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Biomedical subjects

R C Johnson

Publications and source records attributed to R C Johnson.

At least 109 records · Page 6Linked to original sources
PubMed1996

Novel proteins that interact with the COOH-terminal cytosolic routing determinants of an integral membrane peptide-processing enzyme.

The steady state distribution of membrane forms of peptidylglycine alpha-amidating monooxygenase (PAM) in the secretory pathway of neurons and endocrine cells depends on signals in its cytosolic COOH-terminal domain (CD). Mutagenesis studies yielded catalytically active PAM proteins that are not properly localized or internalized. Employing the yeast two-hybrid system, we isolated two distinct cDNAs whose protein products showed a strong interaction with the CD of PAM. The interaction of these novel PAM COOH-terminal interactor proteins (P-CIPs) did not occur with a misrouted CD mutant as bait in the yeast system. Both proteins, P-CIP2 and P-CIP10, were expressed as fusion proteins that interacted in vitro with solubilized integral membrane PAM. P-CIP2 was homologous to several serine/threonine and dual specificity protein kinases, while P-CIP10 contained spectrin-like repeats. Endogenous P-CIP2 was localized to the Golgi region of AtT-20 corticotrope tumor cells, and expression of integral membrane PAM disrupted the distribution of endogenous P-CIP2. Both P-CIP2 and P-CIP10 mRNAs were found to be expressed in rat brain neurons also expressing PAM proteins. P-CIP2 and P-CIP10 may be members of a family of cytosolic proteins involved in the routing of membrane proteins that function in the regulated secretory pathway.

Amino Acid Sequence↗
PubMed1996

Yeast HMG proteins NHP6A/B potentiate promoter-specific transcriptional activation in vivo and assembly of preinitiation complexes in vitro.

Nonhistone proteins 6A and 6B (NHP6A/B) are nonsequence-specific DNA-binding proteins from Saccharomyces cerevisiae that are related structurally and functionally to the mammalian high mobility group proteins 1 and 2. These DNA architectural proteins distort DNA structure severely and have been shown to promote assembly of specialized recombination complexes. Here we show that the yeast NHP6A/B proteins are required for the induction of a subset of genes transcribed by RNA polymerase II (pol II). Activation of the CUP1, CYC1, GAL1, and DDR2 genes was decreased or abolished completely in the delta nhp6A/B strain. No significant change in basal expression was observed for any of the 10 genes examined. Analysis of chimeric gene constructs localized the regions dependent on NHP6A/B to be primarily at the core promoters, although the GAL1 UAS also requires NHP6A/B for activity. In vitro, NHP6A stimulated transcription by pol II at the GAL1 promoter three- to fivefold above the level of activation by GAL4-VP16 alone. Gel mobility shift assays showed that NHP6A promotes the formation of a complex with TBP and TFIIA at the TATA box that has enhanced affinity for TFIIB.

Fungal Proteins↗
PubMed1996

Local inflammatory response around diffusion chambers containing xenografts. Nonspecific destruction of tissues and decreased local vascularization.

Immunoisolation of xenogeneic pancreatic islets within membrane-bound devices has been proposed as an approach to cure diabetes. We examined the local response to implanted xenografts and allografts in comparison with isografts in diffusion chambers with 0.4-microm pore membranes when implanted into epididymal fat pads of rats. These membranes prevented host cell entry into the device but did not prevent passage of large molecules such as IgG and IgM. Well-differentiated allogeneic tissues (Sprague-Dawley rat embryonic lung implanted into Lewis rats) survived for 1 year when implanted in intact devices, but similar tissues were destroyed within 3 weeks when implanted within devices with holes poked in the membrane to allow host cell contact. In contrast, xenografts (CF1 mouse embryonic lung implanted into Lewis rats) were destroyed within 3 weeks even when implanted in devices with intact membranes. The death of the xenogeneic tissues was accompanied by a severe local accumulation of inflammatory cells and a decrease in local vascularization. When isogeneic tissues (Lewis rat embryonic lung implanted in Lewis rats) were mixed with xenogeneic tissues, a local inflammatory response occurred and both iso- and xenogeneic tissues were destroyed within 5 weeks. These results suggest the possibility that xenografts are killed by local accumulation of inflammatory cells, perhaps mediated by the release of antigens from the tissues within the device and presentation by an indirect pathway. The observation that the local response to xenografts is sufficient to kill isografts complicates issues of immunoprotection, suggesting that successful immunoisolation will require membranes that not only provide protection of the encapsulated tissues from the host immune system but also have properties that diminish the release of xenogeneic antigens.

