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Biomedical subjects

D E Johnson

Publications and source records attributed to D E Johnson.

At least 91 records · Page 5Linked to original sources
PubMed1999

Characterization of caspase proteases in cytokine-dependent myeloid progenitor cells using enzyme affinity labeling.

Bone marrow-derived myeloid progenitor cells are dependent on the presence of cytokines such as interleukin-3 (IL-3) for their survival. The withdrawal of IL-3 from IL-3-dependent myeloid progenitors results in death via an apoptotic program. Previous studies have shown that IL-3 withdrawal induces the activities of caspase proteases. However, the molecular identities of myeloid progenitor caspases have not been determined. In this study, we used an affinity labeling reagent (biotin-YVAD-acyloxymethyl ketone) that binds to processed active caspase subunits, to study caspase activation in 32D and FDCP-1 myeloid progenitor cells. After IL-3 withdrawal, we detected affinity labeling of caspase subunits of 20, 17, and 16 kDa in both cell lines. Surprisingly, affinity labeling of the 20- and 17-kDa proteins, but not the 16-kDa protein, was also detected in healthy cells maintained in the presence of IL-3. By contrast, in cytokine-independent cell lines, affinity labeling of caspase subunits was detected only after treatment with an apoptotic stimulus. Immunoblotting experiments showed that caspase-3 constitutes at least a portion of the 20- and 17-kDa affinity-labeled proteins detected in the myeloid progenitor cell lines. Taken together, these data provide direct evidence of caspase activation in cytokine-dependent myeloid progenitors, and suggest that unique apoptotic pathways may exist in these cells.

Affinity Labels↗
PubMed1999

Caspase-mediated degradation of T-cell receptor zeta-chain.

We recently reported an association between loss in T-cell receptor (TcR) zeta-chain expression and tumor-induced apoptosis of T lymphocytes. In this study, the possibility that zeta-chain serves as a direct substrate for activated caspases was investigated. Here, we report that two DXXD motifs, which are putative recognition sequences for caspase-3-related proteases and are present in the amino acid sequence of the zeta-chain, are cleaved in apoptotic Jurkat T lymphocytes. Cleavage of zeta-chain in Jurkat cells ligated by agonistic anti-Fas antibody was inhibited in the presence of peptide inhibitors of caspases, including the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone and N-benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone, an inhibitor of caspase-3-like activity. Fas-induced cleavage of zeta-chain was also inhibited in Jurkat cells overexpressing the intracellular inhibitors of caspase activity, Bcl-2 or cytokine response-modifier A. In vitro translated zeta-chain was cleaved in a similar fashion by recombinant caspase-3 or caspase-7 in a dose-dependent manner. In the presence of N-benzyloxycarbonyl-AspGlu-Val-Asp-fluoromethyl ketone, no cleavage of in vitro translated zeta-chain was observed. These results suggest that the loss of TcR zeta-chain, previously associated with tumor-induced immune dysfunction and more recently associated with tumor-induced apoptosis of T lymphocytes, is mediated by a direct degradation of the zeta-chain by activated caspases. This is the first report of involvement of caspases in degradation of the zeta protein.

Amino Acid Sequence↗
PubMed1999

Identification of microglial signal transduction pathways mediating a neurotoxic response to amyloidogenic fragments of beta-amyloid and prion proteins.

Microglial interaction with amyloid fibrils in the brains of Alzheimer's and prion disease patients results in the inflammatory activation of these cells. We observed that primary microglial cultures and the THP-1 monocytic cell line are stimulated by fibrillar beta-amyloid and prion peptides to activate identical tyrosine kinase-dependent inflammatory signal transduction cascades. The tyrosine kinases Lyn and Syk are activated by the fibrillar peptides and initiate a signaling cascade resulting in a transient release of intracellular calcium that results in the activation of classical PKC and the recently described calcium-sensitive tyrosine kinase PYK2. Activation of the MAP kinases ERK1 and ERK2 follows as a subsequent downstream signaling event. We demonstrate that PYK2 is positioned downstream of Lyn, Syk, and PKC. PKC is a necessary intermediate required for ERK activation. Importantly, the signaling response elicited by beta-amyloid and prion fibrils leads to the production of neurotoxic products. We have demonstrated in a tissue culture model that conditioned media from beta-amyloid- and prion-stimulated microglia or from THP-1 monocytes are neurotoxic to mouse cortical neurons. This toxicity can be ameliorated by treating THP-1 cells with specific enzyme inhibitors that target various components of the signal transduction pathway linked to the inflammatory responses.

