Gly-238-Ser substitution changes the substrate specificity of the SHV class A beta-lactamases

The SHV-type beta-lactamase SHV-2A is related to SHV-1 by a Gly-238-Ser replacement. Strains carrying SHV-2A are resistant to the third generation cephems cefotaxime and ceftizoxime, whereas those that carry SHV-1 are sensitive to these drugs. We present a kinetic analysis of a SHV-1 and SHV-2A enzymes, with the goal of gaining insight into the role of residue 238 in hydrolyzing cefotaxime and ceftizoxime. SHV-2A shows altered kinetic properties for a number of other cephems that also have heterocyclic side chains at the amino position of the 7-aminocephalosporanic acid nucleus (R1 side chain), including a significantly higher kcat/Km than does SHV-1 for cephaloridine, cephalothin, and cefotiam. Two cephems with straight chain R1 substitutions, cephalosporin C and cephacetrile, are not hydrolyzed more efficiently by SHV-2A. These results indicate that the Ser-238-Gly substitution increases the affinity toward cephems with a heterocyclic ring in the R1 side chain. In addition, the data for ampicillin and benzylpenicillin show that addition of a nitrogen to the second carbon of the R1 side chain of a penem results in a lower kcat/Km for SHV-2A relative to SHV-1. These data strongly suggest that the previously proposed hydrogen bond formation between Ser-238 and the second carbon nitrogen of cefotaxime is not an important factor in hydrolysis by SHV-2A. We propose that the Gly-238 to Ser-238 replacement in SHV-2A has altered the hydrophobic pocket so that it can better accommodate cephems with bulky R1 side chains.     

Rat pineal gland phosducin: cDNA isolation, nucleotide sequence, and chromosomal assignment in the mouse

The pineal gland contains a soluble phosphoprotein, phosducin, which is homologous to that of retinal photoreceptors. Phosducin has been shown to bind the beta, gamma subunits of the retinal G-protein transducin. Retinal phosducin has been cloned and now we report a rat pineal cDNA encoding phosducin. A 1217-nucleotide cDNA was isolated from a rat pineal library by DNA-DNA hybridization with a polymerase chain reaction-amplified cDNA of bovine retina mRNA for phosducin. Northern blot analysis demonstrates that the mRNA for phosducin is approximately 1.3 kb in both rat pineal and rat retina. The translated mRNA from rat pineal encodes a protein with 246 amino acids, compared to the 245 amino acids of bovine retina phosducin. The predicted molecular weight of rat pineal phosducin is 28,201. Immunoblot analysis with affinity-purified antibodies against bovine retina phosducin identify a single immunoreactive protein of approximately 33 kDa in both rat retina and rat pineal. The amino acid sequence of rat pineal phosducin is homologous to that of bovine retina phosducin, revealing 89% identity and another 5.7% similarity. Both rat pineal and bovine retina phosducins are acidic proteins with pIs of 4.3 and 4.5, respectively. The translated protein lacks hydrophobic domains that would suggest an integral membrane protein. Rat pineal phosducin has a single consensus phosphorylation domain for protein kinase A that is nearly identical to that of retinal phosducin, which is phosphorylated by protein kinase A in situ. Rat phosducin also contains three potential phosphorylation domains for protein kinase C and nine for casein kinase II as well as a predicted site for N-glycosylation. The cDNA encoding phosducin was used to localize the gene within a linkage group to a large segment of mouse chromosome 1 in a conserved region with the long arm of human chromosome 1 with a panel of DNA samples from an interspecific cross. In keeping with a proposed role of retinal phosducin in down-regulation of the photo-transduction cascade, a modulatory role in signal transduction is proposed for pineal phosducin.     

Mapping anti-müllerian hormone (Amh) and related sequences in the mouse: identification of a new region of homology between MMU10 and HSA19p.

A panel of 78 backcross progeny, BALB/cJ x (BALB/cJ x CAST/Ei)F1, was used to map the gene encoding anti-Müllerian hormone (Amh), also called Müllerian inhibiting substance, to mouse Chromosome 10 (MMU10). This analysis identified a new region of linkage homology between human Chromosome 19p (HSA 19p) and MMU10 and localized an apparent recombinational hot spot in (C57BL/6J x Mus spretus)F1 females [compared with (BALB/cJ x CAST/Ei)F1 males] to the interval between phenylalanine hydroxylase (Pah) and mast cell growth factor (Mgf). In addition, eight unlinked polymorphic sequences, provisionally designated Amh-related sequences (Amh-rs1 through Amh-rs8), were identified by Southern blot analysis using Amh probes. Amh-rs1, -rs2, -rs4, and -rs7 were mapped to MMU1, 13, 12, and 15, respectively, by recombinant inbred (RI) strain and intraspecific backcross analyses. The NXSM RI strain distribution patterns for the four unmapped loci are also presented.     

