Biomedical subjects
Publications and source records attributed to L Wilson.
Antitumor drugs of the vinca alkaloid and taxane classes function by suppressing the dynamics of microtubules in spindles, blocking cell division at metaphase. The drugs bind to various sites on the tubulin dimer and at different positions within the microtubule, suggesting that there are many unexplored targets for the design of novel drugs of this type.
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We have characterized the effects of vinblastine on the dynamic instability behavior of individual microtubules in living BS-C-1 cells microinjected with rhodamine-labeled tubulin and have found that at low concentrations (3-64 nM), vinblastine potently suppresses dynamic instability without causing net microtubule depolymerization. Vinblastine suppressed the rates of microtubule growth and shortening, and decreased the frequency of transitions from growth or pause to shortening, also called catastrophe. In vinblastine-treated cells, both the average duration of a pause (a state of attenuated dynamics where neither growth nor shortening could be detected) and the percentage of total time spent in pause were significantly increased. Vinblastine potently decreased dynamicity, a measure of the overall dynamic activity of microtubules, reducing this parameter by 75% at 32 nM. The present work, consistent with earlier in vitro studies, demonstrates that vinblastine kinetically caps the ends of microtubules in living cells and supports the hypothesis that the potent chemotherapeutic action of vinblastine as an antitumor drug is suppression of mitotic spindle microtubule dynamics. Further, the results indicate that molecules that bind to microtubule ends can regulate microtubule dynamic behavior in living cells and suggest that endogenous regulators of microtubule dynamics that work by similar mechanisms may exist in living cells.
OBJECTIVE: This study examined the applicability of the cognitive-behavioral model for temporomandibular disorders (TMD) by determining whether changes in TMD patient pain-related beliefs and coping over the course of treatment related to improvement in symptoms and disability and whether patients' posttreatment beliefs and coping predicted future pain and functioning. PATIENTS/SETTING: We studied 139 TMD patients at a health maintenance organization or a university dental school who completed a clinical trial comparing usual treatment with or without a brief cognitive-behavioral intervention. OUTCOME MEASURES/DESIGN: Pain, disability, depression, objective physical impairment, and pain beliefs and coping strategies were assessed pretreatment and at 3- and 12-month follow-ups. RESULTS: Increased ability to control pain and decreased Disease Conviction and Passive Coping scores were associated with improved pain, jaw opening, and depression from pretreatment to 3-month follow-up. Patient beliefs and coping at 3-month follow-up did not contribute much to the prediction of pain or physical and psychological functioning at 12-month follow-up after controlling for 3-month pain and functioning scores. However, passive coping and low ability to control pain at 3 months predicted greater activity interference at 12 months. CONCLUSIONS: Pretreatment to 3-month follow-up changes in beliefs and coping are associated modestly with TMD patient improvement after conservative dental treatment with and without a brief cognitive-behavioral intervention. Research is needed to develop interventions that produce greater decreases in disease conviction, passive coping, and perceived inability to control pain and to determine whether these changes mediate symptom and disability improvement.
We have previously shown in a large X-linked pedigree that a deletion removing the dystrophin muscle promoter, the first muscle exon and part of intron 1 caused a severe dilated cardiomyopathy with no associated muscle weakness. Dystrophin expression was present in the muscle of affected males and transcription studies indicated that this dystrophin originated from the brain and Purkinje cell isoforms, upregulated in this skeletal muscle. We have now studied dystrophin transcription and expression in the heart of one member of this family. In contrast to the skeletal muscle, dystrophin transcription and expression were absent in the heart, with the exception of the distal Dp71 dystrophin isoform, normally present in the heart. The 43- and 50-kD dystrophin-associated proteins were severely reduced in the heart, despite the presence of Dp71, but not in skeletal muscle. The absence of dystrophin and the down-regulation of the dystrophin-associated proteins in the heart accounted for the severe cardiomyopathy in this family. The mutation present in these males selectively affects dystrophin expression in the heart; this could be secondary to the removal of cardiac-specific regulatory sequences. This family may represent the first example of a mutation specifically affecting the cardiac expression of a gene, present physiologically in both the skeletal and cardiac muscles.
