Search PubMed⌕ Search

Biomedical subjects

D A Johnson

Publications and source records attributed to D A Johnson.

At least 181 records · Page 10Linked to original sources
PubMed1991

In vivo antitumor activity of a monoclonal antibody-Vinca alkaloid immunoconjugate directed against a solid tumor membrane antigen characterized by heterogeneous expression and noninternalization of antibody-antigen complexes.

It is widely believed that antigen heterogeneity and noninternalization of antigen-antibody complexes will severely limit the antitumor activity of monoclonal antibody-drug conjugates. The B72.3 monoclonal antibody binds to a tumor-associated antigen which is heterogeneously expressed in human carcinomas (J. Schlom, Cancer Res., 46: 3225-3238, 1986). We therefore performed studies to assess the degree of internalization of B72.3 antibody-antigen complexes and the level of in vivo antitumor activity that could be achieved with B72.3 conjugated to 4-desacetyl vinblastine-3-carboxhydrazide. Internalization studies were performed on LS174T colorectal carcinoma and OVCAR-3 ovarian carcinoma cells using iodinated B72.3 as well as an iodinated antibody that binds to the human transferrin receptor, IIB21. These data indicated that, in contrast to HB-21, the B72.3 antigen-antibody complex was not internalized. The B72.3-Vinca alkaloid immunoconjugate demonstrated significant antitumor activity against LS174T xenografts, although complete regressions of established tumors were not achieved. Immunohistochemical analyses indicated that the B72.3 antigen was heterogeneously expressed in the LS174T xenografts and that tumor cells which were not killed by high doses of B72.3-Vinca also expressed the B72.3 antigen. These studies indicated that significant antitumor activity may be achieved by monoclonal antibody-drug conjugates even when antigen heterogeneity and noninternalization of antigen-antibody complexes are encountered. The data also suggested that the formulation of antibody-drug conjugate cocktails to counteract antigen heterogeneity may not be sufficient to eradicate all malignant cells within a solid tumor mass.

Animals↗
PubMed1991

Diazepam administered prior to coronary artery occlusion increases latency to ventricular fibrillation.

Few studies have addressed the antiarrhythmic potential of pretreatment with diazepam in acute myocardial infarction. Thus, the effect of diazepam pretreatment prior to coronary artery occlusion was examined in conscious pigs. Animals were instrumented with aortic catheters to measure arterial pressure, a pulmonary artery catheter for drug administration, and a snare around the left anterior descending coronary artery for permanent occlusion one week later. Diazepam (1 mg/kg iv bolus) or vehicle was administered 10 minutes prior to occlusion. Eight of 14 animals receiving diazepam (57%) and 13 of 22 receiving vehicle animals (59%) developed ventricular fibrillation following coronary occlusion. However, the latency to ventricular fibrillation was significantly shorter (7 +/- 1 min) in animals receiving vehicle compared to animals receiving diazepam (11 +/- 1 min). Significant increases in heart rate were seen up to 5 hours after coronary occlusion only in animals receiving vehicle. The results indicate that diazepam pretreatment can increase ventricular fibrillation latency and prevent heart rate increases following acute myocardial infarction.

Animals↗
PubMed1991

Reversible control of oestradiol-stimulated growth of MCF-7 tumours by tamoxifen in the athymic mouse.

We investigated the ability of high concentrations of oestradiol to reverse the growth inhibitory action of tamoxifen on MCF-7 breast cancer cells in vivo. Tamoxifen inhibits the oestradiol stimulated growth of MCF-7 cells in athymic mice. Using a sustained release preparation of tamoxifen we consistently achieved serum concentrations of the drug in the 40 to 50 ng ml-1 range and much higher levels in tissues. These serum levels are sufficient to inhibit the oestrogen stimulated growth of MCF-7 tumours exposed to physiologic (i.e. 300-600 pg ml-1 serum oestradiol concentrations). However, by administering dosages that increase serum oestradiol concentrations to 900-2000 pg ml-1, mimicking the increase often observed clinically in premenopausal women taking tamoxifen, we show that the growth inhibitory action of tamoxifen can be partially reversed. Serum tamoxifen levels were elevated to nearly 400 ng ml-1 by injecting 1 mg day-1 tamoxifen (IP 3 x weekly); this dosage was more effective at inhibiting oestradiol stimulated tumour growth than subcutaneous tamoxifen capsules alone. Our data suggest that at low serum levels tamoxifen may not act optimally. There may be a need to monitor tamoxifen levels in premenopausal patients to ensure that they are high enough not to be overcome by a tamoxifen induced increase in ovarian steroidogenesis.

