Structure-based virtual screening of hypothetical inhibitors of the enzyme longiborneol synthase-a potential target to reduce Fusarium head blight disease.

Fusarium head blight (FHB) is one of the most destructive diseases of wheat and other cereals worldwide. During infection, the Fusarium fungi produce mycotoxins that represent a high risk to human and animal health. Developing small-molecule inhibitors to specifically reduce mycotoxin levels would be highly beneficial since current treatments unspecifically target the Fusarium pathogen. Culmorin possesses a well-known important synergistically virulence role among mycotoxins, and longiborneol synthase appears to be a key enzyme for its synthesis, thus making longiborneol synthase a particularly interesting target. This study aims to discover potent and less toxic agrochemicals against FHB. These compounds would hamper culmorin synthesis by inhibiting longiborneol synthase. In order to select starting molecules for further investigation, we have conducted a structure-based virtual screening investigation. A longiborneol synthase structural model is first built using homology modeling, followed by molecular dynamics simulations that provided the required input for a protein-ligand ensemble docking procedure. From this strategy, the three most interesting compounds (hits) were selected among the 25 top-ranked docked compounds from a library of 15,000 drug-like compounds. These putative inhibitors of longiborneol synthase provide a sound starting point for further studies involving molecular modeling coupled to biochemical experiments. This process could eventually lead to the development of novel approaches to reduce mycotoxin contamination in harvested grain.

Increased acetylcholine-induced vasodilation in pregnant rats: A role for gap junctional communication.

We have tested the hypothesis that increased gap junctional communication contributes to the augmented endothelium-dependent vasodilation in pregnancy. Contractile force and connexin43 expression were measured in aortic rings from nonpregnant and pregnant rats. Norepinephrine-constricted aortas from pregnant rats were more sensitive to acetylcholine, but not to sodium nitroprusside, compared with those from nonpregnant rats. Vessels from pregnant rats, constricted either with 45 mmol/L KCl or with norepinephrine + 10(-4) mol/L N(G)-monomethyl-L-arginine (L-NMMA), an inhibitor of nitric oxide synthase, also exhibited greater relaxation to acetylcholine. Heptanol, an uncoupler of gap junctional communication, inhibited acetylcholine responses in norepinephrine-constricted aortas from nonpregnant rats but greatly impaired acetylcholine relaxation in aortas from pregnant rats. Heptanol also inhibited in both groups acetylcholine responses in vessels constricted with KCl, only minimally affected acetylcholine relaxation in arteries constricted with norepinephrine + L-NMMA, and did not change sodium nitroprusside-induced relaxation. Tetraethylammonium chloride induced greater contractions in control aortas compared with aortas from pregnant rats. Increased connexin43 mRNA levels were found in the uterus and in the mesenteric, uterine, and thoracic aortic arteries, but not in the heart and brain, from pregnant rats. These results suggest that increased gap junctional communication, possibly due to increased gap junction protein expression, may facilitate the effects of endothelium-derived relaxing factors, contributing to the augmented endothelium-dependent relaxation in arteries from pregnant rats.

A simple RT-PCR-based strategy for screening connexin identity.

Vertebrate gap junctions are aggregates of transmembrane channels which are composed of connexin (Cx) proteins encoded by at least fourteen distinct genes in mammals. Since the same Cx type can be expressed in different tissues and more than one Cx type can be expressed by the same cell, the thorough identification of which connexin is in which cell type and how connexin expression changes after experimental manipulation has become quite laborious. Here we describe an efficient, rapid and simple method by which connexin type(s) can be identified in mammalian tissue and cultured cells using endonuclease cleavage of RT-PCR products generated from "multi primers" (sense primer, degenerate oligonucleotide corresponding to a region of the first extracellular domain; antisense primer, degenerate oligonucleotide complementary to the second extracellular domain) that amplify the cytoplasmic loop regions of all known connexins except Cx36. In addition, we provide sequence information on RT-PCR primers used in our laboratory to screen individual connexins and predictions of extension of the "multi primer" method to several human connexins.

