1-Alkyl-3-amino-5-aryl-1H-[1,2,4]triazoles: novel synthesis via cyclization of N-acyl-S-methylisothioureas with alkylhydrazines and their potent corticotropin-releasing factor-1 (CRF(1)) receptor antagonist activities.

Cyclizations of alkylhydrazines with N-acyl-S-methylisothioureas, readily synthesized from acyl chlorides, sodium thioisocyanate, dialkylamines then methyl iodide in a one-pot reaction, gave 1-alkyl-3-dialkylamino-5-phenyltriazoles 7 as major products. The regioisomers were assigned through the use of NOE NMR experiments. While bearing a N-bis(cyclopropyl)methyl-N-propylamino group, this series of compounds shows very good binding affinity on the human CRF₁ receptor. Among them, 1-methyl-3-[N-bis(cyclopropyl)methyl-N-propylamino]-5-(2,4-dichlorophenyl)-1H-[1,2,4]triazole 7a had the best binding affinity for the CRF₁ receptor (K_i=9 nM).     

Position Effect Variegation in Drosophila Melanogaster: Relationship between Suppression Effect and the Amount of Y Chromosome

Position effect variegation results from chromosome rearrangements which translocate euchromatic genes close to the heterochromatin. The euchromatin-heterochromatin association is responsible for the inactivation of these genes in some cell clones. In Drosophila melanogaster the Y chromosome, which is entirely heterochromatic, is known to suppress variegation of euchromatic genes. In the present work we have investigated the genetic nature of the variegation suppressing property of the D. melanogaster Y chromosome. We have determined the extent to which different cytologically characterized Y chromosome deficiencies and Y fragments suppress three V-type position effects: the Y-suppressed lethality, the white mottled and the brown dominant variegated phenotypes. We find that: (1) chromosomes which are cytologically different and yet retain similar amounts of heterochromatin are equally effective suppressors, and (2) suppression effect is positively related to the size of the Y chromosome deficiencies and fragments that we tested. It increases with increasing amounts of Y heterochromatin up to 60-80% of the entire Y, after which the effect reaches a plateau. These findings suggest suppression is a function of the amount of Y heterochromatin present in the genome and is not attributable to any discrete Y region.     

Expression of endogenous receptors for neoglycoproteins, especially lectins, that allow fiber typing on formaldehyde-fixed, paraffin-embedded muscle biopsy specimens. A glycohistochemical, immunohistochemical, and glycobiochemical study

A panel of biotinylated (neo)glycoproteins was used for specific detection of endogenous sugar receptors, especially lectins, in formaldehyde-fixed, paraffin-embedded muscle biopsy specimens from human deltoid, quadriceps, and biceps muscles, tibial and quadriceps muscles of rat, and bovine masseter muscle. The glycohistochemical probes used consisted of conjugates of a labeled, histochemically inert carrier protein and various covalently linked, histochemically crucial sugar moieties. Specific binding of alpha-L-fucoside, beta-D-galactoside, beta-D-xyloside, and alpha-D-mannoside to muscle sections was detected, showing no species-specific differences. The presence of receptors for the N-acetylated sugars in natural glycoconjugates, and for sugars with a phosphate group, i.e., mannose-6-phosphate and galactose-6-phosphate, was demonstrated glycohistochemically. However, these binding specificities revealed species-specific differences, e.g., the absence of N-acetyl-D-galactosamine-specific receptors or galactose-6-phosphate-specific receptors in rat muscle. Other charged sugars included glucuronic acid and sialic acid, which bound only to ox and rat muscle or failed to reveal their respective receptors in all types of muscle investigated. This different extent of staining with anionic probes served as a further control to ascertain carbohydrate binding specificity. Positive glycohistochemical reaction developed within sarcomeres only at the level of A-bands. Granular staining was observed in the sarcoplasm among the myofibrils and also in the subsarcolemmal regions. Differences in expression of glycohistochemically detectable sugar receptors were noted between type 1, type 2A, and type 2B fibers. The molecular properties of one type of glycohistochemically detectable sugar receptor were inferred both immunohistochemically and biochemically. An antiserum against an endogenous beta-galactoside-specific lectin from muscle tissue localized this lectin within sections consistently similar to (neo)glycoproteins, detecting beta-galactoside-specific receptor(s). This similarity of binding patterns strongly supports the assumption that (neo)glycoproteins with beta-galactoside termini indeed bind to the respective endogenous lectin. The lectin-specific antiserum enabled us to ascertain that glycohistochemical fiber typing corresponds to enzyme histochemical typing. Moreover, biochemical purification using affinity chromatography and subsequent affinity elution revealed only the immunohistochemically detectable beta-galactoside-specific lectin. Consequently, use of a panel of neoglycoproteins, when frozen sections for histochemical analysis are not available, co     

