Effect of N,N-Dicyclohexylcarbodiimide on Acetylcholine Release from Torpedo Synaptosomes and Proteoliposomes Reconstituted with the Proteolipid Mediatophore

The mediatophore is a presynaptic membrane protein that has been shown to translocate acetylcholine (ACh) under calcium stimulation when reconstituted into artificial membranes. The mediatophore subunit, a 15-kDa proteolipid, presents a very high sequence homology with the N,N'-dicyclohexylcarbodiimide (DCCD)-binding proteolipid subunit of the vacuolar-type H(+)-ATPase. This prompted us to study the effect of DCCD, a potent blocker of proton translocation, on calcium-dependent ACh release. The present work shows that DCCD has no effect on ACh translocation either from Torpedo synaptosomes or from proteoliposomes reconstituted with purified mediatophore. However, using [14C]DCCD, we were able to demonstrate that the drug does bind to the 15-kDa proteolipid subunit of the mediatophore. These results suggest that although the 15-kDa proteolipid subunits of the mediatophore and the vacuolar H(+)-ATPase may be identical, different domains of these proteins are involved in proton translocation and calcium-dependent ACh release and that the two proteins have a different membrane organization.     

Collagen concentrations in dissected tissue compartments of rat uterus on days 6, 7 and 8 of pregnancy.

Implantation and non-implantation sites were dissected into myometrial and stromal components; a decidual/embryonic region was obtained on Days 7 and 8 of pregnancy. The concentration of collagen (as a percentage of the dry weight of tissue), measured by hydroxyproline analysis, was significantly lower in the implantation regions than in the non-implant regions in all areas studied. The concentrations in the antimesometrial myometrium and stroma of the implantation region remained the same over the days studied. In contrast, the mesometrial collagen concentration in the implantation region declined from Day 6 to Day 8 of pregnancy. Collagen concentration was low within the decidual/embryonic tissue on Days 7 and 8 of pregnancy. Remodelling of collagen within the embryonic area appears to be an important feature of the uterine response to implantation in rats.     

DNA sequence analysis of the lamB gene from Klebsiella pneumoniae: implications for the topology and the pore functions in maltoporin.

Summary We have determined the sequence of the lamB gene from Klebsiella pneumoniae. It encodes the precursor to the LamB protein, a 429 amino acid polypeptide with maltoporin function. Comparison with the Escherichia coli LamB protein reveals a high degree of homology, with 325 residues strictly identical. The N-terminal third of the protein is the most conserved part of the molecule (1 change in the signal sequence, and 13 changes up to residue 146 of the mature protein). Differences between the two mature proteins are clustered mainly in six regions comprising residues 145–167, 173–187, 197–226, 237–300, 311–329, and 367–387 (K. pneumoniae LamB sequence). The most important changes were found in regions predicted by the two-dimensional model of LamB folding to form loops on the cell surface. In vivo maltose and maltodextrin transport properties of E. coli K 12 and K. pneumoniae strains were identical. However, none of the E. coli K12 LamB-specific phages was able to plaque onto K. pneumoniae. Native K. pneumoniae LamB protein forms highly stable trimers. The protein could be purified by affinity chromatography on starch-Sepharose as efficiently as the E. coli K12 LamB protein, indicating a conservation of the binding site for dextrins. However, none of the monoclonal antibodies directed against native E. coli K12 LamB protein recognized native purified K. pneumoniae LamB protein. These data indicate that most of the variability occurs within exposed regions of the protein and provide additional support for the proposed model of LamB folding. The fact that the N-terminal third of the protein is highly conserved is in agreement with the idea that it is part of, or constitutes, the pore domain located within the transmembranous channel and that it is not accessible from the cell surface.     

Developmental stages of the CD (Sprague-Dawley) rat skeleton after maternal exposure to ethylene glycol.

