Redistribution of cytosolic FGFR1 after induced migration of urothelial cells in culture

The distribution of cytosolic fibroblast growth factor receptor 1 (FGFR1) was studied in correlation to cell migration in urothelial cell line g/G. Cell motility was analysed with a new method using consecutive series of photographs of cells relocated on CELLocate coverslips and with image analysis software. The results confirmed that FGF1 stimulated cell motility only when cells were grown on collagen I coating. During the transition from sessile to motile cell phenotype a complete redistribution of cytosolic FGFR1 was revealed. In sessile cells, FGFR1 had a filamentous distribution and its location matched cytokeratin 7. In cells of the migrating phenotype, the distribution of FGFR1 was diffuse, mainly located in cytosol. Our data reveal that the location of cytosolic FGFR1 depends on the motile characteristics of the cell. The results also indicate that attachment of cells to collagen I is crucial for the induction of urothelial cell motility with FGF1.     

Association of TNF promoter polymorphisms with type 1 diabetes in the South Croatian population

Type 1 diabetes mellitus (TIDM) is an autoimmune disease characterized by the destruction of pancreatic p cells. Tumor necrosis factor (TNF) is a pleotropic cytokine with potent immunomodulatory and inflammatory activity. Association studies of TNF polymorphisms and type 1 diabetes (TIDM) frequently demonstrated TNF involvement with TIDM. Although TNF may play an important role in the pathogenesis of TIDM, the genetic association of TNF región with the disease has not been conclusive because of the strong linkage disequilibrium with HLA genes. In this study, we examined two TNF promoter variants (rs 1800629 at position -308, and rs361525 at position -238) for TIDM association in 233 patients and 144 controls from the population of South Croatia. A higher frequency of TNF -308 A alíele and also, a more frequent specific -308A -238G haplotype in TIDM patients were observed with a limited significance. However, we did not find strong evidence of association of TNF promoter polymorphisms with TIDM. In order to elucidate the trae contribution of TNF to TIDM susceptibility in our population, more comprehensive studies with HLA adjustment in a larger sample are required.     

Cytochemical localization of carbohydrates in intercalated duct and acinar cells of mouse parotid gland

Summary The glycoconjugate composition of mouse intercalated duct and acinar cells of parotid gland has been compared. Mucins containing 1,2-glycols were demonstrated by the tannic acid-uranyl acetate technique. Hexose residues of glycoconjugates were identified using ferritin conjugated withCanavalia ensiformis agglutinin (Con A),Triticum vulgare or wheat germ agglutinin (WGA),Ricinus communis I agglutinin (RCA-I),Phaseolus vulgaris agglutinin (PHA-E) andArachis hypogaea agglutinin (PNA). Whereas qualitative and quantitative differences were observed in sugar residues of secretory granules in intercalated duct and acinar cells, apical plasmalemmae were labelled sparsely and similarly. This indicates that the glycocalyx composition of apical plasma minae in the parotid acinar and intercalated duct cells is little influenced by secretory granule composition.     

De novo DNA synthesis by human DNA polymerase lambda, DNA polymerase mu and terminal deoxyribonucleotidyl transferase

DNA polymerases (pols) catalyse the synthesis of DNA. This reaction requires a primer-template DNA in order to grow from the 3'OH end of the primer along the template. On the other hand terminal deoxyribonucleotidyl transferase (TdT) catalyses the addition of nucleotides at the 3'OH end of a DNA strand, without the need of a template. Pol lambda and pol micro are ubiquitous enzymes, possess both DNA polymerase and terminal deoxyribonucleotidyl transferase activities and belong to pol X family, together with pol beta and TdT. Here we show that pol lambda, pol micro and TdT, all possess the ability to synthesise in vitro short fragments of DNA in the absence of a primer-template or even a primer or a template in the reaction. The DNA synthesised de novo by pol lambda, pol micro and TdT appears to have an unusual structure. Furthermore we found that the amino acid Phe506 of pol lambda is essential for the de novo synthesis. This novel catalytic activity might be related to the proposed functions of these three pol X family members in DNA repair and DNA recombination.     