Animals↗
PubMed1991

Distinctions in ligand binding sites on the nicotinic acetylcholine receptor.

Ligand-gated ion channels possess intrinsic binding sites for noncompetitive inhibitors that differ substantially in ligand specificity and structural characteristics from most binding sites found on globular proteins. We have used the nicotinic acetylcholine receptor to examine the characteristics of such diverse sites because the high-affinity binding site in the proximity of the ion channel has unusual binding interactions and ligand specificity, whereas the site of agonist activation exhibits classical structure-activity characteristics. Noncompetitive inhibitors that bind to the former site show a wide degree of structural variation and appear to associate at separate loci and in distinct orientations in the vicinity of the channel. The receptor structure appears to provide a large domain with multiple hydrophobic crevices that can bind noncompetitive inhibitors, yet binding of these inhibitors is mutually exclusive. The mutually exclusive behavior suggests that association of a single ligand is sufficient to prevent access of additional ligands to distinct sites. This could occur either by physical occlusion to the site of binding or by formation of a conformational state that will not allow entry of additional noncompetitive inhibitors.

Amino Acid Sequence↗
PubMed1991

Behavioral arousal enhances inducibility and rate of ventricular tachycardia.

Behavioral arousal may trigger malignant cardiac arrhythmias. To study the effect of arousal on ventricular tachycardia, pigs were instrumented with catheters to measure mean arterial pressure and sample plasma catecholamines and left anterior descending coronary artery snares for occlusion 1 wk later. Bipolar pacing catheters were placed in the right ventricular apex to induce ventricular tachycardia. One week after occlusion, electrophysiological testing was repeated before and immediately after arousal caused either by restraining and lifting the pig in a canvas sling or by bringing a stall mate into the room. The number of stimuli needed to induce monomorphic ventricular tachycardia was reduced by both types of arousal (P less than 0.05) compared with control conditions. Ventricular tachycardia rate was increased 60 +/- 17 beats/min after lifting stimulation (P less than 0.05). When beta 1-receptor blockade was induced by metoprolol, inducibility of ventricular tachycardia and rate were not different from control. Thus, in pigs, arousal may facilitate arrhythmogenesis. This effect may be mediated by sympathetic neural activity in the heart because it was annulled by beta 1-adrenergic blockade.

Animals↗
PubMed1991

The effect of increased mastication by daily gum-chewing on salivary gland output and dental plaque acidogenicity.

The effect of increased mastication on plaque metabolism and salivary gland function was determined in 11 human subjects who chewed a sugarless gum for ten minutes of each waking hour for two weeks. Prior to and at the conclusion of the gum-chewing regimen, unstimulated whole saliva and 2% citric-acid-stimulated parotid saliva were collected. Flow rates, pH, and buffer capacity were determined on all saliva samples. In addition, parotid saliva was analyzed for protein concentration and the proteins further studied by SDS-PAGE. The plaque pH response to a 10% sucrose rinse was also measured before and after the regimen. Significant increases were observed in the pH and buffer capacity of unstimulated whole saliva as were similar increases in the flow rate, pH, and buffer capacity of stimulated parotid saliva. Protein concentrations and profiles remained unaffected. In addition, the resting plaque pH and minimum plaque pH reached after a sucrose challenge were both raised significantly, with a significant reduction in the cH area. The results of this study indicate that increased masticatory effort by frequent consumption of sugar-free chewing gum over a prolonged time period resulted in a functional increase in the output of stimulated parotid saliva, as well as in increases in pH and buffer capacity of whole and parotid saliva, which may help to reduce plaque acidogenicity.

Acids↗
PubMed1991

Effect of fixation on the amplification of nucleic acids from paraffin-embedded material by the polymerase chain reaction.

