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.     

Full expression of the exaggerated salt appetite of the spontaneously hypertensive rat requires a prenatal factor

1. The spontaneously hypertensive rat (SHR) exhibits a lower bodyweight in utero and an exaggerated salt appetite post partum. To determine whether salt appetite is affected by the perinatal environment, we measured the salt appetite of embryo-cross-transferred SHR and Wistar Kyoto (WKY) rats at maturity. 2. One-cell embryos were collected from the oviducts of donor rats and transferred into the oviducts of recipients through the infundibulum. The salt appetite of the resultant female offspring for 0.10 and 0.15 mol/L saline was measured at 20-30 weeks of age. 3. Salt intake of SHR gestated in WKY rats was significantly lower than that of SHR gestated in SHR, while that of WKY rats gestated in SHR was higher than that of WKY rats gestated in WKY rats. 4. Therefore, some maternal factor plays a role in the development of the exaggerated salt appetite of the SHR. This factor is also able to affect the later salt appetite of WKY rat offspring born to SHR surrogates.     

Diagnosis of fetal renal anomalies

Urinary tract abnormalities are fairly common in the perinatal period. Ultrasonography is an effective diagnostic tool for identifying fetal urinary tract anomalies and can be helpful in establishing prognosis and guiding treatment decisions. The sonographer is encouraged to characterize and localize the fetal urinary tract abnormality when identified. In doing so, the correct diagnosis can be selected from the differential possibilities, providing vital clinical information needed to develop a care plan for both the pregnant woman and her fetus.     

Analysis of regulated exocytosis in adrenal chromaffin cells: insights into NSF/SNAP/SNARE function

In a duplicate study during 1987-1991, 478 24-h duplicate samples from 14 homes for elderly people and 10 homes for youth were investigated for their contents of selected harmful substances. The analyses covered 45 active substances of pesticides, 17 PCB-congeners as well as lead, cadmium, and nitrate contents. Pesticides could be detected only in 15% of the investigated samples. The pesticide contents reached max. 8% of the respective FAO/WHO-limits. As the mean intake of the three most important PCB-congeners (sum of the congeners 138, 153, and 180) values of 0.9 and 1.1 micrograms per day and ration or person, respectively, were found. Also in the worst case the daily PCB intake was below the recommended ADI value of the FDA of 1 microgram/kg of body weight. The daily nutritional intake of lead and cadmium via the investigated daily rations reached about 5.6% and 20% of the Provisionally Tolerably Weekly Intake values of the FAO/WHO. The mean nitrate content of the duplicate portions was 101 mg per day and person (median: 79 mg per day and person). Referred to the median value the WHO limit (3.65 mg/kg body weight and day) was exhausted to about 36%.     

Inversin, a novel gene in the vertebrate left-right axis pathway, is partially deleted in the inv mouse

A DNA fragment containing the recA gene of Gluconobacter oxydans was isolated and further characterized for its nucleotide sequence and ability to functionally complement various recA mutations. When expressed in an Escherichia coli recA host, the G. oxydans recA protein could efficiently function in homologous recombination and DNA damage repair. The recA gene's nucleotide sequence analysis revealed a protein of 344 amino acids with a molecular mass of 38 kDa. We observed an E. coli-like LexA repressor-binding site in the G. oxydans recA gene promoter region, suggesting that a LexA-like mediated response system may exist in G. oxydans. The expression of G. oxydans recA in E. coli RR1, a recA+ strain, surprisingly caused a remarkable reduction of the host wild-type recA gene function, whereas the expression of both Serratia marcescens recA and Pseudomonas aeruginosa recA gene caused only a slight inhibitory effect on function of the host wild-type recA gene product. Compared with the E. coli RecA protein, the identity of the amino acid sequence of G. oxydans RecA protein is much lower than those RecA proteins of both S. marcescens and Pseudomonas aeruginosa. This result suggests that the expression of another wild-type RecA could interfere with host wild-type recA gene's function, and the extent of such an interference is possibly correlated to the identity of the amino acid sequence between the two classes of RecA protein.     

