"In vitro" study on release of cyclic AMP and thyroid hormone in autonomously functioning thyroid nodules

The TSH effect on slice and the incubation medium cyclic AMP levels and T3 and T4 released from 8 autonomously functioning thyroid nodules (AFTN) and their respective perinodular (PN) tissues were examined. The thyroid slices were incubated in Eagle's Medium containing TSH (5 to 100 mU/ml) for 60 min and 300 min for tissue cyclic AMP generation and for cyclic AMP, T3 and T4 release, respectively. Basal cyclic AMP levels were not different either in AFTN and in PN slices or into the incubation medium. In both tissues TSH produced a similar cyclic AMP generation. In contrast, cyclic AMP released into the incubation medium was significantly higher in AFTN than in PN tissues, after TSH stimulation. Basal T3 values and TSH-stimulated T3 release in AFTN were not different from PN tissue. However, basal T4 levels were significantly higher in AFTN than in PN tissue as well as T4 released in response to TSH. In addition, T3/T4 ratio was lower in AFTN than in PN tissues. The cyclic AMP released into the incubation medium correlated with both T3 and T4 release in PN tissue but in the AFTN tissue no correlations were found. These findings suggest that the adenylate cyclase-cyclic AMP system is more sensitive to TSH-stimulation in AFTN when compared with PN tissue and that AFTN tissue has a preferential T4 secretion.     

Expression and characterization of <Emphasis Type="Italic">Trichoderma virens</Emphasis> UKM-1 endochitinase in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

A gene encoding endochitinase from Trichoderma virens UKM-1 was cloned and expressed in E. coli BL21 (DE3). Both the endochitinase gene and its cDNA sequences were obtained. The endochitinase gene encodes 430 amino acids from an open reading frame comprising of 1,690 bp nucleotide sequence with three introns. The endochitinase was expressed as soluble and active enzyme at 20°C when induced with 1 mM IPTG. Maximum activity was observed at 4 h of post-induction time. SDS-PAGE showed that the purified endochitinase exhibited a single band with molecular weight of 42 kDa. Biochemical characterization of the enzyme displayed a near neutral pH characteristic with an optimum pH at 6.0 and optimum temperature at 50°C. The enzyme is stable between pH 3.0–7.0 and is able to retain its activity from 30 to 60°C. The presence of Mg²⁺ and Ca²⁺ ions increased the enzyme activity up to 20%. The purified enzyme has a strong affinity towards colloidal chitin and low effect on ethyl cellulose and D-cellubiose which are non-chitin related substrates. HPLC analysis from the chitin hydrolysis showed the release of (GlcNAc)₃, (GlcNAc)₂ and GlcNAc, in which (GlcNAc)₂ was the main product.     

Novel targets for Huntington's disease in an mTOR-independent autophagy pathway

Autophagy is a major clearance route for intracellular aggregate-prone proteins causing diseases such as Huntington's disease. Autophagy induction with the mTOR inhibitor rapamycin accelerates clearance of these toxic substrates. As rapamycin has nontrivial side effects, we screened FDA-approved drugs to identify new autophagy-inducing pathways. We found that L-type Ca2+ channel antagonists, the K+ATP channel opener minoxidil, and the G(i) signaling activator clonidine induce autophagy. These drugs revealed a cyclical mTOR-independent pathway regulating autophagy, in which cAMP regulates IP3 levels, influencing calpain activity, which completes the cycle by cleaving and activating G(s)alpha, which regulates cAMP levels. This pathway has numerous potential points where autophagy can be induced, and we provide proof of principle for therapeutic relevance in Huntington's disease using mammalian cell, fly and zebrafish models. Our data also suggest that insults that elevate intracytosolic Ca2+ (like excitotoxicity) inhibit autophagy, thus retarding clearance of aggregate-prone proteins.     

Cytokine gene polymorphisms in endemic pemphigus foliaceus: a possible role for IL6 variants

Endemic pemphigus foliaceus (EPF) is a complex autoimmune disease characterized by the presence of antibodies against desmoglein 1, which lead to the loss of adhesion among keratinocytes (acantholysis). Variants of HLA class II genes have been the only genetic factors found to modulate susceptibility to EPF. This study aims at investigating the influence of cytokine genetic variants in the pathogenesis of EPF, since they may affect the expression levels of these immunomodulatory molecules. The sample included 168 patients and 189 controls and was comprised of mostly Caucasoids and Mulattos. The approach consisted of a case-control association study and the alleles were identified by mismatched PCR-RFLP. No associations were found with variants of IL1A, IL1B, IL1RN, IL4R and IL10. There was a weak negative association with the haplotype -1082G -592C (OR=0.49) of the IL10 gene in Mulattos. In regard to polymorphism -590 of the IL4 gene, a positive association with the T/T genotype (OR=2.71) and a negative association with the C variant (OR=0.37) were found. Associations with IL6 -174 variants suggest that the C/C genotype has a protective effect (OR=0.13) while carriers of the G allele are more susceptible (OR=7.66) to EPF.     