The observed increase in phosphotyrosine content of focal adhesion-associated proteins, in response to integrin engagement, indicates a role for integrin-regulatable tyrosine kinase(s) in cytoskeletal re-organisation. The tyrosine kinase pp125FAK, by virtue of its focal adhesion localisation in fibroblasts, represents a prime candidate to perform this function. We have investigated whether pp125FAK performs a similar function in mouse aortic smooth muscle cells (MASMC). MASMC cultured for 16 hours exhibit F-actin stress fibres and focal adhesions. We have shown that vinculin, pp125FAK and tyrosine-phosphorylated proteins are localised in focal adhesions during this time period. MASMC, under these culture conditions exhibit elevated pp125FAK tyrosine kinase activity, as measured by an increased autophosphorylation potential. We investigated the development of F-actin stress fibres and focal adhesions in MASMC in response to adherence to fibronectin, conditions shown to promote cytoskeletal reorganisation in fibroblasts. Within 30 minutes, MASMC exhibited well-developed F-actin stress fibres and prominent focal adhesions which immunostained intensely for vinculin, pp125FAK and phosphotyrosine. Adherence to fibronectin has been reported to activate pp125FAK tyrosine kinase in fibroblasts, leading to the proposal that pp125FAK plays a critical role in focal adhesion formation. Therefore pp125FAK activation, in response to adherence to fibronectin, was investigated in MASMC. Anti-phosphotyrosine immunoblotting and in vitro kinase assays of MASMC lysates have revealed that, under conditions which promote focal adhesion formation, pp125FAK remains inactive. Since overnight cultures of MASMC exhibited elevated pp125FAK tyrosine kinase activity, we investigated whether these cells deposit their own combination of extracellular matrix (ECM) molecules and/or secrete factors into their conditioned medium which are capable of activating pp125FAK tyrosine kinase. Our results indicate that MASMC-elaborated ECM, but not their conditioned medium, supported pp125FAK tyrosine kinase activation. Furthermore, MASMC exposed to MASMC-ECM displayed a poorly defined F-actin stress fibre network and rudimentary focal adhesions. Thus we have demonstrated the existence of two adhesion-mediated situations in MASMC; one in which fibronectin promotes cytoskeletal reorganisation in the absence of pp125FAK tyrosine kinase activity and the other in which cells adhering to MASMC-ECM display elevated pp125FAK tyrosine kinase activity in association with an impaired ability to promote F-actin stress fibre and focal adhesion formation. These results indicate that in MASMC, pp125FAK tyrosine kinase activity is not involved in F-actin stress fibre assembly and focal adhesion formation.
OBJECTIVE: The aim of this study was to measure the impact of two middle-trimester parenting communication classes on postpartum anxiety, marital adjustment, and postpartum adjustment. METHODS: Seventy primiparous, low-risk couples enrolled in prenatal classes in a large urban hospital were randomized to experimental and control groups. An educational intervention consisting of two second-trimester classes was facilitated by two social workers. The classes were based on a previous assessment of the educational needs of postpartum couples and contained didactic sessions, role-playing sessions, and values clarification exercises. The Spielberger State-Trait Anxiety Inventory and the Dyadic Adjustment Scale were administered to both groups prenatally in the second trimester and at 6 weeks and 6 months postpartum. A modified version of the O'Hara Postpartum Adjustment Questionnaire was administered at 6 weeks and 6 months postpartum. RESULTS: Both groups scored comparably in the prenatal period. The experimental group scored significantly lower on anxiety and higher on dyadic adjustment at both postpartum time periods than the control group. The experimental group also indicated a higher degree of postpartum adjustment. CONCLUSION: Prenatal parenting communication classes had a significant impact on postpartum anxiety, postpartum marital satisfaction, and postpartum adjustment.