Amplification of nucleic acids from paraffin-embedded material by the polymerase chain reaction (PCR) is increasingly being used to detect viral genomes and oncogene mutations. To determine the effect of fixation on the preservation of the nucleic acids, we fixed two randomly chosen fresh pathology specimens in formalin, B-5, Bouin's, Zenker's, ethanol, and Omnifix for 6, 24, 48, 72, and 168 hr (1 week), and then embedded the tissue in paraffin. Oligonucleotide primers specific for the cytoplasmic-beta-actin gene were chosen to span an intron such that amplification yielded a product of 250 BP for DNA and 154 BP for RNA. A single 6-microns section was cut from each paraffin block, deparaffinized, and then subjected to 30 rounds of amplification for either DNA or RNA. On amplifying DNA, consistent product was seen in the ethanol and Omnifix specimens up to 72 hr of fixation time, whereas variable product was seen with formalin or Zenker's fixation; all specimens fixed in Bouin's or B-5 were negative. On amplifying RNA, a product could be detected even after 1 week of fixation in ethanol or Omnifix, and after 48 hr in the formalin-fixed tissue. The Zenker's-fixed tissues gave variable results, and the Bouin's and B-5 tissues gave consistent results only after 6 hr of fixation. We therefore conclude that choice of fixative and fixation time are critical factors influencing the outcome of PCR amplification of nucleic acids from paraffin-embedded material.

Base Sequence↗
PubMed1991

Development and evaluation of CHIPASAT, an attention test for children: II. Test-retest reliability and practice effect for a normal sample.

The CHIPASAT (Children's Paced Auditory Serial Addition Task), developed to assess auditory-verbal attention, was administered to a sample of 70 normal school children on two occasions separated by an interval of approximately 4 weeks. A significant practice effect was found, independent of age and sex, with performance improved by approximately 20% on retest. The test-retest reliability of .90 was significant, indicating task stability. The practice effect was consistent with that reported in the literature for the adult PASAT, and reasons for this effect are discussed. Initial test raw scores on WISC--R Arithmetic and Digit Span subtests yielded low but significant correlations of .38 and .35 with CHIPASAT scores.

Adolescent↗
PubMed1991

Diltiazem therapy for symptoms associated with nutcracker esophagus.

UNLABELLED: A randomized double-blind, cross-over prospective trial in 22 patients was designed to evaluate possible effect of an oral calcium channel blocker, diltiazem, on symptoms of chest pain and/or dysphagia in patients with nutcracker esophagus. We studied 22 consecutive patients referred to an esophageal diagnostic center for evaluation of noncardiac chest pain or dysphagia having high amplitude esophageal contractions, 14 of whom completed the study. Diltiazem (60-90 mg qid) was compared with placebo, each being administered for 8 wk. Patients were evaluated with esophageal motility pre- and posttreatment periods and with regular symptom assessment throughout each 8-wk treatment. Active diltiazem therapy resulted in significantly lower (p less than 0.05) mean distal esophageal peristaltic pressure (128 +/- 20 mm Hg; +/- SE) than placebo (158 +/- 16 mm Hg). Mean chest pains scores were significantly (p less than 0.05) lower with diltiazem therapy than with placebo. Only nine of the 14 patients fulfilled presently acceptable criteria for diagnosing nutcracker esophagus, and the diltiazem effect was similar, although not significant, because of the smaller sample. CONCLUSIONS: In this preliminary study involving 14 patients, the oral calcium channel blocker, diltiazem, appeared to improve noncardiac chest pain associated with strong esophageal contraction, the nutcracker esophagus. These improved symptoms were associated with significant decreases in contraction pressure.

Adult↗
PubMed1990

The effect of bovine lactoferrin on muscle growth in vivo and in vitro.

Lactoferrin was found to be a potent stimulator of proliferation for L6 myoblasts. Both apo and holo-forms of lactoferrin were equipotent. By contrast, only the holo-form of transferrin (a structurally related iron binding protein) stimulated proliferation, apo-transferrin was without activity. Holo-transferrin was also less stimulatory than lactoferrin. Purified lactoferrin was administered to mature female rats and to neonatal rats by daily subcutaneous injection to determine if there was a measurable effect on muscle cell growth in vivo. Results from the in vivo studies suggest that lactoferrin has little or no effect on muscle cell growth in the whole animal.

Animals↗
PubMed1990

The Glu residue in the conserved Asn-Glu-Pro sequence of two highly divergent endo-beta-1,4-glucanases is essential for enzymatic activity.

