Bacteriophage P22 as a vehicle for transducing cosmid gene banks between smooth strains of Salmonella typhimurium: use in identifying a role for aroD in attenuating virulent Salmonella strains

Summary A cosmid gene bank of the virulent Salmonella typhimurium C5 was constructed in Escherichia coli K12. The bank was repackaged into bacteriophage heads and transduced into the semi-rough S. typhimurium strain AS68 which expresses the LamB receptor protein. Approximately 6000 ampicillin-resistant transductants were pooled and used as host for the propagation of bacteriophage P22. The P22 lysate was able to transduce cosmid recombinants to smooth strains of S. typhimurium and individual transductants were selected which complemented various S. typhimurium auxotrophic mutations. A stable mutation was introduced into the aroD gene of S. typhimurium C5. The resulting aroD ^- mutant, named CU038, was highly attenuated compared with the wild-type parent strain and BALB/c mice immunised orally with CU038 were well protected against challenge with the virulent C5 parental strain. Using the cosmid bank repackaged into bacteriophage P22 heads it was possible to isolate cosmid recombinants that could complement the aroD mutation of CU038 either by in vitro selection using minimal medium or in vivo selection for restoration of virulence in BALB/c mice. Repackaged P22 cosmid banks could provide a simple system for selecting in vivo for Salmonella virulence determinants. A Salmonella typhi strain harbouring mutations in aroA and aroD was constructed for potential use as a live oral typhoid vaccine in humans.     

Nitrogen Assimilating Enzyme Activities and Enzyme Protein during Development and Senescence of Effective and Plant Gene-Controlled Ineffective Alfalfa Nodules

Effective (N₂-fixing) alfalfa (Medicago sativa L.) and plant-controlled ineffective (non-N₂-fixing) alfalfa recessive for the in₁ gene were compared to determine the effects of the in₁ gene on nodule development, acetylene reduction activity (ARA), and nodule enzymes associated with N assimilation and disease resistance. Effective nodule ARA reached a maximum before activities of glutamine synthetase (GS), glutamate synthase (GOGAT), aspartate aminotransferase (AAT), asparagine synthetase (AS), and phosphoenolpyruvate carboxylase (PEPC) peaked. Ineffective nodule ARA was only 5% of effective nodule ARA. Developmental profiles of GS, GOGAT, AAT, and PEPC activities were similar for effective and ineffective nodules, but activities in ineffective nodules were lower and declined earlier. Little AS activity was detected in developing ineffective nodules. Changes in GS, GOGAT, AAT, and PEPC activities in developing and senescent effective and ineffective nodules generally paralleled amounts of immunologically detectable enzyme polypeptides. Effective nodule GS, GOGAT, AAT, AS, and PEPC activities declined after defoliation. Activities of glutamate dehydrogenase, malate dehydrogenase, phenylalanine ammonia lyase, and caffeic acid-o-methyltransferase were unrelated to nodule effectiveness. Maximum expression of nodule N-assimilating enzymes appeared to require the continued presence of a product associated with effective bacteroids that was lacking in in₁ effective nodules.     

Effect of a high-intensity static magnetic field on sciatic nerve regeneration in the rat

The effect of a high-intensity static magnetic field on peripheral nerve regeneration is evaluated in rat sciatic nerve. Forty-four rats underwent sciatic nerve repair using polyethylene nerve guides. Postoperatively, the animals were exposed to a 1-tesla magnetic field for 12 hours per day for 4 weeks with appropriate controls. Our results demonstrate that a 1-tesla static magnetic field has no statistically significant effect on nerve regeneration as determined by myelinated axon counts and electrophysiologic studies. Also, the specific orientation of the sciatic nerve with respect to the magnetic field has no influence on axonal growth or nerve conduction. Periods of restraint of 12 hours per day for 4 weeks significantly inhibit weight gain but have no effect on peripheral nerve regeneration.     

Approximate matching of regular expressions

Given a sequenceA and regular expressionR, theapproximate regular expression matching problem is to find a sequence matchingR whose optimal alignment withA is the highest scoring of all such sequences. This paper develops an algorithm to solve the problem in timeO(MN), whereM andN are the lengths ofA andR. Thus, the time requirement is asymptotically no worse than for the simpler problem of aligning two fixed sequences. Our method is superior to an earlier algorithm by Wagner and Seiferas in several ways. First, it treats real-valued costs, in addition to integer costs, with no loss of asymptotic efficiency. Second, it requires onlyO(N) space to deliver just the score of the best alignment. Finally, its structure permits implementation techniques that make it extremely fast in practice. We extend the method to accommodate gap penalties, as required for typical applications in molecular biology, and further refine it to search for substrings ofA that strongly align with a sequence inR, as required for typical data base searches. We also show how to deliver an optimal alignment betweenA andR in onlyO(N+logM) space usingO(MN logM) time. Finally, anO(MN(M+N)+N ²logN) time algorithm is presented for alignment scoring schemes where the cost of a gap is an arbitrary increasing function of its length.     

Insulin effects on apolipoprotein B production by normal, diabetic and treated-diabetic rat liver and cultured rat hepatocytes.