Amyloid↗
PubMed1999

In vivo tumor suppression by adenovirus-mediated interferon alpha2b gene delivery.

A replication-deficient adenovirus encoding human interferon alpha2b, driven by the human cytomegalovirus (CMV) promoter, was constructed and characterized. This construct was used to infect human cells derived from different types of cancer. The production of protein and its secretion into the culture medium were tested by Western blotting and immunoassay. Inhibition of cell proliferation and antiviral activity, two of the most important biological activities of interferon, were observed with this construct. PC-3 cells, derived from human prostatic cancer, or Hep3B cells, derived from human hepatocellular carcinoma, were injected subcutaneously to generate and establish in vivo tumors in athymic nude mice. Intratumoral injection with the recombinant adenovirus expressing interferon alpha2b resulted in complete regression of tumor growth. Our results demonstrate that interferon gene delivery using recombinant adenoviral vectors may be a useful approach to treat a variety of cancers.

Adenoviridae↗
PubMed1999

Serum immunoglobulin response and protection from homologous challenge by Proteus mirabilis in a mouse model of ascending urinary tract infection.

We tested the hypothesis that experimental Proteus mirabilis urinary tract infection in mice would protect against homologous bladder rechallenge. Despite production of serum immunoglobulin G (IgG) and IgM (median titers of 1:320 and 1:80, respectively), vaccinated (infected and antibiotic-cured) mice did not show a decrease in mortality upon rechallenge; the survivors experienced only modest protection from infection (mean log(10) number of CFU of P. mirabilis Nal(r) HI4320 per milliliter or gram in vaccinated mice versus sham-vaccinated mice: urine, 100-fold less [3.5 versus 5.5; P = 0.13]; bladder, 100-fold less [3.1 versus 5.1; P = 0.066]; kidneys, 40-fold less [2.7 versus 4.3; P = 0.016]). Western blots using protein from the wild-type strain and isogenic mutants demonstrated antibody responses to MR/P and PMF fimbriae and flagella. There was no correlation between serum IgG or IgM levels and protection from mortality or infection. There was a trend toward elevated serum IgA titers and protection from subsequent challenge (P >/= 0.09), although only a few mice developed significant serum IgA levels. We conclude that prior infection with P. mirabilis does not protect significantly against homologous challenge.

Animals↗
PubMed1999

Expanded safety and immunogenicity of a bivalent, oral, attenuated cholera vaccine, CVD 103-HgR plus CVD 111, in United States military personnel stationed in Panama.

To provide optimum protection against classical and El Tor biotypes of Vibrio cholerae O1, a single-dose, oral cholera vaccine was developed by combining two live, attenuated vaccine strains, CVD 103-HgR (classical, Inaba) and CVD 111 (El Tor, Ogawa). The vaccines were formulated in a double-chamber sachet; one chamber contained lyophilized bacteria, and the other contained buffer. A total of 170 partially-immune American soldiers stationed in Panama received one of the following five formulations: (a) CVD 103-HgR at 10(8) CFU plus CVD 111 at 10(7) CFU, (b) CVD 103-HgR at 10(8) CFU plus CVD 111 at 10(6) CFU, (c) CVD 103-HgR alone at 10(8) CFU, (d) CVD 111 alone at 10(7) CFU, or (e) inactivated Escherichia coli placebo. Among those who received CVD 111 at the high or low dose either alone or in combination with CVD 103-HgR, 8 of 103 had diarrhea, defined as three or more liquid stools. None of the 32 volunteers who received CVD 103-HgR alone or the 35 placebo recipients had diarrhea. CVD 111 was detected in the stools of 46% of the 103 volunteers who received it. About 65% of all persons who received CVD 103-HgR either alone or in combination had a fourfold rise in Inaba vibriocidal titers. The postvaccination geometric mean titers were comparable among groups, ranging from 450 to 550. Ogawa vibriocidal titers were about twice as high in persons who received CVD 111 as in those who received CVD 103-HgR alone (600 versus 300). The addition of CVD 111 improved the overall seroconversion rate and doubled the serum Ogawa vibriocidal titers, suggesting that the combination of an El Tor and a classical cholera strain is desirable. While CVD 111 was previously found to be well tolerated in semiimmune Peruvians, the adverse effects observed in this study indicate that this strain requires further attenuation before it can be safely used in nonimmune populations.

