Discriminatory Role of Detergent-Resistant Membranes in the Dimerization and Endocytosis of Prostate-Specific Membrane Antigen

Prostate-specific membrane antigen (PSMA) is a type-II membrane glycoprotein that was initially identified in LNCaP cells. It is expressed at elevated levels in prostate cancer. In view of the correlation between the expression levels of PSMA and disease grade and stage, PSMA is considered to be one of the most promising biomarkers in the diagnosis and treatment of prostate cancer. In LNCaP cells PSMA undergoes internalization via clathrin-coated pits followed by accumulation in the endosomes. PSMA associates with different types of detergent-resistant membranes (DRMs) along the secretory pathway. Its mature form is mainly insoluble in Lubrol WX, but does not associate with Triton X-100-DRMs. To understand the mechanism of PSMA internalization we investigated its association during internalization with DRMs. For this purpose, internalization was induced by antibody cross-linking. We demonstrate at the biochemical and cell biological levels that: [i] exclusively homodimers of PSMA are associated with Lubrol WX-DRMs, [ii] antibody-induced cross-linking of PSMA molecules results in a time-dependent partitioning into another DRMs type, namely Triton X-100-DRMs, and [iii] concomitant with its association with Triton-X-100-DRMs internalization of PSMA occurs along tubulin filaments. In a previous work (Colombatti et al. (2009) PLoS One 4: e4608) we demonstrated that the small GTPases RAS and RAC1 and the MAPKs p38 and ERK1/2 are activated during antibody cross-linking. As downstream effects of this activation we observed a strong induction of NF-kB associated with an increased expression of IL-6 and CCL5 genes and that IL-6 and CCL5 enhanced the proliferative potential of LNCaP cells synergistically. These observations together with findings reported here hypothesize a fundamental role of DRMs during activation of PSMA as platforms for trafficking, endocytosis and signalling. Understanding these mechanisms constitutes an essential prerequisite for utilization of PSMA as a therapeutically suitable target in prostate cancer.     

Longitudinal Metabolomic Profiling of Amino Acids and Lipids across Healthy Pregnancy

Pregnancy is characterized by a complexity of metabolic processes that may impact fetal development and ultimately, infant health outcomes. However, our understanding of whole body maternal and fetal metabolism during this critical life stage remains incomplete. The objective of this study is to utilize metabolomics to profile longitudinal patterns of fasting maternal metabolites among a cohort of non-diabetic, healthy pregnant women in order to advance our understanding of changes in protein and lipid concentrations across gestation, the biochemical pathways by which they are metabolized and to describe variation in maternal metabolites between ethnic groups. Among 160 pregnant women, amino acids, tricarboxylic acid (TCA) cycle intermediates, keto-bodies and non-esterified fatty acids were detected by liquid chromatography coupled with mass spectrometry, while polar lipids were detected through flow-injected mass spectrometry. The maternal plasma concentration of several essential and non-essential amino acids, long-chain polyunsaturated fatty acids, free carnitine, acetylcarnitine, phosphatidylcholines and sphingomyelins significantly decreased across pregnancy. Concentrations of several TCA intermediates increase as pregnancy progresses, as well as the keto-body β-hydroxybutyrate. Ratios of specific acylcarnitines used as indicators of metabolic pathways suggest a decreased beta-oxidation rate and increased carnitine palmitoyltransferase-1 enzyme activity with advancing gestation. Decreasing amino acid concentrations likely reflects placental uptake and tissue biosynthesis. The absence of any increase in plasma non-esterified fatty acids is unexpected in the catabolic phase of later pregnancy and may reflect enhanced placental fatty acid uptake and utilization for fetal tissue growth. While it appears that energy production through the TCA cycle increases as pregnancy progresses, decreasing patterns of free carnitine and acetylcarnitine as well as increased carnitine palmitoyltransferase-1 rate and β-hydroxybutyrate levels suggest a concomitant upregulation of ketogenesis to ensure sufficient energy supply in the fasting state. Several differences in metabolomic profiles between Hispanic and non-Hispanic women demonstrate phenotypic variations in prenatal metabolism which should be considered in future studies.     

Fragile×expression and×inactivation

Summary The inactive fragile×chromosomes of a 47,fra(X),fra(X),Y male with a typical fragile×phenotype were successfully separated from the active homologues by means of somatic cell hybridization. It was shown by FUdR-induction and caffein-posttreatment that the separated inactive×chromosomes expressed their fragile sites and that the presence of an active mutated \sxchromosome was not a prerequisite for fragile X expression. The fragility seems to be an intrinsic property of the individual fragile site. This result is in favour of the classical concept that the fragile site at Xq27.3 has a primary pathogenetic function in this syndrome, although the fragility itself could represent a secondary phenomenon related to an unknown alteration of the DNA in this chromosome region. It is also concluded that inactivation of the fragile\sxchromosome in females is not responsible for either false negative fragile\sxfindings or the observation of fragile\sxnegative colonies isolated from fragile\sxpositive fibroblasts in heterozygotes.     