A preliminary report on the use of transfer factor for treating stage D3 hormone-unresponsive metastatic prostate cancer

As conventional treatments are unsuccessful, the survival rate of stage D3 prostate cancer patients is poor. Reports have suggested the existence of humoral and cell-mediated immunity (CMI) against prostate cancer tumour-associated antigens (TAA). These observations prompted us to treat stage D3 prostate cancer patients with an in vitro produced transfer factor (TF) able to transfer, in vitro and in vivo, CMI against bladder and prostate TAA. Fifty patients entered this study and received one intramuscular injection of 2–5 units of specific TF monthly. Follow-up, ranging from 1 to 9 years, showed that complete remission was achieved in 2 patients, partial remission in 6, and no progression of metastatic disease in 14. The median survival was 126 weeks, higher than the survival rates reported in the literature for patients of the same stage.     

Effect of infusing branched-chain amino acid during incremental exercise with reduced muscle glycogen content

The aim of this study was to investigate whether, when muscle glycogen is reduced, a pre-exercise infusion of branched-chain amino acids (BCAA) modifies exercise performance or the metabolic and respiratory responses to incremental exercise. Six moderately trained volunteers took part in the following protocol on two occasions. On day 1, at 9 a.m. in the postabsorptive state, they performed a graded incremental exercise (increases of 35 W every 4 min) to exhaustion (Ex-1). A meal of 1,000 kcal (4,200 kJ; 60% protein, 40% fat) was consumed at 12 p.m. No food was then allowed until the end of the experiment (20–21 h later). A 90-min period of exercise at alternating high and moderate intensities, designed to deplete muscle glycogen, was performed between 6 p.m. and 7.30 p.m. The morning after (day 2), the subjects randomly received either a mixed solution of BCAA (260 mg × kg^–1 × h^–1 for 70 min), or saline. They then repeated the graded incremental exercise to exhaustion (Ex-2). Metabolic and respiratory measurements suggested a muscle glycogen-depleted state had been achieved. No significant differences were observed in total work performed, maximal oxygen uptake or plasma ammonia, alanine, and blood pyruvate concentrations in the two treatments. After BCAA infusion, higher blood lactate concentrations were observed at maximal power output in comparison with those during saline [BCAA 4.97 (SEM 0.41) mmol × l^–1, Saline 3.88 (SEM 0.47) mmol × l^–1,P < 0.05]. In summary, in conditions of reduced muscle glycogen content, after a short period of fasting, BCAA infusion had no significant effect on the total work that could be performed during a graded incremental exercise.     

L cells expressing DQ molecules of the DR3 and DR4 haplotypes: reactivity patterns with mAbs

cDNAs coding for the HLA class II DR and DQ and chains of the diabetogenic haplotypes DR3 and DR4 were introduced into a mammalian expression vector and transfected into L-cell mouse fibroblasts to produce cells expressing individual human class II molecules. Stable L transfectants were generated expressing each of the DR or DQ isotypes of the cis-encoded and chains of the DR3 or DR4 haplotypes, as well as the trans-encoded and chains of the DQ molecules of the two haplotypes. However, isotype mismatched combinations (DR/DQ or DQ/DR) did not result in any stable transfectants. The stable DQ L-cell transfectants obtained, along with homozygous B-cell lines expressing the DQ2 and DQ8 specificities, were tested against a large panel of twentyone anti-HLA class II monoclonal antibodies (mAbs). Their unusual reactivity patterns are described including the failure of most pan-DQ mAbs to react with all DQ expressing L-cell transfectants. Interestingly, some mAbs react with certain heterodimers expressed on B-LCL but fail to recognize the same heterodimers expressed on the transfectants. This is suggestive of minor structural modifications that class II molecules undergo depending on the cells they are expressed on.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number U07848. The name DQB1 ^* 0202 was officially assigned by the WHO Nomenclature Committee in April 1994. This follows the agreed policy that, subject to the conditions stated in the most recent Nomenclature Report (Bodmer et al. 1992), names will be assigned to new sequences as they are identified. Lists of such new names will be published in the following WHO Nomenclature Report     

