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Biomedical subjects

K A Johnson

Publications and source records attributed to K A Johnson.

At least 145 records · Page 8Linked to original sources
PubMed1996

Slow rate of phosphodiester bond formation accounts for the strong bias that Taq DNA polymerase shows against 2',3'-dideoxynucleotide terminators.

Taq and T7 DNA polymerases have become basic molecular biology "tools" for DNA sequence analysis. However, Taq, unlike T7 DNA polymerase, is strongly biased against the incorporation of 2',3'-dideoxynucleotide triphosphates (ddNTPs) indicating very different substrate selectivities. Equilibrium binding and rate constants were measured for 2',3'-ddNTPs as well as for several other 3'-substituted terminators and compared to 2'-deoxynucleotide substrates (dNTPs). In steady-state experiments, Taq Pol I was strongly biased in favor of dATP1 over ddATP incorporation by about 700 to 1, in contrast to T7 DNA polymerase which showed a preference of only about 4 to 1. Manganese reduced but did not eliminate selectivity against 2',3'-ddNTPs. Transient kinetic traces indicated different rate-limiting steps for substrate and terminator incorporation. Further mechanistic studies showed that the binding constants for substrates and terminators were equivalent. However, the rate constants for phosphodiester bond formation for 2',3'-ddNTPs were 200-3000-fold lower than for dNTPs. Alternative terminators showed only slight improvements. The data were consistent with a model in which both substrates and terminators undergo ground-state binding followed by formation of a tight-binding Enz.DNA.Nucleotide complex. Immediately after complex formation, substrates undergo a rapid nucleoside phosphoryl transfer reaction. However, the reaction rates for terminators were slower presumably due to misalignment of reactive groups in the active site. Thus, the strong bias that Taq DNA polymerase shows against terminators is due to a very slow "chemistry" step. Such a strong bias has several kinetic consequences for DNA sequence patterns. These consequences are discussed in the text.

Base Sequence↗
PubMed1996

HIV-1 reverse transcriptase resistance to nonnucleoside inhibitors.

The parameters governing the polymerization mechanism of reverse transcriptase containing the tyrosine to cysteine mutation at position 181 (Y181C) were determined using pre-steady-state techniques. The pathway for single nucleotide incorporation catalyzed by Y181C is similar to that determined for wild-type RT where a rate-limiting conformational change precedes fast chemistry and is followed by slow steady-state release of the primer/template. The Y181C mutant enzyme binds a 25/45-mer duplex DNA tightly with a Kd of 11 nM. However, the Y181C mutation weakens the nucleotide affinity 2-3-fold relative to the wild-type complex. We also determined the parameters governing the mechanism of nonnucleoside inhibitor resistance with Y181C. The Kd value of Nevirapine with the mutant E.DNA complex increased approximately 500-fold. The decreased affinity of Nevirapine for the mutant enzyme is a consequence of a faster inhibitor dissociation rate from the enzyme complex of Y181C relative to that of the wild-type. The E.DNA complex of Y181C may be saturated with Nevirapine, and the I.E.DNA complex is capable of a maximum incorporation rate of 0.1 s-1 (a 10-fold faster rate than that of the wild-type I.E.DNA complex). The overall two-step binding of nucleotide to Y181C in the presence of Nevirapine remains unaffected.

Antiviral Agents↗
PubMed1996

Expression of the human mismatch repair gene hMSH2 in normal and neoplastic tissues.

Hereditary nonpolyposis colorectal cancer is caused by inherited mutations of mismatch repair genes. We developed monoclonal antibodies to the prototype human mismatch repair gene hMSH2 and used them to detect an immunoreactive protein of M(r) 100,000 in mismatch-proficient cell lines. In addition, a M(r) 150,000 protein coimmunoprecipitated with the hMSH2 gene product in cell lines expressing hMSH2. Immunohistochemistry demonstrated that the hMSH2 protein was exclusively nuclear. Whereas the hMSH2 protein was expressed in a variety of tissues, the most striking pattern was observed in esophageal and intestinal epithelia, where expression was limited to the replicating compartment. Neoplastic cells within benign and malignant mismatch repair-proficient tumors expressed the protein, but no hMSH2 immunoreactivity was observed in the colorectal tumors of patients with germline hMSH2 mutation. These results have implications for tumorigenic mechanisms and, potentially, for diagnosis.

