Malignant transformation of NIH3T3 cells by overexpression of mot-2 protein

The murine mortalin genes, mot-1 and mot-2, are members of the hsp70 family of proteins and differ from each other by only two amino acid residues. Mot-1 is expressed in normal cells and has pancytosolic cellular distribution whereas mot-2 is found in the perinuclear region of immortal cells. We report here that a high level of expression of mot-2 protein resulted in malignant transformation of cells as analysed by anchorage independent growth and nude mice assays. A high level of protein expression is attributed to the 900 bp 3' untranslated region of the cDNA which does not have any transforming activity per se. Mortalin cDNA clones isolated from human transformed cells were also found to have transforming activity in similar assays and a high level of expression was apparent in some of the human immortalized cells that showed non-pancytosolic mortalin immunofluorescence. Taken together, the data suggest that nonpancytosolic mortalin may have a role in tumorigenesis.     

Caspases cleave focal adhesion kinase during apoptosis to generate a FRNK-like polypeptide

Focal adhesion kinase (Fak) is a non-receptor protein-tyrosine kinase that stimulates cell spreading and motility by promoting the formation of contact sites between the cell and the extracellular matrix (focal adhesions). It suppresses apoptosis by transducing survival signals that emanate from focal adhesions via the clustering of transmembrane integrins by components of the extracellular matrix. We demonstrate that Fak is cleaved by caspases at two distinct sites during apoptosis. The sites were mapped to DQTD772, which was preferentially cleaved by caspase-3, and VSWD704, which was preferentially cleaved by caspase-6 and cytotoxic T lymphocyte-derived granzyme B. The cleavage of Fak during apoptosis separates the tyrosine kinase domain from the focal adhesion targeting (FAT) domain. The carboxyl-terminal fragments that are generated suppress phosphorylation of endogenous Fak and thus resemble a natural variant of Fak, FRNK, that inhibits Fak activity by preventing the localization of Fak to focal adhesions. The cleavage of Fak by caspases may thus play an important role in the execution of the suicide program by disabling the anti-apoptotic function of Fak. Interestingly, rodent Fak lacks an optimal caspase-3 consensus cleavage site although it is cleaved in murine cells undergoing apoptosis at an upstream site. This appears to be the first example of a caspase substrate where the cleavage sites are not conserved between species.     

Age-dependent inaccuracy of asthma death certification in Northern England, 1991-1992

OBJECTIVE: To test the usefulness of a commercial DNA hybridization assay for the detection of high-risk (HR) human papillomavirus (HPV) types in archival cervical smears and to compare the sensitivity with that of polymerase chain reaction (PCR) using consensus primers. STUDY DESIGN: Stained material was scraped from archival slides and the pellet volume noted. DNA was extracted using silica/guanidinium isothiocyanate and the quality checked by amplification of the beta-globin gene. HR-HPV DNA was detected using a commercial hybrid capture assay (HCA) and the results compared with an in-house amplification system with consensus primers. RESULTS: Of 156 archival smears stored for 12-13 years, 20 were positive by HCA using an HR probe cocktail. Ninety-eight were also tested by PCR, and 35 were positive. The percentage of HPV-positive samples increased with the increasing size of the pellet. HR-HCA detected more positives in samples with high grade squamous intraepithelial lesion (moderate/severe dyskaryosis). CONCLUSION: Both hybridization by HCA and amplification by PCR could be used to detect genital HPV in archival smears. The general primers PCR detected more positives than HR-HCA but included HPV 6/11. While variation in sample size and prolonged storage may reduce the quality of DNA, the use of archival material for longitudinal studies of HPV presence is potentially worthwhile.     