Ethylene glycol (EG), a chemical which causes skeletal malformations in rats, was administered by gavage to sperm positive CD rats on gestational days (gd) 6 through 15 at doses of 0 or 2,500 mg/kg/day to assess its effects on the pre- and postnatal skeletal development. Dams and fetuses/pups were killed on gd 18, 20, postnatal day (pnd) 1, 4, 14, 21, or 63, and offspring were double-stained for examination of skeletal malformations and degree of ossification of rapidly developing skeletal districts. No difference in gestational day of delivery between controls and the EG-treated dams was seen. Fetal weights per litter were significantly decreased with EG treatment in both the gd 18 and 20 groups. Pup body weight on pnd 1 was significantly below controls; however, EG had no effect on pup body weight on pnd 4-63. The percentage of fetuses/pups with skeletal malformations per litter was significantly increased after EG exposure for all time points except at pnd 63, with a predominance of axial skeletal defects. The percentages of total ossification, of sternabrae ossified, and of vertebral centra ossified were significantly reduced in the EG groups on gd 20 and on pnd 1-21, but not on gd 18 or on pnd 63. When the ossification data were subjected to statistical analysis with fetal/pup weights as a covariate, the values for EG-exposed pups on gd 20 were not statistically significantly different from the control values. The reduced ossification values for EG-exposed pups on pnd 1-21 retained statistical significance even after covariate analysis. There was no effect of dose or body weight on ossification of fore- or hindlimb digits. In conclusion, the differences in incidence of skeletal alterations observed prenatally and through pnd 21 were not evident by pnd 63, suggesting that perinatal skeletal abnormalities may not always be permanent.     

How predatory mites cope with the web of their tetranychid prey: a functional view on dorsal chaetotaxy in the Phytoseiidae

It has been proposed from behavioural observations that the ability of Phytoseiidae to penetrate the web is related to chaetotaxy. As phytoseiid species differ widely in chaetotaxy, as well as in their ability to cope with complex webs, this hypothesis was tested by studying relations between these two variables. In this paper we develop a simple method to quantify the degree to which the soma is protected from contact with the silk and apply it to phytoseiid species for which behavioural performance in the web is known from the literature. It is found that the better protected species experience less hinder from complex webs, such as produced byTetranychus spp. Moreover, they appear to be more successful in exterminating local populations ofTetranychus spp. Thus, setal patterns in the Phytoseiidae may well be a good indicator for selecting candidates for biological control ofTetranychus spp. Finally, we propose a parsimonious scenario for the evolution of dorsal chaetotaxy in the Phytoseiidae.     

Role of cytochrome P450 IA2 in acetanilide 4-hydroxylation as determined with cDNA expression and monoclonal antibodies

The role of P450 IA2 in the hydroxylation of acetanilide was examined using an inhibitory monoclonal antibody (MAb) 1-7-1 and vaccinia cDNA expression producing murine P450 IA1 (mIA1), murine P450 IA2 (mIA2), or human P450 IA2 (hIA2). Acetanilide hydroxylase (AcOH) activity was measured using an HPLC method with more than 500-fold greater sensitivity than previously described procedures. This method, which does not require the use of radioactive acetanilide, was achieved by optimizing both the gradient system and the amount of enzyme needed to achieve detection by uv light. MAb 1-7-1 inhibits up to 80% of the AcOH activity in both rat liver microsomes and cDNA expressed mouse and human P450 IA2. MAb 1-7-1, which recognizes both P450 IA1 and P450 IA2, completely inhibits the aryl hydrocarbon hydroxylase (AHH) activity of cDNA expressed in IA1. The inhibition of only 80% of the AHH activity present in MC liver microsomes by MAb 1-7-1 suggests that additional P450 forms are contributing to the overall AHH activity present in methylcholanthrene (MC)-liver microsomes as MAb 1-7-1 almost completely inhibits the AHH activity of expressed mIA1. Maximal inhibition of IA2 by 1-7-1 results in an 80% decrease in acetanilide hydroxylase activity in both liver microsomes and expressed mouse and human IA2. The capacity of MAb 1-7-1 to produce identical levels of inhibition of acetanilide hydroxylase activity in rat MC microsomes (80%) and in expressed mouse (81%) and human P450 IA2 (80%) strongly suggests that P450 IA2 is the major and perhaps the only enzyme responsible for the metabolism of acetanilide. These results demonstrate the complementary utility of monoclonal antibodies and cDNA expression for defining the contribution of specific P450 enzymes to the metabolism of a given substrate. This complementary approach allows for a more precise determination of the inhibitory capacity of MAb with respect to the metabolic capacity of the target P450.     

Fidelity of mammalian DNA replication and replicative DNA polymerases.

