Kinetic mechanism of DNA polymerase I (Klenow)

The minimal kinetic scheme for DNA polymerization catalyzed by the Klenow fragment of DNA polymerase I (KF) from Escherichia coli has been determined with short DNA oligomers of defined sequence. A key feature of this scheme is a minimal two-step sequence that interconverts the ternary KF.DNAn.dNTP and KF.DNAn+1.PPi complexes. The rate is not limited by the actual polymerization but by a separate step, possibly important in ensuring fidelity [Mizrahi, V., Henrie, R. N., Marlier, J. F., Johnson, K. A., & Benkovic, S. J. (1985) Biochemistry 24, 4010-4018]. Evidence for this sequence is supplied by the observation of biphasic kinetics in single-turnover pyrophosphorolysis experiments (the microscopic reverse of polymerization). Data analysis then provides an estimate of the internal equilibrium constant. The dissociations of DNA, dNTP, and PPi from the various binary and ternary complexes were measured by partitioning (isotope-trapping) experiments. The rate constant for DNA dissociation from KF is sequence dependent and is rate limiting during nonprocessive DNA synthesis. The combination of single-turnover (both directions) and isotope-trapping experiments provides sufficient information to permit a quantitative evaluation of the kinetic scheme for specific DNA sequences.     

Androgenic regulation of luteinizing hormone secretion: relationship to androgen binding in sheep pituitary

Castrated ram lambs (wethers) were investigated for sensitivity to androgen feedback and to determine whether this feedback inhibition of luteinizing hormone (LH) was associated with changes in pituitary androgen receptors. Administration of Silastic capsules containing either dihydrotestosterone or testosterone was found to produce dose-dependent inhibitory effects on serum LH levels in wethers. Physiological dosages of these androgens (i.e., those that produce serum levels of dihydrotestosterone [0.24 ng/ml] or testosterone [2.1 ng/ml] similar to those of intact rams) resulted in differential inhibition of serum LH and LH content of the anterior pituitary. Whereas the inhibitory effect of dihydrotestosterone on pituitary LH content was much more dramatic than that seen with testosterone, the high dosage of testosterone also produced a substantial decrease in pituitary LH content. Responses of the pituitary to changes in serum androgen were compared to responses of the seminal vesicle, which served as a control androgen target organ. Androgen levels were positively correlated with seminal vesicle weights, but pituitary weights were unaffected by castration and/or androgen replacement. Treatments with dihydrotestosterone were associated with decreased cytosol androgen binding activity (i.e., receptors) in pituitary and seminal vesicle, suggesting that both of these tissues were sites of androgen action. Although testosterone inhibited serum LH levels, pituitary cytosol androgen receptors were not affected by changes in serum testosterone. We conclude from these data that dihydrotestosterone is a physiological regulator of pituitary LH secretion in the ram and that further study is needed to investigate the complex actions of testosterone and its metabolites on pituitary function.     

Interaction of granulosa and theca layers in the control of progesterone secretion in the domestic hen

The ovulatory cycle of the domestic hen is approximately 26 h in length. The hen ovulates an egg each day at a progressively later time until she finally skips a day, resets her "clock" and a new sequence is started. The ovarian component of this unique timing mechanism is the focus of this report. In Experiment 1, we asked whether there was a difference in luteinizing hormone (LH)-stimulated progesterone (P4) secretion by the granulosa layer removed from the largest follicle (F1) that had been the F1 follicle for 8, 12, or 32 h. In Experiment 2, our objective was to determine whether the theca layer of an F1 follicle influenced P4 secretion by the granulosa layer of that follicle and whether such an interaction depended on the maturity of the F1 follicle (had been a F1 follicle for 8 h or 32 h). Results from Experiment 1 revealed that there was no significant difference in LH-stimulated P4 secretion by the granulosa layer in a perifusion system regardless of the length of time the follicle had been the largest follicle. In contrast, in Experiment 2, when granulosa and theca layers from the same follicle were co-incubated in a perifusion system, P4 secretion from the more mature F1 follicle (32 h) increased in response to LH, whereas P4 secretion from the less mature F1 follicle (8 h) was not elevated by LH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Current utilization and further development of the palmyra palm (Borassus flabellifer L., Arecaceae) in Tamil Nadu state,India

The palmyra palm Borassus flabellifer,), a multipurpose tree of great utility, occurs extensively in Tamil Nadu state, India. Figuring in history, literature, and folklore of the state, it is exploited for food from the fruit and tuberous seedlings; beverage and sugar from the sap; fiber from the leaf and leafbase for brushes, cordage, weaving, and plaiting; trunk wood for construction and fuel; and numerous minor products. Increasing exploitation of the palmyra threatens the future supply of palm raw materials so important to rural populations. Integrated development of palmyra products for local and export markets, as well as management/conservation measures, are needed both to maximize the economic value of the products and to assure sustained yield from native stands.     