Replicable unconscious semantic priming

In 4 experiments, subjects classified visually presented target words as pleasant-unpleasant words or male-female first names. Prime words were similar (congruent) or dissimilar (incongruent) in meaning to targets. Brief duration of prime words (17, 33, or 50 ms), along with pre- and postmasking, prevented most subjects from perceiving their physical and semantic properties. By constraining response latencies to fall within a response window--a narrow time band that occurred earlier than subjects would ordinarily respond--these experiments consistently produced subliminal priming effects, indicated by greater error rates for incongruent than congruent priming trials. This conclusion was confirmed by analyzing magnitude of priming as a regression function of prime perceptibility using the method of A. G. Greenwald, M. R. Klinger, and E. S. Schuh (1995). The data of each experiment passed their significant-intercept criterion for demonstrating unconscious cognition.     

Hepatocyte growth factor-stimulated renal tubular mitogenesis: effects on expression of c-myc, c-fos, c-met, VEGF and the VHL tumour-suppressor and related genes

Hepatocyte growth factor (HGF/SF) is a potent renal proximal tubular cell (PTEC) mitogen involved in renal development. HGF/SF is the functional ligand for the c-met proto-oncogene, and germline c-met mutations are associated with familial papillary renal cell carcinoma. Somatic von Hippel-Lindau disease tumour-suppressor gene (VHL) mutations are frequently detected in sporadic clear cell renal cell carcinomas (RCC), and germline VHL mutations are the commonest cause of familial clear cell RCC. pVHL binds to the positive regulatory components of the trimeric elongin (SIII) complex (elongins B and C) and has been observed to deregulate expression of the vascular endothelial growth factor (VEGF) gene. HGF/SF has similarly been reported to up-regulate expression of the VEGF gene in non-renal experimental systems. To investigate the mechanism of HGF/SF action in PTECs and, specifically, to examine potential interactions between the HGF/c-met and the VHL-mediated pathways for renal tubular growth control, we have isolated untransformed PTECs from normal kidneys, developed conditions for their culture in vitro and used these cells to investigate changes in mRNA levels of the VHL, elongin A, B and C, VEGF, c-myc, c-fos and c-met genes after HGF/SF exposure. Significant elevations in the mRNA levels of VEGF, c-myc, c-fos, c-met and elongins A, B and C, but not VHL, were detected after HGF/SF stimulation of human PTECs (P < 0.02), with a consistent order of peak levels observed over successive replicates (c-fos at 1 h, VEGF at 2-4 h, c-myc, at 4 h, followed by c-met and all three elongin subunits at 8 h). This study highlights the spectrum of changes in gene expression observed in PTECs after HGF/SF stimulation and has identified possible candidate mediators of the HGF/SF-induced mitogenic response. Our evidence would suggest that the changes in PTEC VEGF expression induced by HGF/SF are mediated by a VHL-independent pathway.     

Porcine spleen deoxyribonuclease II. Covalent structure, cDNA sequence, molecular cloning, and gene expression

Porcine spleen DNase II, a lysosomal acid hydrolase, is a noncovalently linked alpha.beta heterodimer (Liao, T.-H. (1985) J. Biol. Chem. 260, 10708-10713). The alpha subunit, after disulfide cleavage, yields two chains, alpha1 and alpha2. The complete amino acid sequences of the alpha1, beta, and alpha2 chains were elucidated by protein sequencing, and the pairings of one interchain disulfide between alpha1 and alpha2 and of three intrachain disulfides in alpha2 were assigned. Six carbohydrate attachment sites, two in beta and four in alpha2, were detected by sugar analyses. The cDNA of DNase II was amplified using primers synthesized on the basis of the amino acid sequences determined. The amplified fragments shown to be a cDNA sequence of 1,292 bases. This cDNA sequence has an open reading frame encoding a 364-amino acid polypeptide containing a putative transmembrane peptide at the NH2-end, two small connecting peptides in the middle, and a peptide at the COOH terminus. These are evidently removed to form mature DNase II. Thus, all three chains in the sequence alpha1, beta, and alpha2 are coded by the same cDNA. When Chinese hamster ovary cells were transfected with a cloned plasmid with an inserted cDNA fragment encoding the entire reading frame, the expressed protein was released into the growth medium as an active form of DNase II.     