A simple RT-PCR-based strategy for screening connexin identity

Vertebrate gap junctions are aggregates of transmembrane channels which are composed of connexin (Cx) proteins encoded by at least fourteen distinct genes in mammals. Since the same Cx type can be expressed in different tissues and more than one Cx type can be expressed by the same cell, the thorough identification of which connexin is in which cell type and how connexin expression changes after experimental manipulation has become quite laborious. Here we describe an efficient, rapid and simple method by which connexin type(s) can be identified in mammalian tissue and cultured cells using endonuclease cleavage of RT-PCR products generated from "multi primers" (sense primer, degenerate oligonucleotide corresponding to a region of the first extracellular domain; antisense primer, degenerate oligonucleotide complementary to the second extracellular domain) that amplify the cytoplasmic loop regions of all known connexins except Cx36. In addition, we provide sequence information on RT-PCR primers used in our laboratory to screen individual connexins and predictions of extension of the "multi primer" method to several human connexins

Myosin II-actin interaction in MDCK cells: role in cell shape changes in response to Ca2+ variations.

Cultured MDCK cell monolayers respond to a low level of extracellular calcium ([Ca2+]e < or = 5 microM) with a loss of transepithelial electrical resistance and transport function, and changes in position of a circumferential ring of actin filaments tethered to the plasma membrane at the zonula adhaerens. Keeping this cytoskeletal structure in place seems necessary to preserve the architecture of the tight junctions and therefore their sealing capacity. All three effects are reversible upon restituting normal [Ca2+]e. Recent work provided evidence of actin-myosin interactions at the filament ring, thus suggesting a contraction process involved in the alteration of the actin cytoskeleton. We now report that active contraction does occur and causes an extensive morphological transformation of MDCK cells. A marked increase in cell height simultaneous with a decrease in width and area of contact to the substratum was seen within 10 min of removal of [Ca2+]e; recovery began immediately after replacing calcium, although it took longer for completion. Conventional and confocal epifluorescence studies showed actin colocalized with myosin II at various planes of resting or contracted cells, in particular at the ring level. Electron-micrographs revealed the circumferential actin ring associated with the plasma membrane in a waist-like constriction where Ca2+ was removed from the cultures. Contraction, as well as relaxation, in response to [Ca2+]e variations were inhibited by cytochalasin-D (an actin-filament disrupting drug), by okadaic acid( an inhibitor of myosin light-chain dephosphorylation), and by 2,3-butanedione monoxime (a blocker of myosin II ATPase activity). Similarly, no response was observed in cells previously depleted of metabolic energy by 2,4-dinitrophenol and 2-deoxy-D-glucose preincubation. The actin-myosin mediated reversible structural transformation of MDCK cells in response to [Ca2+]3 poses new questions for the interpretation of in vitro experiments, as well as for the understanding of epithelial function.

[In vivo resistance to strains of Trypanosoma rhodesiense, Mozambique, 1985]. / Resistencia in vivo de estirpes de Trypanosoma rhodesiense, Mozambique, 1985.

We report the existence of high resistance to Melarsoprol and low resistance to Suramin in 11 Trypanosoma rhodesiense species. They were isolated from humans in the Tete province, Mozambique, and kept in mice at the Maputo National Health Institute. The preliminary results obtained with Suramin administered intracranially, are also reported.

Oxandrolone therapy in patients with Turner syndrome.

Long-term, low-dosage androgen treatment of patients with Turner syndrome results in more rapid growth and significantly greater adult height than in control patients who receive only estrogen for pubertal development. Seventeen patients treated with oxandrolone for one year and ten treated for two years had significantly greater growth velocities during than before treatment. Mean adult height of 25 patients treated with oxandrolone, fluoxymesterone, or both was significantly taller than the height of adult patients with Turner syndrome treated with estrogen only. Excessive skeletal maturation was not generally observed.

Familial syndrome of mental retardation, short stature, contractures of the hands, and genital anomalies.

Two teen-age XY brothers with mental retardation, short stature, obesity, genital abnormalities, and contractures of their hands are described. They have generalized osteoporosis and a history of frequent fractures. Their endocrinologic evaluation was normal except for mild glucose intolerance and delayed, but normal puberty. Although these brothers are similar to individuals with Prader-Willi syndrome, their unusual hand contractures, clinically significant osteoporosis, and lack of hypotonia indicate that they represent a different entity.