Human DNA polymerase lambda functionally and physically interacts with proliferating cell nuclear antigen in normal and translesion DNA synthesis

Proliferating cell nuclear antigen (PCNA) has been shown to interact with a variety of DNA polymerases (pol) such as pol delta, pol epsilon, pol iota, pol kappa, pol eta, and pol beta. Here we show that PCNA directly interacts with the newly discovered pol lambda cloned from human cells. This interaction stabilizes the binding of pol lambda to the primer template, thus increasing its affinity for the hydroxyl primer and its processivity in DNA synthesis. However, no effect of PCNA was detected on the rate of nucleotide incorporation or discrimination efficiency by pol lambda. PCNA was found to stimulate efficient synthesis by pol lambda across an abasic (AP) site. When compared with pol delta, human pol lambda showed the ability to incorporate a nucleotide in front of the lesion. Addition of PCNA led to efficient elongation past the AP site by pol lambda but not by pol delta. However, when tested on a template containing a bulky DNA lesion, such as the major cisplatin Pt-d(GpG) adduct, PCNA could not allow translesion synthesis by pol lambda. Our results suggest that the complex between PCNA and pol lambda may play an important role in the bypass of abasic sites in human cells.     

Differentiation of epithelial cells in the urinary tract

Uroplakins, cytokeratins and the apical plasma membrane were studied in the epithelia of mouse urinary tract. In the simple epithelium covering the inner medulla of the renal pelvis, no uroplakins or cytokeratin 20 were detected and cells had microvilli on their apical surface. The epithelium covering the inner band of the outer medulla became pseudostratified, with the upper layer consisting of large cells with stalks connecting them to the basal lamina. Uroplakins and cytokeratin 20 were not expressed in these cells. However, some superficial cells appeared without connections to the basal lamina; these cells expressed uroplakins Ia, Ib, II and III and cytokeratin 20, they contained sparse small uroplakin-positive cytoplasmic vesicles and their apical surface showed both microvilli and ridges. Cytokeratin 20 was seen as dots in the cytoplasm. This epithelium therefore showed partial urothelial differentiation. The epithelium covering the outer band of the outer medulla gradually changed from a two-layered to a three-layered urothelium with typical umbrella cells that contained all four uroplakins. Cytokeratin 20 was organized into a complex network. The epithelium possessed an asymmetric unit membrane at the apical cell surface and fusiform vesicles. Umbrella cells were also observed in the ureter and urinary bladder. In males and females, the urothelium ended in the bladder neck and was continued by a non-keratinized stratified epithelium in the urethra in which no urothelial cell differentiation markers were detected. We thus show here the expression, distribution and organization of specific proteins associated with the various cell types in the urinary tract epithelium.W. Mello Jr. thanks FAPESP, São Paulo, Brazil for financial support.     

Analysis of scores in diagnosis of acute appendicitis in women

Acute appendicitis is a common surgical condition that requires prompt diagnosis. Besides modern imaging techniques, scoring systems, based on clinical signs and symptoms and routine laboratory assessments, have been used as a diagnostic aid. However, differences in sensitivities and specificities were observed if the scores were applied to various populations and clinical settings. The purpose of this paper is to assess validity of three scores (modified Alvarado score, Ohmann score and Eskelinen score) for diagnosis of acute appendicitis in women. 126 female patients admitted for suspicion of acute appendicitis in a tertiary hospital emergency department were analyzed prospectively. Modified Alvarado score, Ohmann score and Eskelinen score were calculated at admission and compared to final diagnosis. All patients with modified Alvarado score 7 or more had acute appendicitis (100% specificity) and it can be used to determine the need for immediate appendectomy. Values of Ohmann score greater than 6 resulted in 0.9% rate of overlooked appendicitis. Besides obvious educational role, scores may help to determine the group of patients who require immediate appendectomy, therefore expediting treatment and avoid unnecessary observation or more lengthy diagnostic procedures that require highly educated and skilled senior staff: No single score may be used alone to dictate or decline surgery. Different cut-off points may also be considered for different subpopulations.     

