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Biomedical subjects

J S Johnson

Publications and source records attributed to J S Johnson.

At least 55 records · Page 3Linked to original sources
PubMed1996

Temperature-regulated expression of the tac/lacl system for overproduction of a fungal xylanase in Escherichia coli.

Temperature-regulated expression of recombinant proteins in the tac promoter (Ptac) system was investigated. Expression levels of fungal xylanase and cellulase from N. patriciarum in E. coli strains containing the natural lacI gene under the control of the Ptac markedly increased with increasing cultivation temperature in the absence of a chemical inducer. The specific activities (units per milligram protein of crude enzyme) of the fungal xylanase and cellulase produced from recombinant E. coli strain pop2136 grown at 42 degrees C were about 4.5 times higher than those of the cells grown at 23 degrees C and were even slightly higher when compared with cells grown in the presence of the inducer isopropyl beta-D-thiogalactopyranoside. The xylanase expression level in the temperature-regulated Ptac system was about 35% of total cellular protein. However, this system can not be applied to E. coli strains containing lacIq, which confers over production of the lac repressor, for high-level expression of recombinant proteins. In comparison with the lambda PL system, the Ptac-based xylanase plasmid in E. coli pop2136 gave a considerably higher specific activity of the xylanase than did the best lambda PL-based construct using the same thermal induction procedure. The high-level expression of the xylanase using the temperature-regulated Ptac system was also obtained in 10-litre fermentation studies using a fed-batch process. These results unambiguously demonstrated that the temperature-modulated Ptac system can be used for overproduction of some non-toxic recombinant proteins.

Base Sequence↗
PubMed1995

Inhibition of matrix metalloproteinases by hydroxamates containing heteroatom-based modifications of the P1' group.

In this study, structure-based drug design of matrix metalloproteinase inhibitors [human fibroblast collagenase (HFC), human fibroblast stromelysin (HFS), and human neutrophil collagenase (HNC)] was utilized in the development of potent hydroxamates which contain novel, heteroatom-based modifications of the P1' group. A series containing a P1' butyramide group resulted in a nanomolar potent and selective HNC inhibitor as well as a dual HFS/HNC inhibitor. Benzylic ethers with a four- or five-carbon methylene linker in the P1' position also produced nanomolar potent HFS/HNC inhibition and micromolar potent HFC inhibition as expected. Surprisingly, the phenolic ethers of the same overall length as the benzylic ethers showed nanomolar potencies against HFC, as well as HFS and HNC. The potency profile of the phenolic ethers was optimized by structure-activity relationships of the phenolic group and the C-terminal amide. These inhibitors may help elucidate the in vivo roles of matrix metalloproteinases in normal and disease states.

Crystallography, X-Ray↗
PubMed1995

Induction and reversal of long-lived specific unresponsiveness to a T-dependent antigen following CTLA4Ig treatment.

We have examined the ability of CTLA4Ig to induce long-lasting Ag-specific tolerance to a T-dependent Ag. Treatment of mice with murine CTLA4Ig following immunization with SRBC induced immune unresponsiveness to SRBC that was only reversible upon repeated Ag exposure. This unresponsiveness was Ag specific, lasted > 90 days, was observed in thymectomized mice, and was maintained even during challenge with another strong T-dependent Ag. A third challenge with SRBC, however, led to an Ab response. Splenic T cells and B cells from unresponsive mice were functional upon transfer to irradiated hosts, indicating that they had been neither depleted nor rendered permanently "tolerant" by CTLA4Ig. The mechanism of immunosuppression by CTLA4Ig was investigated by measuring cytokine transcripts in spleens of immunized mice. CTLA4Ig treatment following primary immunization blocked the induction of IL-2 transcripts in splenic T cells and IL-4 transcripts in both T cells and non-B, non-T cells. Splenocytes from CTLA4Ig-treated, SRBC-unresponsive mice showed altered induction of IL-2 and IL-4 transcripts, but T cells nonetheless became primed for IL-4 mRNA synthesis despite the lack of a measurable Ab response. Anti-IL-4 mAb and human CTLA4Ig were synergistic in their ability to induce unresponsiveness, indicating that incomplete suppression of IL-4 production by CTLA4Ig limited its effectiveness.

