Evidence for the subcellular localization and specificity of chlordane inhibition in the marine bacterium Aeromonas proteolytica.

Sublethal levels (10 to 100 micrograms/ml) of the chlorinated insecticide chlordane (1,2,4,5,6,7,8,8-octachloro-3a,4,7,7a-tetrahydro-4,7-methanoindan) were introduced into the growth medium of the marine bacterium, Aeromonas proteolytica. Chlordane inhibited the synthesis of an extracellular endopeptidase by almost 40% but exhibited no such inhibition of the extracellular aminopeptidase also produced during the growth cycle. Studied with 14C-labeled chlordane demonstrated that the insecticide was not biologically degraded under the test conditions used and that up to 75% of the recoverable chlordane was cell associated within 48 h. Studied with uniformly labeled L[14C]valine and [2-14C]uracil established that neither the transport nor the incorporation of these protein and ribonucleic acid precursors was inhibited by chlordane. Separation of the membrane fractions using isopycnic centrifugation localized 14C-labeled chlordane in the cytoplasmic membrane. Also, chlordane inhibited the membrane-bound adenosine 5'-triphosphatase while the soluble (released) form of this enzyme remained unaffected. These data indicate that chlordane resides in the cytoplasmic membrane and may cause specific alterations in membrane-associated activities.     

Evaluation of the Mast ID and API 50CH systems for identification of Listeria spp

A multipoint inoculation technique (Mast ID) for the identification and species determination of Listeria monocytogenes (sensu strictu) and six other species of the genus Listeria was evaluated. This was compared with the commercially available API 50CH system. Both methods successfully identified all 123 strains tested. The Mast ID system is inexpensive and utilizing a multipoint inoculation technique permits the screening of up to 21 isolates per 9-cm petri dish. The API 50CH system was more expensive and time consuming and is therefore suitable only for the examination of smaller numbers of strains.     

Diverse post-translational modifications of the pannexin family of channel-forming proteins

The pannexin family of channel-forming proteins is composed of 3 distinct but related members called Panx1, Panx2, and Panx3. Pannexins have been implicated in many physiological processes as well as pathological conditions, primarily through their function as ATP release channels. However, it is currently unclear if all pannexins are subject to similar or different post-translational modifications as most studies have focused primarily on Panx1. Using in vitro biochemical assays performed on ectopically expressed pannexins in HEK-293T cells, we confirmed that all 3 pannexins are N-glycosylated to different degrees, but they are not modified by sialylation or O-linked glycosylation in a manner that changes their apparent molecular weight. Using cell-free caspase assays, we also discovered that similar to Panx1, the C-terminus of Panx2 is a substrate for caspase cleavage. Panx3, on the other hand, is not subject to caspase digestion but an in vitro biotin switch assay revealed that it was S-nitrosylated by nitric oxide donors. Taken together, our findings uncover novel and diverse pannexin post-translational modifications suggesting that they may be differentially regulated for distinct or overlapping cellular and physiological functions.     

Phosphorylation of caldesmon77 by protein kinase C in vitro and in intact human platelets

Caldesmon is a widely distributed calmodulin- and actin-binding protein which occurs in different forms depending on the tissue or cell type under examination. On the basis of molecular weight, caldesmon species can be divided into two classes: caldesmon77 (Mr 70,000-80,000) and caldesmon150 (Mr 140,000-150,000). We have examined the phosphorylation of caldesmon77 by protein kinase C (the Ca2+/phospholipid-dependent enzyme) in vitro and in intact platelets. Caldesmon77, purified from bovine liver, could be phosphorylated by purified rat brain protein kinase C to a level of approximately 1.0 mol of phosphate per mol of caldesmon77 monomer. Two-dimensional tryptic peptide mapping and phosphoamino acid analysis reveals that caldesmon77 is phosphorylated at two major sites exclusively on serine residues. Following treatment of platelets with tumor-promoting phorbol ester, caldesmon77 phosphorylation was elevated 4-fold. Tryptic peptide mapping of phosphorylated platelet caldesmon77 demonstrates that phosphorylation is most significantly enhanced on two peptides which had migration patterns identical with those of the two major phosphopeptides of bovine liver caldesmon77 phosphorylated in vitro. The results of this study indicate that protein kinase C can phosphorylate caldesmon77 in vitro and in intact platelets, suggesting a role for protein kinase C in the regulation of caldesmon77 function or localization.     

Stimulation by Hyphopichia burtonii and Bacillus amyloliquefaciens of aflatoxin production by Aspergillus flavus in irradiated maize and rice grains.

Aspergillus flavus was grown on maize and rice extract agars and on irradiated viable cracked maize and rice grains, either in pure culture or in dual culture with wild strains of either Hyphopichia burtonii or Bacillus amyloliquefaciens. Aflatoxin production by A. flavus and its growth and interactions with the other microorganisms were studied at three water activities (aw) (0.98, 0.95, and 0.90) and two temperatures (25 and 16 degrees C). Both H. burtonii and B. amyloliquefaciens markedly stimulated growth and aflatoxin production by A. flavus on cracked maize, especially at 25 degrees C and 0.95 and 0.98 aw. No aflatoxin was detected in pure cultures of A. flavus on cracked rice after 12 days of incubation at 25 degrees C, but some was produced by mixed cultures at 16 degrees C and 0.98 aw. The morphological interactions among A. flavus, H. burtonii, and B. amyloliquefaciens were also examined on maize and rice extract agars under similar controlled conditions.     

