The CDKN2a common variants: 148 Ala/Thr and 500 C/G in 3' UTR, and their association with clinical course of melanoma

Changes in CDKN2a gene are known to be linked with sporadic melanoma and hereditary predisposition to this cancer. In the Polish population mutations in the coding region of the CDKN2a gene are rather rare, therefore the attention has been focused on polymorphisms and alterations in uncoding regions such as 3' UTR. The aim of this study was to analyze two common polymorphisms, Ala148Thr and 500 C/G, and correlate them with the clinical course of melanoma. DNA from 285 patients was analyzed and found polymorphisms were correlated with the clinical parameters employing statistical methods. The obtained results allow us to conclude: (i) survival times of 500 C/G carriers vs. cumulating proportion surviving was not statistically significant; (ii) CDKN2a polymorphism 500 C/G correlated with Ala148Thr; (iii) no correlation was observed between the 500 C/G polymorphism and age of diagnosis, localization of primary melanoma and survival time; (iv) we did not find correlation between 500 C/G and type of cancer in the family; (v) changes in the CDKN2a gene were not found in patients with second cancer.     

Endogenous peptides from biophysical and biochemical fractionation of serum analyzed by matrix-assisted laser desorption/ionization and electrospray ionization hybrid quadrupole time-of-flight

Blood peptides can be concentrated, extracted, and analyzed with strong signal-to-noise ratios by precipitation in organic solvents followed by extraction in water. Matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) hybrid quadrupole time-of-flight (Qq-TOF) were used to analyze the precipitated and extracted endogenous peptides from fetal calf serum. C18 solid-phase extraction with or without prior precipitation in ammonium sulfate, size exclusion chromatography, dealbuminization, dye affinity chromatography, ultrafiltration, and differential precipitation in organic solvents were compared. Hundreds of different ions could be observed by MALDI in the various fractions. It appeared that some peptides were freely dissolved and that not all peptides in blood were obliged to remain bound to albumin or other high-molecular-mass proteins. Mass spectra with high signal-to-noise ratios were obtained from polypeptides precipitated with organic solvents followed by extraction of the peptides from the pellet with water. The peptides extracted from organic precipitates were analyzed by nano liquid chromatography (LC)-ESI-Qq-TOF. In addition to many commonly abundant serum proteins, apparent low-abundance peptides associated with cancer biology from proteins such as insulin-like growth factor II, thymosin beta4 and beta9, plasminogen, coagulation factors, and extracellular matrix protein 1 were observed.     

Expression of cell survival/death genes: Bcl-2 and Bax at the rate of colon cancer prognosis

The rate of tumour growth is dependent on the balance between proliferation and apoptosis at all stages of carcinogenesis. Apoptosis inhibition, in turn, depends partly on the balance between expression of two cell death regulatory genes, Bcl-2 and Bax. Colon cancer has long been associated with disturbances in apoptosis regulation. The aim of our study was to determine the expression levels of Bcl-2 and Bax mRNAs in 1 microg sample of total RNA obtained from normal colon and colon adenocarcinoma. This study was intended to evaluate possible differences in Bcl-2 and Bax mRNA levels at particular stages of colon adenocarcinoma classified according to Duke's system. The apoptotic frequency (represented by Bax mRNA copy number) was inversely proportional to the decrease of Bcl-2 gene expression. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) was performed to confirm apoptosis.     

Intronic CA-repeat and CA-rich elements: a new class of regulators of mammalian alternative splicing

We have recently identified an intronic polymorphic CA-repeat region in the human endothelial nitric oxide synthase (eNOS) gene as an important determinant of the splicing efficiency, requiring specific binding of hnRNP L. Here, we analyzed the position requirements of this CA-repeat element, which revealed its potential role in alternative splicing. In addition, we defined the RNA binding specificity of hnRNP L by SELEX: not only regular CA repeats are recognized with high affinity but also certain CA-rich clusters. Therefore, we have systematically searched the human genome databases for CA-repeat and CA-rich elements associated with alternative 5' splice sites (5'ss), followed by minigene transfection assays. Surprisingly, in several specific human genes that we tested, intronic CA RNA elements could function either as splicing enhancers or silencers, depending on their proximity to the alternative 5'ss. HnRNP L was detected specifically bound to these diverse CA elements. These data demonstrated that intronic CA sequences constitute novel and widespread regulatory elements of alternative splicing.     

Fish track wastewater pollution to estuaries

Excess nitrogen is a forceful agent of ecological change in coastal waters, and wastewater is a prominent source of nitrogen. In catchments where multiple sources of nitrogen pollution co-exist, biological indicators are needed to gauge the degree to which wastewater-N can propagate through the receiving food webs. The purpose of this study was to test whether estuarine fish are suitable as indicators of sewage-N pollution. Fish were analysed from three estuaries within a 100-km strip on the Australian East Coast. The estuaries differ substantially in wastewater loading: (1) the Maroochy Estuary receives a large fraction of the local shire’s treated sewage, (2) the Mooloolah Estuary has no licensed treated wastewater outfalls but marinas/harbours and stormwater may contribute nitrogen, and (3) the Noosa Estuary which neither receives licensed discharges nor has suspected wastewater loads. Sampling for fish included both high rainfall (‘wet’ season) and low rainfall (‘dry’ season) periods. Muscle-δ¹⁵N was the variable predicted to respond to treated wastewater loading, reflecting the relative enrichment in ¹⁵N resulting from the treatment process and distinguishing it from alternative N sources such as fertiliser and natural nitrogen inputs (both ¹⁵N-depleted). Of the 19 fish species occurring in all three estuaries, those from the Maroochy Estuary had significantly elevated δ¹⁵N values (up to 9.9‰), and inter-estuarine differences in fish-δ¹⁵N were consistent across seasons. Furthermore, not only did all fish from the estuary receiving treated wastewater carry a very distinctive sewage-N tissue signal, but enriched muscle-δ¹⁵N was also evident in all species sampled from the one estuary in which sewage contamination was previously only suspected (i.e. the Mooloolah Estuary: 0.2–4.8‰ enrichment over fish from reference system). Thus, fish-δ¹⁵N is a suitable indicator of wastewater-N not only in systems that receive large loads, but also for the detection of more subtle nitrogen inputs. Arguably, fish may be preferred indicators of sewage-N contamination because they: (1) integrate nitrogen inputs over long time periods, (2) have an element of ‘ecological relevance’ because fish muscle-δ¹⁵N reflect movement of sewage-N through the food chain, and (3) pollution assessments can usually be based on evidence from multiple species.     

