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Biomedical subjects

D E Johnson

Publications and source records attributed to D E Johnson.

At least 109 records · Page 6Linked to original sources
PubMed1998

Suppression of tumorigenicity of glioblastoma cells by adenovirus-mediated MMAC1/PTEN gene transfer.

Mutated in multiple advanced cancers 1/phosphatase and tensin homologue (MMAC1/PTEN) is a novel tumor suppressor gene candidate located on chromosome 10 that is commonly mutated in human glioblastoma multiforme and several other cancer types. To evaluate the function of this gene as a tumor suppressor, we constructed a replication-defective adenovirus (MMCB) for efficient, transient transduction of MMAC1 into tumor cells. Infection of MMAC1-mutated U87MG glioblastoma cells with MMCB resulted in dose-dependent exogenous MMAC1 protein expression as detected by Western blotting of cell lysates. In vitro proliferation of U87MG cells was inhibited by MMCB in comparison to several control adenoviruses at equal viral doses, implying a specific effect of MMAC1 expression. Anchorage-independent growth in soft agar was also inhibited by MMCB compared to control adenovirus. Tumorigenicity in nude mice of transiently transduced mass cell cultures was then assessed. MMCB-infected U87MG cells were almost completely nontumorigenic compared to untreated and several control adenovirus-treated cells at equal viral doses. These data support an in vivo tumor suppression activity of MMAC1/PTEN and suggest that in vivo gene transfer with this recombinant adenoviral vector has a potential use in cancer gene therapy.

Adenoviridae↗
PubMed1998

Regulation of survival pathways by IL-3 and induction of apoptosis following IL-3 withdrawal.

The production of hematopoietic cells is regulated by the availability of key cytokines such as interleukin-3 (IL-3). IL-3 promotes the survival and proliferation of bone marrow-derived hematopoietic cells. Recent studies using IL-3-dependent cell lines have begun to shed light on the regulation of apoptosis in cytokine-dependent cells. These studies indicate that IL-3 inhibits apoptosis by stimulating the activation of cellular kinases, including phophatidylinositol (PI) 3-kinase and Akt kinase. On the other hand, withdrawal of IL-3 leads to the activation of caspase proteases and a commitment to cell death. This review will discuss the current state of knowledge regarding the molecular mechanisms of: a) suppression of apoptosis by IL-3, and b) activation of apoptosis following IL-3 withdrawal.

Animals↗
PubMed1998

Toxicity of subcutaneously administered recombinant human interleukin-2 in rats.

Recombinant human interleukin-2 (rIL-2) was administered subcutaneously to rats at doses of 0.3-10 mg/kg/day in a range-finding study and 0.03-0.3 mg/kg/day in a 4-week toxicity study. Treatment-related effects were assessed by hematology, clinical chemistry, anti-rIL-2 antibody production, and gross and histopathologic evaluations. Doses of 1 mg/kg/day or above were not tolerated, resulting in death or moribund termination by Day 7. Slight decreases in red blood cell counts (including hematocrit and hemoglobin) were observed at >/= 0.1 mg/kg/day. White blood cells counts increased in a dose-dependent manner; increases were primarily due to increases in lymphocytes and eosinophils. Hepatic abnormalities, including increases in aspartate aminotransferase and bilirubin, were noted at 0.3 mg/kg/day. Histologic findings were evident primarily in the spleen, liver, lung, and injection sites, with dose-related increases in inflammatory cell foci/infiltrates noted in these sites. Findings in the liver also included biliary hyperplasia, hepatocellular degeneration, necrosis, vascular mural thickening in the portal triads, and fibrosis. Red and white pulp hyperplasia and capsular fibrosis occurred in the spleen. Most clinical and histopathologic findings were reversible within 4 weeks after termination of treatment. Anti-rIL-2 antibodies were detected beginning on Day 19 and were still present on Day 56. The pharmacological and toxicological effects associated with subcutaneous administration of rIL-2 are comparable to those reported after intravenous administration, indicating that subcutaneous dosing may be an alternative to the current clinical iv regimens.

Animals↗
PubMed1998

Exposure-based safety evaluation of recombinant human macrophage colony-stimulating factor (M-CSF) in cynomolgus monkeys.

