The NodA proteins of Rhizobium meliloti and Rhizobium tropici specify the N-acylation of Nod factors by different fatty acids

Rhizobia synthesize mono- N -acylated chitooligosaccharide signals, called Nod factors, that are required for the specific infection and nodulation of their legume hosts. The biosynthesis of Nod factors is under the control of nodulation ( nod ) genes, including the nodABC genes present in all rhizobial species. The N -acyl substitution can vary between species and can play a role in host specificity. In Rhizobium meliloti , an alfalfa symbiont, the acyl chain is a C16 unsaturated or a (ω-1) hydroxylated fatty acid, whereas in Rhizobium tropici , a bean symbiont, it is vaccenic acid (C18:1). We constructed R. meliloti derivatives having a non-polar deletion of nodA , and carrying a plasmid with either the R. meliloti or the R. tropici nodA gene. The strain with the R. tropici nodA gene produced Nod factors acylated by vaccenic acid, instead of the C16 unsaturated or hydroxylated fatty acids characteristic of R. meliloti Nod factors, and infected and nodulated alfalfa with a significant delay. These results show that NodA proteins of R. meliloti and R. tropici specify the N -acylation of Nod factors by different fatty acids, and that allelic variation of the common nodA gene can contribute to the determination of host range.     

The effect of immunization of heifers against alpha 1-26 inhibin conjugate on the superovulatory response to pFSH

A total of 121 heifers was blocked by time and diet and then randomly assigned, within block, to an inhibin-immunized (I) or a control (C) group. Immunized heifers (n = 61) received a primary immunization (Day 0) with 0.33 mg of an alpha 1-26 bovine inhibin fragment-human serum albumin (HSA) conjugate injected with non-ulcerative Freund's and DEAE-dextran adjuvants. Booster injections were given on Days 28 and 56. Control heifers (n = 60) received HSA and adjuvants. On Days 56 and 83 the ovaries of heifers were examined by ultrasound to determine the ovulation rate, and blood samples were collected for antibody titer determination. On Day 84, 61 heifers (C, n = 30; I, n = 31) received a total of 24 mg of porcine follicle stimulating hormone (pFSH), while 60 heifers (C, n = 30; I, n = 30) received 12 mg im pFSH, which was administered twice daily for 4 d in decreasing doses during the mid-luteal phase of the estrous cycle. Luteolysis was induced with prostaglandin F(2alpha) analog. The heifers were artificially inseminated and were slaughtered 7 d after estrus. Embryos were recovered and morphologically graded on a scale of 1 to 5 (1 = excellent; 5 = degenerated). Antibody titers (percentage binding at 1:125 serum dilution) differed (P < 0.01) between Group C and I heifers at Days 56 (0.1 vs 30%) and 83 (0.2 vs 37%), and 26% of Group I and 1% of Group C heifers (P < 0.01) had twin ovulations on Day 83. The mean number of embryos recovered was reduced (P = 0.02) in Group I heifers (8.9 +/- 1.2) compared with C heifers (12.1 +/- 1.1); however, the mean number of freezable embryos (Grades 1 and 2) was not affected (P = 0.61) by immunization, and there was no interaction with pFSH (P = 0.36). Ovulation rate as well as embryo yield and quality were not different (P > 0.10) between Group C and I heifers when 12 mg pFSH were administered; however, immunization decreased the superovulatory response to 24 mg of pFSH.     

Relationships Between the Body Mass Index and Body Composition

The aims of this study were to evaluate the Body Mass Index (BMI) (weight/stature²) as a proxy for percent body fat (%BF) and to determine its association with fat-free mass (FFM). Multivariate analysis of variance and partial correlations were used to examine relationships between BMI and %BF and FFM from densitometry for 504 men and 511 women, aged 20 to 45 years. Sensitivity/specificity analyses used cut offs of 28 kg/m² in men and 26 kg/m² in women for BMI, and 25% in men and 33% in women for %BF. Significantly higher associations existed in each gender between BMI and %BF in the upper BMI tertile than in the lower BMI tertiles. In the lower BMI tertiles, correlations between BMI and FFM were approximately twice as large as those between BMI and %BF. The BMI correctly identified about 44% of obese men, and 52% of obese women when obesity was determined from %BF. BMI is an uncertain diagnostic index of obesity. Results of Receiver Operator Characteristic (ROC) analyses using %BF and total body fat, both provided a BMI of 25 kg/m² in men and 23 kg/m² in women as diagnostic screening cut offs for obesity.     