Animals↗
PubMed1996

Azithromycin compared with amoxicillin in the treatment of erythema migrans. A double-blind, randomized, controlled trial.

OBJECTIVE: To determine whether azithromycin or amoxicillin is more efficacious for the treatment of erythema migrans skin lesions, which are characteristic of Lyme disease. DESIGN: Randomized, double-blind, double-dummy, multicenter study. Acute manifestations and sequelae were assessed using a standardized format. Baseline clinical characteristics and response were correlated with serologic results. Patients were followed for 180 days. SETTING: 12 outpatient centers in eight states. PATIENTS: 246 adult patients with erythema migrans lesions at least 5 cm in diameter were enrolled and were stratified by the presence of flu-like symptoms (such as fever, chills, headache, malaise, fatigue, arthralgias, and myalgias) before randomization. INTERVENTION: Oral treatment with either amoxicillin, 500 mg three times daily for 20 days, or azithromycin, 500 mg once daily for 7 days. Patients who received azithromycin also received a dummy placebo so that the dosing schedules were identical. RESULTS: Of 217 evaluable patients, those treated with amoxicillin were significantly more likely than those treated with azithromycin to achieve complete resolution of disease at day 20, the end of therapy (88% compared with 76%; P=0.024). More azithromycin recipients (16%) than amoxicillin recipients (4%) had relapse (P=0.005). A partial response at day 20 was highly predictive of relapse (27% of partial responders had relapse compared with 6% of complete responders; P<0.001). For patients treated with azithromycin, development of an antibody response increased the possibility of achieving a complete response (81% of seropositive patients achieved a complete response compared with 60% of seronegative patients; P=0.043). Patients with multiple erythema migrans lesions were more likely than patients with single erythema migrans lesions (P<0.001) to have a positive antibody titer at baseline (63% compared with 17% for IgM; 39% compared with 16% for IgG). Fifty-seven percent of patients who had relapse were seronegative at the time of relapse. CONCLUSIONS: A 20-day course of amoxicillin was found to be an effective regimen for erythema migrans. Most patients were seronegative for Borrelia burgdorferi at the time of presentation with erythema migrans (65%) and at the time of relapse (57%).

Adult↗
PubMed1996

Identification of new Fis binding sites by DNA scission with Fis-1,10-phenanthroline-copper(I) chimeras.

The chimeric nuclease Fis-OP has been used to identify novel Fis binding sites. Tethering the chemical nuclease OP-Cu+ to position 73 of the protein with a newly developed longer acetyl-beta-alanylamino spacer has facilitated the localization of two high-affinity Fis binding sequences in a 3 kb pUC19 plasmid. The shorter acetamido linker has allowed the chimeric nuclease to locate two strong Fis binding sites in the 50 kb phage lambda genome. All four sites reside in biologically interesting loci and have been confirmed by gel-retardation and DNase I footprint analyses. A newly discovered site resides in the lac operon of Escherichia coli. The binding of Fis to this site may antagonize repression by the LacI repressor. These studies demonstrate the feasibility of applying chimeric chemical nucleases to the task of identifying functional protein binding sites of biological interest within genomes without any assumption about their sequence preference.

Bacteriophage lambda↗
PubMed1996

Defects in hemostasis in P-selectin-deficient mice.

Recently, our laboratory showed that platelets, like leukocytes, roll on activated endothelium expressing P-selectin, thus suggesting a role for P-selectin in hemostasis (Frenette et at, Proc Natl Acad Sci USA 92:7450, 1995). We report here that the P-selectin--deficient mice show a 40% prolongation of the bleeding time on amputation of the tip of the tail. Moreover, defective hemostasis was observed in a local Shwartzman-like reaction induced by skin injections of lipopolysaccharide followed by tumor necrosis factor-alpha in the P-selectin--deficient mice. The hemorrhagic lesions, quantitated both macroscopically and microscopically, were twofold larger in the P-selectin--deficient mice. This was also confirmed by measuring the radioactivity in the skin using chromium-labeled red blood cells. Therefore, it is evident that P-selectin plays a role in hemostasis as suggested by its support of platelet rolling.

