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Biomedical subjects

A L Johnson

Publications and source records attributed to A L Johnson.

At least 55 records · Page 3Linked to original sources
PubMed2000

Caspase-3 and -6 expression and enzyme activity in hen granulosa cells.

We have cloned and sequenced cDNAs corresponding to the complete coding regions of the chicken homologues to mammalian caspase-3 and caspase-6. Both caspases are included among members of the cysteine protease (caspase) family that are most closely identified with mediating apoptosis. The deduced amino acid sequences for chicken caspase-3 and -6 show 65% and 68% identity with the respective human sequences, with complete conservation found within the QACRG active peptide region. Both caspase-3 and -6 are widely expressed within various tissues from the hen. Within the ovary, levels of caspase-3 and caspase-6 mRNA and protein do not change significantly in theca tissue during follicle development. On the other hand, procaspase-3 and -6 protein levels are elevated by 2- to 5-fold in preovulatory, compared to prehierarchal (6- to 8-mm diameter), follicle granulosa cells. Nevertheless, the function of this family of cell death-inducing proteins requires activation of the proenzyme caspase, which occurs after cleavage at predictable sites within the N-terminal domain. Accordingly, it was determined that okadaic acid, a pharmacologic inducer of apoptotic cell death in cultured apoptosis-resistant, preovulatory follicle granulosa cells, induced both caspase-3- and caspase-6-like activity within 8-16 h of treatment. By comparison, spontaneous apoptotic cell death that occurs in apoptosis-sensitive, prehierarchal follicle granulosa cells after short-term suspension culture is accompanied by a more rapid increase (within 2 h) in both caspase-3- and -6-like activity. Treatment with 8-bromo-cAMP, which has previously been shown to attenuate, or at least slow, the onset of apoptosis in prehierarchal follicle granulosa cells, mitigates this suspension culture-induced increase in caspase activity. While the present results provide further support for the relationship between caspase activation and apoptotic cell death in hen granulosa cells, the molecular ordering of enzymatic events and the caspase-specific substrates remain to be elucidated.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed2000

Effects of cognitive therapy on psychological symptoms and social functioning in residual depression.

BACKGROUND: About 30% of psychiatric out-patients with major depression demonstrate partial remission. AIMS: To explore whether the addition of cognitive therapy (CT) had any differential effect on residual symptoms or social adjustment. METHOD: Patients with residual symptoms of major depression (n=158) were randomised to receive clinical management (CM) alone, or CM plus 18 sessions of CT. Subjects' depressive symptoms and social functioning were assessed regularly over 16 months. RESULTS: The addition of CT produced statistically significant differential effects on: two out of four measures of overall severity of depression; specific psychological symptoms (guilt, self-esteem and hopelessness); and social functioning (including dependency, interpersonal behaviour and friction). CONCLUSIONS: In patients showing only partial response to antidepressants, the addition of CT produced modest improvements in social and psychological functioning. The implications for research on the mechanisms of action of CT are discussed.

Adult↗
PubMed2000

Factors predicting prognosis of epilepsy after presentation with seizures.

The objective of this study was to identify the factors, at the time of diagnosis, that determine the prognosis for remission of epilepsy. A prospective community-based cohort study of 792 patients recruited at the time of their first diagnosis of epileptic seizures was undertaken; in those classified 6 months after presentation, the median follow-up period was 7.2 years (quartiles at 6.2 and 8.2 years) after presentation. We analyzed data from 6 months after the first identified seizure, which prompted the diagnosis of epilepsy, to allow us to factor in those aspects contingent on a diagnostic assessment Baseline clinical and demographic data were analyzed using the Cox proportional hazards regression model with remission of epilepsy for 1, 2, 3, and 5 years as outcome measures. The dominant clinical feature predicting remission was the number of seizures in the 6-month diagnostic assessment period. Thus, the chance of entering 1 year of remission by 6 years for a patient who had 2 seizures during this initial 6 months was 95%; for 5 years of remission, it was 47% as opposed to 75% for 1 year of remission and 24% for 5 years of remission if there had been 10 or more seizures during this period. The number of seizures in the early phase of epilepsy (here, taken as the first 6 months after presentation) is the single most important predictive factor for both early and long-term remission of seizures.

Child↗
PubMed1999

Effect of perioperative prophylactic antimicrobial treatment in dogs undergoing elective orthopedic surgery.