High-Pitch CT Pulmonary Angiography in Third Generation Dual-Source CT: Image Quality in an Unselected Patient Population

Objectives

To investigate the feasibility of high-pitch CT pulmonary angiography (CTPA) in 3^rd generation dual-source CT (DSCT) in unselected patients.

Methods

Forty-seven patients with suspected pulmonary embolism underwent high-pitch CTPA on a 3^rd generation dual-source CT scanner. CT dose index (CTDI_vol) and dose length product (DLP) were obtained. Objective image quality was analyzed by calculating signal-to-noise-ratio (SNR) and contrast-to-noise ratio (CNR). Subjective image quality on the central, lobar, segmental and subsegmental level was rated by two experienced radiologists.

Results

Median CTDI was 8.1 mGy and median DLP was 274 mGy*cm. Median SNR was 32.9 in the central and 31.9 in the segmental pulmonary arteries. CNR was 29.2 in the central and 28.2 in the segmental pulmonary arteries. Median image quality was “excellent” in central and lobar arteries and “good” in subsegmental arteries according to both readers. Segmental arteries varied between “excellent” and “good”. Image quality was non-diagnostic in one case (2%), beginning in the lobar arteries. Thirteen patients (28%) showed minor motion artifacts.

Conclusions

In third-generation dual-source CT, high-pitch CTPA is feasible for unselected patients. It yields excellent image quality with minimal motion artifacts. However, compared to standard-pitch cohorts, no distinct decrease in radiation dose was observed.     

Strategic role of the ubiquitin-dependent segregase p97 (VCP or Cdc48) in DNA replication

Genome amplification (DNA synthesis) is one of the most demanding cellular processes in all proliferative cells. The DNA replication machinery (also known as the replisome) orchestrates genome amplification during S-phase of the cell cycle. Genetic material is particularly vulnerable to various events that can challenge the replisome during its assembly, activation (firing), progression (elongation) and disassembly from chromatin (termination). Any disturbance of the replisome leads to stalling of the DNA replication fork and firing of dormant replication origins, a process known as DNA replication stress. DNA replication stress is considered to be one of the main causes of sporadic cancers and other pathologies related to tissue degeneration and ageing. The mechanisms of replisome assembly and elongation during DNA synthesis are well understood. However, once DNA synthesis is complete, the process of replisome disassembly, and its removal from chromatin, remains unclear. In recent years, a growing body of evidence has alluded to a central role in replisome regulation for the ubiquitin-dependent protein segregase p97, also known as valosin-containing protein (VCP) in metazoans and Cdc48 in lower eukaryotes. By orchestrating the spatiotemporal turnover of the replisome, p97 plays an essential role in DNA replication. In this review, we will summarise our current knowledge about how p97 controls the replisome from replication initiation, to elongation and finally termination. We will also further examine the more recent findings concerning the role of p97 and how mutations in p97 cofactors, also known as adaptors, cause DNA replication stress induced genomic instability that leads to cancer and accelerated ageing. To our knowledge, this is the first comprehensive review concerning the mechanisms involved in the regulation of DNA replication by p97.     

A unified model for the G1/S cell cycle transition

Efficient S phase entry is essential for development, tissue repair, and immune defences. However, hyperactive or expedited S phase entry causes replication stress, DNA damage and oncogenesis, highlighting the need for strict regulation. Recent paradigm shifts and conflicting reports demonstrate the requirement for a discussion of the G1/S transition literature. Here, we review the recent studies, and propose a unified model for the S phase entry decision. In this model, competition between mitogen and DNA damage signalling over the course of the mother cell cycle constitutes the predominant control mechanism for S phase entry of daughter cells. Mitogens and DNA damage have distinct sensing periods, giving rise to three Commitment Points for S phase entry (CP1-3). S phase entry is mitogen-independent in the daughter G1 phase, but remains sensitive to DNA damage, such as single strand breaks, the most frequently-occurring lesions that uniquely threaten DNA replication. To control CP1-3, dedicated hubs integrate the antagonistic mitogenic and DNA damage signals, regulating the stoichiometric cyclin: CDK inhibitor ratio for ultrasensitive control of CDK4/6 and CDK2. This unified model for the G1/S cell cycle transition combines the findings of decades of study, and provides an updated foundation for cell cycle research.