Animals↗
PubMed1996

The Hin dimer interface is critical for Fis-mediated activation of the catalytic steps of site-specific DNA inversion.

BACKGROUND: Hin is a member of an extended family of site-specific recombinases--the DNA invertase/resolvase family--that catalyze inversion or deletion of DNA. DNA inversion by Hin occurs between two recombination sites and requires the regulatory protein Fis, which associates with a cis-acting recombinational enhancer sequence. Hin recombinase dimers bind to the two recombination sites and assemble onto the Fis-bound enhancer to generate an invertasome structure, at which time they become competent to catalyze DNA cleavage and strand exchange. In this report, we investigate the role of the Hin dimer interface in the activation of its catalytic functions. RESULTS: We show that the Hin dimer is formed at an interface that contains putative amphipathic alpha-helices in a manner that is very similar to gamma delta resolvase. Certain detergents weakened cooperative interactions between the subunits of the Hin dimer and dramatically increased the rate of the first chemical step of the reaction--double-strand cleavage events at the center of the recombination sites. Amino-acid substitutions within the dimer interface led to profound changes in the catalytic properties of the recombinase. Nearly all mutations strongly affected the ability of the dimer to cleave DNA and most abolished DNA strand exchange in vitro. Some amino-acid substitutions altered the concerted nature of the DNA cleavage events within both recombination sites, and two mutations resulted in cleavage activity that was independent of Fis activation in vitro. Disulfide-linked Hin dimers were catalytically inactive; however, subsequent to the addition of the Fis-bound enhancer sequence, catalytic activity was no longer affected by the presence of oxidizing agents. CONCLUSIONS: The combined results demonstrate that the Hin dimer interface is of critical importance for the activation of catalysis and imply that interactions with the Fis-bound enhancer may trigger a conformational adjustment within the region that is important for concerted DNA cleavage within both recombination sites, and possibly for the subsequent exchange of DNA strands.

Binding Sites↗
PubMed1996

Hypoxia-induced exocytosis of endothelial cell Weibel-Palade bodies. A mechanism for rapid neutrophil recruitment after cardiac preservation.

The period of hypoxia is an important priming event for the vascular dysfunction that accompanies reperfusion, with endothelial cells (ECs) and neutrophils (PMNs) playing a central role. We hypothesized that EC Weibel-Palade (WP) body exocytosis during the hypoxic/ischemic period during organ preservation permits brisk PMN recruitment into postischemic tissue, a process further amplified in an oxidant-rich milieu. Exposure of human umbilical vein ECs to a hypoxic environment (pO2 approximately 20 torr) stimulated release of von Willebrand factor (vWF), stored in EC WP bodies, as well as increased expression of the WP body-derived PMN adhesion molecule P-selectin at the EC surface. Increased binding of 111In-labeled PMNs to hypoxic EC monolayers (compared with normoxic controls) was blocked with a blocking antibody to P-selectin, but was not affected by a nonblocking control antibody. Although increased P-selectin expression and vWF release were also noted during reoxygenation, hypoxia alone (even in the presence of antioxidants) was sufficient to increase WP body exocytosis. To determine the relevance of these observations to hypothermic cardiac preservation, during which the pO2 within the cardiac vasculature declines to similarly low levels, experiments were performed in a rodent (rat and mouse) cardiac preservation/transplantation model. Immunodepletion of recipient PMNs or administration of a blocking anti-P-selectin antibody before transplantation resulted in reduced graft neutrophil infiltration and improved graft survival, compared with identically preserved hearts transplanted into control recipients. To establish the important role of endothelial P-selectin expression on the donor vasculature, murine cardiac transplants were performed using homozygous P-selectin deficient and wild-type control donor hearts flushed free of blood/platelets before preservation/transplantation. P-selectin-null hearts transplanted into wild-type recipients demonstrated a marked (13-fold) reduction in graft neutrophil infiltration and increased graft survival compared with wild-type hearts transplanted into wild-type recipients. To determine whether coronary endothelial WP exocytosis may occur during cardiac preservation in humans, the release of vWF into the coronary sinus (CS) was measured in 32 patients during open heart surgery. CS samples obtained at the start and conclusion of the ischemic period demonstrated an increase in CS vWF antigen (by ELISA) consisting of predominantly high molecular weight multimers (by immunoelectrophoresis). These data suggest that EC WP exocytosis occurs during hypothermic cardiac preservation, priming the vasculature to recruit PMNs rapidly during reperfusion.