Preparation of enteric-coated microspheres of Mycoplasma hyopneumoniae vaccine with cellulose acetate phthalate: The effect of swine serum on micromeritic properties

Summary Cellulose acetate phthalate was used to prepare the Mycoplasma hyopneumoniae vaccine (MHV) microspheres using a solvent evaporation method. Swine serum was used as an additive in the antigen to form the core materials. The addition of serum had a significant effect on surface topography of the MHV microspheres. By using this modified solvent evaporation method, the recoveries of antigens in the MHV microspheres were generally over 90% of the weight and antigenicity of antigens originally added in the formulation.     

Comparison of an electrochemiluminescent homogenous immunoassay and an ELISA for monitoring productivity in mammalian cell bioreactors

Summary An Enzyme Linked Immuno Sorbent Assays (ELISA) and an Electro-chemiluminescent Immuno Assay (CIA) are compared for the purpose of monitoring product formation in mammalian cell bioreactors. The ELISA had a relative standard deviation of 10%, compared to 8% for the CIA . The CIA was found to be a fast and accurate alternative to the ELISA. The use of more than one immuno assay format was also shown to provide additional insight into process performance.     

Tendency to inspect predators predicts mortality risk in the guppy (Poecilia reticulata)

Although predator inspection behavior in fishes has become amodel system for examining game theoretical strategies suchas Tit for Tat, the direct costs of inspection behavior havenot been quantified. To begin quantifying such costs, I conductedan experiment that examined mortality due to predation as afunction of predator inspection in the guppy (Poecilia reticulata).Before being subjected to a "survivorship" experiment, guppieswere assayed for their tendency to inspect a predator. Groupswere then composed of six guppies that differed in their tendencyto inspect. These groups were placed into a pool containinga predator, and survivorship of guppies with different inspectiontendencies was noted 36 and 60 h later. Results indicate thatindividuals that display high degrees of inspection behaviorsuffer greater mortality than their noninspecting shoalmates.     

Restoration of vigor and rooting competence in stem tissues of mature citrus by repeated grafting of their shoot apices onto freshly germinated seedlings in vitro

Summary Repeated grafting of 0.2-cm shoot tips from fruiting-age trees ofCitrus reticulata Blanco ‘Ponkan’ mandarin andC. sinensis Osbeck ‘Liu Tseng’ sweet orange onto freshly germinated ‘Troyer’ citrange [Poncirus trifoliata (L.) Raf. X.C. sinensis Osbeck] seedlings in vitro resulted in progressive restoration of rooting competence and vigor of regenerated roots and shoots. The restored traits were retained through the course of the investigation and suggested a phase reversal phenomenon.     

Effects of acute oligohydramnios on respiratory system of fetal sheep.

Prolonged oligohydramnios, or a lack of amniotic fluid, is associated with pulmonary hypoplasia and subsequent perinatal morbidity, but it is unclear whether short-term or acute oligohydramnios has any effect on the fetal respiratory system. To investigate the acute effects of removal of amniotic fluid, we studied nine chronically catheterized fetal sheep at 122-127 days gestation. During a control period, we measured the volume of fluid in the fetal potential airways and air spaces (VL), production rate of that fluid, incidence and amplitude of fetal breathing movements, tracheal pressures, and fetal plasma concentrations of cortisol, epinephrine, and norepinephrine. We then drained the amniotic fluid for a short period of time [24-48 h, 30.0 +/- 4.0 (SE) h] and repeated the above measurements. The volume of fluid drained for the initial studies was 1,004 +/- 236 ml. Acute oligohydramnios decreased VL from 35.4 +/- 2.9 ml/kg during control to 22.0 +/- 1.6 after oligohydramnios (P less than 0.004). Acute oligohydramnios did not affect the fetal lung fluid production rate, fetal breathing movements, or any of the other measured variables. Seven repeat studies were performed in six of the fetuses after reaccumulation of the amniotic fluid at 130-138 days, and in four of these studies the lung volume also decreased, although the overall mean for the repeat studies was not significantly different (27.0 +/- 5.2 ml/kg for control vs. 25.5 +/- 5.5 ml/kg for oligohydramnios). Again, none of the other measured variables were altered by oligohydramnios in the repeat studies.(ABSTRACT TRUNCATED AT 250 WORDS)