Calcium Release at Fertilization in Starfish Eggs Is Mediated by Phospholipase Cγ

Although inositol trisphosphate (IP₃) functions in releasing Ca²⁺ in eggs at fertilization, it is not known how fertilization activates the phospholipase C that produces IP₃. To distinguish between a role for PLCγ, which is activated when its two src homology-2 (SH2) domains bind to an activated tyrosine kinase, and PLCβ, which is activated by a G protein, we injected starfish eggs with a PLCγ SH2 domain fusion protein that inhibits activation of PLCγ. In these eggs, Ca²⁺ release at fertilization was delayed, or with a high concentration of protein and a low concentration of sperm, completely inhibited. The PLCγSH2 protein is a specific inhibitor of PLCγ in the egg, since it did not inhibit PLCβ activation of Ca²⁺ release initiated by the serotonin 2c receptor, or activation of Ca²⁺ release by IP₃ injection. Furthermore, injection of a PLCγ SH2 domain protein mutated at its phosphotyrosine binding site, or the SH2 domains of another protein (the phosphatase SHP2), did not inhibit Ca²⁺ release at fertilization. These results indicate that during fertilization of starfish eggs, activation of phospholipase Cγ by an SH2 domain-mediated process stimulates the production of IP₃ that causes intracellular Ca²⁺ release.     

Microbial Oxidation of Vinyl Sulfides to Chiral sulfoxides

A variety of bacteria, yeasts and fungi were screened for the preparation of enantiomers of chiral sulfoxides from aryl-aryl or alkyl-aryl (E) and (Z) vinyl sulfides 1. The asymmetric oxidation was studied on a model substrate, (E)-methyl-(2-phenyl)-vinylsulfide la. Along with various amounts of sulfone, all strains tested gave the corresponding sulfoxide, optically active, albeit with different chemical and optical yields. The stereochemical outcome of the reaction appears to be quite invariant and both enantiomers can be obtained. The high sensitivity towards substrate structure observed in a variety of sulfides is related to the toxicity of the substrates and the microbial oxidation is therefore assumed to be a detoxication reaction.     

High-affinity binding of an influenza hemagglutinin-derived peptide to purified HLA-DR

Immunogenic peptides have been shown to bind detergent-solubilized class II (Ia) molecules from mice. In this investigation, we report that highly purified HLA-DR (DR) molecules in detergent solution are capable of binding a synthetic peptide (HAp) derived from the influenza hemagglutinin sequence. Although the presentation of this peptide has been demonstrated only to DR1-restricted Th cells, the association rate constants for the formation of HAp-DR1, -DR5, and -DR8 complexes were essentially identical (ka = 1.1 x 10(2) to 1.6 x 10(2) M-1 s-1). By contrast, the value of the rate constants for the dissociation of preformed HAp-DR1, -DR5, and -DR8 complexes varied nearly threefold (kd = 1.6 x 10(6) to 4.4 x 10(-6) s-1). The value of the equilibrium dissociation constants (KD) derived from these rate constants were 13 nM, 24 nM, and 28 nM, for HAp-DR1, -DR5, and -DR8 complexes, respectively. Scatchard analysis demonstrated that the KD obtained from the rate constants for the HAp-DR1 reaction was in excellent agreement with that obtained under equilibrium conditions. SDS-PAGE confirmed that the HAp-DR complexes were remarkably stable, as HAp remained associated with the DR alpha beta heterodimer after treatment of the complexes with SDS and beta-mercaptoethanol. Steady-state binding studies demonstrated that 18% of all DR1 molecules had bound HAp at equilibrium, whereas only 3.8% of all DR8 molecules had bound HAp under identical conditions. The slight differences in the KD for HAp-DR complexes suggest that differences in the affinity of a peptide for DR alleles alone may not always explain the process of MHC restriction.     

Superovulation in heifers given FSH initiated either at day 2 or day 10 of the estrous cycle

The aim of this study was to determine if initiation of superovulation in heifers during the time of development of the first dominant follicle (Days 2 to 6) would give equivalent ovulation and embryo production rates as treatment initiated at mid-cycle. Estrus was synchronized in 60 beef heifers using luprostiol (PG) and they were randomly allocated to treatment with 4.5, 3.5, 2.5 and 1.5 mg of porcine follicle stimulating hormone (FSH) administered twice daily, either on Days 2, 4, 5 and 6 (Day-2 group), respectively, or with similar doses at four consecutive days during mid-cycle (Day-10 group, initiation on Day 9 to 11). All heifers received 500 mug cloprostenol at the fifth FSH injection and 250 mug at the sixth injection. Blood samples for progesterone determination were collected at the time of FSH injections. Heifers were slaughtered 7 d post estrus, and the number of ovulations and large follicles (>/=10mm) were determined on visual inspection of the ovary. Following flushing of the uterine horns the quality of embryos and the fertilization rate were determined. Significant differences between treatments were determined using a two-sided t-test, and frequency distributions were compared using Chi-square tests. The mean number (+/-SEM) of ovulations for heifers in the Day-10 group was 12.9+/-1.0, and 8.5+/-0.9 embryos were recovered. Both the number of ovulations (6.7+/-0.8) and embryos recovered (4.1+/-0.6) were lower (P=0.0001) in heifers in the Day-2 group. Following grading based on a morphological basis, a higher number (P=0.002) of embryos was categorized as Grades 1 and 2 (4.1+/-0.6) and Grade 3 (2.1+/-0.4) in Day-10 heifers than in the Day-2 group (Grade 1 and 2, 1.9+/-0.3; Grade 3, 0.7+/-0.2). The number of Grade 4 and 5 embryos (Day 10, 1.6+/-0.2; Day 2, 1.4+/-0.2) and the number of unfertilized ova (Day 10, 0.7+/-0.4; Day 2, 0.2+/-0.1) did not differ between treatments. Progesterone concentrations were lower (P=0.0001) in Day-2 heifers at FSH treatment prior to prostaglandin, and the decline was more rapid following prostaglandin injection at Day 5 (P=0.02). Results of this study indicate that the number of ovulations and embryos recovered was lower in heifers when FSH treatment was initiated on Day 2 compared with Day 10 of the estrous cycle.     