Abatacept↗
PubMed1995

Modification of a xylanase cDNA isolated from an anaerobic fungus Neocallimastix patriciarum for high-level expression in Escherichia coli.

A Neocallimastix patriciarum xylanase cDNA with the core coding sequence essentially identical to xynA was isolated and modified for high-level expression in Escherichia coli. The xylanase cDNA was truncated into individual catalytic domains, which were modified at the N-terminus. These modified xylanases were synthesised as non-fusion proteins under the control of the tac promoter. High-level expression was obtained with the modified domain II construct, accounting for approx. 25% of total cellular protein. However, with the same vector and expression cassette, expression levels of constructs containing domain I or domains I and II fused in tandem were very low. RNA analysis revealed that the striking difference in expression levels of these three constructs was not due to transcription efficiency, but was mainly related to transcript stability. Further analysis of the domain II construct revealed that the high-level expression of the domain II xylanase was largely attributed to the presence of a favourable N-terminal coding sequence, as mutation at the N-terminus of the domain II dramatically reduced the expression level. The modified domain II xylanase produced in E. coli had a specific activity of 1229 U mg-1 protein at pH 7 and 50 degrees C without purification. The availability of a recombinant fungal xylanase with high specific activity and in high yield offers a potentially attractive source of xylanase for industrial applications.

Animals↗
PubMed1995

In situ lymphoproliferation in renal transplant biopsies.

A double immunohistochemical labelling procedure in paraffin-embedded renal tissue is reported in which CD3 was targeted as a T cell marker and Ki67 as a marker of cell proliferation. Proliferating and quiescent T cells were unequivocally identified in situ, and their precise location within the kidney was clarified by the use of periodic acid-Schiff counterstaining to outline the basement membranes. Proliferating tubular epithelial cells were also clearly identified. The results showed that T lymphocytes proliferate within the tubular compartment during acute renal allograft rejection. Preliminary evaluation of the method in routine transplant biopsies indicated significant correlations between histologically defined rejection grade and mean intratubular T lymphocytes per tubular cross section and between proliferation of tubular epithelial cells and of intratubular T lymphocytes. The associated tubular epithelial cell proliferation may be a response to local damage.

Antigens, Differentiation↗
PubMed1994

CTLA4Ig treatment ameliorates the lethality of murine graft-versus-host disease across major histocompatibility complex barriers.

Graft-versus-host disease (GVHD), a pathological condition associated with BMT, results from activation of donor T lymphocytes by host tissues. CD28 and CTLA-4 are structurally related T cell receptors for members of the B7 (CD80) gene family, which transmit important costimulatory signals for T cell activation in vitro and in vivo. Here we have investigated the effects of CTLA4Ig, a soluble form of CTLA-4, on lethal GVHD in a murine model. Lethal GVHD was induced by transfer of parent C57BL/6 bone marrow and spleen cells into lethally irradiated (C57BL/6 x DBA/2)F1 recipients. Short courses of treatment with CTLA4Ig did not block engraftment, but prolonged survival of BMT recipients even when administration was delayed for 6 days after transplantation. CTLA4Ig-treated survivors of GVHD maintained body weight and did not exhibit visible signs of GVHD. However, treatment regimens that maximally prolonged survival did not detectably prevent T cell-mediated hematological abnormalities associated with GVHD, including pancytopenia and abnormal cellular composition of the spleen. Our data thus show that the lethality of acute GVHD in this model system is more dependent upon CD28/CTLA-4 costimulation than are other GVHD-associated abnormalities, and can be blocked for an extended period by brief treatment with CTLA4Ig.

Abatacept↗
PubMed1994

1.56 A structure of mature truncated human fibroblast collagenase.

The X-ray crystal structure of a 19 kDa active fragment of human fibroblast collagenase has been determined by the multiple isomorphous replacement method and refined at 1.56 A resolution to an R-factor of 17.4%. The current structure includes a bound hydroxamate inhibitor, 88 waters and three metal atoms (two zincs and a calcium). The overall topology of the enzyme, comprised of a five stranded beta-sheet and three alpha-helices, is similar to the thermolysin-like metalloproteinases. There are some important differences between the collagenase and thermolysin families of enzymes. The active site zinc ligands are all histidines (His-218, His-222, and His-228). The presence of a second zinc ion in a structural role is a unique feature of the matrix metalloproteinases. The binding properties of the active site cleft are more dependent on the main chain conformation of the enzyme (and substrate) compared with thermolysin. A mechanism of action for peptide cleavage similar to that of thermolysin is proposed for fibroblast collagenase.