Cholera↗
PubMed1999

Requirement of MrpH for mannose-resistant Proteus-like fimbria-mediated hemagglutination by Proteus mirabilis.

Two new genes, mrpH and mrpJ, were identified downstream of mrpG in the mrp gene cluster encoding mannose-resistant Proteus-like (MR/P) fimbriae of uropathogenic Proteus mirabilis. Since the predicted MrpH has 30% amino acid sequence identity to PapG, the Galalpha(1-4)Gal-binding adhesin of Escherichia coli P fimbriae, we hypothesized that mrpH encodes the functional MR/P hemagglutinin. MR/P fimbriae, expressed in E. coli DH5alpha, conferred on bacteria both the ability to cause mannose-resistant hemagglutination and the ability to aggregate to form pellicles on the broth surface. Both a DeltamrpH mutant expressed in E. coli DH5alpha and an isogenic mrpH::aphA mutant of P. mirabilis were unable to produce normal MR/P fimbriae efficiently, suggesting that MrpH was involved in fimbrial assembly. Amino acid residue substitution of the N-terminal cysteine residues (C66S and C128S) of MrpH abolished the receptor-binding activity (hemagglutinating ability) of MrpH but allowed normal fimbrial assembly, supporting the notion that MrpH was the functional MR/P hemagglutinin. Immunogold electron microscopy of P. mirabilis HI4320 revealed that MrpH was located at the tip of MR/P fimbriae, also consistent with its role in receptor binding. The isogenic mrpH::aphA mutant of HI4320 was less able to colonize the urine, bladder, and kidneys in a mouse model of ascending urinary tract infection (P < 0.01), and therefore MR/P fimbriae contribute significantly to bacterial colonization in mice. While there are similarities between P. mirabilis MR/P and E. coli P fimbriae, there are more notable differences: (i) synthesis of the MrpH adhesin is required to initiate fimbrial assembly, (ii) MR/P fimbriae confer an aggregation phenotype, (iii) site-directed mutation of specific residues can abolish receptor binding but allows fimbrial assembly, and (iv) mutation of the adhesin gene abolishes virulence in a mouse model of ascending urinary tract infection.

Adhesins, Bacterial↗
PubMed1999

Effects of level and source of carbohydrate and level of degradable intake protein on intake and digestion of low-quality tallgrass-prairie hay by beef steers.

Ruminally fistulated steers (n = 13; 263 kg) were used in an incomplete Latin square with 13 treatments and four periods to evaluate the effects of level and source of supplemental carbohydrate (CHO) and level of degradable intake protein (DIP) on the utilization of low-quality, tallgrass-prairie hay. Steers were given ad libitum access to forage (5.7% CP, 2.6% DIP, and 74.9% NDF). The supplementation treatments were fashioned as a 2x3x2 factorial arrangement plus a negative control (NC; no supplement). The factors included two DIP levels (.031 and .122% BW) and three CHO sources (starch, glucose, and fiber) fed at two levels (.15 and .30% BW) within each level of DIP supplementation. The effect of supplementation on forage OM intake (FOMI) was dependent (P<.01) on level and source of CHO and level of DIP fed. When DIP was low, forage, total, and digestible OM intakes were generally greater for the starch treatment than for the nonstarch treatments. However, when the DIP level was high, intakes were greater for the nonstarch (i.e., fiber and glucose) treatments. Generally, FOMI decreased (P<.01) when more supplemental CHO was provided. Supplementation typically increased fiber digestion, but the response was dependent (P<.01) on level and source of CHO and level of DIP. Generally, supplements with low levels of CHO improved NDF digestion (NDFD). However, supplements with the high level of CHO decreased NDFD, except for fiber at the high level of DIP. Organic matter digestion was increased by supplementation, but the impact of increasing CHO was dependent (P<.01) on source of CHO and level of DIP. Supplementation treatments had significant impact on ruminal pH, NH3 N, and the total concentration of organic acids as well as their relative proportions. In conclusion, supplemental DIP enhanced the use of low-quality forage; however, the impact of supplemental CHO on low-quality forage use was dependent on source and level of CHO offered, as well as the level of DIP provided.

