2) and arjungenin. Compound 2 was previously obtained as a product from enzymatic hydrolysis, and it is reported for the first time as a natural product. The structure of compound 1 was determined as 2alpha,3beta, 19alpha,23,24-pentahydroxyurs-12-ene by extensive use of 1D and 2D NMR spectroscopic methods. Compound 1 exhibited moderate antifungal activity against Cladosporium cladosporioides.

Antifungal Agents↗
PubMed1998

Probing the binding region of the single-stranded DNA-binding domain of rat DNA polymerase beta using nanosecond-pulse laser-induced cross-linking and mass spectrometry.

In recent years, there has been a significant number of studies in which UV light has been used as a reagent to induce cross-links in nucleic acid-protein complexes. An area of considerable interest among those interested in structural biology is the garnering of information about the sites of cross-linking within the protein and nucleic acid members of photolinked conjugates, under the assumption that such knowledge should lead to identification of contact regions or sites within the native complexes. In this paper, we present our results from a photocross-linking study of the complex of the single-stranded DNA-binding domain of rat DNA polymerase beta (pol beta-ss) with the oligonucleotide d(ATATATA). In this study, we have used single nanosecond laser pulses as the cross-linking reagent and matrix-assisted laser desorption/ionization-time of flight mass spectrometry as an analytical tool to identify cross-linked peptides purified from proteolytic digests of the cross-linked complex. Six cross-linked peptides have been identified in tryptic digests of the protein-oligonucleotide conjugates that result from irradiation of the pol beta-ss-d(ATATATA) complex with a single laser pulse. Comparisons with NMR data in the literature for the same complex show that each of the cross-linked peptides contains amino acids that are in contact with the nucleic acid component of the complex.

Amino Acid Sequence↗
PubMed1997

Structural analyses of gp45 sliding clamp interactions during assembly of the bacteriophage T4 DNA polymerase holoenzyme. I. Conformational changes within the gp44/62-gp45-ATP complex during clamp loading.

A multisubunit ring-shaped protein complex is used to tether the polymerase to the DNA at the primer-template junction in most DNA replication systems. This "sliding clamp" interacts with the polymerase, completely encircles the DNA duplex, and is assembled onto the DNA by a specific clamp loading complex in an ATP-driven process. Site-specific mutagenesis has been used to introduce single cysteine residues as reactive sites for adduct formation within each of the three subunits of the bacteriophage T4-coded sliding clamp complex (gp45). Two such mutants, gp45S19C and gp45K81C, are reacted with the cysteine-specific photoactivable cross-linker TFPAM-3 and used to track the changes in the relative positioning of the gp45 subunits with one another and with the other components of the clamp loading complex (gp44/62) in the various stages of the loading process. Cross-linking interactions performed in the presence of nucleotide cofactors show that ATP binding and hydrolysis, interaction with primer-template DNA, and release of ADP all result in significant conformational changes within the clamp loading cycle. A structural model is presented to account for the observed rearrangements of intersubunit contacts within the complex during the loading process.

Adenosine Triphosphate↗
PubMed1997

Immune deficiencies in chronic intestinal pseudo-obstruction.

AIM: Chronic intestinal pseudo-obstruction has been associated with urinary disorders, myopathy, and ophthalmoplegia in adults and cholelithiasis in children. We observed a high percentage of total-parenteral-nutrition-dependent patients with pseudo-obstruction and recurrent infections requiring gammaglobulin infusions. METHODS: All records for 23 children with chronic intestinal pseudo-obstruction (10 females and 13 males, mean age 9.8 y +/- 4.9 y, range 4-24 y) referred for a nutritional evaluation from 1992 to 1995 were reviewed. Chronic intestinal pseudo-obstruction was diagnosed by clinical, radiographic findings and antroduodenal manometry. Intestinal full-thickness biopsies were performed in seven children. RESULTS: Hypogammaglobulinemia was diagnosed in 18 patients (78%): 16 patients had various immunoglobulin deficiencies and 2 had selective antibody deficiency. Intravenous gammaglobulin was administered in 14 patients. Other medical conditions affecting the children are summarized as follows: autonomic dysfunction in 10 patients (43%), recurrent hypoglycemia in 9 (39%), asthma in 9 (39%), cholecystitis in 7 (30%), low serum carnitine level in 6 (26%), urinary dysfunction in 6 (26%), pancreatitis in 5 (22%), behavioral problems in 5 (22%), myopathy in 2 (9%), idiopathic thrombocytopenia in 2 (8%), velopharyngeal insufficiency in 1 (4%), oculocutaneous albinism in 1 (4%), Pierre-Robin syndrome in 1 (4%), and protein C deficiency in 1 (4%). Munchausen syndrome was suspected in two patients. CONCLUSIONS: Chronic intestinal pseudo-obstruction appears to be associated with immune deficiencies. It is unclear if the immune deficiencies, intestinal pseudo-obstruction, and the other medical conditions have a common underlying etiology. Repeated infections may be due to impaired immune function in children with chronic intestinal pseudo-obstruction. We recommend screening for immune deficiencies in children with chronic intestinal pseudo-obstruction.