Heterozygous expression of X-linked chondrodysplasia punctata

Summary Two females showing partial expression of X-linked chondrodysplasia punctata were identified in a family. Bone dysplasia was caused by an aberrant X chromosome that had an inverse duplication of the segment Xp21.2–Xp22.2 and a deletion of Xp22.3-Xpter. To characterise the aberrant X chromosome, dosage blots were performed on genomic DNA from a carrier using a number of X-linked probes. Anonymous sequences from Xp21.2–Xp22.2 to which probes D2, 99.61, C7, pERT87-15, and 754 bind were duplicated on the aberrant X chromosome. The proposita was heterozygous for all these markers. Dosage blots also showed that the loci for steroid sulfatase and the cell surface antigen 12E7 (MIC2) were deleted as expected from the cytogenetic results. Mouse human cell hybrids were constructed that retained the normal X in the active state. Analysis of these hybrid clones for the markers from Xp21.2–Xp22.2 revealed that all the alleles of the informative markers, present in a single dosage in the genomic DNA, were carried on the normal X chromosome of the proposita. The duplicated X chromosome therefore had two identical alleles, indicating that the aberration resulted from an intrachromosomal rearrangement.     

The spectrum of CFTR mutations in south-west German cystic fibrosis patients

The cystic fibrosis transmembrane conductance regulator (CFTR) gene of 110 cystic fibrosis (CF) patients from the south-west of Germany was screened for 12 different mutations. This analysis resulted in an identification of 79% of all CF mutations and a complete genotype in 66% of the families. The most common mutation found was F508 (67%). Another 5 mutations accounted for a further 12.5% (4% G542X; 3% R553X; 3% N1303K; 2% 1717-1 GA; 0.5% G551D) whereas 6 mutations (R117H, A455E, I507, S549I, S549N, and R1162X) were not found. Fifty-four (49%) patients were AF508 homozygotes and 18 (16.5%) were compound heterozygotes for F508 and one of the rarer mutations. These frequencies differ slightly from those found in the north of Germany and considerably from those reported from the south of Europe, which seems to be consistent with a north to south decline of the relative abundance of F508. Two patients, age 6 and 25 years, were compound heterozygotes for G542X and N1303K. The clinical features of the 6 year old were characterised by severe gastrointestinal and as yet only mild pulmonary complications whereas the 25 year old manifested severe pulmonary and gastrointestinal symptoms indicating that the N1303K mutation of the C-terminal CFTR nucleotide binding fold significantly impairs protein function in both the pancreas and the lungs.     

Cytogenetic study of F1 hybrids betweenCrepis dinarica andCrepis froelichiana (Asteraceae)

Crepis dinarica andC. froelichiana are two closely related species of theC. praemorsa complex. Even though they exhibit the same chromosome number (2n = 8) and similar idiogram shape, they differ widely in quantity and distribution of heterochromatin bands. The hybrids between these two species comprise three morphological types. Parental genomes were distinguished in hybrids by Giemsa differential staining (C-banding). Although meiosis presents only a few abnormalities (about 2.4%), the percentage of aborted pollen grains is very high (90%).     

Oxygen transport and peripheral microcirculation in long-term diabetes

The purpose of this investigation was to evaluate the impact of long-term diabetes on muscle blood flow (MBF) and oxygen transport (vO2) during exercise. Twelve male patients (58 +/- 8 years, mean +/- SD), with at least a 10-year history of diabetes controlled by insulin, and seven age-matched controls (56 +/- 5 years, mean +/- SD) participated in this study. No patient had been clinically diagnosed as having peripheral vascular disease, and on the average resting ankle/arm systolic blood pressure ratios were normal. Following a baseline period, 5 min of cycle ergometer exercises at 75 W were performed in the upright position and, after 1-hr recovery, in the supine position. Continuous vO2 was determined via breath-by-breath analysis. MBF was measured in the vastus lateralis (VL) and tibialis anterior (TA) by 133Xe clearance. In the erect position, the diabetic group (compared with the control group, respectively) exhibited significantly (P less than 0.05) lower exercise MBF [ml. (100 g.min)-1] in both VL (19 +/- 2.5 vs 30.9 +/- 2) and TA (13.7 +/- 2 vs 22.0 +/- 4), a lower steady-state VO2 (1.3 +/- 0.3 vs 1.7 +/- 0.2 liters.min-1) during exercise including the values in the last 15 sec of exercise, and greater accumulation of blood lactate (35 +/- 2 vs 22.0 +/- 2 mg/100 ml). The same trends in the data were observed during supine exercise; however, the blood pressure of the diabetics was significantly elevated during exercise when compared with that of controls. The reduced exercise MBF in the TA and VL demonstrated that impaired microvascular flow, without clinically overt peripheral vascular disease, in long-term diabetics leads to reduced oxygen delivery and exercise tolerance.     

DNA recovery and direct detection of Tn5 sequences from soil

Specific Tn5 sequences inserted in the genome of Enterobacter agglomerans were detected in EcoRI digested DNA directly recovered from soil 70 d after its inoculation with the bacteria, when these were no longer culturable on agar medium. A new method of DNA extraction from soil was used. No amplification of DNA sequences by PCR was needed.