OBJECTIVE: To determine whether perioperative antimicrobial prophylaxis would reduce incidence of postoperative infection among dogs undergoing elective orthopedic procedures. DESIGN: Randomized, controlled, blinded, intention clinical trial. ANIMALS: Dogs of any breed, sex, or age undergoing elective orthopedic surgery at a veterinary teaching hospital. PROCEDURES: Dogs were randomly assigned to 1 of 3 groups: treatment with saline solution, treatment with potassium penicillin G, and treatment with cefazolin. Treatments were intended to be administered within 30 minutes prior to surgery; a second dose was administered if surgery lasted > 90 minutes. Dogs were monitored for 10 to 14 days after surgery for evidence of infection. RESULTS: After the first 112 dogs were enrolled in the study, it was found that infection rate for control dogs (5/32 dogs) was significantly higher than the rate for dogs treated with antimicrobials (3/80 dogs). Therefore, no more dogs were enrolled in the study. A total of 126 dogs completed the study. Monte Carlo simulations indicated that compared with dogs that received antimicrobials prophylactically, dogs that received saline solution developed infections significantly more frequently. Difference in efficacy, however, was not observed between the 2 antimicrobial drugs used. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that perioperative antimicrobial prophylaxis decreased postoperative infection rate in dogs undergoing elective orthopedic surgery, compared with infection rate in control dogs. Cefazolin was not more efficacious than potassium penicillin G in these dogs.

Animals↗
PubMed1999

A Bub2p-dependent spindle checkpoint pathway regulates the Dbf2p kinase in budding yeast.

Exit from mitosis in all eukaroytes requires inactivation of the mitotic kinase. This occurs principally by ubiquitin-mediated proteolysis of the cyclin subunit controlled by the anaphase-promoting complex (APC). However, an abnormal spindle and/or unattached kinetochores activates a conserved spindle checkpoint that blocks APC function. This leads to high mitotic kinase activity and prevents mitotic exit. DBF2 belongs to a group of budding yeast cell cycle genes that when mutated prevent cyclin degradation and block exit from mitosis. DBF2 encodes a protein kinase which is cell cycle regulated, peaking in metaphase-anaphase B/telophase, but its function remains unknown. Here, we show the Dbf2p kinase activity to be a target of the spindle checkpoint. It is controlled specifically by Bub2p, one of the checkpoint components that is conserved in fission yeast and higher eukaroytic cells. Significantly, in budding yeast, Bub2p shows few genetic or biochemical interactions with other members of the spindle checkpoint. Our data now point to the protein kinase Mps1p triggering a new parallel branch of the spindle checkpoint in which Bub2p blocks Dbf2p function.

Anaphase↗
PubMed1999

Prevention of relapse in residual depression by cognitive therapy: a controlled trial.

BACKGROUND: Previous studies indicate that depressed patients with partial remission and residual symptoms following antidepressant treatment are common and have high rates of relapse. There is evidence that cognitive therapy may reduce relapse rates in depression. METHODS: One hundred fifty-eight patients with recent major depression, partially remitted with antidepressant treatment (mean daily doses equivalent to 185 mg of amitriptyline or 33 mg of fluoxetine) but with residual symptoms of 2 to 18 months' duration, were included in a controlled trial. Subjects were randomized to receive clinical management alone or clinical management plus cognitive therapy for 16 sessions during 20 weeks, with 2 subsequent booster sessions. Subjects were assessed regularly throughout the 20 weeks' treatment and for a further year. They received continuation and maintenance antidepressants at the same dose throughout. RESULTS: Cognitive therapy reduced relapse rates for acute major depression and persistent severe residual symptoms, in both intention to treat and treated per protocol samples. The cumulative relapse rate at 68 weeks was reduced significantly, from 47% in the clinical management control group to 29% with cognitive therapy (hazard ratio 0.54; 95% confidence interval, 0.32-0.93; intention to treat analysis). Cognitive therapy also increased full remission rates at 20 weeks but did not significantly improve symptom ratings. CONCLUSION: In this difficult-to-treat group of patients with residual depression who showed only partial response despite antidepressant treatment, cognitive therapy produced worthwhile benefit.

Adult↗
PubMed1999

External skeletal fixation. Linear fixators.