We initially aligned 28 different cellulase sequences in pairwise fashion and found half of them have the sequence -Asn-Glu-Pro- located in a region flanked by hydrophobic-rich amino acids. Based on lysozyme as a model, the glutamate residue could be essential for enzyme function. We tested this possibility by site-directed mutagenesis of the genes coding Bacillus polymyxa and Bacillus subtilis endo-beta-1,4-glucanases. The genes and amino acid sequences of these two enzymes show very little similarity. Change of Glu-194 and Glu-169 to the isosteric glutamine form in these respective enzymes resulted in a dramatic loss of CMCase activity which could be restored by reverse mutation. Similar mutations to less-conserved residues, Glu-72 and Glu-147, of the B. subtilis enzyme did not cause any loss of activity.

Amino Acid Sequence↗
PubMed1990

Orientation of cobra alpha-toxin on the nicotinic acetylcholine receptor. Fluorescence studies.

Four flourescein isothiocyanate (FITC) derivatives of Naja naja siamemsis 3 neurotoxin (alpha-toxin), labeled at the epsilon-amino groups of Lys-23, Lys-35, Lys-49, or Lys-69, and a tetramethylrhodamine isothiocyanate (TRITC) derivative, labeled at epsilon-amino group of Lys-23, were prepared and used to analyze the orientation of cobra alpha-toxin on the nicotinic acetylcholine receptor (AcChR) relative to both the plane of the membrane and the central ion channel. Fluorescence-quenching studies of the AcChR-bound FITC derivatives indicated significant solute accessibility to each site of labeling and suggested that none of the sites of FITC labeling is included in the binding surface of the alpha-toxin. Labeling of Lys-23 with TRITC did not affect the affinity of the alpha-toxin toward the AcChR and confirmed, contrary to some previous reports, a minimal role of Lys-23 in the binding surface of the alpha-toxin. Measurements of energy transfer between the lipid-membrane surface and the sites of labeling on receptor-bound alpha-toxin derivatives show that the relative distances of closest approach between the surface of the lipid membrane domain and the sites of labeling are in the order Lys-23 less than or equal to Lys-49 less than Lys-35 less than or equal to Lys-69. Energy transfer between AcChR tryptophans and the sites of labeling of bound derivatives was about 50% greater to Lys-49 than to Lys-23, Lys-35, or Lys-69, suggesting that Lys-49 is closer to receptor tryptophans and to the center of the extracellular domain of the receptor than Lys-23, Lys-35, or Lys-69. Combined with previous observations that the tip of the central loop of the alpha-toxin directly interacts with the AcChR, the above results suggest a model of the approximate orientation of the snake neurotoxins on the receptor. This model shows the tip of the central loop of the toxin directly interacting with the receptor surface and the major axis of the neurotoxin tilting from a perpendicular projection from the membrane. The surface of the alpha-toxin that includes Lys-23 projects away from the central ion channel and the surface that includes Lys-35 and Lys-69 faces the ion channel.

Amino Acid Sequence↗
PubMed1990

Antitumor activity of L/1C2-4-desacetylvinblastine-3-carboxhydrazide immunoconjugate in xenografts.

The murine IgG3 monoclonal antibody L/1C2 is reactive with a high percentage of human carcinomas and has preferentially strong reactivity with tumors of squamous differentiation. This antibody was tested for antitumor activity in vitro and in xenograft models as a carbohydrate-linked immunoconjugate with the Vinca derivative 4-desacetylvinblastine-3-carboxhydrazide (DAVLBHYD). The conjugate retained good immunoreactivity and was highly active in a cytotoxicity assay. In human tumor nude mouse xenograft studies, L/1C2-DAVLBHYD antitumor activity was superior to that seen with free drug, free antibody, mixtures of free drug and free antibody, or control DAVLBHYD conjugates prepared with non-tumor-binding IgGs. With well-established tumors, potent antitumor activity was observed, including the ability to specifically regress greater than 400-mg tumors to 0 mg. In some cases, apparent long-term cures were effected. In studies using six different human tumor xenografts, the level of potency of L/1C2-DAVLBHYD was related to L/1C2 antigen expression, although the growth rate probably also contributes to the conjugate sensitivity of the tumors.

Animals↗
PubMed1990

Temperature-dependent lateral and transverse distribution of the epidermal growth factor receptor in A431 plasma membranes.