Amino Acid Sequence↗
PubMed1993

In a drug discrimination procedure isolation-reared rats generalize to lower doses of cocaine and amphetamine than rats reared in an enriched environment.

Rats with different behavioral histories, defined by rearing and housing in either an enriched condition (EC) or an isolation condition (IC), were trained in a two-lever operant procedure to discriminate 5.0 mg/kg cocaine from saline. In cocaine dose-generalization tests, the IC rats exhibited an ED50 (1.01 mg/kg) significantly lower than the EC rats (ED50: 1.55 mg/kg). The cocaine-appropriate responding was emitted when the rats were treated with d-amphetamine, and for the d-amphetamine test doses the ED50 (0.19 mg/kg) was again significantly lower for the IC rats compared to the ECs (ED50: 0.33 mg/kg). These data suggest that IC rats are more sensitive to the stimulus properties of indirect dopaminergic agonists than EC rats and highlight the importance of environmental variables in governing an organism's response to the stimulus properties of abused drugs.

Animals↗
PubMed1993

Superficial tinea infections.

Dermatophytes commonly cause superficial dermatoses, especially in children, but these infections are frequently misdiagnosed as nonfungal disease. Superficial tinea infections can vary widely in appearance, ranging from a single, small, circular lesion with mild erythema and a scaling active border to multiple large areas of marked inflammation with vesicle formation. Therefore, laboratory tests, particularly the potassium hydroxide slide preparation, are often necessary to establish the diagnosis. The culture becomes particularly important if the smear is negative but a superficial dermatosis is strongly suspected. Tinea capitis is frequently diagnosed only by culture. Since many agents are available to treat superficial dermatoses, cost should be an important consideration in the selection of a topical antifungal agent. Oral agents are recommended for scalp and nail infections, as well as extensive body infections. Griseofulvin is the oral agent of choice because of its efficacy and low toxicity.

Humans↗
PubMed1992

Evaluation of a dual-channel full dynamic range compression system for people with sensorineural hearing loss.

This article describes an evaluation of an in the ear hearing aid, which applies fast-acting full dynamic range compression independently in two frequency bands. This can compensate for the loudness recruitment typically associated with sensorineural hearing loss. The crossover frequency between the two bands and the gain and compression ratio in each band are programmable to suit the individual patient. Twenty subjects with moderate sensorineural hearing loss were tested in a counterbalanced order using the aid programmed as a linear amplifier (condition L) and as a two-band compressor (condition C). All subjects were fitted binaurally. Subjects were also tested without hearing aids (condition U) and using the hearing aids that they normally wore (condition Own). Speech intelligibility was measured in quiet at three sound levels (50, 65, and 80 dB SPL), and speech reception thresholds (SRTs) in 12-talker babble were measured under monaurally and binaurally aided conditions, with the speech and babble both coincident and spatially separated. In condition C, speech intelligibility in quiet was high at all sound levels. Speech intelligibility at the two lower levels decreased in condition L, and decreased still further in conditions Own and U. Condition C gave, on average, better speech intelligibility in babble (lower SRTs) than conditions L, Own, or U. The advantage of condition C over condition L varied across subjects and was correlated with the dynamic range for tones at high frequencies; small dynamic ranges were associated with greater benefit from compression. A significant advantage for binaural aiding was found both when the speech and noise were spatially separated and when they were coincident. The binaural advantage was similar for the C and L conditions, indicating that the independent compression at the two ears did not adversely affect the use of binaural cues. Questionnaires on the subjects' experiences with the aids in everyday life indicated that they generally preferred condition C over condition L.

Adult↗
PubMed1992

Profuse diarrhea after misoprostol use in a patient with a history of Crohn's disease.