Effect of dietary intake on pattern of growth of dominant follicles during the oestrous cycle in beef heifers

Friesian x Hereford heifers (n = 19; mean +/- s.e.m. body weight (BW) = 375 +/- 5 kg) were used in a randomized incomplete block design. Heifers were fed 0.7 (n = 7; L), 1.1 (n = 7; M) or 1.8% (n = 5; G) of BW in dry matter (DM)/day for 10 weeks. Ovaries were examined by ultrasound, for one oestrous cycle, from week 5 of treatment. Maximum diameter of dominant follicles was smaller (P less than 0.05) in L (11.8 +/- 0.1 mm) than in M (13.7 +/- 0.2 mm) or G (13.2 +/- 0.3 mm) heifers. Growth rate (mm/day) of dominant follicles during the oestrous cycle was not affected (P greater than 0.05) by dietary intake. Persistence of dominant follicles was shorter (P less than 0.05) in L (9.8 +/- 0.2 days) than in M (11.9 +/- 0.3 days) or G (12.7 +/- 0.4 days) heifers. Three dominant follicles were identified during the oestrous cycle of 5 of 7 L, 3 of 7 M and 1 of 5 G heifers (P less than 0.10); 2 dominant follicles were identified in the remaining heifers (n = 2 of 7, 4 of 7 and 4 of 5, respectively). Length of the luteal phase and luteal-phase concentrations of progesterone were not affected (P greater than 0.05) by treatment. Low dietary intake reduced the diameter and persistence of dominant follicles during the oestrous cycle of beef heifers and tended to increase the proportion of oestrous cycles with 3 dominant follicles.     

Characterization of muscarinic acetylcholine receptors on the rat pancreatic gastrin-producing cell line B6 RIN

The mechanisms of cholinergic stimulation of gastrin cells were studied in the rat pancreatic cell line B6 RIN. Carbachol induced an increase in intracellular Ca²⁺ and stimulated gastrin release in a dose-dependent manner over the range 10⁻⁵-10⁻³ M. These effects were completely abolished by atropine, suggesting the implication of muscarinic cholinergic receptors. The binding properties of these receptors were investigated. [N-Methyl-³H]scopolamine ([³h]nms) binding on cell homogenates was time-dependent, saturable and consistent with a single high-affinity binding class (K_d = 39.5 pM, and B_max = 7.9 fmol/mg DNA). Carbachol competitively inhibited [³H]NMS binding. The potency of inhibition of [³H]NMS binding by subtype selective antagonists was hexahydrodifenidol> pirenzepine> AF-DX 116. These results suggest the M₃, muscarinic receptors may be involved in the carbachol-induced gastrin release from B6 RIN cells.     

Molecular characterization of a repeat element causing large-scale size variation in the mitochondrial DNA of the sea scallop Placopecten magellanicus

The scallop Placopecten magellanicus has the largest reported animal mitochondrial DNA (average 35 kb) and exhibits large inter- and intraindividual length variation owing to the varying copy number of a repeated element. We have characterized the repeat array by using restriction mapping and sequence analysis. The repeated element consists of 1,442 bp flanked on either side by the sequence ACTTTCC in a direct orientation. The array contains two to eight copies of the repeated element arranged in a direct orientation and in tandem. Only complete copies of the element are present in the array. The repeat element contains three regions with characteristic nucleotide sequences: a 10-bp inverted repeat shown to extrude into a cruciform in a supercoiled DNA plasmid, a 120-bp tract rich in G/C (70%) and adjacent to the inverted repeat, and periodically interspersed homopolymer runs of A and T occurring near the middle of the element which induce DNA curvature in dimeric constructs of the element. The element appears to be unique to P. magellanicus. The structural properties of the repeat element and its organization in an array of repeats may be important in explaining the generation and maintenance of large-scale mitochondrial DNA size variation observed in many animal species.