Transductional analysis of chromosome replication time

Summary Following transduction of exponentially growing cultures of Escherichia coli with phage P1, cells with recombinant phenotype begin to increase in number after an initial lag of about one generation time. We show that transductants for markers located at different positions on the chromosome begin to increse at different times, in reverse order to that in which they are replicated. The period over which this happens is equal in duration to the time taken to replicate the chromosome and we have used this relationship to calculate the C-period of E. coli K12 growing at 30°C. We exclude transduction-induced filamentation as the cause of the initial lag and suggest that the lag may result from the way in which donor DNA is inherited.     

Thiamine-induced Formation of the Monopyrrole Moiety of Prodigiosin

Thiamine stimulates the production of a red pigment, which is chromatographically and spectrophotometrically identical to prodigiosin, by growing cultures of Serratia marcescens mutant 9-3-3. This mutant is blocked in the formation of 2-methyl-3-amylpyrrole (MAP), the monopyrrole moiety of prodigiosin, but accumulates 4-methoxy-2,2,'-bipyrrole-5-carboxaldehyde (MBC) and can couple this compound with MAP to form prodigiosin. Addition of thiamine caused production of MAP, and as little as 0.02 mg of thiamine per ml in a peptone-glycerol medium stimulated production of measurable amounts of prodigiosin. Phosphate salts and another type of peptone decreased the thiamine-induced formation of prodigiosin; yeast extract and glycerol enhanced the formation of this substance. Thiamine also enhanced production of prodigiosin by wild-type strain Nima of S. marcescens. The thiamine antagonists, oxythiamine and pyrithiamine, inhibited thiamine-induced production of MAP and of prodigiosin by the mutant strain 9-3-3, formation of prodigiosin by the wild-type strain Nima, and production of MAP by another mutant, strain WF. The pyrimidine moiety of thiamine was only 10% as effective as the vitamin; the thiazole moiety, only 4%; and the two moieties together, 25%. Various other vitamins tested did not stimulate formation of prodigiosin by strain 9-3-3. Thiamine did not stimulate production of prodigiosin by a single-step mutant that showed the same phenotypic block in prodigiosin biosynthesis as strain 9-3-3. This is not surprising since strain 9-3-3 originated as a result of two mutational events. One event may involve thiamine directly, and the other may involve the biosynthesis of MAP. Thiamine is probably involved in the regulation of the biosynthesis of MAP, because the vitamin or inhibitory antagonists must be added during the early phases of growth in order to be effective.     

A possible role for the pcnB gene product of Escherichia coli in modulating RNA: RNA interactions.

Summary The sequence of the PcnB protein of Escherichia coli, a protein required for copy number maintenance of ColE1-related plasmids, was compared with the PIR sequence database. Strong local similarities to the sequence of the E. coli protein tRNA nucleotidyltransferase were found. Since a substrate of the latter protein, tRNA, structurally resembles the RNAs that control ColE1 copy number we believe that we may have identified a region in PcnB that interacts with these RNAs. Consistent with this idea is our observation that PcnB is required for the replication of R1, a plasmid whose replication is also regulated by a small RNA.     

Recovery of Cholinergic Phenotype in the Injured Rat Neostriatum: Roles for Endogenous and Exogenous Nerve Growth Factor

Polyclonal antibodies against recombinant human nerve growth factor (rhNGF) potently inhibited PC12 neurite outgrowth, blocked high-affinity 125I-rhNGF binding but not its receptor, and cross-reacted with rat, mouse, and human nerve growth factor (NGF) but not with brain-derived neurotrophic factor, neurotrophin-3, ciliary neurotrophic factor, insulin-like growth factor, epidermal growth factor, or activin A. Immunocytochemistry revealed many NGF-positive neurons in the rat neostriatum. The NGF-positive neurons disappeared by 3 days after mechanical injury to the neostriatum and were replaced by intensely NGF- and glial fibrillary acidic protein-positive astrocytes. Enzyme-linked immunosorbent assay measurements revealed that the NGF content of the injured striatum was elevated by eightfold 3 days postinjury and by twofold 2 weeks later. The high-affinity choline uptake (HACU) into cholinergic nerve terminals was decreased by 23% at 2 and 4 weeks postinjury, yet choline acetyltransferase (ChAT) activity in these neurons was unchanged at 2 weeks and decreased by 14% at 4 weeks. Daily infusion of 1 microgram of rhNGF into the injury area did not alter the loss of HACU. However, this treatment elevated ChAT activity by 23-29% above intact neostriatal levels and by 53-65% relative to HACU at both survival times. Thus, lesion-induced increases in NGF levels within astrocytes are associated with maintenance of striatal ChAT activity at normal levels following cholinergic injury, even with decreases in HACU. Pharmacologic doses of rhNGF can further augment ChAT activity in damaged cholinergic neurons, showing the usefulness of exogenous NGF even when endogenous NGF is elevated in response to injury.     