The safety of M-CSF was assessed in cynomolgus monkeys in an intravenous dosing regimen. Exposure (AUC0-24) multiples (monkey vs human) were calculated using the no observable adverse effect level (NOAEL) observed in this study and correlated with known M-CSF-induced toxicities in a previous continuous intravenous infusion (civ) study in monkeys. M-CSF was administered by daily intravenous infusion (2 h) to cynomolgus monkeys (2/sex/ group) at 0.1, 0.3, 0.7, and 1.0 mg/kg/day, for 28 consecutive days. Control animals (2/sex) received placebo. The 0.7 mg/kg/day group was held for an additional 4-week recovery period. Criteria evaluated included physical observations, ophthalmoscopy, electrocardiography, body weight, food consumption, clinical pathology, antibody formation, pharmacokinetics, necropsy, organ weights, and histopathology. The only effect previously seen in monkeys after intravenously administered M-CSF occurred in animals in the 0.7 and 1.0 mg/kg/day groups. They exhibited a slight decrease in platelets between days 4 and 12 with subsequent recovery. No effects related to M-CSF administration were evident in macroscopic or microscopic evaluations and there was no evidence of anti-M-CSF antibody production. M-CSF at all dose levels was completely eliminated within each dosing interval with no accumulation. Clearance of M-CSF was enhanced during the first week of dosing, but returned to baseline clearance levels by day 27. This dosing regimen was shown to be remarkably free of toxicities noted in a previous monkey study where M-CSF was given by civ at similar daily doses. At the high dose, which was considered to be the NOAEL, the AUC0-24 was 40-fold greater than the AUC0-24 in clinical trials where 2.0 mg/m2 was administered by a daily 2-h infusion.

Animals↗
PubMed1998

Early biochemical detection of delayed neurotoxicity resulting from developmental exposure to chloropyrifos.

Developing animals are more sensitive than adults to the delayed neurotoxicity caused by chlorpyrifos exposure. In developing rat brain, chlorpyrifos doses that cause no discernible systemic toxicity and only a minor degree of cholinesterase inhibition, nevertheless evoke alterations in cell function and number that appear after several days' delay. In the current study, neonatal rats were exposed to subtoxic doses of chlorpyrifos (no weight loss or mortality) on either postnatal days 1-4, or postnatal days 11-14, and the effects on cellular RNA levels were determined in two brain regions that are targeted for delayed neurotoxicity-the brainstem and forebrain. In both regions, chlorpyrifos exposure evoked significant alterations in RNA concentration and content, variables that are ordinarily very tightly controlled in the developing brain. The effects on RNA appeared well before the deficits in cell function and number and showed a regional selectivity similar to that of subsequent, delayed neurotoxicity. Deficits in RNA were more prominent in the brainstem, an early-developing brain region, than in the forebrain, which develops later. These results suggest that chlorpyrifos can elicit delayed developmental neurotoxicity by targeting the pivotal macromolecules that control cell differentiation in a critical postmitotic period. The lower threshold for these cellular effects compared to that for systemic toxicity indicates that the developing brain is a selective target for chlorpyrifos, effects that should be considered in assessing safety thresholds.

Animals↗
PubMed1998

Sphincter preservation in rectal cancer. Surgical considerations for local excision.

The principles involved in selecting patients for local excision of rectal cancers, as well as the various techniques and principles for local resection, are detailed in this article. The assessment of the current role of this technique awaits further maturation of data from phase II clinical trials. The addition of chemotherapy and radiation therapy may allow treatment of more advanced rectal cancers by these techniques.

Abdomen↗
PubMed1998

IL-3 withdrawal activates a CrmA-insensitive poly(ADP-ribose) polymerase cleavage enzyme in factor-dependent myeloid progenitor cells.