Animals↗
PubMed1996

Expression of the APC tumor suppressor protein in oligodendroglia.

Mutations of the adenomatous polyposis coli (APC) tumor suppressor gene have been linked to familial polyposis, an inherited predisposition to colon cancer, and a high percentage of sporadic colon adenomas. Although this gene is best known for its role in development of bowel neoplasms, in recent studies we have found that APC mRNA levels are greatly enriched in brain compared with peripheral tissues. To help define its role in the nervous system, in this study we have determined its cellular localization immunohistochemically in adult rat brain sections and have detected intense APC immunoreactivity in oligodendrocytes. Since prominent APC immunostaining is detected in cell bodies of mature oligodendrocytes, these antibodies may provide a useful addition to available oligodendrocyte markers. Although the cellular function of APC remains undefined, previous biochemical studies have demonstrated that APC is associated with catenins, cytoplasmic proteins involved in regulating cell-cell adhesion. We propose that, in addition to its critical role in ensuring normal maturation of colonic epithelial cells, the APC tumor suppressor protein also regulates the adhesive properties of oligodendrocytes.

Adenomatous Polyposis Coli↗
PubMed1996

Subchronic toxicity of ingested 1,3-dichloropropene in rats and mice.

Male and female Fischer 344 rats and B6C3F1 mice (10/sex/ dose group) were given 0, 5, 15, 50, or 100 mg/kg/day (rats) or 0, 15, 50, 100, or 175 (mice) mg/kg/day racemic 1,3-dichloropropene (1,3-D), respectively, via their diets for 13 weeks. Satellite groups of rats (recovery = 10 rats/sex/group) ingesting 0 or 100 mg/kg/ day 1,3-D were provided control feed for an additional 4 weeks to examine recovery. The test material was stabilized in the feed by microencapsulation in a starch/sucrose matrix (80/20). The body weights of male and female rats ingesting > or = 5 and > or = 15 mg/kg/ day, respectively, and of all treatment groups of mice were decreased relative to controls. The terminal body weights of high dose group rats and mice were decreased approximately 13-16%. A number of changes in serum biochemical parameters and decreases in organ weights accompanied the depressed body weights of these animals. Histologically, the only treatment-related change observed was a slight degree of basal cell hyperplasia and hyperkeratosis in the nonglandular portion of the stomachs of a majority of male and female rats ingesting > or = mg/kg/day. After the 4-week recovery period, most treatment-related changes were noted to be reversible in nature. No treatment-related histopathological changes were observed in the tissues of treated mice. Based upon relatively slight depressions in body weights at the lowest dosages tested, the no-observed-adverse-effect levels for male rats and both sexes of mice were determined to be 5 mg/kg/day and 15 mg/kg/ day, respectively. A no-observed-effect level of 5 mg/kg/day was established for female rats.

Administration, Oral↗
PubMed1996

Chronic dietary toxicity/oncogenicity studies on 2,4-dichlorophenoxyacetic acid in rodents.

Forms of 2,4-dichlorophenoxyacetic acid (collectively known as 2,4-D) are herbicides used to control a wide variety of broadleaf and woody plants. Doses in the 2-year chronic/oncogenicity rat study were 0, 5, 75, and 150 mg/kg/day. The chronic toxicity paralleled subchronic findings, and a NOEL of 5 mg/kg/day was established. A slight increase in astrocytomas observed (in males only) at 45 mg/kg/day in a previously conducted chronic rat study was not confirmed in the present study at the high dose of 150 mg/kg/ day. Doses in the 2-year mouse oncogenicity studies were 0, 5, 150, and 300 mg/kg/day for females and 0, 5, 62.5, and 125 mg/ kg/day for males. No oncogenic effect was noted in the study. In summary, the findings of these studies indicate low chronic toxicity of 2,4-D and the lack of oncogenic response to 2,4-D following chronic dietary exposure of 2,4-D in the rat and mouse.

