Deletion analysis of gene minE which encodes the topological specificity factor of cell division in Escherichia coli

The proton NMR spin lattice relaxation times of bound water (T1b) were studied on frozen rat liver samples during the precancerous step of cancer induction by diethylnitrosamine. Two components of the T1b were found, which show a different temperature dependence of their relative magnetization. Four different steps were identified during the experimental cancer induction according to 1H NMR findings, while only three were distinguished after histological examination. The results point to the influence of cross-relaxation and corroborate previous reports on differences observed between the heat capacity of normal and tumorous tissues.     

Deletion in the CMT1A locus on chromosome 17p11.2 in hereditary neuropathy with liability to pressure palsies

Hereditary neuropathy with liability to pressure palsies (NHPP) is an autosomal dominant disease of peripheral nerves, characterized by recurrent focal neuropathies often with an underlying asymptomatic polyneuropathy. We report the clinical, electrophysiological, and histopathological findings in three families with HNPP and confirm the presence of a deletion on chromosome 17p11.2, including all the markers known to be duplicated in Charcot-Marie-Tooth disease type 1A. This deletion appears to be the underlying molecular deficit in this disease and provides additional evidence for the importance of this locus for peripheral nerve function.     

Abnormalities in the p34cdc2-related PITSLRE protein kinase gene complex (CDC2L) on chromosome band 1p36 in melanoma

Recently, several clusters of hepatitis A have been observed among hemophiliacs linked to factor VIII concentrates treated for virus inactivation solely with the solvent/detergent (S/D) method, a procedure that does not affect nonenveloped viruses such as the hepatitis A virus (HAV). A new outbreak of hepatitis A in six hemophiliacs treated with the same lot of a factor VIII preparation occurred recently in Germany. The objective of the study was to clarify whether these diseases were caused by the administration of the S/D-treated plasma product, rather than a community-acquired infection. Polymerase chain reactions designed to detect HAV nucleic acid have been carried out in the implicated factor VIII lots, in the corresponding plasma pools, and in serum samples of four out of six infected individuals. The nucleic acid sequences were determined in samples that resulted in positive amplification products. HAV sequences were found in one of the two plasma pools used for manufacture of the incriminated product, in the incriminated lot itself, and in all recipient sera tested so far, although the latter were collected up to 7 weeks after the onset of jaundice. The sequences obtained were completely identical, revealing a unique HAV strain of genotype IA. This study provides conclusive evidence that hepatitis A can be transmitted by factor VIII concentrates treated solely by the S/D procedure for virus inactivation. This inactivation method is not effective against nonenveloped viruses. Since a number of hepatitis A transmission episodes have been described with such preparations during the past 10 years, their continued use seems to be questionable unless additional virus removal or inactivation steps are introduced to prevent the transmission of nonenveloped viruses. Molecular approaches again proved to be reliable tools for elucidating the chain of virus transmission.     

A modifying locus for familial adenomatous polyposis may be present on chromosome 1p35-p36

Sixteen healthy male dogs were used at random in this protocol. The dogs were anaesthetized with isoflurane in oxygen. Eight of the dogs received 0.25 mg/kg of butorphanol (group B) and the others an equal volume of isotonic saline (group S) administered by a catheter inserted in the lumbosacral epidural space. Butorphanol concentrations in plasma and cerebrospinal fluid (CSF) were measured using high-performance liquid chromatography with electrochemical detection. Maximum concentration of butorphanol and time to obtain this concentration were 42.28 ng/mL at 13.88 min in blood, and 18.03 ng/mL at 30 min in CSF. Volume of distribution, clearance, mean distribution and elimination half-lives were respectively 4.39 L/kg, 2.02 L/h.kg, 16.5 min and 189.1 min. Mean isoflurane minimal alveolar concentration values for group B obtained following hind- or forelimb stimulation decreased by 31% after epidural butorphanol. Cutaneous analgesia (to pin-prick test) persisted for 3 h after the end of isoflurane anaesthesia in group B and was in correlation with the plasmatic analgesic dose of butorphanol (9 ng/mL). These results suggested that analgesia was predominantly obtained by action of butorphanol on the supraspinal structures following its vascular systemic absorption.     

Deletion and down-regulation of mts1/p16 gene in human gastric cancer]

An adult male diver developed limb pains and peripheral neurologic signs following a series of dives. He was treated for acute neurologic decompression illness (DCI) and responded well, but subsequently he deteriorated and developed features of a progressive multisystem disease. Investigation confirmed a clinical diagnosis of polyarteritis nodosa complicated by a mononeuritis multiplex. Vasculitis is uncommon and its masquerade as neurologic DCI may be unique. However, this case empha-sizes the importance of careful clinical assessment and illustrates the potentially wide differential diagnosis of DCI.     