Calcitonin gene-related peptide immunoreactivity in airway epithelial cells of the human fetus and infant

Summary Calcitonin gene-related peptide-immunoreactive cells were identified within the epithelium of distal conducting airways in the human fetus and infant. Several peptides and amines, including calcitonin, have been identified previously within a specific population of airway epithelial cells. These cells, referred to as pulmonary neuroendocrine cells, are postulated to be airway chemoreceptors responsible for changes in ventilation and perfusion in response to changes in airway gas composition. Calcitonin gene-related peptide immunoreactive cells could be identified throughout the period of development studies (20 weeks gestation to 3 months of age), but were present in only limited numbers in less than 50% of individuals (n=23). In contrast, large numbers of calcitonin gene-related peptide immunoreactive cells were identified in 100% of infants (1–3 months, n=5) with bronchopulmonary dysplasia. The differential processing of mRNA transcribed from the calcitonin gene in neural and non-neural tissue suggests that calcitonin, rather than calcitonin gene-related peptide, is the primary product of translation in pulmonary neuroendocrine cells. However, considering the potent vasodilatory and bronchoconstrictive effects of calcitonin gene-related peptide, its presence in pulmonary neuroendocrine cells, even in small amounts, may be important in controlling pulmonary vaso- and/or bronchomotor tone. The presence of large numbers of calcitonin gene-related peptide immunoreactive cells in infants with bronchopulmonary dysplasia suggests that calcitonin gene-related peptide may be one further agent contributing to the pulmonary pathophysiology seen in this disease.     

Immediate breast reconstruction: reducing the risks

One-hundred and sixty-five consecutive immediate breast reconstructions in 157 patients were reviewed. Reconstructions were performed with tissue expanders (53 percent) or immediate gel prostheses (47 percent). Immediate reconstruction was associated with an 18 percent rate of implant loss. Certain risk factors were identified at the p less than 0.05 level using immediate gel implants: failure to achieve complete muscle coverage of the implant, smoking at the time of surgery, initial gel implants of 400 ml or more volume, and age. Expander loss was increased by detaching the pectoralis major (p less than 0.05) and probably by lack of complete muscle coverage in general. Chemotherapy, history of previous smoking, and clinical stage of the carcinoma did not seem to affect reconstructive success. Smoking and patient age should be considered during patient selection for immediate reconstruction. Muscle coverage of the prosthesis should always be attempted. Muscle coverage is mandatory in the smoker. Gel implants of 400 ml or more volume are to be avoided at the initial operation. This approach should enable all surgeons to achieve lower rates of implant loss.     

Thrombolysis using liposomal-encapsulated streptokinase: an in vitro study

The clot-lysing ability of streptokinase (SK) was examined using membrane-bound thrombi. Encapsulation of SK in large unilamellar phospholipid vesicles (liposomes) resulted in entrapping approximately 30% of its original activity. Measurements of streptokinase activity for liposomal-encapsulated streptokinase (LESK) indicated little loss of activity or leakage in Tris-buffered saline over a 24-hr period at temperatures of 4 and 23 degrees C. However, incubation of free SK and LESK in platelet-poor plasma (PPP) at 37 degrees C resulted in a decrease of SK activity. The retention of SK activity in LESK was considerably higher than that of unentrapped SK. Clot-dissolving time (CDT) was measured by monitoring the pressure drop during slow filtration in plasma through membrane-bound thrombi. The results indicated that both LESK and free SK were able to activate the fibrinolytic system. Without prior incubation in PPP at 37 degrees C, the CDT of a SK and PPP mixture (SK/PPP) was 10.7 +/- 1.9 min (n = 12), while that of a LESK and PPP mixture (LESK/PPP) was 12.4 +/- 1.7 min (n = 12). The CDT-detected clot-lysing abilities of both SK and LESK were diminished by incubation in PPP, but to different extents. After 15- and 30-min incubations, the CDT of SK/PPP increased significantly to 15.5 +/- 1.5 and 24.1 +/- 2.4 min (n = 5, P less than 0.05), respectively. In contrast, the CDT of LESK/PPP increased to 13.3 +/- 0.8 min (n = 5) after 15 min of incubation and to 16.0 +/- 1.1 min (n = 5, P less than 0.05) after a 30-min incubation. These results suggest that entrapment of SK in liposomes preserves the thrombolytic potential of the plasminogen activator by limiting its exposure to the components of the plasma.     

Diffusion of the Interspecies Electron Carriers H(2) and Formate in Methanogenic Ecosystems and Its Implications in the Measurement of K(m) for H(2) or Formate Uptake

We calculated the potential H(2) and formate diffusion between microbes and found that at H(2) concentrations commonly found in nature, H(2) could not diffuse rapidly enough to dispersed methanogenic cells to account for the rate of methane synthesis but formate could. Our calculations were based on individual organisms dispersed in the medium, as supported by microscopic observations of butyrate-degrading cocultures. We isolated an axenic culture of Syntrophomonas wolfei and cultivated it on butyrate in syntrophic coculture with Methanobacterium formicicum; during growth the H(2) concentration was 63 nM (10.6 Pa). S. wolfei contained formate dehydrogenase activity (as does M. formicicum), which would allow interspecies formate transfer in that coculture. Thus, interspecies formate transfer may be the predominant mechanism of syntrophy. Our diffusion calculations also indicated that H(2) concentration at the cell surface of H(2)-consuming organisms was low but increased to approximately the bulk-fluid concentration at a distance of about 10 mum from the surface. Thus, routine estimation of kinetic parameters would greatly overestimate the K(m) for H(2) or formate.     

Aliphatic hydrocarbons obtained from iron carbides: Possible stellar and meteoritic implications

Origins of Life and Evolution of Biospheres -