2,4-Dichlorophenoxyacetic Acid↗
PubMed1996

Metabolic rate, organ mass, and mitochondrial proton leak variations in lean and obese rats.

The purpose of this study was to determine if differences in metabolic rate between lean and obese strains of rats were associated with differences in proton leak across the inner mitochondrial membrane. Metabolic rates were determined for each of five obese Zucker, Sprague-Dawley, and Fisher 344 rats and three lean Zucker rats by 24-hour indirect respiration calorimetry measurements. Feed intakes were different (P < 0.05) among all strains, with the obese Zucker rats having the greatest intakes. Adjusted to a common dietary intake, the obese Zucker rats had at least 21% lower heat productions than the lean strains of rats. Following the calorimetry measurements, the rats were sacrificed, internal organs were removed and weighed and mitochondria were isolated from the liver. Internal organs composed a larger proportion of lean body mass in obese compared to lean rats. Respiration rates and membrane potentials of the mitochondria were then determined. Proton leak kinetics were visualized by plotting proton leak (calculated from respiration rate) against membrane potential. The lean rats had a 2-3-fold higher proton leak rate than the obese Zucker rats at the same membrane potential. A low mitochondrial proton leak rate may explain part of the abnormal heat productions and bioenergetic efficiencies in the obese Zucker rat.

Animals↗
PubMed1996

Ultrasound imaging of the appendix testis and appendix epididymis.

The appendix testis and epididymis are well visualized on ultrasound examination. It is important to recognize the normal anatomy of the appendages to exclude them as a cause of pathology and confirm their presence as normal structures. We describe the appearances of the appendices testis and epididymis in fifty-one consecutive patients presenting to the Southampton Radiology Department. The appendix testis was identified on 80% of testes examined by ultrasound and the appendix epididymis on 6% of testes.

Adult↗
PubMed1996

Zygomatic osteoma with atypical heterogeneity in a dog.

An osteoma of the zygomatic bone in a young dog is described. It had large, centralized radiolucent regions consisting of fatty bone marrow and sparse trabeculae. A discrete, proliferative nodule within the osteoma consisted of closely-packed woven bone trabeculae and pleomorphic osteoblasts associated with haphazard osteoid deposits, resembling osteosarcoma-like change. These heterogeneous structural features were at variance with more classic reports of osteoma, which usually describe a uniform cancellous or cortical bone architecture.

Animals↗
PubMed1996

4-Phenylcyclohexene: 2-week inhalation toxicity and neurotoxicity studies in Swiss-Webster mice.

4-Phenylcyclohexene (4-PCH) is a by-product formed during the polymerization of styrene-butadiene latex used in carpet backing. Limited reports suggest that exposure to very low levels of 4-PCH or other emission products following new carpet installation may result in health complaints. Significantly, it has been claimed that Swiss-Webster mice held in neck restraints and exposed head-only to approximately 0.4 ppm 4-PCH for a few hours suffered severe toxicity including death. A 2-wk inhalation and neurotoxicity study was therefore conducted in Swiss-Webster mice using standard methods of toxicity testing. Groups of 40 mice were exposed to 0, 7, 18 or 71 ppm (near-saturated atmosphere) 4-PCH vapour, 6 hr/day for 9 consecutive days. Data were collected on a wide variety of clinical, neurological and histopathological parameters including extensive neurohistopathology. All animals survived the exposures, and there were no treatment-related effects. Because of the occurrence of spontaneous lesions in two high-dose group mice, 40 additional males were exposed to 0 ppm or a near-saturated atmosphere of 4-PCH under the same exposure regimen. No treatment-related lesions were observed in the follow-up study, confirming the conclusions of the original study. These findings, consistent with the reported lack of toxicity of inhaled 4-PCH in rats, do not suggest a direct, organic, association between low-level 4-PCH exposure and human complaints. Further, the results of this study suggest that positive findings in mice may have been due to methodological problems and not to exposure to 4-PCH.