Concurrent disruption of cell cycle associated genes in mantle cell lymphoma: a genotypic and phenotypic study of cyclin D1, p16, p15, p53 and pRb

Mantle cell lymphomas (MCL) are morphologically and immunophenotypically distinctive lymphoid neoplasms characterised by overexpression of cyclin D1. Recent studies have suggested that co-operating aberrations of cell cycle associated genes may provide a growth advantage to a tumour. To address this issue further, we investigated five typical and three aggressive (blastoid) MCL for alterations in the cell cycle regulating genes p15, p16, CDK4, Rb and p53. In 3/3 aggressive cases with cyclin D1 overexpression we found aberration of at least one additional gene. One case showed diminished expression of the retinoblastoma protein (pRb); one case harboured deletion of both p15 and p16; and one case exhibited both deletion of p16 and point mutation of p53. However, we also identified two typical cases which in addition to cyclin D1 overexpression exhibited diminished pRb expression and p15 and p16 hypermethylation, respectively. Our findings confirm and extend other recent investigations and indicate that co-operating genetic alterations of cell cycle-associated genes may contribute to the pathogenesis of MCL.     

Activation of p21ras is not sufficient to ensure a complete G1 phase of the cell division cycle in mouse fibroblasts

In the mouse BP-A31 fibroblasts, mRNAs coding the three isoforms (Ha, Ki, N) of ras are expressed, and there are no activating mutations in the codons 12, 13 or 61. We have produced a subline (Ras2) expressing an oestrogen-inducible v-Ha-ras gene. The contribution of v-Ha-ras to the overall p21ras-GTP content was evaluated by metabolic labelling with 32P. Surprisingly, p21ras-GTP complexes were predominant in the serum-deprived BP-A31 cells as well as in the Ras2 cells. The excess of p21ras-GTP was not due to the lack of the GTPase activating protein. In transient transfection experiments, the serum response element (SRE)-directed CAT was expressed in serum-deprived BP-A31 cells, and insulin caused a further two- to threefold increase in CAT activity. A dominant negative ras mutant (Ha-Ras Asn-17) cancelled both the basal and insulin-induced CAT expression in the BP-A31 but not in the Ras2 cells. Expression of v-Ha-ras in Ras2 cells did not relax their growth factor-dependence and oestradiol had only a minor mitogenic effect. We conclude that p21ras activation does not ensure a complete cell division cycle in these cells, and does not entirely account for the transduction of the mitogenic signal initiated by insulin.     

Multiple breakpoints within the BCL-1 locus in B-cell lymphoma: rearrangements of the cyclin D1 gene

Centrocytic lymphoma (CC) and intermediately differentiated lymphocytic lymphoma (IDL) are B-cell non-Hodgkin's lymphomas composed of lymphocytes presumably derived from follicle mantle cells. In these lymphomas, a specific chromosomal translocation, t(11;14)(q13;q32), has been described. Previous studies suggested an association between t(11;14) chromosomal translocations and BCL-1 rearrangements. To evaluate the association between BCL-1 rearrangements and CC/IDL, Southern blot analysis was performed on a panel of 20 cases of CC/IDL, 22 cases of morphologically similar non-Hodgkin's lymphomas, 11 cases of chronic B-cell leukemias, and 2 cases of myelomas. We used various probes covering a considerable proportion of the 120-kilobase BCL-1 locus, and rearrangements in 50% of CC/IDL (10 of 20) were detected. In CC, all 4 breakpoints were located at the major translocation cluster (MTC). In contrast, in IDL, rearrangements were detected in 3 different cluster regions: 2 cases in the MTC, 2 cases with a breakpoint 24 kilobases outside the MTC, and 2 additional cases with breakpoints found 3 kilobases 5' of the first exon of the PRAD1/CCND1 gene, which is located 120 kilobases outside the MTC. In addition, one leukemia showed a breakpoint 63 kilobases outside the MTC. In all cases, there was comigration of the rearranged 11q13 fragment and the immunoglobulin heavy chain-joining gene complex, indicating a t(11;14)(q13;q32) chromosomal rearrangement. Our results show that Southern blot analysis is helpful to identify CC/IDL, but multiple breakpoints are present over a large region, and therefore, many probes are necessary to cover all breakpoints.     