A paclitaxel-resistant cell line, KPTA5, was established by co-selecting the parental erythroleukemic cell line K562 with stepwise increased concentrations of paclitaxel (Taxol) in the presence of the cyclosporin D analogue PSC 833 (2 microM), a potent modulator of the multidrug resistance phenotype. KPTA5 cells are 9-fold resistant to paclitaxel and taxotere, but do not exhibit significant resistance to Vinca alkaloids, etoposide, anthracyclines, antimetabolites, or alkylating agents. Doubling time and morphology were similar to the parental K562 cells. Reverse transcriptase-polymerase chain reaction (rt-PCR) analysis revealed no alterations in the expression of the mdr1 and MRP genes. Cellular paclitaxel accumulation was unchanged. Cell cycle analyses showed that at 20 nM there was a significantly higher proportion of K562 cells blocked in G2/M, in comparison with KPTA5 cells. In both cases, disruption of the mitotic spindles and the presence of multiple mitotic asters were comparable but occurred at lower paclitaxel concentrations in K562 cells than in KPTA5 cells. There was no difference in total tubulin content between K562 and KPTA5 cells as analyzed by immunoblotting with an anti-beta-tubulin monoclonal antibody. However, we found that KPTA5 cells presented a 2-fold increase both in 5 beta-tubulin mRNA expression and in the corresponding tubulin protein Class IV isotype content, as evaluated by rt-PCR and immunostaining. In conclusion, the KPTA5 cell line displays a novel mechanism of resistance to paclitaxel which does not involve altered cellular drug accumulation. The data presented suggest that alterations in expression of the 5 beta-tubulin gene may be involved in paclitaxel resistance.
The growing and shortening dynamics of individual bovine brain microtubules at their plus ends at steady state in vitro, assembled from isotypically pure alpha beta II, alpha beta III, or alpha beta IV tubulin dimers, were determined by differential interference contrast video microscopy. Microtubules assembled from the purified alpha beta III isotype were considerably more dynamic than microtubules made from the alpha beta II or alpha beta IV isotypes or from unfractionated phosphocellulose-purified tubulin. Furthermore, increasing the proportion of the alpha beta II isotype in a mixture of the alpha beta II and alpha beta III isotypes suppressed microtubule dynamics, demonstrating that microtubule dynamics can be influenced by the tubulin isotype composition. The data support the hypothesis that cells might determine the dynamic properties and functions of its microtubules in part by altering the relative amounts of the different tubulin isotypes.
1. This study was designed to determine whether cutaneous receptors in the hand exert reflex effects on fusimotor neurones innervating relaxed muscles. Recordings were made from fifty-four muscle spindle afferents in the radial nerve while the arm was held relaxed in a supporting frame. Cutaneous afferents were activated by trains of stimuli at non-noxious levels to the superficial radial nerve or to the palmar surface of the fingers. 2. For the population of muscle spindle afferents, the mean discharge rate was 7.1 +/- 6.4 Hz (range 0-24 Hz). Thirty-three per cent had no background discharge, and this occurred significantly more often in finger extensors than wrist extensors. 3. Trains of cutaneous stimuli produced no change in the discharge rates of the majority of spindle endings irrespective of whether the spindle afferent had a background discharge or was given one by muscle stretch. However, with two of forty afferents, the stimuli produced an increase in discharge at latencies of 135 and 155 ms. 4. With a further fourteen muscle spindle endings, the dynamic responses to stretch were measured 100-400 ms after the trains of cutaneous stimuli. For four spindle afferents there was a statistically significant change in the dynamic response to stretch occurring at conditioned-stretch intervals of 100-200 ms. For two afferents the dynamic response decreased by 17 and 26% and for two others it increased by about 24 and 37%. 5. While these results support the view that the level of background fusimotor drive is low in the relaxed state, they suggest that there is some dynamic fusimotor drive to completely relaxed muscles operating on the human hand, and that this drive can be altered reflexly by cutaneous afferent inputs from the hand.
A proportion of rats exposed to inescapable tailshock stress displayed a performance deficit, termed learned helplessness, in a subsequent shuttlebox avoidance task. The technique of in vivo microdialysis was used to determine hippocampal norepinephrine levels in learned helpless, nonhelpless and nonprestressed control rats. Similar basal norepinephrine levels were detected in samples between rat groups. Following an exposure to a milder form of inescapable shock, an increase in norepinephrine output was detected in learned helpless rats, which was significantly greater than nonhelpless, nonprestressed, or control animals. Thus, inescapable stress appears to sensitize the hippocampus to increase norepinephrine release in response to a subsequent smaller stressor. This hypersensitivity might underlie the avoidance impairment of learned helplessness. Therefore, the possibility exists that similar neurochemical changes may also be responsible for some of the symptoms of human posttraumatic stress disorder (PTSD), such as the poor coping associated with seemingly mild stress.
