Biomedical subjects
Publications and source records attributed to S W Clark.
We demonstrate the use of a prism-grating sequence to reduce third-order dispersion inside a mode-locked Yb fiber laser. This laser generates pulses as short as 33 fs with extremely clean temporal and spectral profiles. Nanojoule pulse energies are possible.
For murine adenosine deaminase, we have determined that a single zinc or cobalt cofactor bound in a high affinity site is required for catalytic function while metal ions bound at an additional site(s) inhibit the enzyme. A catalytically inactive apoenzyme of murine adenosine deaminase was produced by dialysis in the presence of specific zinc chelators in an acidic buffer. This represents the first production of the apoenzyme and demonstrates a rigorous method for removing the occult cofactor. Restoration to the holoenzyme is achieved with stoichiometric amounts of either Zn2+ or Co2+ yielding at least 95% of initial activity. Far UV CD and fluorescence spectra are the same for both the apo- and holoenzyme, providing evidence that removal of the cofactor does not alter secondary or tertiary structure. The substrate binding site remains functional as determined by similar quenching measured by tryptophan fluorescence of apo- or holoenzyme upon mixing with the transition state analog, deoxycoformycin. Excess levels of adenosine or N6- methyladenosine incubated with the apoenzyme prior to the addition of metal prevent restoration, suggesting that the cofactor adds through the substrate binding cleft. The cations Ca2+, Cd2+, Cr2+, Cu+, Cu2+, Mn2+, Fe2+, Fe3+, Pb2+, or Mg2+ did not restore adenosine deaminase activity to the apoenzyme. Mn2+, Cu2+, and Zn2+ were found to be competitive inhibitors of the holoenzyme with respect to substrate and Cd2+ and Co2+ were noncompetitive inhibitors. Weak inhibition (Ki > or = 1000 microM) was noted for Ca2+, Fe2+, and Fe3+.
The biological basis for failure of the human anterior cruciate ligament to heal after rupture is unknown. Since this failure could be influenced by abnormalities in matrix protein production or degradation, or both, several diverse matrix protein markers were utilized to survey the state of these extracellular proteins in intrinsic anterior cruciate ligament fibroblasts. Matrix gene expression was visualized by in situ hybridization 9 to 365 days after rupture using probes for type-I collagen, collagenase, 72kDa-gelatinase, and tissue inhibitor of metalloproteinase. Remnants of anterior cruciate ligament were biopsied arthroscopically from 20 patients at reconstruction, fixed in 4% paraformaldehyde, and processed with cDNA probes for the aforementioned mRNAs. mRNA expression of type-I collagen was detected in all specimens, was equally distributed throughout the remnants, and remained evident even at 1 year after injury. Neither of the matrix-degrading enzymes nor their inhibitor (tissue inhibitor of metalloproteinase) was expressed at substantial levels at any time point. Collagen expression within the anterior cruciate ligament confirmed the viability of the ligament remnants for as long as 1 year after rupture. The lack of significant expression of the two matrix-degrading metalloproteinases by the fibroblasts is not consistent with an autodegradation of the remaining ruptured ligament tissue, and whether the lack of matrix remodelling may account, at least in part, for the poor healing response of the anterior cruciate ligament remains to be determined. This initial investigation by in situ hybridization techniques provides a descriptive profile of matrix gene expression in the damaged human anterior cruciate ligament.
BACKGROUND: The reliability and validity of Axis II diagnoses were investigated in a sample of 108 patients with nonpsychotic Axis I disorders. METHODS: Patients were assessed for personality disorders (PDs) with either the Personality Disorder Examination (PDE) or the Structured Interview for DSM-III-R Personality (SIDP-R). Validity was examined by comparing interview diagnoses with "best-estimate" consensus diagnoses assigned by a panel of judges. RESULTS: Interrater reliabilities were excellent when using continuous data (eg, total or cluster scores; intra-class correlation coefficients, .82 to .92); they were lower with categorical diagnoses (eg, any PD vs no PD; kappa = 0.55 and 0.58 with the two interviews). Validity coefficients (ie, kappa values reflecting agreement between the interviews and the consensus diagnosis) for the decision of any PD vs no PD were 0.18 (56% agreement) with the PDE and 0.37 (75% agreement) with the SIDP-R; validity coefficients for identifying cases of "marked" PD were 0.21 (62% agreement) with the PDE and 0.24 (60% agreement) with the SIDP-R. CONCLUSIONS: There have been important advances in the development of structured interviews for Axis II diagnoses, but the findings suggest a continued need to be thoughtful about their strengths and weaknesses before accepting their results as definitive diagnostic tests. The findings also demonstrated some of the advantages of continuous vs categorical data.