To elucidate further the structure and molecular dynamics of the epidermal growth factor receptor, temperature-dependent aggregation and extracellular protrusion of the epidermal growth factor receptor in isolated plasma membranes from A431 cells were examined by fluorescence energy-transfer techniques. Epidermal growth factor was labeled at the amino terminus with either fluorescein isothiocyanate or tetramethylrhodamine isothiocyanate. A radionuclide receptor displacement assay demonstrated the bioactivity of these derivatives. Aggregation of the epidermal growth factor receptor was measured by determining the increase in fluorescence energy transfer between receptor-bound fluorescein and tetramethylrhodamine-labeled epidermal growth factor. Energy transfer between receptor-bound fluorescent derivatives was reversibly greater at 37 than 4 degrees C, indicating temperature-dependent aggregation of the receptor. The extracellular protrusion of the epidermal growth factor receptor was calculated from the magnitude of energy transfer between receptor-bound fluorescein labeled epidermal growth factor and 5-(N-dodecanoylamino)-eosin partitioned into the lipid membrane at 4 and 37 degrees C. No significant change in the distance of closest approach between the N-terminus of epidermal growth factor and the plasma membrane was observed at 4 degrees C (69 +/- 2 A) and 37 degrees C (67 +/- 2 A). Thus, the extracellular protrusion of the occupied epidermal growth factor receptor did not change detectably upon receptor aggregation.

Animals↗
PubMed1990

Genetic structure of natural populations of the nitrogen-fixing bacterium Rhizobium meliloti.

The genetic structure of populations of the symbiotic nitrogen-fixing soil bacterium Rhizobium meliloti was examined by analysis of electrophoretically demonstrable allelic variation in 14 metabolic, presumably chromosomal, enzyme genes. A total of 232 strains were examined, most of which were isolated from southwest Asia, where there is an unsurpassed number of indigenous host species for R. meliloti. The collection consisted of 115 isolates recovered from annual species of Medicago in Syria, Turkey, and Jordan; 85 isolates cultured from two perennial species of Medicago (M. sativa [alfalfa] and M. falcata) in northern Pakistan and Nepal; and 32 isolates collected at various localities in North and South America, Europe, South Africa, New Zealand, and Australia, largely from M. sativa. Fifty distinctive multilocus genotypes (electrophoretic types [ETs]) were identified, and cluster analysis revealed two primary phylogenetic divisions separated at a genetic distance of 0.83. By the criterion of genetic differentiation conventionally applied in defining species limits among members of the family Enterobacteriaceae and certain other bacteria, the two primary divisions of R. meliloti represent distinct evolutionary species. Division A included 35 ETs represented by 209 strains from the eastern Mediterranean basin, northern Pakistan, Nepal, and various other localities worldwide. This division contained the nine commercial alfalfa inoculant strains examined. Division B included 15 ETs represented by 23 isolates, 21 of which were isolated from annual medic species growing in previously uninoculated soils in the eastern Mediterranean basin. The two remaining strains in division B, both representing the same ET, were isolated in the United States and Australia.(ABSTRACT TRUNCATED AT 250 WORDS)

Alleles↗
PubMed1990

Molecular cloning, expression, and characterization of endo-beta-1,4-glucanase genes from Bacillus polymyxa and Bacillus circulans.

Endo-beta-1,4-glucanase genes from Bacillus circulans and from B. polymyxa were cloned by direct expression by using bacteriophage M13mp9 as the vector. The enzymatic activity of the gene products was detected by using either the Congo red assay or hydroxyethyl cellulose dyed with Ostazin Brilliant Red H-3B. The B. circulans and B. subtilis PAP115 endo-beta-1,4-glucanase genes were shown to be homologous by the use of restriction endonuclease site mapping, DNA-DNA hybridization, S1 nuclease digestion after heteroduplex formation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the protein products. Analysis of the nucleotide sequence of 3.1 kilobase pairs of cloned B. polymyxa DNA revealed two convergently transcribed open reading frames (ORFs) consisting of 398 codons (endoglucanase) and 187 codons (ORF2) and separated by 374 nucleotides. The coding region of the B. polymyxa endoglucanase gene would theoretically produce a 44-kilodalton preprotein. Expression of the B. polymyxa endoglucanase in Escherichia coli was due to a fusion of the endoglucanase gene at codon 30 with codon 9 of the lacZ alpha-peptide gene. The B. polymyxa endoglucanase has 34% amino acid similarity to the Clostridium thermocellum celB endoglucanase sequence but very little similarity to endoglucanases from other Bacillus species. ORF2 has 28% amino acid similarity to the NH2-terminal half of the E. coli lac repressor protein, which is responsible for DNA binding.

Amino Acid Sequence↗