BACKGROUND: The purpose of this study is to determine the impact of adjuvant systemic chemotherapy and adjuvant hormonal therapy on local relapse in the conservatively treated breast. MATERIALS AND METHODS: Before December 1989, 548 patients underwent lumpectomy with axillary dissection followed by radiation therapy to the intact breast. Adjuvant systemic therapy was administered as clinically indicated. The majority of patients with pathologically involved lymph nodes received adjuvant systemic therapy, whereas those with pathologically negative lymph nodes received no adjuvant systemic therapy. The majority of patients received a course of radiation therapy either concomitant with or before systemic therapy. In only nine cases was radiation therapy delayed more than 16 weeks after surgery. RESULTS: As of June 1992, the 548 patients had a median follow-up of 6.4 years. In univariate and multivariate Cox regression analysis, patient age and adjuvant systemic chemotherapy were statistically significant independent prognostic factors relating to breast relapse. Those patients who received adjuvant systemic chemotherapy had a lower breast relapse than those who did not. Among patients who received tamoxifen, there was a statistically insignificant trend toward a lower relapse rate compared with those who did not receive tamoxifen. CONCLUSIONS: It appears from this retrospective analysis that patients who received adjuvant systemic therapy, either concomitantly or after their course of radiation therapy, had a lower relapse rate in the conservatively treated breast than those patients who received no adjuvant systemic therapy.
4-Nitro-o-phenylenediamine (NOP) is a powerful direct-acting mutagen which demonstrates significant enhancement in mutagenicity when exposed to plant enzymatic systems. Evidence implicating the involvement of peroxidactic oxidation in NOP activation has been obtained from plant-cell suspension and isolated enzyme experiments. Using selected cytochrome P450 and peroxidase enzyme inhibitors in conjunction with Salmonella typhimurium strain TA98 and intact plant-cell activating systems as well as isolated horseradish peroxidase enzyme we have further investigated NOP activation by plant systems. The activation of NOP by both plant cells and by horseradish peroxidase was suppressed by the P450 inhibitors methimazole and (+)-catechin and by the peroxidase inhibitors diethyldithiocarbamate and potassium cyanide, but was not suppressed by the P450 inhibitors metyrapone and 7,8-benzoflavone. In addition, peroxidase enzymatic activity was measured and found to be inhibited by methimazole, diethyldithiocarbamate and potassium cyanide but not by (+)-catechin. The data strongly support the involvement of exogenous peroxidase in the plant activation of NOP, but point to a complex metabolic system that requires multistep processing before full mutagenic potential of the plant-activated component of NOP is expressed.
The cysteine proteinases synthesized by the adult stage of the trematode Fasciola hepatica were found to be a very heterogeneous group of proteins as demonstrated by one- and two-dimensional gel analyses. N-terminal amino acid sequencing indicated the presence of at least two distinct gene products among the secreted cysteine proteinases. Enzymic studies and peptide sequence analysis of the excreted/secreted cysteine proteinases suggested a close relationship to the plant thiol cathepsins and the mammalian cathepsin L subfamily. The cloning of a representative cDNA for a putative Fasciola cathepsin confirmed similarities to the cathepsin L subfamily but revealed low identity with the cathepsin-like proteinases of the related trematode, Schistosoma, nematode cathepsins and the mammalian cathepsin B subfamily. Furthermore, peptide and protein sequencing revealed the modification of certain highly conserved prolines to unusual 3-hydroxyproline derivatives. This is the first report of modified prolines in any proteinase. This finding, as well as the high activities of these cathepsins at neutral to alkaline pH values, raises a number of questions as to the physiological function of these thiol cathepsins and their interaction with host tissues.