As part of our ongoing efforts to understand the functional role of vertebrate centractins, we have identified a new member of the actin-related family of proteins in the yeast Saccharomyces cerevisiae using a PCR-based approach. Consistent with the current nomenclature for actin-related proteins in yeast, we propose to denote this locus ACT3. The primary amino acid sequence of Act3p is most similar to canine and human alpha-centractin (73% similarity/54% identity). The sequence of a genomic clone indicates ACT3 lies adjacent to and is transcribed convergently with respect to FUR1 on chromosome VIII. Molecular genetic analysis indicates ACT3 is represented by a single gene from which the corresponding mRNA is expressed at a low level compared to ACT1. Tetrad analysis of heterozygotes harboring a TRP1 replacement of the ACT3-coding region indicates ACT3 is nonessential for growth under normal conditions and at extremes of temperature and osmolarity. However, growth at 14 degrees C indicates a spindle orientation defect similar to phenotypes recently described for yeast harboring mutations in actin, tubulin, or cytoplasmic dynein. Taken together, our data suggest that ACT3 is the S. cerevisiae homologue of vertebrate centractins.
An examination of human-expressed sequence tags indicated the existence of an isoform of centractin, an actin-related protein localized to microtubule-associated structures. Using one of these tags, we isolated and determined the nucleotide sequence of a full-length cDNA clone. The protein encoded represents the first example of multiple isoforms of an actin-related protein in a single organism. Northern analysis using centractin-specific probes revealed three species of mRNA in HeLa cells that could encode centractin isoforms. One mRNA encodes the previously-identified centractin (now referred to as alpha-centractin). The full-length cDNA clone isolated using the expressed sequence tag encodes a new member of the centractin family, beta-centractin. A probe specific for alpha-centractin hybridized to the third species of mRNA observed (referred to as gamma-centractin). Comparisons of Northern blots of human tissues indicated that alpha-centractin and beta-centractin mRNAs are equally distributed in all populations of mRNA examined, whereas the expression of gamma-centractin appears to be tissue specific. The amino acid sequence of beta-centractin, deduced from the cDNA, indicates a 91% identity with alpha-centractin, increasing to 96% similarity when conservative amino acid changes are taken into account. As antibodies previously raised against alpha-centractin reacted only poorly with beta-centractin, new antibodies were produced and combined with two-dimensional gel electrophoresis to discriminate the two isoforms. Using this system, the subcellular distribution of the alpha- and beta-isoforms were determined. Both isoforms were found predominantly in the cytosolic fraction as a part of a previously identified 20S complex (referred to as the dynactin complex) with no evidence for a free pool of either isoform. The isoforms were found in a constant ratio of approximately 15:1 (alpha:beta) in the dynactin complex.
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Actin is one of the most ubiquitous, abundant and well-conserved proteins of eukaryotes, participating in many crucial cellular processes including the maintenance of cell shape, motility and cell division. Actins from the most divergent sources still share amino-acid identities in excess of 70% (ref. 3). This may well explain why low-abundance homologues of actin have been difficult to isolate. Genes encoding distant relatives of actin in budding and fisson yeast have now been cloned. We report here the discovery of a vertebrate actin-like protein, which we name centractin. A full-length complementary DNA clone was isolated whose sequence reveals amino-acid identities with actin of over 50%, increasing to more than 70% when conservative amino-acid changes are considered. Northern analysis and western blotting indicate a ubiquitous tissue and species distribution. Morphological and biochemical criteria show that centractin is associated with centrosomes.