Regional 5-hydroxyindoleacetic acid production in humans

Veno-arterial plasma concentration differences and regional organ plasma flows were used to quantify the relative amounts of 5-hydroxyindoleacetic acid (5-HIAA) contributed by various sites into the peripheral circulation. Positive venoarterial concentration gradients were found in the hepatosplanchnic, forearm, cardiac and jugular vessels in the healthy subjects. The renal circulation was determined to be the principal site of 5-HIAA clearance, extracting 18 +/- 2 nmol/min. The gut was the greatest contributor to the total 5-HIAA plasma pool with the relative contributions of the various organs being as follows: hepatosplanchnic organs 58%, skeletal muscle 26%, brain 6% and the heart 3%. The source of 5-HIAA stemming from these regional beds remains unknown, it may derive from serotonin taken up by and deaminated in ubiquitous endothelial cells, enterochromaffin cells of the gut, peripheral serotonergic nerves, serotonin turnover in platelets or perhaps the metabolism of serotonin taken up by sympathetic nerves. To test the latter hypothesis we examined 23 patients with chronic congestive heart failure and 9 patients with pure autonomic failure to investigate the possible effects of sympathetic nervous system overactivity and underactivity on peripheral 5-HIAA production and plasma 5-HIAA concentration. The resting arterial plasma 5-HIAA concentration in the heart failure patients was increased three-fold. This elevated plasma 5-HIAA concentration was attributable to an increased rate of whole body 5-HIAA production. The arterial 5-HIAA plasma concentration in the autonomic failure patients was paradoxically elevated, being 70% greater than that of the healthy subjects. The increased 5-HIAA plasma concentration in these patients was accounted for by a reduction in 5-HIAA plasma clearance. In all subjects studied there was a weak relationship only between total body norepinephrine spillover to plasma and the arterial 5-HIAA plasma concentration. We found that in healthy subjects the overflow of 5-HIAA into the hepatic vein was significantly related to the underlying degree of sympathetic activity. It can be concluded that 5-HIAA is produced at a number of sites throughout the body with the arterial plasma concentration being dependent on both the level of production and plasma clearance. By far the majority of 5-HIAA in plasma is derived from the gut with only minimal contribution from the brain.     

Radiation doses in the population of the coastal area of the Kakhovsk Water Reservoir

Central and cerebral hemodynamics was studied using echocardiography and transcranial dopplerography in 200 poststroke patients over 60 in the course of 5-year follow-up. It was established that cardiac hypodynamic syndrome, i.e. continuous or paroxysmal fall of minute blood volume below 3 l, is the most significant contributing factor in the onset of cerebral ischemia. Drugs are recommended for prevention of ischemic stroke. Those lowering cardiac hemodynamic reserve should be excluded in patients at high risk of acute failure of cerebral circulation.     

Is there a correlation between raised erythropoietin and thrombotic events in sickle-cell anaemia?

Children with sickle-cell anaemia are predisposed to thrombotic strokes, the aetiology of which is unclear. We propose that erythropoietin, produced in response to chronic anaemia, is responsible for changes in platelet reactivity with a resulting increase in thromboses. This hypothesis is based on reports of enhanced aggregability of erythropoietin-driven platelets and an increased rate of thrombosis in patients receiving large doses of recombinant erythropoietin. Experiments in animals have shown that erythropoietin stimulates synthesis of platelets, that erythropoietin-driven platelets are hyper-reactive compared with age-matched control platelets, and that erythropoietin is pro-thrombotic. These data suggest that erythropoietin-dependent changes in platelet reactivity may potentiate thrombosis in sickle-cell anaemia, particularly in children who, compared with adults, have markedly higher erythropoietin concentrations and incidence of strokes.     

Process Requirements for Cost-Effective Precision Grinding

Costs in precision cylindrical grinding are compared for different abrasives, machines and grinding conditions. The analysis is for repeated batch production. Account is taken of machine cost and abrasive cost. Cost comparisons were based on extensive trials to assess re-dress life against workpiece quality requirements. Experiments show that different workpiece materials require different strategies to reduce costs. Easy-to-grind AISI 52100 and difficult-to-grind Inconel 718 materials were ground at conventional speeds and at high speeds. It is shown that wheel speed affects production rate through acceptable values of re-dress life, removal rate and dwell time. Advantages were gained using vitrified CBN at conventional speed and at high speed. For both materials, vitrified CBN wheels used at high speed, gave better quality at lower cost than conventional abrasives. Wheel costs became negligible and labour costs greatly reduced. Re-dress life trials, usually neglected, are shown to be essential to reduce costs and maintain quality [1].