Comparative chromosomal localization of the canine-derived BAC clones containing LEP and IGF1 genes in four species of the family Canidae

In the present report we show the chromosomal localization of two BAC clones, carrying the leptin (LEP) and insuline-like growth factor 1 (IGF1) genes, respectively, in four species belonging to the family Canidae: the dog, red fox, arctic fox and the Chinese raccoon dog. The assignments are in agreement with earlier data obtained from comparative chromosome painting for the dog, red fox and arctic fox.     

Extracorporeal shock waves stimulate frog sciatic nerves indirectly via a cavitation-mediated mechanism.

Shock waves (SWs) are single pressure pulses with amplitudes up to over 100 MPa, a rise time of only a few nanoseconds, and a short duration of approximately 2 microseconds. Their clinical application for stone destruction causes pain, indicating nerve stimulation by SWs. To examine this phenomenon, sciatic nerves of frogs were exposed to SWs in an organ bath. The SWs were generated with an experimental Dornier lithotripter model XL1 at an operating voltage of 15 kV. The nerves were mounted in a chamber which allowed electrical nerve stimulation and the registration of electrically and SW-induced compound action potentials (SWCAPs). The chamber was filled with frog Ringer's solution. In a standardized protocol. The first experiment established that 95.0 +/- 4.7% of administered SWs induced action potentials which were lower in amplitude (1.45 +/- 1.14 versus 1.95 +/- 0.95 mV, p = 0.004) but similar in shape to electrically induced compound action potentials. In a second experiment, it was shown that the site of origin of the SWCAPs could be correctly determined by simultaneous recording of action potentials at both ends of the nerve. The mechanism of shock wave stimulation was examined by experiments 3 and 4. In experiment 3, in contrast to the previous experiments, SW exposure of the nerves was performed 6 cm outside the shock wave focus. This resulted in a mean probability of inducing a SWCAP of only 4%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nucleoside binding site of herpes simplex type 1 thymidine kinase analyzed by X-ray crystallography

The crystal structures of the full-length Herpes simplex virus type 1 thymidine kinase in its unligated form and in a complex with an adenine analogue have been determined at 1.9 A resolution. The unligated enzyme contains four water molecules in the thymidine pocket and reveals a small induced fit on substrate binding. The structure of the ligated enzyme shows for the first time a bound adenine analogue after numerous complexes with thymine and guanine analogues have been reported. The adenine analogue constitutes a new lead compound for enzyme-prodrug gene therapy. In addition, the structure of mutant Q125N modifying the binding site of the natural substrate thymidine in complex with this substrate has been established at 2.5 A resolution. It reveals that neither the binding mode of thymidine nor the polypeptide backbone conformation is altered, except that the two major hydrogen bonds to thymidine are replaced by a single water-mediated hydrogen bond, which improves the relative acceptance of the prodrugs aciclovir and ganciclovir compared with the natural substrate. Accordingly, the mutant structure represents a first step toward improving the virus-directed enzyme-prodrug gene therapy by enzyme engineering.     

Junctional adhesion molecule a serves as a receptor for prototype and field-isolate strains of mammalian reovirus

Reovirus infections are initiated by the binding of viral attachment protein sigma1 to receptors on the surface of host cells. The sigma1 protein is an elongated fiber comprised of an N-terminal tail that inserts into the virion and a C-terminal head that extends from the virion surface. The prototype reovirus strains type 1 Lang/53 (T1L/53) and type 3 Dearing/55 (T3D/55) use junctional adhesion molecule A (JAM-A) as a receptor. The C-terminal half of the T3D/55 sigma1 protein interacts directly with JAM-A, but the determinants of receptor-binding specificity have not been identified. In this study, we investigated whether JAM-A also mediates the attachment of the prototype reovirus strain type 2 Jones/55 (T2J/55) and a panel of field-isolate strains representing each of the three serotypes. Antibodies specific for JAM-A were capable of inhibiting infections of HeLa cells by T1L/53, T2J/55, and T3D/55, demonstrating that strains of all three serotypes use JAM-A as a receptor. To corroborate these findings, we introduced JAM-A or the structurally related JAM family members JAM-B and JAM-C into Chinese hamster ovary cells, which are poorly permissive for reovirus infection. Both prototype and field-isolate reovirus strains were capable of infecting cells transfected with JAM-A but not those transfected with JAM-B or JAM-C. A sequence analysis of the sigma1-encoding S1 gene segment of the strains chosen for study revealed little conservation in the deduced sigma1 amino acid sequences among the three serotypes. This contrasts markedly with the observed sequence variability within each serotype, which is confined to a small number of amino acids. Mapping of these residues onto the crystal structure of sigma1 identified regions of conservation and variability, suggesting a likely mode of JAM-A binding via a conserved surface at the base of the sigma1 head domain.     

Consequences of disease-causing mutations on lubricin protein synthesis, secretion, and post-translational processing

Lubricin, a protein product of the gene PRG4, is a secreted mucin-like proteoglycan that is a major lubricant in articulating joints. Mutations in PRG4 cause the autosomal recessive, human disorder camptodactyly-arthropathy-coxa vara-pericarditis syndrome. We developed rabbit polyclonal antibodies against human lubricin to determine the consequence of disease-causing mutations at the protein level and to study the protein's normal post-translational processing. Antiserum generated against an epitope in the amino-terminal portion of lubricin detected protein in wild-type synovial fluid and in conditioned media from wild-type cultured synoviocytes. However, the antiserum did not detect lubricin in synovial fluid or cultured synoviocytes from several patients with frameshift or nonsense mutations in PRG4. Antiserum generated against an epitope in the protein's carboxyl-terminal, hemopexin-like domain identified a post-translational cleavage event in wild-type lubricin, mediated by a subtilisin-like proprotein convertase (SPC). Interestingly, in contrast to wild-type lubricin, one disease-causing mutation that removes the last 8 amino acids of the protein, including a conserved cysteine residue, was not cleaved within the hemopexin-like domain when expressed in COS-7 cells. This suggests that formation of an intrachain disulfide bond is required for SPC-mediated cleavage and that SPC-mediated cleavage is essential to protein function.     

The renin and iso-renin-angiotensin system in rats with experimental pitutary tumors (38585).

Renin, iso-renin, angiotensin I. angiotensin-converting enzyme, and angiotensinases were measured in plasma and in various extrarenal tissues of rats. Despite complete suppression of plasma renin in rats bearing pituitary tumors iso-renin and all other components of the renin-angiotensin system were found to be at or above control concentrations. The results strongly suggest that there is local synthesis of iso-renin in extrarenal tissues.