Linear external skeletal fixators offer some unique advantages over other fixation systems; they are biomechanically versatile, minimize surgical trauma to the fracture environment, and allow for simple staged disassembly to help promote bone healing. Historically, the use of linear external skeletal fixators has been reported to result in numerous complications, primarily related to the pin-bone interface. External skeletal fixators have experienced a resurgence in the past two decades. Increased knowledge of proper pin insertion techniques and advancements in pin design have greatly enhanced the longevity of the pin-bone interface, resulting in fewer complications. This article reviews some of the potential advantages of linear external skeletal fixators and principles of application to help strengthen the pin-bone interface and minimize complications.

Animals↗
PubMed1999

Association of the cell cycle transcription factor Mbp1 with the Skn7 response regulator in budding yeast.

We previously isolated the SKN7 gene in a screen designed to isolate new components of the G1-S cell cycle transcription machinery in budding yeast. We have now found that Skn7 associates with Mbp1, the DNA-binding component of the G1-S transcription factor DSC1/MBF. SKN7 and MBP1 show several genetic interactions. Skn7 overexpression is lethal and is suppressed by a mutation in MBP1. Similarly, high overexpression of Mbp1 is lethal and can be suppressed by skn7 mutations. SKN7 is also required for MBP1 function in a mutant compromised for G1-specific transcription. Gel-retardation assays indicate that Skn7 is not an integral part of MBF. However, a physical interaction between Skn7 and Mbp1 was detected using two-hybrid assays and GST pulldowns. Thus, Skn7 and Mbp1 seem to form a transcription factor independent of MBF. Genetic data suggest that this new transcription factor could be involved in the bud-emergence process.

Cell Cycle↗
PubMed1999

Febrile convulsions in 220 children--neurological sequelae at 12 years follow-up.

We report a 12-year follow-up study of children with febrile convulsions (FCs). The National General Practice Study of Epilepsy (NGPSE) is a large prospective community-based cohort study of 1,195 patients of all ages from first presentation with an identified seizure. Two hundred and twenty children with a first febrile convulsion were identified from the above study between 1984 and 1987. Children were prospectively followed up to ascertain subsequent seizures, neurological problems and treatment. Two hundred and seven patients were followed for a minimum of 8.4 years (median 11.2 years). In the FC cohort, 6% of the children developed subsequent epilepsy, which compares with a population risk of about 1.4%. Ten percent had neurological sequelae. Eleven percent of the children had received medication to prevent recurrence of FC, and in one third of these cases, this was for simple FCs. Using a time-dependent covariate Cox proportional hazards model, the number of FCs was associated with an increased risk of epilepsy (hazard ratio 2.48; 95% confidence limits, CL 1.68, 3.65) up to a limit of 4. A statistically significant association between occurrence of complex FC and subsequent epilepsy was not found, but a review of other studies quantified the odds ratio for epilepsy after a complex first FC as 3.4 (95% CL 2.1, 5.4). Epilepsy is a significant if infrequent sequel to FCs. Factors associated with subsequent epilepsy are the number of FCs or a complex first FC. Overtreatment of this condition continues.

Child↗
PubMed1999

A method to monitor DNA transfer during transfection.

This report describes a method for monitoring the transfer of DNA during transfection. This method involves random labeling of plasmid DNA with fluorescein-12-dUTP, flow cytometric detection and sorting of the fluorescent transfected cells, and laser confocal fluorescence microscopic determination of the intracellular location of the plasmid DNA. By this method, >95% of the sorted cells were labeled, indicating a >95% specificity of the sorting procedure. The sorted cells were viable, as indicated by their ability to exclude trypan blue dye (>98% cells excluded the dye) and to maintain cell growth. The results of the kinetics of the Lipofectin transfection technology show that the fraction of the cells that internalized plasmid DNA increased from 10% at 1 hr after initiation of the transfection procedures to 18% at 3 hr. This method does not require protein expression, does not require the use of selection pressure such as drug treatment to isolate the cells that internalized DNA, and can be used to study the early events of DNA transfection.

Cell Survival↗
PubMed1999

Bcl-X(LONG) protein expression and phosphorylation in granulosa cells.