Murine myeloid progenitor cells that are dependent on interleukin-3 (IL-3) undergo apoptosis when this essential cytokine is withdrawn. To determine whether IL-3 withdrawal leads to the activation of caspase proteases, known mediators of apoptosis, we studied proteolytic cleavage of the caspase substrate protein poly(ADP-ribose) polymerase (PARP) in two IL3-dependent myeloid progenitor cell lines, 32D and FDCP-1. We observed that IL-3 withdrawal leads to PARP cleavage in both cell lines, with complete cleavage occurring by 24 h after cytokine removal. The induced PARP cleavage activities were blocked by the caspase inhibitors z-DEVD-fluoromethyl ketone (z-DEVD-FMK) and z-VAD-fluoromethyl ketone (z-VAD-FMK), or by overexpression in 32D cells of Bcl-2 or BCR/ABL. By contrast, overexpression in 32D cells of cowpox virus CrmA protein, an inhibitor of Fas-mediated PARP cleavage, failed to inhibit PARP cleavage following IL-3 withdrawal. CrmA also failed to block DNA fragmentation and loss of cell viability. We propose that a CrmA-insensitive caspase protease is activated in the IL-3-deprived myeloid precursors, and that activation of this protease may direct the cells on a path towards commitment to death.

Animals↗
PubMed1998

Use of green fluorescent protein to assess urease gene expression by uropathogenic Proteus mirabilis during experimental ascending urinary tract infection.

Proteus mirabilis, a cause of complicated urinary tract infection, expresses urease when exposed to urea. While it is recognized that the positive transcriptional activator UreR induces gene expression, the levels of expression of the enzyme during experimental infection are not known. To investigate in vivo expression of P. mirabilis urease, the gene encoding green fluorescent protein (GFP) was used to construct reporter fusions. Translational fusions of urease accessory gene ureD, which is preceded by a urea-inducible promoter, were made with gfp (modified to express S65T/V68L/S72A [B. P. Cormack et al. Gene 173:33-38, 1996]). Constructs were confirmed by sequencing of the fusion junctions. UreD-GFP fusion protein was induced by urea in both Escherichia coli DH5alpha and P. mirabilis HI4320. By using Western blotting with antiserum raised against GFP, expression level was shown to correlate with urea concentration (tested from 0 to 500 mM), with highest induction at 200 to 500 mM urea. Fluorescent E. coli and P. mirabilis bacteria were observed by fluorescence microscopy following urea induction, and the fluorescence intensity of GFP in cell lysates was measured by spectrophotofluorimetry. P. mirabilis HI4320 carrying the UreD-GFP fusion plasmid was transurethrally inoculated into the bladders of CBA mice. One week postchallenge, fluorescent bacteria were detected in thin sections of both bladder and kidney samples; the fluorescence intensity of bacteria in bladder tissue was higher than that in the kidney. Kidneys were primarily infected with single-cell-form fluorescent bacteria, while aggregated bacterial clusters were observed in the bladder. Elongated swarmer cells were only rarely observed. These observations demonstrate that urease is expressed in vivo and that using GFP as a reporter protein is a viable approach to investigate in vivo expression of P. mirabilis virulence genes in experimental urinary tract infection.

Animals↗
PubMed1998

Comparison of Escherichia coli strains recovered from human cystitis and pyelonephritis infections in transurethrally challenged mice.

Urinary tract infection, most frequently caused by Escherichia coli, is one of the most common bacterial infections in humans. A vast amount of literature regarding the mechanisms through which E. coli induces pyelonephritis has accumulated. Although cystitis accounts for 95% of visits to physicians for symptoms of urinary tract infections, few in vivo studies have investigated possible differences between E. coli recovered from patients with clinical symptoms of cystitis and that from patients with symptoms of pyelonephritis. Epidemiological studies indicate that cystitis-associated strains appear to differ from pyelonephritis-associated strains in elaboration of some putative virulence factors. With transurethrally challenged mice we studied possible differences using three each of the most virulent pyelonephritis and cystitis E. coli strains in our collection. The results indicate that cystitis strains colonize the bladder more rapidly than do pyelonephritis strains, while the rates of kidney colonization are similar. Cystitis strains colonize the bladder in higher numbers, induce more pronounced histologic changes in the bladder, and are more rapidly eliminated from the mouse urinary tract than pyelonephritis strains. These results provide evidence that cystitis strains differ from pyelonephritis strains in this model, that this model is useful for the study of the uropathogenicity of cystitis strains, and that it would be unwise to use pyelonephritis strains to study putative virulence factors important in the development of cystitis.

