Cleavage of Müllerian inhibiting substance activates antiproliferative effects in vivo

Müllerian inhibiting substance (MIS), an inhibitor of growth and development of the female reproductive ducts in male fetuses, requires precise proteolytic cleavage to yield its biologically active species. Human plasmin is now used to cleave and, thereby, activate immunoaffinity-purified recombinant human MIS at its monobasic arginine-serine site at residues 427-428. To avoid the need for exogenous enzymatic cleavage and to simplify purification, we created an arginine-arginine dibasic cleavage site (MIS RR) using site-directed mutagenesis to change the serine at position 428 (AGC) to an arginine (cGC). The mutant cDNA was then stably transfected into a MIS-responsive ocular melanoma cell line, OM431, followed by cloning for amplified expression to test its biological activity in vitro and in vivo. Media from each clone were assayed for production of MIS RR by a sensitive ELISA for holo-MIS, and high- and low-producing clones were selected for further study. Media from the highest MIS RR producer caused Müllerian duct regression in an organ culture bioassay. Other transfections were done with an empty vector (pcDNAI Neo) or a construct lacking the leader sequence and thus failing to secrete MIS, to serve as controls. The OM431 clones containing the MIS RR mutant were growth inhibited in monolayer culture. The high- and low-producing MIS RR OM431 clones, along with transfected OM431 controls, were injected into the tail veins of immunosuppressed severe combined immunodeficiency mice for in vivo analyses. Four to 6 weeks later, pulmonary metastases were counted in uniformly inflated lungs. OM431 clones containing the more easily cleaved MIS RR displayed a significant dose-dependent reduction in pulmonary metastases when compared to the lungs of animals given injections of OM431 clones containing empty vector, leaderless MIS, or wild-type MIS that requires activation by plasmin cleavage. Since the purification protocol of MIS RR is less complicated than that for wild-type MIS, which requires subsequent enzymatic activation, MIS RR can be used for scale-up production with increased yields for further therapeutic trials against MIS-sensitive tumors.     

The effect of Pseudomonas aeruginosa infection on clinical parameters in steady-state bronchiectasis

STUDY OBJECTIVE: To investigate the effect of Pseudomonas aeruginosa infection on clinical parameters in Chinese patients with noncystic fibrosis and steady-state bronchiectasis. DESIGN: Prospective, cross-sectional clinicomicrobiological study with informed consent. SETTING: Consecutive outpatient recruitment from a specialist bronchiectasis respiratory clinic. PATIENTS: Outpatients (n = 100; 62 women; 55.1+/-16.7 years old; FEV1/FVC 1.4+/-0.7/2.1+/-0.9 L), who had stable respiratory symptoms for more than 3 weeks. MEASUREMENTS AND RESULTS: Respiratory pathogens isolated from the sputum were: Pseudomonas aeruginosa (33), Haemophilus influenzae (10), Moraxella catarrhalis (2), other Gram-negative bacilli (5), Streptococcus pneumoniae (6), Staphylococcus aureus (5), mycobacteria (3), and yeast (1). Clinical parameters in patients with positive isolation of P aeruginosa were compared with those without the organism in the sputum culture (non-P aeruginosa). In the P aeruginosa group, the FEV1/FVC ratio and sputum volume were lower (p < 0.005) and higher (p < 0.0001), respectively, than those of the non-P aeruginosa group. The FEV1/FVC ratio (< 60%) and sputum volume (grading > 5) were independently associated with a positive sputum isolation of P aeruginosa with odds ratios of 3.1 (confidence interval [CI] 1.2 to 8.4; p < 0.01) and 4.7 (CI 1.6 to 13.3; p < 0.001), respectively. CONCLUSIONS: P aeruginosa is the predominant respiratory pathogen isolated in the sputum of Chinese patients with steady-state bronchiectasis, and its isolation is associated with high sputum output (> or = 75th quartile) and moderately severe airflow obstruction (FEV1/FVC < 60%).     

Injections of D-amphetamine into the ventral pallidum increase locomotor activity and responding for conditioned reward: a comparison with injections into the nucleus accumbens

The nucleus accumbens and ventral pallidum receive dopamine (DA) projections from the mesencephalon. Although DA inputs to the nucleus accumbens are implicated in both locomotion and reward processes, little is known of the behavioural significance of DA in the ventral pallidum. These studies examined the effects of D-amphetamine injected into the nucleus accumbens or ventral pallidum on locomotor activity and responding for a conditioned reward (CR). In the nucleus accumbens D-amphetamine dose dependently (1, 3 and 10 microg) increased locomotion within 5-10 min of injection. Intra-ventral pallidum microinjections of D-amphetamine also increased activity in this dose range, but the effect occurred with a longer latency (5-20 min). The magnitude of the response evoked by ventral pallidum injections was lower than that evoked by nucleus accumbens injections. The GABAA antagonist picrotoxin (0.1 microg) stimulated activity when injected into the ventral pallidum but not the nucleus accumbens, providing a pharmacological dissociation between the two injection sites. In the CR studies, D-amphetamine injected into both sites potentiated responding for a CR previously paired with food delivery, without altering responding on an inactive lever. Picrotoxin injected into the ventral pallidum reduced responding and abolished the selectivity of responding for CR. The results show that DA release in the ventral pallidum enhances locomotion and responding for a CR, providing evidence that DA in the ventral pallidum plays a significant role in the mediation of the effects of D-amphetamine. The failure of picrotoxin to elevate responding for CR despite increasing locomotor activity indicates that pharmacologically-induced blockade of GABAA receptors in the ventral pallidum disrupts goal-directed responding.     