Mutants of Clostridium acetobutylicum ATCC 824 exhibiting resistance to 2-bromobutyrate or rifampin were isolated after nitrosoguanidine treatment. Mutants were screened for solvent production by using an automated alcohol test system. Isolates were analyzed for levels of butanol, ethanol, acetone, butyrate, acetate, and acetoin in stationary-phase batch cultures. The specific activities of NADH- and NADPH-dependent butanol dehydrogenase and butyraldehyde dehydrogenase as well as those of acetoacetyl-coenzyme A:acetate/butyrate:coenzyme A-transferase (butyrate-acetoacetate coenzyme A-transferase [EC 2.8.3.9]) (CoA-transferase), butyrate kinase, and phosphotransbutyrylase were measured at the onset of stationary phase. Rifampin-resistant strain D10 and 2-bromobutyrate mutant R were found to be deficient in only CoA-transferase, while several other mutants exhibited reduced butyraldehyde dehydrogenase and butanol dehydrogenase activities as well. The colony morphology of 2-bromobutyrate mutant R was similar to that of the parent on RCM medium; however, it had about 1/10 the level of CoA-transferase and increased levels of butanol dehydrogenase and butyraldehyde dehydrogenase. A nonsporulating, spontaneously derived degenerated strain exhibited reduced levels of butyraldehyde dehydrogenase, butanol, dehydrogenase, and CoA-transferase compared with those of the original strain. When C. acetobutylicum ATCC 824 was grown on medium containing low levels of 2-bromobutyrate, an altered colony morphology was observed. Not all strains resistant to 2-bromobutyrate (12 mM) were non-solvent-producing strains.
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A two-year Vermont program identified 494 (7 per cent) of 6,982 premarital female serologies that were seronegative (less than 1:8) to rubella by hemagglutination inhibition (HI) titer. All 494 susceptible patients and their physicians were notified of their results by letter. The State Health Department received reports that a total of 194 (39 per cent) of the susceptible patients had received rubella vaccinations as a result of their notifications. Intensive follow-up of susceptibles appears to be important factor in the success of premarital rubella screening programs.
Virus-specific, early appearing neutralizing activities (ENA) and interferon (IFN) were detected in tears collected from patients during epidemics of acute hemorrhagic conjunctivitis (AHC). In one study, ENA that neutralized enterovirus type 70 (EV70) was detected in tears collected from 114 of 130 AHC patients in Florida. In another study, ENA against coxsackievirus type A24 (CA24) was detected in tears collected from 39 of 57 patients in Singapore with AHC caused by CA24. No tear samples contained ENAs to both EV70 and CA24. Tear samples from uninfected eyes did not contain ENA to EV70 or CA24. ENA to EV70 was detected in 6 of 11 patients 1-6h before the onset of AHC. In addition, tears of 68% of patients seen on the day of onset produced tears that contained ENA to EV70. Thus, ENA to EV70 may be detected less than 24h after infection (based on 24h incubation period). IFN beta was detected in 30% of tear samples collected from patients on the day of onset of AHC caused by EV70. This finding suggested that ENA and IFN could act together to inhibit primary infections of AHC. It was found that the combination of ENA and IFN inhibited virus replication synergistically (greater than or equal to 300 fold reduction) in preinfected cells. Our findings suggest that ENA represents a previously unreported early defense mechanism of the eye, that endogenous ENA and endogenous IFN could inhibit viruses synergistically in vivo, and that ENA in tears could be useful in identifying the agent causing AHC.
An epidemic of acute hemorrhagic conjunctivitis in Miami, Florida, involved approximately 800 documented cases and more than 2,500 suspected cases. This epidemic was caused by an enterovirus 70 infection affecting primarily young black people residing within a high-risk area. Acute hemorrhagic conjunctivitis is characterized by the rapid onset of swollen eyelids, foreign-body sensation, burning, watery discharge, and, usually, bilateral ocular involvement. Signs include distinctive bulbar conjunctival hemorrhages and a follicular conjunctival reaction with only mild and infrequent corneal involvement. This infection is short in duration, self-limited, and free of significant ocular sequelae. Symptomatic treatment appears to be as effective as various topical medical regimens for relief of symptoms. Secondary bacterial infections (occurring in individuals who used urine as an eyewash) and one case of a transient acute Bell's palsy were the only complications associated with this acute hemorrhagic conjunctivitis epidemic.
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A 32-year-old man developed a recurrent nonrhegmatogenous retinal detachment, uveal effusion, and visual loss as a result of latent secondary syphilis. Treponemes were found in the subretinal fluid and the Treponema pallidum hemagglutination test demonstrated substantially higher titers in the subretinal fluid than in the serum (1: 2,569 vs 1: 16). Despite scleral dissection and a scleral implant and treatment with penicillin, the patient's visual loss persisted and the last examination showed a thickened choroid, a flat nonrhegmatogenous retinal detachment, and reaccumulation of subretinal fluid.
1. The hepatopancreas of terrestial snails actively synthesizes purines from labelled formic acid. 2. The guanine nucleotides are initially labelled to the highest specific activity followed by adenine and hypoxanthine. 3. The labelling patterns suggest that uric acid synthesis is the primary catabolic process for nitrogen excretion while guanine excretion is due to an inability to reutilize the base.