Animals↗
PubMed1998

In vivo phase variation of Escherichia coli type 1 fimbrial genes in women with urinary tract infection.

Type 1 fimbriae, expressed by most Escherichia coli strains, are thought to attach to human uroepithelium as an initial step in the pathogenesis of urinary tract infections (UTI). Numerous reports using both in vitro and murine models support this role for type 1 fimbriae in colonization. Unfortunately, only a limited number of studies have directly examined the expression of fimbriae in vivo. To determine whether type 1 fimbrial genes are transcribed during an acute UTI, we employed a modification of an established method. The orientation (ON or OFF) of the invertible promoter element, which drives transcription of type 1 fimbrial genes, was determined by PCR amplification using primers that flank the invertible element, followed by SnaBI digestion. The orientation of the type 1 fimbrial switch was determined under three experimental conditions. First, E. coli strains from different clinical sources (acute pyelonephritis patients, cystitis patients, and fecal controls) were tested under different in vitro culture conditions (agar versus broth; aerated versus static). The genes in the more-virulent strains (those causing acute pyelonephritis) demonstrated a resistance, in aerated broth, to switching from OFF to ON, while those in fecal strains readily switched from OFF to ON. Second, bladder and kidney tissue from CBA mice transurethrally inoculated with E. coli CFT073 (an established murine model of ascending UTI) was assayed. The switches directly amplified from infected bladder and kidney tissues were estimated to be 33 and 39% ON, respectively, by using a standard curve. Finally, bacteria present in urine samples collected from women with cystitis were tested for type 1 fimbria switch orientation. For all 11 cases, an average of only 4% of the switches in the bacteria in the urine were ON. In 7 of the 11 cases, we found that all of the visible type 1 fimbrial switches were in the OFF position (upper limit of detection of assay, 98% OFF). Strains recovered from these urine samples, however, were shown after culture in vitro to be capable of switching the fimbrial gene to the ON position and expressing mannose-sensitive hemagglutinin. The results from experimental infections and cases of cystitis in women suggest that type 1 fimbrial genes are transcribed both in the bladder and in the kidney. However, those bacteria found in the urine and not attached to the uroepithelium are not transcriptionally active for type 1 fimbrial genes.

Adolescent↗
PubMed1998

Diet energy requirements of growing Holsteins.

The conceptual bases of recent NRC publications of nutrient requirements to meet dietary energy needs of growing Holsteins, emphasizing heifers gaining 0.75 kg/d, are described and compared with several world systems and with body composition data. Requirements of Holsteins for net energy for maintenance are generally thought to be higher (from 7 to 20%) than those of British beef breeds. Total estimates of metabolizable energy requirements are quite similar for most energy systems for cattle at 200 to 400 kg of body weight. For heavier cattle, the energy density estimates based on beef breed equations project higher metabolizable energy for gain and total metabolizable energy requirements. Extrapolations from the chemical composition of growing Holstein heifers indicate that, at the slower growth that is typical of replacements, energy densities are lower than for more rapidly growing heifers and that net energy requirements are consistent with current US standards.

Animals↗
PubMed1998

Bcl-2- and CrmA-inhibitable dephosphorylation and cleavage of retinoblastoma protein during etoposide-induced apoptosis.

Cell numbers are regulated by a balance between proliferation and apoptosis (programmed cell death). Recent evidence suggests that proteins regulating cell proliferation also mediate apoptosis. Therefore, cellular fate might be determined by cross talk between regulators of cell cycle progression and apoptosis. Previously, we had found that during DNA damage-induced apoptosis, retinoblastoma protein (RB), an important G1/S regulator and tumor suppressor, became dephosphorylated and then immediately cleaved into p48 and p68 fragments. Here, we report that expression of the Bcl-2 oncoprotein, an inhibitor of caspases (interleukin 1 -converting enzyme-like proteases), blocked RB dephosphorylation, RB cleavage and apoptosis in etoposide-treated human Jurkat T cells. In addition, expression of the cowpox virus CrmA protein, a direct inhibitor of caspases, also inhibited both RB changes and apoptosis. Taken together, our findings demonstrate important roles for caspases in the processes of etoposide-induced RB dephosphorylation, RB proteolysis and apoptosis.

Animals↗