Administration, Inhalation↗
PubMed1996

Methylmethacrylate and bone screw repair of seventh lumbar vertebral fracture-luxations in dogs.

Fracture-luxations of the seventh lumbar vertebrae in two large dogs were stabilised with bone screws and methylmethacrylate. Screws inserted bilaterally into the bodies of the lumbar vertebrae and ilial wings were left protruding by about 2 cm, and embedded in methylmethacrylate bone cement. Implant failure occurred in one dog six days after surgery, necessitating a revision of the fixation. Fracture healing and return of normal ambulation occurred in both dogs. Implants were removed in one dog because of screw loosening and discomfort.

Animals↗
PubMed1996

Life in a changing world: TCH gene regulation of expression and responses to environmental signals.

The Arabidopsis TCH genes were discovered as a consequence of their marked upregulation of expression in response to seemingly innocuous stimuli such as touch. Further analyses have indicated that these genes are upregulated by a variety of diverse stimuli. Understanding the mechanism(s) and factors that control TCH gene regulation will shed light on the signaling pathways that enable plants to respond to changing environmental conditions. The TCH proteins include calmodulin, calmodulin-related proteins and a xyloglucan endotransglycosylase. Expression analyses and localization of protein accumulation indicate that the potential sites of TCH protein function include expanding cells and tissues under mechanical strain. We hypothesize that the TCH proteins may collaborate in cell wall biogenesis.

Arabidopsis↗
PubMed1995

Detection of p21ras mutations in colorectal adenomas and carcinomas by enzyme-linked immunosorbent assay.

BACKGROUND: Point mutations of the ras protooncogene, primarily within codons 12 and 13, are commonly identified in colorectal carcinomas and large adenomas. Despite data suggesting that ras genotyping may have clinical significance with respect to colorectal cancer screening and prognosis, more widespread use has been limited because of the lack of a suitable assay system. The principal objective of this study was to assess the feasibility and validity of a qualitative enzyme-linked immunosorbent assay (ELISA) for detecting the four most common ras mutations in human colorectal tumors at the protein (p21ras) level. METHODS: Tissue homogenates (11-121 micrograms) from endoscopically or surgically resected colorectal adenomas, carcinomas, and normal mucosae were evaluated by a commercially available ELISA (Oncogene Science, Inc. Cambridge, MA) for mutant p21ras containing arginine position 12 (arg12), valine position 12 (val12), aspartate position 12 (asp12), and aspartate position 13 (asp13) amino acid substitutions. Portions of the same tissue from an initial series of 27 specimens also were subjected to mutant-enriched polymerase chain reaction (PCR) and/or PCR amplification with subsequent DNA sequence analysis to validate the ELISA data. RESULTS: Forty-seven adenomas, 9 carcinomas, and 14 normal mucosae were assayed. Mutations were identified in 16 (34%) of the adenomas (7-asp12, 7-val12, 2-asp13), 3 (33%) of the carcinomas (1-asp12, 1-arg12, 1-asp13), and none of the normal mucosae by ELISA: Polymerase Chain Reaction and DNA sequencing analyses demonstrated identical results for 21 of the 23 (91%) and 14 of 16 (88%) homogenates tested, respectively. The ELISA demonstrated an overall sensitivity of 80-86%, specificity of 90-92%, positive predictive value of 86-100%, and negative predictive value of 86-91%. CONCLUSIONS: The ELISA is a feasible and valid approach for identifying p21ras mutations in human colorectal adenomas and carcinomas.