Inhibition of platelet function by A02131-1, a novel inhibitor of cGMP-specific phosphodiesterase, in vitro and in vivo

The effect of A02131-1 [3-(5'-hydroxymethyl-2'-furyl)-1-benzyl thieno (3,2-c)pyrazole], a cGMP-specific phosphodiesterase (PDE) inhibitor, on platelet function was investigated. The compound was found to inhibit the aggregation of and adenosine triphosphate (ATP) release from human platelet-rich plasma and washed platelets that were induced by aggregation inducing drugs such as arachidonic acid (AA), collagen, U46619, platelet-activating factor (PAF), adenosine diphosphate (ADP) and A23187, and the inhibitory effect was concentration-dependent. A02131-1 also disaggregated the performed platelet aggregates induced by these inducers. Thromboxane B2 (TXB2) formations caused by collagen, PAF, ADP, and A23187 were inhibited by A02131-1 at concentrations that did not affect the AA-induced formation of TXB2 and prostaglandin D2 (PGD2). A02131-1 suppressed both the generation of inositol 1,4,5-triphosphate (IP3) and the increase of intracellular Ca2+ concentration stimulated by these aggregation inducers. A02131-1 was shown to increase the cAMP and cGMP levels in platelets and the extent was found to be dependent on concentration as well as time. A02131-1 increased the cAMP level much more slowly than the cGMP level. Activities of adenylate cyclase, guanylate cyclase, and PDEs (type I and III) were not altered by A02131-1. However, the activity of cGMP-specific PDE (type V) was inhibited by A02131-1. The antiplatelet aggregation activity and the effect on raising cAMP level of A02131-1 were both potentiated by prostaglandin E1 (PGE1). In the mouse tail bleeding test, A02131-1 was clearly shown to be more effective than dipyridamole in prolonging the tail bleeding time of conscious mice. These data indicate that A02131-1 is a cGMP-specific PDE (type V) inhibitor in human platelets.     

Clear cell papulosis of the skin

Clear cell papulosis is a new entity first described in 1987. To date, six patients have been reported: all were young Taiwanese children. The disease is characterized clinically by multiple small, whitish maculopapules distributed along the milk line and by the presence of large, benign pagetoid cells in the epidermis resembling the clear cell of the nipple. The significance of this entity lies in its potential histogenetic link with Paget's disease of the skin. We report four new Taiwanese patients, three girls and one boy, aged between 21 months and 4 years. Two were sisters. Small hypopigmented macules first appeared on the pubis. They were eventually distributed bilaterally along the milk line but were most numerous in the public area. The disease may easily be overlooked when the macules are tiny or few in number and thus display no clear milk-line distribution, or when they occur in white-skinned individuals. Histologically, solitary large clear cells with large, round pale nuclei were detected in the basal layer of the hypomelaninized epidermis. The numbers of clear cells varied on haematoxylin and eosin staining and were only small in two patients. The cytoplasm of the clear cells was decorated by antikeratin AE1 and anticarcinoembryonic antigen antibodies. AE1 was the best marker of the clear cell. Some of the AE1-positive cells were tadpole-like in shape and were situated well above the basal layer. Ultrastructurally, large clumps of disintegrated or vacuolated mucin granules were present in the cytoplasm of the clear cells. The melanocytes appeared normal; the suprabasal keratinocytes were essentially devoid of melanosomes. The pathological findings in the present study support the hypothesis that these clear cells are an aberrant derivative of sweat gland cells in the epidermis and are potentially the precursor cells giving rise to mammary and extramammary Paget's disease. The differential diagnosis includes chicken pox scars, idiopathic guttate hypomelanosis, hypomelanotic tinea versicolor, anetoderma and early, hypopigmented lesions of Paget's disease.