OBJECTIVE: To report a case of profuse diarrhea after misoprostol use in a patient with a history of Crohn's disease and to discuss the role of eicosanoids in Crohn's disease. DATA SOURCES: Patient medical records, case reports, review articles identified by MEDLINE, and personal communication with the physician, patient, and manufacturer. DATA EXTRACTION: From interviews, the manufacturer, and pertinent published sources by one author and reviewed by the others. DATA SYNTHESIS: A 55-year-old woman presented to clinic complaining of multiple joint pains. Her medical history was significant for peptic ulcer disease, hypertension, and Crohn's disease in remission since May 1989. Her joint pains were treated with ibuprofen 600 mg po qid and misoprostol 200 micrograms po qid (after meals and at bedtime). Following the administration of three doses of ibuprofen and misoprostol, the patient experienced abdominal cramps, pain, and voluminous, watery diarrhea for two days. Upon discontinuation of the ibuprofen and misoprostol, all of her gastrointestinal symptoms resolved within 12 hours. Rechallenge with ibuprofen alone failed to produce a recurrence of symptoms. Enhanced synthesis of intestinal eicosanoids has been demonstrated in Crohn's disease. Misoprostol, a synthetic analog of one of the eicosanoids, could induce a flare-up of Crohn's disease as suggested in this patient. CONCLUSIONS: Misoprostol should be used with caution in patients with known inflammatory bowel disease.

Crohn Disease↗
PubMed1992

Patient experiences with multiband full dynamic range compression.

This investigation compared performance for 18 experienced hearing aid patients with a multiband full dynamic range compression hearing device and their previously worn linear or automatic gain control hearing aids. Performance was evaluated using a variety of clinical tests. These included functional gain and dynamic range measurements, speech recognition, and real ear measurements. Patients rated their performance with the multiband compression device and their previously worn aids on a rating scale pertaining to a variety of real life listening situations. Statistical analysis of data derived from test results shows significantly improved performance on all measures using the multiband compression aid over their conventional aids. Patients rated their performance higher with the compression instrument than with the conventional aids. Differences in performance on clinical tests and subjective patient responses appear to be related to the different signal processing schemes used in the two groups of devices.

Adolescent↗
PubMed1991

Critical period effects on universal properties of language: the status of subjacency in the acquisition of a second language.

Recent studies have shown clear evidence for critical period effects for both first and second language acquisition on a broad range of learned, language-specific grammatical properties. The present studies ask whether and to what degree critical period effects can also be found for universal properties of language considered to be innate. To address this issue, native Chinese speakers who learned English as a second language were tested on the universal principle subjacency as it applies to wh-question formation in English. Subjects arrived in the U.S.A. between the ages of 4 and 38 years. They were immersed in English for a number of years (a minimum of 5) and were adults at the time of testing. Non-native performance on subjacency was found for subjects of all ages of arrival. Performance declined continuously over age of arrival until adulthood, (r = -.63). When immersion occurred as late as adulthood, performance dropped to levels slightly above chance. In all of the analyses performed, subjacency did not differ from language-specific structures in the degree or manner in which it was affected by maturation. These results suggest that whatever the nature of the endowment that allows humans to learn language, it undergoes a very broad deterioration as learners become increasingly mature.

Adolescent↗
PubMed1991

The small GTP-binding protein Rho1p is localized on the Golgi apparatus and post-Golgi vesicles in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae the ras-related protein Rho1p is essentially the only target for ADP-ribosylation by exoenzyme C3 of Clostridium botulinum. Using C3 to detect Rho1p in subcellular fractions, Rho1p was found primarily in the 10,000 g pellet (P2) containing large organelles; small amounts also were detected in the 100,000 g pellet (P3), and cytosol. When P2 organelles were separated in sucrose density gradients Rho1p comigrated with the Kex-2 activity, a late Golgi marker. Rho1p distribution was shifted from P2 to P3 in several mutants that accumulate post-Golgi vesicles. Rho1p comigrated with post-Golgi transport vesicles during fractionation of P3 organelles from wild-type or sec6 cells. Vesicles containing Rho1p were of the same size but different density than those bearing Sec4p, a ras-related protein located both on post-Golgi vesicles and the plasma membrane. Immunofluorescence microscopy detected Rho1p as a punctate pattern, with signal concentrated towards the cell periphery and in the bud. Thus, in S. cerevisiae Rho1p resides primarily in the Golgi apparatus, and also in vesicles that are likely to be early post-Golgi vesicles.

Blotting, Western↗