Further investigation of the primary mechanism of benzimidazole resistance in Haemonchus contortus

The binding of the [³H] benzimidazole carbamates (BZCs)—albendazole (ABZ), oxibendazole (OBZ), parbendazole (PBZ), mebendazole (MBZ), fenbendazole (FBZ) and oxfendazole (OFZ)—to tubulin from three ecologically-related isolates of adult Haemonchus contortus has been examined. The extent of binding of each BZC was inversely proportional to the known resistance status of the isolate. Biochemically, the change in the formation of the BZC-tubulin complex was due to a reduction in the amount of drug bound to resistant tubulin, with no significant change in the association constant of the complex. The resistance factors derived from the binding data support the hypothesis that the complex is ligand-dependent, with the aryl-substituted BZCs—MBZ, OFZ and FBZ—demonstrating lower resistance factors than those of the alkyl-substituted BZCs—ABZ, OBZ and PBZ. Examination of the slope derived from plots of binding against protein concentration demonstrated that the failure of resistant or partially resistant isolates to bind was due to either a decrease in the number of binding sites or, more likely, to reduced stability of the BZC-tubulin complex rendering it unstable to charcoal extraction.     

Complete amino acid sequence of human cartilage link protein (CRTL1) deduced from cDNA clones and chromosomal assignment of the gene

Little is known about the primary amino acid structure of human cartilage link protein (CRTL1). We screened a human genomic library with a cDNA encoding the 3' untranslated region and the adjoining B1 domain of chicken link protein. One clone was isolated and characterized. A 3.5-kb EcoRI-KpnI fragment from this genomic clone that contains the human B1 exon was used to map the gene to chromosome 5q13----q14.1. The same fragment was used to screen a cDNA library prepared from mRNA of Caco-2, a human colon tumor cell line. Two overlapping clones were isolated and shown to encode all of CRTL1. The deduced amino acid sequence is 354 residues long. The amino acid sequence shows a striking degree of identity to the porcine (96%), rat (96%), and chicken (85%) link protein sequences. Furthermore, there is greater than 86% homology between the 3' untranslated region of the genes encoding human and porcine link proteins. These results indicate that there has been strong evolutionary pressure against changes in the coding and 3' untranslated regions of the gene encoding cartilage link protein.     

Evolution ofE.coli tRNATrp

Earlier studies (1) have shown there are direct correlations between the hydrophobicity ranking of most amino acids and their anticodonic nucleotides. However, four anticodonic assignments, i.e. those for Trp, Tyr, Ile and the XGA anticodons for Ser, did not correlate. It was our proposal that this failure to correlate was due to the fact that these assignments were made late, relative to the bulk of the assignments, in evolution through the mutation of existing tRNAs. We have shown (2) thatE. coli tRNA^{Ile 1} and tRNA^{Ile 2} were likely derived from tRNA^{Val 1} and tRNA^Lys respectively andE. coli tRNA^Tyr was possibly derived fromE. coli 5s rRNA or a common precursor with 5s rRNA (3). The fact that quite high homologies were observed in these comparisons is consistent with the late evolution of the tRNAs in question. We now examine the evolution ofE. coli tRNA^Trp by comparing its homology with otherE. coli tRNAs. The data suggest a possible evolutionary relationship withE. coli tRNA^Gly or tRNA^Arg. The data support the idea of the late assignment of anticodons to Trp.Deceased.     

Experimental formulations ofBacillus sphaericus for the control of anopheline and culicine larvae

Summary Four experimental formulations ofBacillus sphaericus Neide (2362 isolate) were evaluated for larvicidal activity against culicine and anopheline larvae in several natural and artificial habitats. A granular formulation (5% primary powder) was tested against natural populations of mosquitoes in two simulated habitats in Florida and in maturing and reflooded rice fields in Louisiana. Larvae ofCulex quinquefasciatus Say were reduced by 97 and 99% after application of the granules at the rate of 10 kg/ha to polluted tanks and 2.5 kg/ha to sod-lined potholes, respectively. Anopheline andPsorophora columbiae (Dyar and Knab) larvae were reduced by 68 and 92–100%, respectively, after application of 5 kg granules/ha to rice fields. A flowable concentrate (12.8% primary powder) applied to unpolluted and organically enriched habitats in Florida at 0.25 kg/ha reduced populations ofCulex spp. by 93–100% and 99%, respectively. Sustained-release briquets (5% primary powder) applied at the rate of one half briquet/1.8 m² sod-lined potholes reduced larval populations ofCx. quinquefasciatus by 88–95% for up to 2 weeks in open sunlight. Sustained-release pellets (30% primary powder) applied to small woodland pools in Memphis, TN at the rate of four pellets/pool virtually eliminated larval populations ofCx. restuans Theobald for over 8 days. Variable persistance of larvicidal activity was noted for the other treatments depending on the formulation, target species and habitat.