Animals↗
PubMed1996

A case-control study to assess possible triggers and cofactors in chronic fatigue syndrome.

PURPOSE: To assess possible triggers and cofactors for chronic fatigue syndrome (CFS) and to compare levels of selected cytokines between cases and an appropriately matched control group. PATIENTS AND METHODS: We conducted a case-control study of 47 cases of CFS obtained through a regional CFS research program maintained at a tertiary care medical center. One age-, gender-, and neighborhood-matched control was identified for each case through systematic community telephone sampling. Standardized questionnaires were administered to cases and controls. Sera were assayed for transforming growth factor-beta (TGF-beta), interleukin-1 beta, interleukin-6, tumor necrosis factor-alpha, and antibody to Borrelia burgdorferi and Babesia microti. RESULTS: Cases were more likely to have exercised regularly before illness onset than controls (67% versus 40%; matched odds ratio (MOR) = 3.4; 95% CI = 1.2 to 11.8; P = 0.02). Female cases were more likely to be nulliparous prior to onset of CFS than controls (51% versus 31%; MOR = 8.0; 95% CI = 1.03 to 170; P = 0.05). History of other major factors, including silicone-gel breast implants (one female case and one female control), pre-morbid history of depression (15% of cases, 11% of controls) and history of allergies (66% of cases, 51% of controls) were similar for cases and controls. However, cases were more likely to have a diagnosis of depression subsequent to their diagnosis of CFS compared to a similar time frame for controls (MOR = undefined; 95% CI lower bound = 2.5; P < 0.001). Positive antibody titers to B burgdorferi (one case and one control) and B microti (zero cases and two controls) were also similar. CONCLUSIONS: Further investigation into the role of prior routine exercise as a cofactor for CFS is warranted. This study supports the concurrence of CFS and depression, although pre-morbid history of depression was similar for both groups.

Adolescent↗
PubMed1996

Results of peripheral endovascular procedures in the operating room.

PURPOSE: This study presents the results of closed (percutaneous) and open endovascular procedures performed exclusively by vascular surgeons in the operating room and compares them with results from combined series from the literature, including primarily closed procedures in radiology or cardiology facilities. METHODS: Retrospective review of 607 consecutive peripheral arterial and venous angioplasties, stents, thrombolytic cases, and inferior vena caval filters in 446 patients was analyzed for immediate success rate, complication rate, and 1-year life table patency rate. RESULTS: The incidence of initial technical success was: aorta, 89%; iliac artery, 91%; superficial femoral artery, 90%; popliteal artery, 91%; tibial arteries, 79%; arm arteries and veins, 86%; renal arteries, 100%; IVC filters, 98%; and iliofemoral veins, 100%. The 1-year primary patency rates, including technical failures, were 70.3% in 113 femoropopliteal procedures and 83.7% in 194 iliac arteries. Advantages to the use of the operating room included: (1) simultaneous angioplasty during a bypass operation for abnormalities proximal or distal to the graft, (2) correction of lesions first discovered during thrombectomy, and (3) optimum patient monitoring and sedation in the operating room. CONCLUSIONS: Endovascular procedures performed by vascular surgeons in the operating room lead to results comparable with procedures performed in nonsurgical interventional suites, and the use of the operating room has advantages.

Adult↗
PubMed1996

Structure and function of macroencapsulated human and rodent pancreatic islets transplanted into nude mice.