The anaerobic oxidation of hydrazine: a novel reaction in microbial nitrogen metabolism

PURPOSE: Pulmonary involvement (PI) is common in leptospiral infection, usually characterized by hemoptysis and diffuse bilateral infiltrates on chest radiographs. Alveolar haemorrhage (AH) has already been proved by autopsy and some case-reports with fiberoptic bronchoscopy (FB) and bronchoalveolar lavage (BAL). The purpose of this study was 1/to evaluate the incidence of AH in leptospirosis 2/to define the impact of BAL on the early diagnosis of the infection. PATIENTS AND METHODS: FB with BAL were performed in 23 consecutive patients with leptospirosis (13 patients with patent signs of PI: group 1, 10 patients without: group 2). AH was defined by a percentage of siderophages > or = 20% and/or a Golde score > 100 and/or an haemorrhagic aspect of BAL fluid. Culture tests were performed on specific medium. RESULTS: We diagnosed AH in all patients of group 1 and in 7 patients of group 2. Filaments were seen in 6 specimens of BAL fluid, initially thought to be leptospires, but culture tests were negative. CONCLUSION: AH is identified in all cases of leptospirosis with PI. Occult AH often occurs to patients without any respiratory symptom. Physicians should consider leptospiral infection in the differential diagnosis of AH. Culture-tests for leptospirosis in BAL do no help in diagnosing leptospirosis.     

Effects of accelerated testing on porcine bioprosthetic heart valve fiber architecture

We undertook the following study to quantitatively assess the changes in porcine bioprosthetic heart valve (PBHV) fiber architecture to increasing levels of fatigue damage using an in vitro accelerated test model. PBHVs were subjected to 0-500 million test cycles at 16 Hz, and small-angle light scattering (SALS) was used to quantify the gross fiber structure of the cusps. The degree of gross fiber alignment remained essentially constant from 0 to 500 million cycles over the entire cusp. Increasing fiber orientation randomness, indicative of local damage, was observed only in the vicinity of the nodulus of Arantii after 50 million cycles. The SALS data from the damaged regions suggested shearing between fiber layers, which may be part of the failure process and accelerates valve failure. Histological analysis revealed a relatively intact gross fiber structure with the collagen fiber crimp remaining, although delamination and de-registration of the crimp was also observed. Accelerated tested PBHVs also demonstrated a pronounced 'sagging', which began at the earliest cycle number tested (1.4 million cycles) and whose rate decreased logarithmically with cycle number. Results of this study suggest that PBHV cusps can alter their shape without any visually apparent material yielding or fiber failure under continual cyclic loading. Further, while most of the 4 mmHg pressure fixed PBHV's gross fiber architecture remains unchanged after 500 million cycles of accelerated testing, localized accumulated fiber damage can occur on a sub-visual structural level as early as 50 million cycles.     

Uncertainty of discrete stochastic systems: general theory andstatistical inference

Uncertainty is defined in a new manner, as a function of discrete probability distributions satisfying a simple and intuitively appealing weak monotonicity condition. It is shown that every uncertainty is Schur-concave and conversely, every Schur-concave function of distributions is an uncertainty. General properties of uncertainties are systematically studied. Many characteristics of distributions introduced previously in statistical physics, mathematical statistics, econometrics and information theory are shown to be particular examples of uncertainties. New examples are introduced, and traditional as well as some new methods for obtaining uncertainties are discussed. The information defined by decrease of uncertainty resulting from an observation is investigated and related to previous concepts of information. Further, statistical inference about uncertainties is investigated, based on independent observations of system states. In particular, asymptotic distributions of maximum likelihood estimates of uncertainties and uncertainty-related functions are derived, and asymptotically α-level Neyman-Pearson tests of hypotheses about these system characteristics are presented     

Protective factors in adolescent health behavior

The role of psychosocial protective factors in adolescent health-enhancing behaviors--healthy diet, regular exercise, adequate sleep, good dental hygiene, and seatbelt use--was investigated among 1,493 Hispanic, White, and Black high school students in a large, urban school district. Both proximal (health-related) and distal (conventionality-related) protective factors have significant positive relations with health-enhancing behavior and with the development of health-enhancing behavior. In addition, in cross-sectional analyses, protection was shown to moderate risk. Key proximal protective factors are value on health, perceived effects of health-compromising behavior, and parents who model health behavior. Key distal protective factors are positive orientation to school, friends who model conventional behavior, involvement in prosocial activities, and church attendance. The findings suggest the importance of individual differences on a dimension of conventionality-unconventionality. Strengthening both proximal and distal protective factors may help to promote healthful behaviors in adolescence.     