A major difference between the divergence patterns within the lines-1 families in mice and voles

L1 retroposons are represented in mice by subfamilies of interspersed sequences of varied abundance. Previous analyses have indicated that subfamilies are generated by duplicative transposition of a small number of members of the L1 family, the progeny of which then become a major component of the murine L1 population, and are not due to any active processes generating homology within preexisting groups of elements in a particular species. In mice, more than a third of the L1 elements belong to a clade that became active approximately 5 Mya and whose elements are > or = 95% identical. We have collected sequence information from 13 L1 elements isolated from two species of voles (Rodentia: Microtinae: Microtus and Arvicola) and have found that divergence within the vole L1 population is quite different from that in mice, in that there is no abundant subfamily of homologous elements. Individual L1 elements from voles are very divergent from one another and belong to a clade that began a period of elevated duplicative transposition approximately 13 Mya. Sequence analyses of portions of these divergent L1 elements (approximately 250 bp each) gave no evidence for concerted evolution having acted on the vole L1 elements since the split of the two vole lineages approximately 3.5 Mya; that is, the observed interspecific divergence (6.7%-24.7%) is not larger than the intraspecific divergence (7.9%-27.2%), and phylogenetic analyses showed no clustering into Arvicola and Microtus clades.      

Immunohistochemical localization of irisin in skin,eye, and thyroid and pineal glands of the crested porcupine (Hystrix cristata)

Irisin was first identified in muscle cells. We detected irisin immunoreactivity in various organs of the crested porcupine (Hystrix cristata). In the epidermis, irisin immunoreactivity was localized mainly in stratum basale, stratum spinosum and stratum granulosum layers; immunoreactivity was not observed in the stratum corneum. In the dermis, irisin was found in the external and internal root sheath, cortex and medulla of hair follicles, and in sebaceous glands. Irisin immunoreactivity was found in the neural retina and skeletal muscle fibers associated with the eye. The pineal and thyroid glands also exhibited irisin immunoreactivity.     

Accuracy of genomic selection for age at puberty in a multi‐breed population of tropically adapted beef cattle

Genomic selection is becoming a standard tool in livestock breeding programs, particularly for traits that are hard to measure. Accuracy of genomic selection can be improved by increasing the quantity and quality of data and potentially by improving analytical methods. Adding genotypes and phenotypes from additional breeds or crosses often improves the accuracy of genomic predictions but requires specific methodology. A model was developed to incorporate breed composition estimated from genotypes into genomic selection models. This method was applied to age at puberty data in female beef cattle (as estimated from age at first observation of a corpus luteum) from a mix of Brahman and Tropical Composite beef cattle. In this dataset, the new model incorporating breed composition did not increase the accuracy of genomic selection. However, the breeding values exhibited slightly less bias (as assessed by deviation of regression of phenotype on genomic breeding values from the expected value of 1). Adding additional Brahman animals to the Tropical Composite analysis increased the accuracy of genomic predictions and did not affect the accuracy of the Brahman predictions.     

Mdm12p, a Component Required for Mitochondrial Inheritance That Is Conserved between Budding and Fission Yeast

Saccharomyces cerevisiae cells lacking the MDM12 gene product display temperature-sensitive growth and possess abnormally large, round mitochondria that are defective for inheritance by daughter buds. Analysis of the wild-type MDM12 gene revealed its product to be a 31-kD polypeptide that is homologous to a protein of the fission yeast Schizosaccharomyces pombe. When expressed in S. cerevisiae, the S. pombe Mdm12p homolog conferred a dominant-negative phenotype of giant mitochondria and aberrant mitochondrial distribution, suggesting partial functional conservation of Mdm12p activity between budding and fission yeast. The S. cerevisiae Mdm12p was localized by indirect immunofluorescence microscopy and by subcellular fractionation and immunodetection to the mitochondrial outer membrane and displayed biochemical properties of an integral membrane protein. Mdm12p is the third mitochondrial outer membrane protein required for normal mitochondrial morphology and distribution to be identified in S. cerevisiae and the first such mitochondrial component that is conserved between two different species.