Macroencapsulation of human pancreatic islets inside biomembranes is a promising approach to maintain islet allografts in the diabetic recipient without immunosuppression. In order to test this possibility islets isolated from human pancreata were kept in culture before macroencapsulation in a tissue chamber device. The device consisted of two titanium rings, which supported two flat membranes. These membranes have previously been shown to protect pancreatic islets and fetal lung tissue from allograft rejection and also to promote neovascularization at the membrane surface. In a first series of experiments macroencapsulated human islets were implanted into the epididymal fat pad of athymic, nude mice concomitant to an injection from the same batch of islets under the kidney capsule. Light microscopy of encapsulated and subcapsularly grafted human islets showed that the survival inside the membranes was as good as under the kidney capsule. There was an extensive formation of new blood vessels at the membrane outer surface. In a second series of experiments insulin was extracted from encapsulated human islets implanted either into the epididymal fat pad or subcutaneously. The encapsulated human islets contained as much insulin as the non-encapsulated ones. In these experiments mouse and rat islets were also used. Rodent islets, however, survived less well than the human islets as evidenced by the markedly reduced insulin content values. In a third series of experiments human islets were loaded into the chambers and transplanted into nude mice without the concomitant implantation of non-encapsulated islets under the kidney capsule of the recipienets. Measurements of human C-peptide in serum samples obtained 4 to 8 weeks post-implantation showed considerable concentrations (0.70-185 ng/ml) in all animals. We conclude that isolated human islets survive when implanted into nude mice and continue to release insulin for several weeks. There are, however, species differences suggesting that rodent islets are much more susceptible to the environmental stress inside the membranes than human islets.

Adult↗
PubMed1996

Presence of common antigenic epitope in outer surface protein (Osp) A and OspB of Japanese isolates identified as Borrelia garinii.

Japanese Borrelia strains FujiP2, AP83, NT24, NT29 and HT2 which had a 31-kilodalton protein non-reactive with monoclonal antibody (MAb) H5332 to outer surface protein A (OspA) were identified as B. garinii by the DNA hybridization method. MAb P3134 raised to strain NT24 reacted with OspA and the OspB-ranging protein of these isolates and cross-reacted with the OspB-ranging protein of some other isolates. Since the reactive protein was extracted by the Triton X-114 phase partitioning method, the MAb recognized the common epitope present in OspA and OspB. To our knowledge, this is the first report of an MAb reactive to both OspA and OspB.

Antibodies, Bacterial↗
PubMed1996

Comparison of OspA serotypes for Borrelia burgdorferi sensu lato from Japan, Europe and North America.

UNLABELLED: Sixty-one Borrelia burgdorferi sensu lato strains from various sources (ticks, human, and wild animals) in Japan and two strains from ticks in Far Eastern Russia were classified on the basis of reactivity with 16 monoclonal antibodies (mAb) to outer surface protein A (OspA) and by DNA-DNA hybridization assay. Eleven OspA serotypes (J1 to J11) were recognized among the Japanese and the Far East Russian isolates (serotypes J1 to J9 were identified as B. garinii, serotype J10 was identified as B. afzelii, and serotype J11 corresponded to B. japonica), whereas 7 OspA serotypes for North American and European isolates previously reported (Bettina Wilske et al, J. Clin. Microbiol. 31:340-350,1993) were not observed except for OspA serotype 2 which showed identical reactivity with OspA serotype J10. This finding provides helpful information for understanding the geographical distribution of Lyme disease borrelia and the development of vaccine and diagnostic tests. IN CONCLUSION: 1.B. burgdorferi sensu stricto has not been observed in Japan, 2. Japanese B. afzelii isolates are closely related to those from Europe, 3. B. garinii isolates from Japan are highly heterogeneous and apparently different from European B. garinii isolates.

Animals↗
PubMed1996

Resistance to tick-borne spirochete challenge induced by Borrelia burgdorferi strains that differ in expression of outer surface proteins.