Leiomyosarcoma of the esophagus: results of surgical treatment

BACKGROUND: This study examined the results of surgical treatment of leiomyosarcoma of the esophagus. METHODS: Between January 1920 and December 1996, 17 patients (9 men and 8 women) with leiomyosarcoma of the esophagus were treated surgically at the Mayo Clinic. Median age was 58 years and ranged from 26 to 76 years. Symptoms included dysphagia in 11 patients (64.7%) and odynophagia in 6 (35.3%). The tumor was located in the middle third of the esophagus in 10 patients (58.8%) and in the cervical esophagus in 7 (41.2%). Procedures performed included esophagogastrectomy in 9 patients (Ivor Lewis in 5, left thoracoabdominal in 3, and transhiatal in 1), enucleation in 3, transgastric excision in 1, and exploration without resection in 4. RESULTS: The procedure was considered curative in 11 patients (64.7%). There was one operative death (mortality, 5.9%). Complications occurred in 3 patients (17.6%) and included anastomotic leak in 2 and bleeding requiring reoperation in 1. Growth pattern was infiltrating in 7, polypoid in 5, and intramural in 5. Histologically, the tumor was grade 1 in 6 patients, grade 2 in 2, grade 3 in 7, and grade 4 in 2. The tumor was postsurgically classified as stage I in 2 patients, stage IIA in 7, stage IIB in 1, stage IIIA in 5, stage IV in 1, and unknown in 1. Six patients (35.3%) received adjuvant treatment. Follow-up was complete in 16 patients (94.1%) and ranged from 1 to 182 months (median, 48 months). Five- and 10-year actuarial survivals were 47.0% and 31.0%, respectively. Seven patients (41.2%) are currently alive (median survival, 72 months); all underwent curative resection. Factors affecting survival included completeness of resection, growth pattern, postsurgical stage, tumor grade, and tumor location (p < 0.05). CONCLUSIONS: We conclude that leiomyosarcoma of the esophagus is rare. Complete resection provides long-term survival.     

Incidence, etiology, location, prevention and treatment of positive surgical margins after radical prostatectomy for prostate cancer

PURPOSE: During radical prostatectomy for prostate cancer tumor at the surgical margin is a relatively frequent finding. We summarize the literature on the incidence, etiology, location, prevention and treatment of positive surgical margins after radical prostatectomy. MATERIALS AND METHODS: The literature was reviewed for data on positive margins during radical prostatectomy for prostate cancer. RESULTS: Positive surgical margins may result from artifacts induced by tissue processing, incising inadvertently into the prostate or incising into extraprostatic tumor that has extended beyond the limits of resection. Patients with 10 ng./ml. or greater preoperative prostate specific antigen, biopsy Gleason score 7, multiple positive biopsies, or clinical stage T2b, T2c or T3 cancer have a higher risk of positive margins. Preoperative endorectal magnetic resonance imaging may be useful in staging a select group of patients. Neoadjuvant androgen deprivation reduces the incidence of positive margins but does not appear to delay progression or improve survival. The surgical approach, retropubic or perineal, may influence the location and etiology of positive margins. In general, nerve and bladder neck sparing procedures do not compromise tumor removal in appropriately selected patients. Positive margins increase the risk of progression and correlate with decreased cancer specific and overall survival. There is no consensus on the management of positive margins. External beam radiation and androgen deprivation may be administered as adjuvant therapy or at the time of recurrence. CONCLUSIONS: Tumor at the specimen edge is an adverse prognostic factor. With appropriate patient selection and meticulous surgical technique some positive margins can be prevented. Controlled prospective randomized studies of postoperative therapy are needed before definitive recommendations can be made for treating positive margins.     

Evaluation of fine needle capillary sampling in superficial and deep-seated lesions. An analysis of 670 cases

The gene rafY from the plasmid pRSD2, which enables Escherichia coli to grow on raffinose, was transferred into expression plasmid pUSL77. The protein was expressed in the porin-deficient Escherichia coli strain KS26 and was isolated and purified to homogeneity. The pure protein was reconstituted into lipid bilayer membranes. It formed an ion-permeable channel with a single-channel conductance of 2.9 nS of the open state in 1 M KCl, which is approximately twice of that of the general diffusion pores OmpF and OmpC of E. coli outer membrane. At lower pH the channel exhibited rapid flickering between three substates of the open channel. The RafY channel appears to be wide and water filled and has a small selectivity for cations over anions. Although RafY is part of an uptake and fermentation system for raffinose it does not contain a binding site for carbohydrates. Our results suggest that RafY is a general diffusion pore with a diameter, larger than that of the general diffusion porins OmpF and OmpC, that allows the diffusion of high-molecular-mass carbohydrates through the outer membrane.