Expression of Bcl-x protein was evaluated in hen ovarian follicles, relative to stage of development, and in cultured granulosa cells after treatment with various apoptosis-suppressing or -inducing agents. Using Western blot analysis, Bcl-X(LONG) was most frequently observed migrating as a doublet with a molecular mass of approximately 28 kDa; the apparent higher molecular mass band of this doublet was determined to represent a phosphorylated form. Consistent with previous findings reported for bcl-x messenger RNA, only the Bcl-X(LONG) (apoptosis-suppressing) form of protein was detected in the hen granulosa cells, and highest levels of Bcl-X(LONG) protein (sum of the protein doublet) expression were found in granulosa from preovulatory follicles together with tissues with immune function (e.g. spleen and bone marrow). Evidence for Bcl-X(SHORT) expression was found only in the theca and several nonovarian tissues. Immunocytochemical analysis of preovulatory vs. prehierarchal follicles confirmed the comparatively greater expression of cytoplasmic Bcl-X(LONG), particularly in preovulatory follicle granulosa. Levels of Bcl-X(LONG) were significantly increased after 20 h of culture in the presence of 8-bromo-cAMP (8-br-cAMP; compared with culture in control medium) in granulosa cells from both stages of follicle development. Such results are correlated with the protein's proposed function to protect against cell death in apoptosis-resistant, preovulatory follicle granulosa cells and are consistent with the ability of this cAMP agonist to increase bcl-X(LONG) messenger RNA levels in cultured cells. Furthermore, several factors that have previously been demonstrated to suppress apoptosis in granulosa cells, in vitro, (e.g. 8-br-cAMP, LH, FSH) were found to rapidly (within 15 min) increase levels of phosphorylated Bcl-X(LONG), compared with control cells, whereas an inhibitor of protein kinase A (H-89) blocked such phosphorylation. By comparison, transforming growth factor alpha, a factor previously found to attenuate apoptosis and apoptosis-inducing agents (e.g. paclitaxel, C8-ceramide, daunorubicin, UV irradiation) failed to phosphorylate Bcl-X(LONG). From these studies, it is concluded that both the phosphorylation of Bcl-X(LONG) (a short-term response) and increased levels of Bcl-X(LONG) (a comparatively slower response) in hen granulosa cells are promoted by gonadotropins via the adenylyl cyclase/cAMP signaling pathway. Moreover, elevated levels of chicken Bcl-X(LONG) protein expression and its phosphorylated state are correlated with resistance to apoptotic cell death in granulosa cells in vitro and ultimately a resistance to ovarian follicle atresia in vivo.

Animals↗
PubMed1999

The nocebo effect: do no harm.

The nocebo effect creates negative expectations about symptoms and can have devastating influence on patient recovery. Just as the placebo effect works by making patients believe they will get better, the nocebo effect can serve to make patients worse. Two case histories are presented in which patients were assigned diagnoses without objective physical findings. This resulted in poor outcomes. Physicians should avoid assigning a diagnosis without objective physical evidence and thus avoid creating the nocebo effect in patients.

Adult↗
PubMed1998

Fragment reconstruction and bone plate fixation versus bridging plate fixation for treating highly comminuted femoral fractures in dogs: 35 cases (1987-1997).

OBJECTIVE: To compare fragment reconstruction and bone plate fixation versus bridging plate fixation for treating highly comminuted (> 4 fragments) femoral fractures in dogs. DESIGN: Retrospective study. ANIMALS: 35 dogs with highly comminuted fractures of the femur. PROCEDURE: Medical records of all dogs included in this study were reviewed. Dogs had been treated with fragment reconstruction and bone plate application (n = 20) or major fragment alignment and bridging plate fixation (15). Postoperative and follow-up radiographs were evaluated. Operating and hospitalization times, bone alignment, bone healing, and complications were considered. RESULTS: There were no differences in hospitalization times, limb alignment, and complications between dogs with fractures treated with fragment reconstruction and dogs with fractures treated with bridging plate fixation. Dogs with fractures treated with bridging plate fixation had shorter operative times and faster times to radiographic evidence of bone healing. CLINICAL IMPLICATIONS: Bridging plate fixation is quicker to perform and results in faster healing than fragment reconstruction and bone plate fixation when used to treat comminuted femoral fractures.

Age Factors↗
PubMed1998

Characterization of the chicken interleukin-1beta converting enzyme (caspase-1) cDNA and expression of caspase-1 mRNA in the hen.