To assess the pattern of development of serologic markers during acute hepatitis B, levels of HBsAg, HBeAg, and hepatitis B virus (HBV) DNA were assayed in stored serum samples obtained sequentially from 12 subjects infected with HBV during experimental studies conducted in the 1950s. Six patients developed acute self-limited hepatitis, three developed chronic hepatitis, and three had an asymptomatic infection without HBsAg. HBsAg was the first serologic marker detected (mean = 52 days after exposure), followed by HBeAg (62 days) and HBV DNA (72 days). Peak HBsAg levels occurred before onset of symptoms and correlated with peak titers of HBeAg and HBV DNA. Patients who developed chronic hepatitis had higher peak levels of viral markers than those with self-limited disease: HBsAg (30 versus 5.4 micrograms/ml), HBeAg (1:2,000 versus 1:60 titer) and HBV DNA (3,192 versus 444 pg/ml). Thus, chronic HBV infection is characterized by high levels of viral replication appearing early during the acute phase of infection.
There has been some evidence from several parasite systems that proteinases might have potential as protective antigens against parasitic infection. A cysteine proteinase complex identified in the regurgitant of adult F. hepatica was examined in this context. The thiol-cathepsin-related proteinases of M(r) 28,000 were purified and tested in vaccine trials of sheep infected with liver fluke. Ten animals were immunised with the purified proteinases and developed antibodies to the cysteine proteinases prior to challenge with F. hepatica metacercariae. Infection appeared to cause a boost in antibody response by Week 4 into infection, and antibody levels were generally sustained throughout infection. The cysteine proteinases are not novel antigens, since low-level antibody titres were also detected in nonimmunised controls by late infection. On completion of the trial, there was no difference in worm burden between the two groups. However, faecal egg counts and therefore worm fecundity were significantly decreased.
A library of the equine herpesvirus 4 (EHV-4) genome was constructed in the lambda gt11 expression vector. Recombinant bacteriophage expressing EHV-4 antigens as beta-galactosidase fusion proteins were detected with rabbit antiserum raised against EHV-4 virions and convalescent horse serum. EHV-4 DNA sequences contained in the immunopositive recombinants were used as hybridization probes for mapping the genes encoding the antigens on the viral genome. The DNA sequence of the probes was determined. Screening the library with rabbit antiserum led to the identification of 40 recombinants, 26 of which were further characterized. Determination of the DNA sequence of the EHV-4 inserts revealed that 23 of the recombinants encode an identical portion of glycoprotein gB. Two of the recombinants encode a portion of the previously unidentified EHV-4 homologue of the EHV-1 immediate early protein. The EHV-4 insert of the remaining recombinant encodes a portion of the previously unidentified EHV-4 homologue of herpes simplex virus 1 (HSV-1) UL36, a tegument protein. Screening the library with horse serum led to the identification of three recombinants, one of which encodes the same gB sequence as the gB recombinant recognized with the rabbit serum. The other two contain overlapping sequences that encode a portion of EHV-4 gX.
OBJECTIVE: A potent, long-acting oxytocin antagonist produced in our laboratory (ANTAG-III) can inhibit uterine response to oxytocin in the rat and baboon for hours and even days. The purpose of this study was to evaluate uterine response to prostaglandins subsequent to the administration of ANTAG-III. STUDY DESIGN: For the rat study one cannula was inserted in the jugular vein, and another cannula to measure uterine activity was inserted in the uterus. In study 1 saline solution or 5 micrograms of ANTAG-III was administered to five rats each, followed by 100 mU of oxytocin at 0.1, 1, and 2 hours. In study 2 six rats each were infused with saline solution of 5 micrograms of ANTAG-III, followed 1 hour later by 5 micrograms of 15-methyl-prostaglandin F2 alpha and uterine activity monitored. After baseline activity returned to normal 100 mU of oxytocin was infused and the uterine response reassessed. For the baboon study ANTAG-III was administered into the aorta of tethered pregnant baboons (n = 2). An oxytocin challenge test was performed starting with 10 mU/min and going up to 400 mU/min. After a significant uterine contractile response was established and activity returned to baseline, a 15-methyl-prostaglandin F2 alpha challenge test was performed. RESULTS: During the period in which the response to oxytocin was inhibited the uterine response to 15-methyl-prostaglandin F2 alpha of the estrous rat and pregnant baboon was maintained. CONCLUSIONS: The inhibition of the estrous rat and pregnant baboon uterus to oxytocin caused by ANTAG-III may be prolonged. During this period uterine response to prostaglandins is not altered.