Short Term Interactions with Long Term Consequences: Modulation of Chimeric Vessels by Neural Progenitors

Vessels are a critical and necessary component of most tissues, and there has been substantial research investigating vessel formation and stabilization. Several groups have investigated coculturing endothelial cells with a second cell type to promote formation and stabilization of vessels. Some have noted that long-term vessels derived from implanted cocultures are often chimeric consisting of both host and donor cells. The questions arise as to whether the coculture cell might impact the chimeric nature of the microvessels and can modulate the density of donor cells over time. If long-term engineered microvessels are primarily of host origin, any impairment of the host''s angiogenic ability has significant implications for the long-term success of the implant. If one can modulate the host versus donor response, one may be able to overcome a host''s angiogenic impairment. Furthermore, if one can modulate the donor contribution, one may be able to engineer microvascular networks to deliver molecules a patient lacks systemically for long times. To investigate the impact of the cocultured cell on the host versus donor contributions of endothelial cells in engineered microvascular networks, we varied the ratio of the neural progenitors to endothelial cells in subcutaneously implanted poly(ethylene glycol)/poly-L-lysine hydrogels. We found that the coculture of neural progenitors with endothelial cells led to the formation of chimeric host-donor vessels, and the ratio of neural progenitors has a significant impact on the long term residence of donor endothelial cells in engineered microvascular networks in vivo even though the neural progenitors are only present transiently in the system. We attribute this to the short term paracrine signaling between the two cell types. This suggests that one can modulate the host versus donor contributions using short-term paracrine signaling which has broad implications for the application of engineered microvascular networks and cellular therapy more broadly.     

Electron density mapping of triblock copolymers associated with model biomembranes: insights into conformational states and effect on bilayer structure

One-dimensional electron-density profiles derived from synchrotron small-angle X-ray scattering (SAXS) have been constructed and used to determine the conformational state of selected poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymers and the region of their association with a lipid bilayer. The number of molecular repeat units in the hydrophobic PPO block has been found to determine both the nature of triblock polymer-membrane association and the conformational state of the symmetric, flanking hydrophilic PEO units. For DMPC-based biomembranes, polymers whose PPO chain length is less than that of the bilayer thickness insert weakly into the membrane with the PEO blocks on the same side of the bilayer, leading to delocalization of the PEO at the membrane-water interface. This polymer architecture has been found not to alter the membrane fluidity and roughness. Conversely, polymers whose chain length is sufficient to span the lipid bilayer are tightly integrated, projecting their PEO chains into the water channels on opposing sides of the bilayer. The coiled conformational state of the PEO chains produces steric pressure on the bilayer, causing a thinning of the membrane and leading to a rigid, less-mobile bilayer than systems where the polymer is introduced as the lipid conjugate.     

Body Mass Index and the Prevalence of Hypertension and Dyslipidemia

Objective: To describe and evaluate relationships between body mass index (BMI) and blood pressure, cholesterol, high‐density lipoprotein‐cholesterol (HDL‐C), and hypertension and dyslipidemia. Research Methods and Procedures: A national survey of adults in the United States that included measurement of height, weight, blood pressure, and lipids (National Health and Nutrition Examination Survey III 1988–1994). Crude age‐adjusted, age‐specific means and proportions, and multivariate odds ratios that quantify the association between hypertension or dyslipidemia and BMI, controlling for race/ethnicity, education, and smoking habits are presented. Results: More than one‐half of the adult population is overweight (BMI of 25 to 29.9) or obese (BMI of ≥30). The prevalence of high blood pressure and mean levels of systolic and diastolic blood pressure increased as BMI increased at ages younger than 60 years. The prevalence of high blood cholesterol and mean levels of cholesterol were higher at BMI levels over 25 rather than below 25 but did not increase consistently with increasing BMI above 25. Rates of low HDL‐C increased and mean levels of HDL‐C decreased as levels of BMI increased. The associations of BMI with high blood pressure and abnormal lipids were statistically significant after controlling for age, race or ethnicity, education, and smoking; odds ratios were highest at ages 20 to 39 but most trends were apparent at older ages. Within BMI categories, hypertension was more prevalent and HDL‐C levels were higher in black than white or Mexican American men and women. Discussion: These data quantify the strong associations of BMI with hypertension and abnormal lipids. They are consistent with the national emphasis on prevention and control of overweight and obesity and indicate that blood pressure and cholesterol measurement and control are especially important for overweight and obese people.