D-Alanyl-D-lactate and D-alanyl-D-alanine synthesis by D-alanyl-D-alanine ligase from vancomycin-resistant Leuconostoc mesenteroides. Effects of a phenylalanine 261 to tyrosine mutation

The Gram-positive bacterium Leuconostoc mesenteroides, ATCC 8293, is intrinsically resistant to the antibiotic vancomycin. This phenotype correlates with substitution of D-Ala-D-lactate (D-Ala-D-Lac) termini for D-Ala-D-Ala termini in peptidoglycan intermediates in which the depsipeptide has much lower affinity than the dipeptide for vancomycin binding. Overproduction of the L. mesenteroides D-Ala-D-Ala ligase (LmDdl) 2 in E. coli and its purification to approximately 90% homogeneity allow demonstration that the LmDdl2 does have both depsipeptide and dipeptide ligase activity. Recently, we reported that mutation of an active site tyrosine (Tyr), Tyr216, to phenylalanine (Phe) in the E. coli DdlB leads to gain of D-Ala-D-Lac depsipeptide ligase activity in that enzyme. The vancomycin-resistant LmDdl2 has a Phe at the equivalent site, Phe261. To test the prediction that a Tyr residue predicts dipeptide ligase while an Phe residue predicts both depsipeptide and dipeptide ligase activity, the F261Y mutant protein of LmDdl2 was constructed and purified to approximately 90% purity. F216Y LmDdl2 showed complete loss of the ability to couple D-Lac but retained D-Ala-D-Ala dipeptide ligase activity. The Tyr-->Phe substitution on the active site omega-loop in D-Ala-D-Ala ligases is thus a molecular indicator of both the ability to make D-Ala-D-Lac and intrinsic resistance to the vancomycin class of glycopeptide antibiotics.     

Avoidance learning in rats: Genetic evidence for two distinct behavioral processes in the shuttle box.

Used an 8 * 8 diallel cross of rats (Roman high, low, and control avoidance; Maudsley reactive and nonreactive; Tryon maze bright and dull; and Wistar albino) to analyze sequentially a conditioned avoidance response (CAR) within a session of 30 trials. On early conditioning trials, the F1 hybrids scored fewer avoidances than the parental strains, whereas on later trials the hybrids were superior to parents. This finding was confirmed within a session of 100 trials in a 3 * 3 diallel and interpreted to mean that 2 distinct adaptive behavior patterns underlie shuttle-box CAR acquisition in rats. There appears to be a tendency to develop an adaptive inactivity or "freezing" behavior pattern and later, as conditioning proceeds, to develop an equally adaptive but more active behavior pattern of "fleeing." Implications for 2-process theories of avoidance conditioning are considered briefly. (20 ref.) (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Mutational analysis of the Mu transposase. Contributions of two distinct regions of domain II to recombination

Mu transposase is a member of a protein family that includes many transposases and the retroviral integrases. These recombinases catalyze the DNA cleavage and joining reactions essential for transpositional recombination. Here we demonstrate that, consistent with structural predictions, aspartate 336 of Mu transposase is required for catalysis of both DNA cleavage and DNA joining. This residue, although located 55 rather than 35 residues NH2-terminal of the essential glutamate, is undoubtedly the analog of the second aspartate of the Asp-Asp-35-Glu motif found in other family members. The core domain of Mu transposase consists of two subdomains: the NH2-terminal subdomain (IIA) contains the conserved Asp-Asp-Glu motif residues, whereas the smaller COOH-terminal subdomain (IIB) contains a large positively charged region exposed on its surface. To probe the function of domain IIB, we constructed mutant proteins carrying deletion or substitution mutations within this region. The activity of the deletion proteins revealed that domains IIA and IIB can be provided by different subunits in the transposase tetramer. Substitution mutations at two pairs of exposed lysine residues within the positively charged surface of domain IIB render transposase defective in transposition at a reaction step after DNA cleavage but prior to DNA joining. The severity of this defect depends on the structure of the DNA flanking the cleavage site. Thus, these data suggest that domain IIB is involved in manipulating the DNA near the cleavage site and that this function is important during the transition between the DNA cleavage and the DNA joining steps of recombination.     

Re: A comparison of pulpal response to freeze-dried bone, calcium-hydroxide, and zinc oxide-eugenol in primary teeth in two cynomolgus monkeys. S Fadavi, AW Anderson. Pediatr Dent 18:52-56, 1996

OBJECTIVE: To explore the clinical value of the dura mater implantation in ocular plastic surgeries. METHODS: Alcohol preserved homologous dura mater was implanted into the upper fornix under the conjunctiva of rabbit eye and light, electron microscopic histological and immunological examinations were made for the implant. 38 eyes consisting of 30 eyes of blepharoptosis, 4 eyes of blepharo-coloboma, 3 eyes of hollow socket and 1 eye of teleepicanthus were treated by homologous dura mater implantation. RESULTS: In the follow-up periods ranging from 1.5 to 2.5 years, 26 eyes in the treated eyes were cured, 9 eyes were markedly improved, 3 eyes failed. Histological findings showed that after 1 week of implantation there was cell infiltration around the implant, at 2 weeks new collagenous tissue developed at the margin of the implant and 2-3 months after transplantation, the grafts were replace by the patient's own connective tissue. The histological examination of 3 failure cases showed that the cause of the failure was due to the ischemia of homograft. The results of unidirectional mixed lymphocyte culture showed that the stimulation index before implantation in comparison with that at 96 and 120 hours after implantation had statistical significant differences, while in comparison with that at 144 hours after implantation had no such difference. Before the implantation, the stimulation index of the lymphocyte culture at 96 hours was significantly different from that at 144 hours statistically (P < 0.05). In the comparison of specific killing activity before and after implantation, there was statistical significant difference. CONCLUSION: The effects of transplantation of homologous dura mater employed in ocular plastic surgeries of blepharoptosis, blepharocoloboma, teleepicanthus and hollow socket are fairly good.