1. The effect of insulin on apolipoprotein (apo B) secretion was studied in 24 h recirculating liver perfusions of isolated normal, diabetic and insulin-treated diabetic rats. In single perfusions from each group apo B accumulated in the media in a linear fashion. 2 In perfusions of normal rat livers, when the medium contained insulin plus cortisol, apo B production was significantly inhibited (by 35.8%), demonstrating a hormone effect on apo B secretion. 3. In perfusions of diabetic-rat livers, apo B production was decreased to 11.8% of normal when the medium contained no hormones, and was not significantly changed by the addition of insulin plus cortisol to the medium, suggesting that the hormone effect on apo B secretion is missing in long-term hypoinsulinaemic states. 4. Treatment of diabetic rats with daily insulin injection restored apo B production and restored the effect of insulin plus cortisol in the medium to inhibit apo B secretion during perfusion. 5. Parallel studies of apo B secretion with insulin alone, cortisol alone and insulin plus cortisol in the medium were performed in primary cultures of hepatocytes to compare results from liver perfusions. 6. Apo B secretion by hepatocytes from normal, diabetic and treated-diabetic rats was inhibited (by 36.8%, 57.1% and 57.9% respectively) when insulin alone was added to the medium. 7. Insulin plus cortisol inhibited apo B secretion by hepatocytes from normal and treated diabetic rats (by 30.2% and 47.2% respectively), but failed to inhibit apo B secretion by hepatocytes from diabetic rats.     

Sex differences in vasopressin neurons in the bed nucleus of the stria terminalis by in situ hybridization

To determine whether a sex difference exists in the biosynthetic capacity of vasopressingergic (AVP) neurons in the bed nucleus of the stria terminalis (BNST), we have used in situ hybridization and quantitative autoradiography to measure propressophysin messenger RNA levels in these cells from adult male and female rats. We have found that significantly more (p less than 0.01) neurons are labeled in male rats than in female rats and that these labeled cells averaged more grains/cell (p less than 0.05) in males than in females. Therefore, the sexual dimorphism of AVP pathways in the BNST and lateral septum recently shown by immunohistochemistry results from a sex difference in the biosynthetic capacity of these AVP neurons.     

Electric field-induced lateral mobility of photosystem I in the photosynthetic membrane: A study by electrophotoluminescence

Electrophoretic movement of photosystem I (PS I) along the photosynthetic membrane of hypotonically swollen thylakoid vesicles was studied by analyzing the electric field-stimulated delayed luminescence (electrophotoluminescence) emitted from PS I. The electrophoretic mobility was inferred from the differences in electrophotoluminescence (EPL) of the photosynthetic vesicles in presence and absence of trains of low amplitude (<80 V/cm) prepulses of 1 ms duration at 4 ms spacing. The average apparent electric mobility, determined from the time course of EPL increase on one hemisphere or its decrease on the other one, as function of prepulse length and intensity was of the order of 3 · 10^-5 cm²V^-1s^-1. The assymetric distribution of the PS I reached a steady state when the diffusional, electrostatic, and elastic forces balanced the electrophoretic driving force. A lateral diffusion coefficient of ~5 · 10^-9 cm²s^-1 was found for the PS I complex from the diffusional relaxation after cessation of the electric field pulse train. Experimental conditions such as concentration, temperature, and viscosity of the aqueous solution were not critical for the effect. Between 23 and 150 electron charges per moving particle were estimated from the measured electrophoretic mobility.     

Transient responses of hybridoma cells to nutrient additions in continuous culture: I. Glucose pulse and step changes

Glucose and glutamine are the main nutrients used by mammalian cells in culture. Each provides unique biosynthetic precursors but are complementary for production of other metabolites and energy. The transient and steady-state responses of hybridoma growth and metabolism to glucose pulse and step changes have been examined. Metabolic quotients are reported for oxygen, glucose, lactate, ammonia, glutamine, alanine, and other amino acids. The glucose consumption rate increased by 100-200% immediately after glucose was added to the reactor, and the increased glycolytic ATP production appears to be responsible for the concurrent rapid decrease in the oxygen consumption rate. The effects on glutamine consumption were delayed, probably due to buffering by the TCA cycle and interrelated pathways. A period of increased biosynthetic activity, as evidenced by an increase in the estimated specific ATP production rate and lower by-product yields from glutamine, preceded the increase in cell concentration after the glucose step change. The biosynthetic yield of cells from ATP was calculated, and it was estimated that maintenance accounted for about 60% of the energy used by the cells at a specific growth rate of 0.66 day(-1). The estimated 22% ATP production due to glycoysis was twice as great as that before the step change.     

Bactericidal effect of lactoferrin on Legionella pneumophila: effect of the physiological state of the organism

Lactoferrin has been previously shown to be bactericidal for Legionella pneumophila. The current study showed that CaCl2, Mg(NO3)2, and MgCl2, but not NaCl, blocked killing. Activity was pH dependent with the greatest activity at 5.0. Sensitivity of the organism was dramatically affected by the growth conditions. Log phase 12 h, broth-grown cells were most sensitive, with older cultures becoming more resistant. Plate-grown cells were completely resistant. Lactoferrin binding, as detected by immunofluorescence microscopy, was temperature dependent (no binding at 4 degrees C), but was independent of killing.