ETR2 is an ETR1-like gene involved in ethylene signaling in Arabidopsis

The plant hormone ethylene regulates a variety of processes of growth and development. To identify components in the ethylene signal transduction pathway, we screened for ethylene-insensitive mutants in Arabidopsis thaliana and isolated a dominant etr2-1 mutant. The etr2-1 mutation confers ethylene insensitivity in several processes, including etiolated seedling elongation, leaf expansion, and leaf senescence. Double mutant analysis indicates that ETR2 acts upstream of CTR1, which codes for a Raf-related protein kinase. We cloned the ETR2 gene on the basis of its map position, and we found that it exhibits sequence homology to the ethylene receptor gene ETR1 and the ETR1-like ERS gene. ETR2 may thus encode a third ethylene receptor in Arabidopsis, transducing the hormonal signal through its "two-component" structure. Expression studies show that ETR2 is ubiquitously expressed and has a higher expression in some tissues, including inflorescence and floral meristems, petals, and ovules.     

A cdc2-like kinase associated with commitment to division in Paramecium tetraurelia

Cell division in higher eukaryotes is mainly controlled by p34cdc2 or related kinases and by other components of these kinase complexes. We present evidence that cdc2-like kinases also occur in Paramecium. Two polypeptides reacted with an antibody directed against the perfectly conserved PSTAIR region found in cdc2 kinases in other eukaryotes. Only the less abundant peptide bound to p13suc1 from Schizosaccharomyces pombe. Using centrifugal elutriation to select cells on the basis of size, we isolated highly synchronous Paramecium G1 cells. With this procedure, we demonstrated that the p13suc1-associated cdc2-like histone H1 kinase was activated before cell division at the point of commitment to division in Paramecium. Further, we show that Paramecium cdc2-like proteins occurred principally as monomers and that these monomers were active as histone H1 kinases in vitro.     

Therapy of highly malignant non-Hodgkin lymphoma with high-dose chemotherapy and stem cell transplantation

Patients with intermediate or high-grade non-Hodgkin's lymphoma are rarely cured of their disease after the failure of conventional therapy. High-dose chemotherapy followed by transplantation of either autologous marrow (ABMT) or peripheral stem cells (PBSCT) offers such patients a new possibility of cure. To patients younger than 60 years and without contraindications high-dose chemotherapy should be offered in case of relapse and bad prognosis. The PARMA-study demonstrated, that high-dose chemotherapy with subsequent autologous stem cell transplants is superior to conventional chemotherapy for relapsing patients. However high-dose chemotherapy in first complete or partial remission followed by ABMT and PBSCT as post-remission therapy for adult NHL has yielded only similar results randomized to those achieved with conventional chemotherapy in several prospectively studies. This approach should be limited to patients with bad prognostic factors (high-intermediate or high-risk groups according to the International Prognostic Factor Project). The success of ABMT and PBSCT in treating these diseases mandates exploration of the best high-dose chemotherapy, the use of autologous or allogenic bone marrow resp. peripheral stem cell for haematopoietic reconstitution and technical aspects such as purging of tumor cells, the short- and long-term transplant-related mortality continues to be a major concern.     

Role of polo kinase and Mid1p in determining the site of cell division in fission yeast

The fission yeast Schizosaccharomyces pombe divides symmetrically using a medial F-actin- based contractile ring to produce equal-sized daughter cells. Mutants defective in two previously described genes, mid1 and pom1, frequently divide asymmetrically. Here we present the identification of three new temperature-sensitive mutants defective in localization of the division plane. All three mutants have mutations in the polo kinase gene, plo1, and show defects very similar to those of mid1 mutants in both the placement and organization of the medial ring. In both cases, ring formation is frequently initiated near the cell poles, indicating that Mid1p and Plo1p function in recruiting medial ring components to the cell center. It has been reported previously that during mitosis Mid1p becomes hyperphosphorylated and relocates from the nucleus to a medial ring. Here we show that Mid1p first forms a diffuse cortical band during spindle formation and then coalesces into a ring before anaphase. Plo1p is required for Mid1p to exit the nucleus and form a ring, and Pom1p is required for proper placement of the Mid1p ring. Upon overexpression of Plo1p, Mid1p exits the nucleus prematurely and displays a reduced mobility on gels similar to that of the hyperphosphorylated form observed previously in mitotic cells. Genetic and two-hybrid analyses suggest that Plo1p and Mid1p act in a common pathway distinct from that involving Pom1p. Plo1p localizes to the spindle pole bodies and spindles of mitotic cells and also to the medial ring at the time of its formation. Taken together, the data indicate that Plo1p plays a role in the positioning of division sites by regulating Mid1p. Given its previously known functions in mitosis and the timing of cytokinesis, Plo1p is thus implicated as a key molecule in the spatial and temporal coordination of cytokinesis with mitosis.     

