Genetic organization and nucleotide sequence of the stability locus of IncFII plasmid NR1

The stability (stb) locus of IncFII plasmid NR1 was mapped to a 1700 base-pair NaeI-TaqI restriction fragment. A series of unstable plasmids that contained insertion, deletion, and point mutations that inactivated the stability function was isolated. The unstable point mutants examined were all stabilized (complemented) in trans by a copy of the wild-type stb locus, suggesting that the mutations had inactivated diffusible gene products. The nucleotide sequence of the stb locus contained two tandem open reading frames, designated stbA and stbB, that encoded essential trans-acting protein products with predicted sizes of 36,000 Mr and 13,000 Mr, respectively. A third open reading frame, stbC, that could encode a peptide of 8000 Mr was contained within stbB in the complementary DNA strand. Plasmid-encoded proteins of 36,000 Mr and 13,000 Mr were identified in minicell experiments as the products of stbA and stbB, respectively. Unstable deletion mutants that retained the promoter proximal region of the stb locus upstream from stbA but had deleted both stbA and stbB were stabilized in trans by plasmids that could supply StbA and StbB. In contrast, deletion mutants that had lost the stbAB promoter region were not complemented in trans, indicating that this region contained an essential cis-acting site (or sites). Unlike some other loci that mediate stable plasmid inheritance, cloned copies of the wild-type stb locus of NR1 did not exert strong incompatibility (i.e. trans destabilization) against other stb+ derivatives of plasmid NR1 present in the same cell.     

Reduction of tumor growth following treatment with a glutamine antimetabolite

Assessment of arterial-venous differences across transplanted methylcholanthrene-induced sarcomas in rats revealed significant decreases in plasma concentrations of glutamine, serine and glucose. Treatment with the glutamine antimetabolite, acivicin, significantly reduced tumor weights by 65% at the conclusion of the experiment 34 days after tumor induction. These results suggest that glutamine is an essential metabolic substrate for tumor growth and that blockade of glutamine utilization can inhibit the growth of these transplantable sarcomas.     

Measurement of total lung aerosol deposition as an index of lung abnormality

Total aerosol deposition in the lung was measured in 100 subjects with various lung conditions. The subjects consisted of 40 normals (N), 15 asymptomatic smokers (S), 10 smokers with small airway disease (SAD), 20 with chronic simple bronchitis (SB), and 15 with chronic obstructive bronchitis (COPD), and a relationship of total aerosol deposition to degree of lung abnormality was investigated. The subjects were categorized by medical history and a battery of pulmonary function tests, including spirometry, body plethysmography, and single and multiple N2 washout measurements. Subjects repeatedly breathed a monodisperse test aerosol (1.0 micron diam) from a collapsible rebreathing bag (0.5 liter volume) at a rate of 30 breaths/min, while inhaled and exhaled aerosol concentrations were continuously monitored by a laser aerosol photometer in situ and recorded on a strip-chart recorder. The number of rebreathing breaths resulting in 90% aerosol loss from the bag (N90) was determined, and percent predicted N90 values were then determined from the results of computer simulation and used as a deposition index. The percent predicted N90 values were 99.7 +/- 14, 86.5 +/- 15, 66.9 +/- 17, 51 +/- 12, and 30.9 +/- 9, respectively, for N, S, SAD, SB, and COPD. All of these values were significantly different from each other (P less than 0.05). There was no difference between male and female but percent predicted N90 values were slightly higher in young than in old normals. Percent predicted N90 values showed a strong linear correlation with spirometric measurements of forced expiratory volume in 1 s and maximum midexpiratory flow rate. However, many of the SAD and SB with normal spirometry showed abnormal N90. These results suggest that total lung aerosol deposition is a sensitive index of lung abnormality and may be of potential use for nonspecific general patient screening.     

Sodium-dependent lysine flux across bullfrog alveolar epithelium

Amino acid transport across the alveolar epithelial barrier was studied by measuring radiolabeled lysine fluxes across bullfrog lungs in an Ussing chamber. In the absence of a transmural electrical gradient, L-[14C]lysine was instilled into the upstream reservoir and the rate of appearance of the radiolabel in the downstream reservoir was determined. Two lungs from the same animal were used simultaneously to determine tracer fluxes both into and out of the alveolar bath. Results showed that the radiolabel flux measured in the alveolar to the pleural direction was greater than that measured in the opposite direction in the presence of sodium in the bathing fluids. The net flux of L-[14C]lysine was saturable with [Na+], with an apparent transport coefficient (Kt) of 28 mM for Na+. Hill analysis of [14C]lysine flux vs. [Na+] indicated a coupling ratio of 1:1 between sodium and radiolabeled L-lysine. Total L-lysine flux as a function of [L-lysine] was also saturable, with Kt of 7.3 mM for L-lysine. Ouabain significantly decreased absorptive (alveolar-to-pleural) radiolabel flux, while slightly increasing the flux observed in the opposite direction. L-leucine completely inhibited absorptive net flux of L-[14C]lysine. alpha-Methylaminoisobutyric acid (MeAIB), on the other hand, only slightly reduced net flux of L-[14C]lysine from the control value. The presence of a net absorptive, Na+-dependent amino acid flux across the alveolar epithelial barrier indicates that the tissue is capable of removing amino acids and sodium from the alveolar fluid by a coupled cotransport mechanism, which may be important for both protein metabolism and fluid balance by alveolar epithelium.     