Hamsters were immunized with thimerosal-killed Borrelia burgdorferi 297 or a mutant of 297 (M297) that lacks the 49-kb linear plasmid and expression of outer surface proteins A and B (OspA and OspB). Ixodes scapularis nymphs infected with either the B. burgdorferi sensu stricto strain 297 or JMNT, similar in OspA and OspB but differing in OspC expression, were used to evaluate protection. In a homologous challenge, 24 hamsters were vaccinated, 8 each with 297 or M297 and 8 sham (adjuvant)-vaccinated controls. Hamsters vaccinated with either bacterin were completely protected against a natural tick bite or subcutaneous (s.c.) inoculation of 297. Borreliae were effectively eliminated from 80 to 90% of the 297-infected ticks that fed on four hamsters immunized with the 297 bacterin. Cultures of spirochetes isolated from the ticks that remained infected were infectious and induced joint inflammation in naive hamsters. There was no reduction of strain 297 spirochetes in ticks that fed on four hamsters immunized with M297, but the hamsters were protected. Results with the M297 bacterin indicate that proteins other than OspA or OspB can protect hamsters against a tick challenge without eliminating B. burgdorferi in the tick. In a heterologous challenge, 36 hamsters were vaccinated, 12 with each bacterin and 12 controls. None of the hamsters immunized with either bacterin were protected from a challenge involving JMNT-infected ticks, while two of four were protected against an s.c. challenge. Hamsters challenged s.c. with strain 297 spirochetes were protected. There was partial elimination of JMNT spirochetes in ticks that fed on the group of four hamsters immunized with the 297 bacterin, and infection rates were reduced by 50 to 60%. JMNT spirochetes reisolated from the ticks that fed on 297-vaccinated hamsters also remained infectious for hamsters. In the JMNT-infected ticks that fed on four M297-immunized hamsters, there was no decline in the proportion of infected ticks. Destruction of spirochetes in ticks that fed on the hamsters vaccinated with the 297 bacterin suggests that antibodies to OspA and OspB may have been responsible, since the mutant did not induce this activity.

Animals↗
PubMed1996

Correlation of plasmids with infectivity of Borrelia burgdorferi sensu stricto type strain B31.

The correlation of plasmid profiles with infectivity was investigated by using five clones of Borrelia burgdorferi sensu stricto strain B31 (ATCC 35210). Plasmid profiles were determined by pulsed-field and two-dimensional gel electrophoresis. The 50% infectious dose (ID50) in hamsters was determined. The ID50 of the clone that possessed a full complement of eight linear and three circular plasmids was 10(3) cells. The loss of the 27.5- and 40-kb linear plasmids did not decrease the infectivity of these cells. Rather, the loss of the 27.5-kb linear plasmid was associated with a more disseminated infection. A moderate decrease of the ID50 from 10(3) to 10(5) cells correlated with the loss of the 9.0-kb circular plasmid and the 27.5-kb linear plasmid. A major loss of infectivity (ID50 > 10(3) cells) occurred with cells that lost the 24.7- and 27.5-kb linear plasmids and the 9.0-kb circular plasmid. A 3.0-kb HindIII fragment of the 24.7-kb linear plasmid was used as a probe to determine the presence of the homologous sequences in the three genospecies of Lyme disease spirochetes. An analysis of 21 infectious strains of B. burgdorferi sensu stricto, B. garinii, and B. afzelii revealed a consistent association of infectivity with strains possessing a linear plasmid (size range, 24 to 36 kb) that hybridized with the HindIII fragment. Western immunoblotting with hamster antisera against infectious B31 clone C-3 revealed two proteins with molecular masses of 28 and 43 kDa that were absent in the noninfectious B31 clone C-1. Additionally, a 14-kDa protein was absent in C-1 but present in infectious clone C-9 as shown by two-dimensional polyacrylamide gel electrophoresis.

Animals↗
PubMed1996

Is an outpatient appointment required for patients referred for a minor operation under local anaesthetic?

The necessity of an outpatient (OP) consultation for patients referred for minor surgery under local anaesthetic (LA) was examined. Two separate prospective studies were performed. The first comprised 107 patients referred for minor surgery, who were assessed in the outpatient department (OPD), before being booked for minor operations. The second study comprised 106 patients whose names were placed directly on a minor operations list on the basis of the GP referral letter alone. In the first study, 97 (91 per cent) patients went on to undergo minor surgery. Five were not suitable for LA and in five an operation was considered unnecessary. Patients not suitable for LA included children, neck swellings, pre-auricular swellings and swellings described as lymph nodes. In the second study, the GP referral letters were screened to exclude the above and of 106 referrals 93 (88 per cent) underwent a minor operation. The benefit of the second study was twofold. First, 106 OP slots were available for other referrals and secondly, patients avoided the OP waiting list and did not lose time from work as a result of the OP visit. There were no adverse effects demonstrated during the second study. We believe the OP consultation can be avoided if the referral letters are carefully screened.

Ambulatory Surgical Procedures↗