We have cloned and sequenced a chicken homolog to the mammalian interleukin-1beta (IL-1beta)-converting enzyme (caspase-1) cDNA and have evaluated caspase-1 mRNA expression in various tissues from the domestic hen, including ovarian follicle granulosa and theca layers. The deduced amino acid (aa) sequence of chicken caspase-1 is 44.9% identical to human caspase-1, and contains an active site pentapeptide that is characteristic of the caspase family of cysteine proteases. Of interest, however, is that the putative chicken caspase-1 cDNA is predicted to encode a comparatively short (19aa) N-terminal prodomain, as well as two Cys residues within the active pentapeptide (QC162C163RG) as compared to the QACRG pentapeptide found in the mammalian caspase-1 sequence. While the chicken caspase-1 mRNA transcript is widely expressed among different tissues, levels are particularly high in the bursa of Fabricius and comparatively low in ovarian follicles at all stages of development. Finally, treatment of granulosa cells with IL-1beta, the primary if not sole product of caspase-1 activity, fails to either promote apoptotic cell death or enhance viability in granulosa cells. Considering the relatively low levels of caspase-1 mRNA expression in ovarian follicle tissues plus the inability of IL-1beta to alter granulosa cell viability, in vitro, it is concluded that caspase-1 is not an integral part of the apoptotic pathway in granulosa cells. However, the pattern of mRNA expression is consistent with a requirement for caspase-1 mediated IL-1beta production in chicken immune tissues.

Amino Acid Sequence↗
PubMed1998

Pharmacodynamics of immediate and delayed effects of paclitaxel: role of slow apoptosis and intracellular drug retention.

The kinetics of the time-dependent antitumor effects of paclitaxel are not fully understood; some literature reports indicate a higher activity by prolonging treatment durations, whereas other reports indicate no enhancement under in vitro conditions. The present study was designed to address this controversy and to determine the mechanism of the higher cytotoxicity associated with longer treatment durations. Six human epithelial cancer cell lines (bladder RT4, breast MCF7, pharynx FaDu, ovarian SKOV3, and prostate PC3 and DU145) were used. To determine whether the higher activity observed for the longer treatment durations is due to a delayed exhibition of drug effects and/or a reflection of cumulative effects that required a continuous drug exposure, cells were treated with paclitaxel for 3-96 h and then either: (a) immediately processed for drug effect measurement; or (b) washed, incubated in drug-free medium, and processed for drug effect measurement at 96 h. The overall drug effect (i.e., combination of cytostatic and apoptotic effects) was determined by the sulforhodamine B assay, which measures the cellular protein. In addition, to determine whether apoptosis occurs with a time delay, apoptosis was measured in cells that were collected immediately after drug treatment for various durations or in cells that were treated with drugs for 3 h but collected at later time points. Apoptosis was determined using agarose gel electrophoresis and by measuring the cytoplasmic DNA-histone complex using ELISA. The contribution of the intracellularly retained drug to the delayed drug effect was studied by characterizing the kinetics of cellular drug uptake and efflux and by examining the effect of removal of the intracellularly retained drug. All six cell lines showed similar results, as follows: (a) paclitaxel produced cytotoxicity that was exhibited immediately after treatment (immediate effect) and after treatment was terminated (delayed effect); (b) the immediate and delayed effects showed different pharmacodynamics. The immediate effect increased with treatment duration and drug concentration. For the delayed effect, all treatments produced the same maximum effect at 96 h, although treatments for < or = 12 h showed higher IC50s than longer treatments, whereas treatments for > or = 24 h showed indistinguishable IC50s; (c) treatment for as brief as 3 h was sufficient to induce apoptosis, which occurred with a lag time of about 24 h, although longer treatments produced a greater extent of apoptosis; (d) The intracellular and extracellular concentrations reached an equilibrium at approximately 5 h, which rules out slow and/or insufficient uptake as the cause of the lower effects at shorter treatment times (i.e., < 24 h); (e) upon removal of drug-containing medium, the amount of drug retained intracellularly was about 10% of the applied dose and was reduced to approximately 0.5% after three successive washes, separated by 3-h equilibration periods; and (f) the delayed effect of the 3-h treatment was largely due to the drug retained intracellularly, whereas the delayed effect of the 24 h treatment was independent of the drug retained intracellularly. In conclusion, in human epithelial cancer cells, paclitaxel-induced cytotoxicity occurred after termination of drug treatment, which was partly due to the slow manifestation of apoptosis and partly due to the significant amount of drug retained intracellularly. Based on these findings and recognizing that some previous studies measured the immediate effect whereas the other studies measured the delayed effect, we propose that the conflicting data in the literature regarding the effect of treatment duration on paclitaxel activity under in vitro conditions are in part due to the different pharmacodynamics of the immediate and delayed drug effects. Furthermore, differences in the delayed effects for treatments of < 24 h and the minimal differences for treatments of > or = 24 h indicate that th

Animals↗