Novel, flexible, and conformationally defined analogs of gepirone: synthesis and 5-HT1A, 5-HT2A, and D2 receptor activity

Novel, flexible arylpiperazine gepirone analogs (1a-3a) with a mixed 5-HT1A/5-HT2A receptor profile, low D2 receptor affinity, and agonistic (2a) or partial agonistic (1a, 3a) activity toward 5-HT1A receptor sites were synthesized. Their conformationally restricted counterparts (1b-3b) were selective 5-HT1A ligands (over 5-HT2A and D2 receptors), which turned out to be agonists (2b, 3b), or partial agonist (1b) of 5-HT1A receptors.     

Do “glanduliferous” larvae of Galerucinae (Coleoptera: Chrysomelidae) possess defensive glands? A scanning electron microscopic study

Larvae of the chrysomelid taxon Galerucinae, Sermylini, are known to release a fluid from dorsolateral segmental openings when disturbed. The release of this fluid resembles the discharge of secretion from well-studied exocrine defensive glands in larvae of Chrysomelinae, the putative sister group of Galerucinae. Thus, Sermylini larvae have been named “glanduliferous” ones. However, no comparative analyses of the internal structures of the segmental openings in Sermylini larvae have been available prior to this study. Therefore, segmental larval openings in 10 Sermylini species were investigated by scanning and light microscopy. Two types of segmental openings were detected: (1) the opening is visible as an integumental slit which can be opened and closed by muscles. No specific glandular structures are associated with this slit. (2) The opening is covered internally by a cuticular sac which can be everted to the outside. A cuticular duct of a gland is leading into this sac. Haemolymph is discharged from both types of openings. These structures are discussed with respect to reflex bleeding in other taxa. Furthermore, a common origin of the segmental openings in larvae of Sermylini and the segmental exocrine glands in chrysomeline larvae is critically questioned.     

The novel protein PTPIP51 exhibits tissue- and cell-specific expression

The expression patterns of both mRNA and protein of the novel protein tyrosine phosphatase interacting protein 51 (PTPIP51) were studied in various organs by in situ hybridization, immunoblotting, and immunocytochemistry. The protein was found in all mammalian species investigated: guinea pig, rat, mouse, pig, and human. The presence of the protein was, however, restricted to specific organs. High levels of PTPIP51 were found in epidermis and seminiferous epithelium. The expression appears to be associated with distinct stages of differentiation. While basal cells in the epidermis and spermatogonia showed no perceptible amount of PTPIP51, keratinocytes of suprabasal layers and differentiating first-order spermatocytes up to spermatids exhibited high expression. In skeletal muscle, the presence of PTPIP51 was restricted to fibers of the fast twitch type. In surface epithelia containing ciliated cells, the protein was associated with the microtubular structures responsible for ciliary movement. Furthermore, specific structures of the central nervous system, for example, neurons of the hippocampal region, ganglion cells of the autonomic nervous system, and axons of the peripheral nervous system showed a distinct staining pattern with the antibody to PTPIP51. Our data suggest that PTPIP51 might be involved in the regulation of cellular processes associated with differentiation, movement, or cytoskeletal organization.Tobias Kajosch died on August 9th 2004     

Sources of infection in childhood tuberculosis in a region of east Croatia

Possible sources of tuberculosis (TBC) infection in children have been assessed in a retrospective epidemiological study covering a north-east region of Croatia in which the incidence of childhood tuberculosis has been increasing since the war in 1991-1995. During the past decade (1993-2003), 271 children up to 18 years of age have been referred for hospital care because of known contacts with tuberculosis (142 children, group A) or because of indicative clinical signs and symptoms (129 children, group B). Possible sources of infection were identified on the basis of medical documentation and field investigations. Frequencies of source identification for different age groups were compared. In group A, the exposure took place most often within the family (parents, grandparents, siblings, 129 of 142 children, 90.87%). Relatives, neighbors, friends and schoolmates accounted for 9.2%. In group B, possible sources of infection were identified for 44 of 129 children (34.1%) and were within the family for 16 of those 44 (36.4%). Evidenced contact with tuberculosis was more usual among younger children (0-9 years of age, 65.5%) in group A than among the older ones (10-18 years of age, 34.5%). In group B, contacts with tuberculosis were equally distributed (50.0%) among younger and older children. High proportion of unrecognized contacts in children having clinical signs and symptoms indicative of tuberculosis (group B, 85 of 129, 65.9%) opens the possibility that extra-familial exposure to tuberculosis occurs more often than expected regardless of the age of children.