Animals↗
PubMed1999

Effects of ruminal administration of supplemental degradable intake protein and starch on utilization of low-quality warm-season grass hay by beef steers.

Hereford x Angus steers were used in a 13-treatment, four-period, incomplete Latin square design to examine the effects of starch and degradable intake protein (DIP) supplements on forage utilization and ruminal function. Steers were given ad libitum access to low-quality hay (4.9% CP) and were not supplemented (NS) or received different amounts of starch (cornstarch grits; 0, .15, and .3% of initial BW) and DIP (Na-caseinate; .03, .06, .09, and .12% of initial BW) administered via ruminal fistulae in a 3 x 4 factorial arrangement of treatments. Supplemented steers consumed more (P < .01) forage OM, total OM, NDF, and digestible OM (DOM) than NS steers. Forage OM, total OM, NDF, and DOM intakes increased linearly (P < .01) as the amount of supplemental DIP increased. The addition of starch to supplements linearly decreased ( P < .01) the intake of forage OM, NDF, and DOM. The digestion of DM, OM, and NDF increased linearly (P < .01) with supplemental DIP and decreased linearly (P < or = .06) with supplemental starch. Particulate and liquid passages generally increased with DIP; however, starch level influenced the nature of the response (P = .03 and .06, respectively). Similarly, ruminal acid detergent-insoluble ash content generally decreased as starch increased, but the effect was dependent on DIP level (P < .01). Supplementation increased (P < .01) ruminal NH3 and total VFA and decreased (P < .01) ruminal pH relative to NS. All treatments supported average pH values in a range (6.3 to 6.7) unlikely to inhibit fibrolytic bacteria. Ruminal NH3 concentration increased quadratically (P = .03) with DIP and decreased linearly (P = .02) with starch. As DIP increased, total VFA concentration increased linearly (P = .02). Providing supplemental DIP to steers fed low-quality forage increased OM intake and digestion, whereas addition of starch to supplements decreased forage intake and digestion.

Animal Feed↗
PubMed1999

Integrated systems reassess strategies.

Integrated health care delivery systems are reassessing their strategies and goals in light of recent experience. The latest data on HMOs suggests more are at least partially owned by integrated systems and hospitals and that integrated systems' hospitals are gaining market share, writes Donald E. L. Johnson.

Contract Services↗
PubMed1999

HMOs may be ready to drop costly physicians.

After expanding their physician panels for several years in an effort to attract new enrollees, HMOs appear to be reversing course in an effort to control expenditures. In some areas, 20% to 30% of physicians could lose their HMO contracts, writes Donald E. L. Johnson.

Cost Control↗
PubMed1999

A randomized double-blind study assessing 4 versus 8 mg. doxazosin for benign prostatic hyperplasia.

PURPOSE: We compare the efficacy of 4 versus 8 mg. doxazosin for benign prostatic hyperplasia. MATERIALS AND METHODS: A total of 82 patients with benign prostatic hyperplasia successfully treated with 4 mg. doxazosin were randomized in a double-blind fashion to take 4 or 8 mg. Patients were evaluated with American Urological Association (AUA) symptom score, Boyarsky score, uroflowmetry and side effect profile before, and 1 and 3 months following randomization. RESULTS: Of the patients 42 and 40 were randomized to receive 4 and 8 mg. doxazosin, respectively. Both groups were similar with respect to patient age, baseline Boyarsky and AUA symptom scores, and baseline maximum urinary flow rate. At 3 months mean improvement from baseline plus or minus standard deviation in Boyarsky score was 0.6 +/- 6.5 and 4.9 +/- 6.6 in the 4 and 8 mg. groups (p <0.05), respectively, mean improvement in AUA symptom score was 1.6 +/- 5.3 and 5.3 +/- 8.0 (p <0.05), and mean maximum flow rate difference was -0.6 +/- 6.4 and +1.4 +/- 7.9 (p >0.05). Of the patients 7 and 8 in the 4 and 8 mg. groups dropped out of the study, and there were no statistical differences in side effects between dosages. CONCLUSIONS: A dose of 8 mg. doxazosin was more efficacious than 4 mg. and the side effects associated with both dosages appeared to be similar. The 8 mg. dose should be tried in patients who have not achieved an adequate therapeutic response to 4 mg. and are tolerating the medication. Consideration should be given to increasing the dosage to 8 mg. in patients who are clinically improved at lower dosages.

Adrenergic alpha-Antagonists↗