Adolescent↗
PubMed1996

Fluorescence monitoring of T4 polymerase holoenzyme accessory protein interactions during loading of the sliding clamp onto the template-primer junction.

Assembly of the T4 polymerase holoenzyme requires coordinated interactions among the core polymerase and the clamp loader (gp44/62) and sliding clamp (gp45) accessory proteins. Here we describe the creation of a mutant of gp45 that can be uniquely modified by fluorescent probes within each protein monomer at a site-specific cysteine residue. The fluorescently labeled gp45 was shown to have the same biological activity as the wild-type protein. These strike "labeled" gp45 adducts were then used in steady-state and fluorescence polarization studies to monitor the interaction of gp45 with the gp44/62 clamp-loading complex and template-primer DNA in the presence and absence of ATP, and of the non-hydrolyzable analog, adenosine 5'-O-(3-thiotriphosphate). We find that a complex of ATP-activated gp44/62 with appropriately labeled gp45 shows significant fluorescent enhancement (and an increase in fluorescent anisotropy), that can be partially reversed by interaction with template-primer DNA. Fluorescence-monitored binding curves between gp45 and ATP-activated gp44/62 reveal that the two protein complexes bind with a 1:1 stoichiometry. Analysis shows that these methods can be used to follow the ATP-driven loading of gp45 onto the template-primer by the gp44/62 clamp-loading complex, and in combination with the kinetic data presented in the companion article, provide insight into the rate-limiting steps during clamp assembly on template-primer DNA. A reaction pathway for this processivity clamp-loading process is proposed.

Adenosine Triphosphatases↗
PubMed1996

The kinetic mechanism of formation of the bacteriophage T4 DNA polymerase sliding clamp.

DNA replication in bacteriophage T4 requires the assembly of a structure called the "sliding clamp" near the 3' end of the DNA strand that is to be extended. This structure is a trimer ring of the T4 gene 45 product (gp45) and serves to regulate the processivity of the DNA polymerase within the T4 DNA replication system. The placement of this ring is performed by an ATPase complex of the products of T4 genes 44 and 62 (gp44/62) that consists of four gp44 subunits and one gp62 subunit. In an effort to understand the role of ATP hydrolysis in processes occurring during the formation of the phage T4 DNA sliding clamp, we have performed direct substrate and product binding experiments and steady-state and presteady kinetic experiments on the gp44/62-gp45 system. Substrate (ATP) and product (ADP) binding studies show that the gp44/62 complex binds 4(+/-1) ATP molecules with a Kd of 34(+/-12) microM, and 3.7(+/-0.3) ADP molecules with a Kd of 14(+/-7) microM. The binding of the other reaction product (inorganic orthophosphate) could not be detected. Presteady-state kinetic analysis of ATP hydrolysis during the sliding-clamp-loading process indicates a biphasic progress curve, consisting of an initial rapid "burst" phase with an amplitude of four ATP molecules per gp44/62 complex and a rate of 15 s(-1), followed by a second slower phase corresponding to the steady-state rate of ATP hydrolysis by this complex. The rate of the burst phase is kinetically consistent with the previously observed rate of T4 DNA polymerase holoenzyme formation. The burst amplitude depends solely on the concentration of gp44/62 ATP binding sites present. These results suggest that the formation of a single T4 sliding clamp requires the hydrolysis of four ATP molecules by one gp44/62 complex in a process requiring 0.5 to 1 second. A model describing the clamp-loading process is discussed in the context of these results.

Adenosine Diphosphate↗