Active caspase-3 is removed from cells by release of caspase-3-enriched vesicles

AbstractCleavage of Rho associated Coiled Coil kinase I (ROCK I) by caspase-3 contributes to membrane blebbing. Whether caspase-3 and ROCK I also play a role in the release of membrane vesicles is unknown. Therefore, we transfected a human breast cancer cell line (MCF-7) that is caspase-3 deficient, lacks membrane blebbing, and does not release membrane vesicles, with caspase-3. Cells expressing caspase-3 demonstrate both ROCK I-mediated membrane blebbing, and release of small (400–600nm) membrane vesicles in a ROCK I-independent manner. These membrane vesicles contain caspase-3, and are enriched in caspase-3 activity compared to the releasing cells. Caspase-3-containing vesicles are taken up by untransfected cells but the cells do not show any sign of apoptosis. In conclusion, we show that the release of caspase-3-enriched membrane vesicles and membrane blebbing are two differentially regulated processes. Furthermore, we hypothesize that packaging of caspase-3 into membrane vesicles contributes to cellular homeostasis by the removal of caspase-3, and concurrently, protects the cells' environment from direct exposure to caspase-3 activity

Androgen receptor mutations

Male sexual differentiation and development proceed under direct control of androgens. Androgen action is mediated by the intracellular androgen receptor, which belongs to the superfamily of ligand-dependent transcription factors. At least three pathological situations are associated with abnormal androgen receptor structure and function: androgen insensitivity syndrome (AIS), spinal and bulbar muscular atrophy (SBMA) and prostate cancer. In the X-linked androgen insensitivity syndrome, defects in the androgen receptor gene have prevented the normal development of both internal and external male structures in 46,XY individuals. Complete or gross deletions of the androgen receptor gene have not been found frequently in persons with complete androgen insensitivity syndrome. Point mutations at several different sites in exons 2–8 encoding the DNA- and androgen-binding domain, have been reported for partial and complete forms of androgen insensitivity. A relatively high number of mutations were reported in two different clusters in exon 5 and in exon 7. The number of mutations in exon 1 is extremely low and no mutations have been reported in the hinge region, located between the DNA-binding domain and the ligand-binding domain and which is encoded by the first half of exon 4. Androgen receptor gene mutations in prostate cancer are very rare and are reported only in exons 4–8. The X-linked spinal and bulbar muscle atrophy (SBMA; Kennedy's disease) is associated with an expanded length (> 40 residues) of one of the polyglutamine stretches in the N-terminal domain of the androgen receptor

The androgen receptor: Functional structure and expression in transplanted human prostate tumors and prostate tumor cell lines

Abstract The growth of the majority of prostate tumors is androgen-dependent, for which the presence of a functional androgen receptor is a prerequisite. Tumor growth can be inhibited by blockade of androgen receptor action. However, this inhibition is transient. To study the role of the androgen receptor in androgen-dependent and androgen-independent prostate tumor cell growth, androgen receptor mRNA expression was monitored in six different human prostate tumor cell lines and tumors, which were grown either in vitro or by transplantation on (male) nude mice. Androgen receptor mRNA was clearly detectable in three androgen-dependent (sensitive) tumors and absent or low in three androgen-independent tumors. Growth of the LNCaP prostate tumor cell line can be stimulated both by androgens and by fetal calf serum. In the former situation androgen receptor mRNA expression is downregulated, whereas in the latter no effect on androgen receptor mRNA levels can be demonstrated. Sequence analysis showed that the androgen receptor gene from LNCaP cells contains a point mutation in the region encoding the steroid-binding domain, which confers an ACT coVon encoding a threonine residue to GCT, encoding alanine

Androgen receptor abnormalities

The human androgen receptor is a member of the superfamily of steroid hormone receptors. Proper functioning of this protein is a prerequisite for normal male sexual differentiation and development. The cloning of the human androgen receptor cDNA and the elucidation of the genomic organization of the corresponding gene has enabled us to study androgen receptors in subjects with the clinical manifestation of androgen insensitivity and in a human prostate carcinoma cell line (LNCaP). Using PCR amplification, subcloning and sequencing of exons 2–8, we identified a G → T mutation in the androgen receptor gene of a subject with the complete form of androgen insensitivity, which inactivates the splice donor site at the exon 4/intron 4 boundary. This mutation causes the inactivation of a cryptic splice donor site in exon 4, which results in the deletion of 41 amino acids from the steroid binding domain. In two other independently arising cases we identified two different nucleotide alterations in codon 686 (GAC; aspartic acid) located in exon 4. One mutation (G → C) results in an aspartic acid → histidine substitution (with negligible androgen binding), whereas the other mutation (G → A) leads to an aspartic acid → asparagine substitution (normal androgen binding, but a rapidly dissociating androgen receptor complex). Sequence analysis of the androgen receptor in human LNCaP-cells (lymph node carcinoma of the prostate) revealed a point mutation (A → G) in codon 868 in exon 8 resulting in the substitution of threonine by alanine. This mutation is the cause of the altered steroid binding specificity of the LNCaP-cell androgen receptor. The functional consequences of the observed mutations with respect to protein expression, specific ligand binding and transcriptional activation, were established after transient expression of the mutant receptors in COS and HeLa cells. These findings illustrate that functional error

Genetic Analyses in Small for Gestational Age Newborns

Context: Small for gestational age (SGA) can be a result of fetal growth restriction, associated with perinatal morbidity and mortality. Mechanisms that control prenatal growth are poorly understood. Objective: The aim of the present study was to gain more insight into prenatal growth failure and determine an effective diagnostic approach in SGA newborns. We hypothesized that one or more CNVs and disturbed methylation and sequence variants may be present in genes known to be associated with fetal growth. Design: A prospective cohort study of subjects with a low birthweight for gestational age. Setting: The study was conducted at an academic pediatric research institute. Patients: A total of 21 SGA newborns with a mean birthweight below the 1st centile and a control cohort of 24 appropriate for gestational age newborns were studied. Intervention: Array comparative genomic hybridization, genome-wide methylation studies and exome sequencing were performed. Main Outcome Measures The numbers of copy number variations, methylation disturbances and sequence variants. Results: The genetic analyses demonstrated three CNVs, one systematically disturbed methylation pattern and one sequence variant explaining the SGA. Additional methylation disturbances and sequence variants were present 20 patients. In 19 patients, multiple abnormalities were found. Conclusion: Our results confirm the influence of a large number of mechanisms explaining dysregulation of fetal growth. We conclude that copy number variations, methylation disturbances and sequence variants all contribute to prenatal growth failure. Such genetic workup can be an effective diagnostic approach in SGA newborns

Основные положения формирования нового единого сельскохозяйственного налога

Обосновывается введение единого сельскохозяйственного налога как постоянной ставки от стоимости валового дохода предприятий.Обгрунтовується введене єдиного сільськогосподарського податку як постійної ставки до вартості валового доходу підприємств.Introduction of the united agricultural tax is grounded as a permanent size to the cost of gross profit of enterprises