Adenoma↗
PubMed1995

Reevaluating glyphosate as a transition-state inhibitor of EPSP synthase: identification of an EPSP synthase.EPSP.glyphosate ternary complex.

Numerous studies have confirmed that glyphosate forms a tight ternary complex with EPSP synthase and shikimate 3-phosphate. It has been proposed [Anton, D., Hedstrom, L., Fish, S., & Abeles, R. (1983) Biochemistry 22, 5903-5908; Steinrücken, H. C., & Amrhein, N. (1984) Eur. J. Biochem. 143, 351-357] that in this complex glyphosate functions as a transition-state analog of the putative phosphoenolpyruvoyl oxonium ion. For this to be true, glyphosate must occupy the space in the enzyme active site that is normally associated with PEP and, through turnover, the carboxyvinyl group of the product EPSP. According to this model, one would predict that, in the reverse EPSP synthase reaction with EPSP and phosphate as substrates, there should be little if any interaction of glyphosate with enzyme or enzyme.substrate complexes. In contrast to this expectation, rapid gel filtration experiments provided direct evidence that glyphosate could be trapped on the enzyme in the presence of EPSP to form a ternary complex of EPSPS.EPSP.glyphosate. The experimentally determined stoichiometry for this complex, 0.62 equiv of glyphosate/mole of EPSPS, is similar to that found for the EPSPS.S3P.glyphosate ternary complex (0.66). This direct binding result was corroborated and quantitated by fluorescence titration experiments which demonstrated that glyphosate forms a reasonably tight (Kd = 56 +/- 1 microM) ternary complex with enzyme and EPSP. This finding was further verified, and its impact on substrate turnover analyzed, by steady-state kinetics. Glyphosate was found to be an uncompetitive inhibitor versus EPSP with Kii(app) = 54 +/- 2 microM.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1995

Sedimentation studies on the kinesin motor domain constructs K401, K366, and K341.

Bacterial expressed kinesin motor domains hydrolyze ATP and promote microtubule-dependent motility. It has routinely been assumed that motor domain preparations are monomeric on the basis of the presumption that dimerization is mediated by the stalk region. However, experimental verification of the oligomeric state of the kinesin construct is required to interpret the results from single-molecule motility assays as well as presteady-state kinetic experiments. We have measured directly the state of assembly of three conventional kinesin motor domain constructs-K401, K366, and K341, comprising the N-terminal 401, 366, and 341 amino acids, respectively, of the Drosophila kinesin heavy chain-by sedimentation velocity and sedimentation equilibrium methods in an analytical ultracentrifuge. K401 (MW of ADP complex, 45,532) is a predominantly a dimer with a sedimentation coefficient, s020,w, of 5.06 S, but it is able to self-associate by means of a 1-2-4 mechanism into higher oligomers. Molecular weight measurements establish the dissociation constant for dimerization at 37 +/- 17 nM in the presence of ATP. The dissociation constant in the presence of ADP is 35 +/- 26 nM and in the presence of AMPPNP is 42 +/- 28 nM. The construct K366 (MW of ADP complex, 41,404) is a monomer (measured MW, 41,768 +/- 1219) at concentrations below 4 microM K366, with a sedimentation coefficient, s020,w, of 3.25 S. At higher concentrations, there is evidence for a weak association of K366 to a 1-2-4-8 model with a slight preference for octamer formation. The smallest construct, K341 (MW of ADP complex, 38,274), is a monomer (measured MW, 38,191 +/- 734) up to at least 10 microM total K341 concentration with a sedimentation coefficient, s020,w, of 2.9 S. Thus, the dimerization domain either is between amino acid residues 367 and 401 or is strongly affected by the removal of this region. Higher oligomers of K401 form by a mechanism involving dimers of dimers, and suggest that native kinesin may also undergo self-association. These results have important implications for the interpretation of ATP-dependent motility assays.

Adenosine Diphosphate↗