Cis-Effects of Heterochromatin on Heterochromatic and Euchromatic Gene Activity in Drosophila Melanogaster

Chromosomal rearrangements that juxtapose heterochromatin and euchromatin can result in mosaic inactivation of heterochromatic and euchromatic genes. This phenomenon, position effect variegation (PEV), suggests that heterochromatic and euchromatic genes differ in their regulatory requirements. This report describes a novel method for mapping regions required for heterochromatic genes, and those that induce PEV of a euchromatic gene. P transposase mutagenesis was used to generate derivatives of a translocation that variegated for the light(+) (lt(+)) gene and carried the euchromatic white(+) (w(+)) gene on a transposon near the heterochromatin-euchromatin junction. Cytogenetic and genetic analyses of the derivatives showed that P mutagenesis resulted in deletions of several megabases of heterochromatin. Genetic and molecular studies showed that the derivatives shared a euchromatic breakpoint but differed in their heterochromatic breakpoint and their effects on seven heterochromatic genes and the w(+) gene. Heterochromatic genes differed in their response to deletions. The lt(+) gene was sensitive to the amount of heterochromatin at the breakpoint but the heterochromatic 40Fa gene was not. The severity of variegated w(+) phenotype did not depend on the amount of heterochromatin in cis, but varied with local heterochromatic environment. These data are relevant for considering mechanisms of PEV of both heterochromatic and euchromatic genes.     

Cytogenetic Analysis of the Second Chromosome Heterochromatin of Drosophila Melanogaster

This paper reports the cytogenetic characterization of the second chromosome heterochromatin of Drosophila melanogaster. High resolution cytological analysis of a sample of translocations, inversions, deficiencies and free duplications involving the pericentric regions of the second chromosome was achieved by applying sequential Hoechst 33258 and N-chromosome banding techniques to larval neuroblast prometaphase chromosomes. Heterochromatic rearrangements were employed in a series of complementation assays and the genetic elements previously reported to be within or near the second chromosome heterochromatin were thus precisely assigned to specific heterochromatic bands. The results of this analysis reveal a nonhomogeneous distribution of loci along the second chromosome heterochromatin. The l(2)41Aa, l(2)41Ab, rolled (l(2)41Ac) and l(2)41Ad loci are located within the proximal heterochromatin of 2R, while the nine remaining loci in the left arm and two (l(2)41Ae and l(2)41Ah) in the right arm map to h35 and to h46, respectively, the most distal heterochromatic regions. In addition, a common feature of these loci revealed by the cytogenetic analysis is that they map to specific heterochromatic blocks but do not correspond to the blocks themselves, suggesting that they are not as large as the Y fertility factors or the Rsp locus. Mutations of the proximal most heterochromatic loci, l(2)41Aa and rolled, were also examined for their phenotypic effects. Extensive cell death during imaginal disc development was observed in individuals hemizygous for either the EMS 31 and rolled mutations, leading to a pattern of phenotypic defects of adult structures.     

Expression of anchorin CII mRNA by cultured chondrocytes

The establishment of a cell culture system promoting chondrocyte differentiation has been utilized to better characterize phenotypic stages of chondrogenesis at the cellular level. Although the expression of the type II collagen gene has been studied during “in vitro” chondrocyte differentiation, little is known about the expression of the gene coding for its receptor: anchorin CII. The modulation of the anchorin mRNA steady state level in chick embryo chondrocytes at different developmental stages is described here.The anchorin mRNA level was low in dedifferentiated chondrocytes, progressively increased after the cell transfer into suspension (a condition promoting differentiation), reached its maximal value after 4 weeks and decreased after 5 weeks.Therefore anchorin CII mRNA reaches its maximum level in hypertrophic stage II chondrocytes.