Further study of effects of synthetic peptides on identifiable giant neurones of an African giant snail (Achatina fulica Férussac)

1. Effects of the following peptides at 10(-4) M on identifiable giant neurones of Achatina fulica Férussac were examined: physalaemin, eledoisin, bradykinin, neurokinin A, neurokinin B, neuromedin B, gastrin releasing peptide decapeptide (neuromedin C), gastrin releasing peptide (14-27), cholecystokinin tetrapeptide, cholecystokinin octapeptide, thyrotropin releasing hormone, Arg-vasotocin, gamma-melanocyte stimulating hormone. 2. The six neurones tested were as follows: PON (periodically oscillating neurone), TAN (tonically autoactive neurone), RAPN (right anterior pallial neurone), d-RPLN (dorsal-right parietal large neurone), VIN (visceral intermittently firing neurone) and d-VLN (dorsal-visceral large neurone). 3. Of the peptides examined, only Arg-vasotocin at 10(-4) M produced the excitatory effects on PON, VIN and d-VLN. Physalaemin showed slight inhibitory effects on TAN; this substance was sometimes almost ineffective on the neurone. 4. The other peptides examined were completely ineffective on all of the neurones tested.     

Divergent changes in the concentrations of gonadotropin beta-subunit messenger ribonucleic acid during the estrous cycle of sheep

Cyclic changes in the production of the pituitary gonadotrophic hormones, LH and FSH are essential events in the maintenance of the reproductive system of female mammals. While studies have examined changes in the secretion of LH and FSH during the estrous cycle and demonstrated the importance of these hormones in regulation of ovarian development and gametogenesis, considerably less is known concerning the regulation of the biosynthesis of these hormones. Although initial studies have examined changes in LH subunit mRNA concentrations during the rat and ovine estrous cycles, no information concerning the physiological regulation of FSH beta mRNA concentrations has been available. In the present study we have examined the relationship between pituitary concentrations of LH and FSH subunit mRNAs and the serum concentrations of these gonadotropins. The results demonstrate a very different pattern of change for FSH beta subunit mRNA than that observed for alpha and LH beta subunit mRNAs. In fact, FSH beta mRNA concentration decline substantially during the preovulatory period, reaching minimal values at a time when alpha and LH beta mRNA levels are near maximal. Furthermore, this decline in FSH beta mRNA amounts occurs when serum FSH concentrations are maximal. Thus, FSH beta mRNA concentrations follow a very different pattern than that of serum FSH. In contrast, LH beta mRNA and serum LH concentrations tend to increase at the same time. These findings provide evidence that concentrations of LH beta and FSH beta mRNAs are likely regulated by different mechanisms.     

Cardiac transplantation in severely ill patients requiring intensive support in hospital

Sixty four patients were referred for cardiac transplantation from a single cardiac team at this hospital between October 1984 and December 1986. Of these patients, 33 were referred for urgent transplantation, all of whom required intensive treatment in hospital with intravenous infusions of cardiac drugs, intra-aortic balloon counterpulsation, peritoneal dialysis, ventilation, or any combination of these to sustain life. Of these 33 patients, six died while awaiting transplantation, one was removed from the waiting list for a transplant, and 26 received cardiac transplants. There were five deaths within 24 hours of operation and one death 10 days after the operation. Twenty of those who had surgery had a successful outcome of transplantation, but there was one late death 10 weeks postoperatively and a further death 31 months after surgery. Eighteen patients were alive and well 10 to 33 months (mean 19·4 months) after transplantation, with an overall survival rate after surgery of 69%.Provided that surgery can be performed before renal failure has progressed such that renal transplantation is necessary, the results are excellent (surgical survival 85·5%) and, we believe, justify the expenditure and staffing requirements necessary to treat these terminally ill patients.     

The purified yeast pre-mRNA splicing factor PRP2 is an RNA-dependent NTPase.

Unlike autocatalyzed self-splicing reactions, nuclear pre-mRNA splicing requires transacting macromolecules and ATP. A protein encoded by the PRP2 gene of Saccharomyces cerevisiae is required, in conjunction with ATP, for the first cleavage-ligation reaction of pre-mRNA splicing. In this study, we have purified two forms of the PRP2 gene product with apparent molecular weights of 100 kDa and 92 kDa, from a yeast strain overproducing the protein. Both proteins were indistinguishable in their ability to complement extracts derived from a heat-sensitive prp2 mutant. Furthermore, we show that the PRP2 protein is capable of hydrolyzing nucleoside triphosphates in the presence of single-stranded RNAs such as poly(U). However, purified PRP2 by itself did not unwind double-stranded RNA substrates. The fact that an RNA-dependent NTPase activity is intrinsic to PRP2 may account for the ATP requirement in the first catalytic reaction of pre-mRNA splicing.