Current models suggest that two or more DNA polymerases may be required for high-fidelity semiconservative DNA replication in eukaryotic cells. In the present study, we directly compare the fidelity of SV40 origin-dependent DNA replication in human cell extracts to the fidelity of mammalian DNA polymerases alpha, delta, and epsilon using lacZ alpha of M13mp2 as a reporter gene. Their fidelity, in decreasing order, is replication greater than or equal to pol epsilon greater than pol delta greater than pol alpha. DNA sequence analysis of mutants derived from extract reactions suggests that replication is accurate when considering single-base substitutions, single-base frameshifts, and larger deletions. The exonuclease-containing calf thymus DNA polymerase epsilon is also highly accurate. When high concentrations of deoxynucleoside triphosphates and deoxyguanosine monophosphate are included in the pol epsilon reaction, both base substitution and frameshift error rates increase. This response suggests that exonucleolytic proofreading contributes to the high base substitution and frameshift fidelity. Exonuclease-containing calf thymus DNA polymerase delta, which requires proliferating cell nuclear antigen for efficient synthesis, is significantly less accurate than pol epsilon. In contrast to pol epsilon, pol delta generates errors during synthesis at a relatively modest concentration of deoxynucleoside triphosphates (100 microM), and the error rate did not increase upon addition of adenosine monophosphate. Thus, we are as yet unable to demonstrate that exonucleolytic proofreading contributes to accuracy during synthesis by DNA polymerase delta. The four-subunit DNA polymerase alpha-primase complex from both HeLa cells and calf thymus is the least accurate replicative polymerase. Fidelity is similar whether the enzyme is assayed immediately after purification or after being stored frozen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of imazaquin on absorption,translocation, and pattern of distribution of chlormequat chloride in winter wheat

The role of imazaquin in the absorption, translocation, and distribution of chlormequat chloride in CYCOCEL^* CL has been studied in winter wheat. Three treatments were applied to the 5th leaf of the main stem at growth stage 5 (Feekes Large scale): (1)¹⁴C-chlormequat chloride, (2) CYCOCEL^* CL containing¹⁴C-chlormequat chloride, and (3) CYCOCEL^* CL containing¹⁴C-imazaquin. Tracing of the radioactivity was followed in the treated leaf, main stem, tillers, and roots. Results showed that more than 85% of the radioactivity absorbed remained in the treated leaf. Ten days after the application of chlormequat chloride alone, 94.4% of the¹⁴C-chlormequat was found in the treated leaf, 2.9% in the main stem, 1.2% in the tillers, and 1.4% in the root system versus 88.2, 8.2, 2.1, and 1.4%, respectively, for the chlormequat chloride plus imazaquin treatment. It was concluded that imazaquin increases the mobility and the pattern of distribution of chlormequat chloride in the plant.     

The genetic defect in multiple endocrine neoplasia type 2A maps next to the centromere of chromosome 10

Multiple endocrine neoplasia type 2A (MEN2A) is a rare cancer syndrome that is inherited in an apparently autosomal dominant fashion. Previous linkage studies had assigned the MEN2A locus to chromosome 10 in the pericentromeric region. We recently have described several new easily scorable RFLPs for the chromosome 10-specific alpha satellite DNA (the D10Z1) locus that is known, on the basis of previous in situ hybridization experiments, to lie at the centromere. We report here tight linkage between MEN2A and D10Z1, as demonstrated by a maximum lod score of 12.02 at the recombination frequency of zero (1-lod-unit support interval 0-4 cM), indicating that the genetic defect in MEN2A lies in the immediate vicinity of the centromere. By means of a set of ordered polymorphic DNA markers from the pericentromeric region, multipoint as well as pairwise linkage analyses place the MEN2A locus at the middle of a small region (approximately 11 cM) bracketing the centromere with FNRB (at 10p11.2) and RBP3 (at 10q11.2) on either side, providing further support for the centromeric location of the MEN2A locus. Marked sex difference in recombination frequencies exists in this pericentromeric region: significantly (P less than .01) more female than male crossovers were observed across all of the adjacent intervals D10S24-FNRB, FNRB-D10Z1, and D10Z1-RBP3. However, a sex difference was not seen in the 7-cM interval from RBP3 to D10S5, suggesting that large variation in the sex difference in recombination can occur over small chromosomal regions.(ABSTRACT TRUNCATED AT 250 WORDS)