High-intensity Raf signal causes cell cycle arrest mediated by p21Cip1

Activated Raf has been linked to such opposing cellular responses as the induction of DNA synthesis and the inhibition of proliferation. However, it remains unclear how such a switch in signal specificity is regulated. We have addressed this question with a regulatable Raf-androgen receptor fusion protein in murine fibroblasts. We show that Raf can cause a G1-specific cell cycle arrest through induction of p21Cip1. This in turn leads to inhibition of cyclin D- and cyclin E-dependent kinases and an accumulation of hypophosphorylated Rb. Importantly, this behavior can be observed only in response to a strong Raf signal. In contrast, moderate Raf activity induces DNA synthesis and is sufficient to induce cyclin D expression. Therefore, Raf signal specificity can be determined by modulation of signal strength presumably through the induction of distinct protein expression patterns. Similar to induction of Raf, a strong induction of activated Ras via a tetracycline-dependent promoter also causes inhibition of proliferation and p21Cip1 induction at high expression levels. Thus, p21Cip1 plays a key role in determining cellular responses to Ras and Raf signalling. As predicted by this finding we show that Ras and loss of p21 cooperate to confer a proliferative advantage to mouse embryo fibroblasts.     

Genetic control of the cell division cycle in the fission yeast Schizosaccharomyces pombe

Differentiated cells have been established in monolayer culture from adult rat liver and their ultrastructural and biochemical features characterized after 20-30 generations. Hepatocytes were isolated by enzyme perfusion of the liver followed by treatment with papain, which allowed cultures to be established more readily and to be cloned at an early stage. Ultrastructural studies indicated that the cells were derived largely from hepatic parenchymal cells. The cells showed structural modifications during primary culture but were stable thereafter. The cultured cells retained some differentiated functions unique to liver cells, including the synthesis of ornithine form arginine and the secretion of serum proteins, albumin, chi- and beta-globulins.     

bcl-2 inhibits wild-type p53-triggered apoptosis but not G1 cell cycle arrest and transactivation of WAF1 and bax

We have earlier shown that wild-type (wt) p53 expressed from a temperature-sensitive construct (ts p53) triggers apoptosis in the v-myc retrovirus-induced, p53-negative T-cell lymphoma line J3D (Y. Wang et al., Cell Growth & Differ., 4: 467-473, 1993). We also found that constitutive bcl-2 expression inhibits wt p53-triggered apoptosis in these cells (Y. Wang et al., Oncogene, 8: 3427-3431, 1993). Here we demonstrate that more than 90% of the ts p53-transfected J3D cells were arrested in G1 at 18 h after induction of wt p53 expression by temperature shift to 32 degrees C. At this time, at least 80% of the cells remained viable. After 30 h at 32 degrees C, around 50% of the cells had died by apoptosis, while most of the remaining cells were still alive in G1, indicating that p53-induced apoptosis occurred following G1 arrest. The G1 cell cycle arrest at 18 h after temperature shift to 32 degrees C was reversible, as shown by the fact that the cells readily resumed exponential growth following temperature shift back to 37 degrees C, although viability dropped from around 80 to 65%. Expression of both WAF1 and bax mRNA was induced by wt p53 in both the ts p53 and ts p53/bcl-2 transfected cells. The kinetics of G1 cell cycle arrest at 32 degrees C was similar in both the ts p53 and the ts p53/bcl-2 double transfectants.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other human cancers

We describe a gene encoding p73, a protein that shares considerable homology with the tumor suppressor p53. p73 maps to 1p36, a region frequently deleted in neuroblastoma and other tumors and thought to contain multiple tumor suppressor genes. Our analysis of neuroblastoma cell lines with 1p and p73 loss of heterozygosity failed to detect coding sequence mutations in remaining p73 alleles. However, the demonstration that p73 is monoallelically expressed supports the notion that it is a candidate gene in neuroblastoma. p73 also has the potential to activate p53 target genes and to interact with p53. We propose that the disregulation of p73 contributes to tumorigenesis and that p53-related proteins operate in a network of developmental and cell cycle controls.     

Combination gene delivery of the cell cycle inhibitor p27 with thymidine kinase enhances prodrug cytotoxicity

Cytoxicity induced by the herpesvirus thymidine kinase (TK) gene in combination with prodrugs is dependent on cell growth and leads to the elimination of genetically modified cells, thus limiting the duration of expression and efficacy of this treatment in vivo. Here, an effort was made to enhance TK/prodrug efficacy by coexpression of a cyclin-dependent kinase inhibitor (CKI), p27, to render cells resistant to TK/prodrug by inhibiting DNA synthesis. Expression of p27 by transfection substantially reduced cell cycle progression, and its activity was enhanced by mutations designed to stabilize the protein. Coexpression of p27 and TK or a p27/TK fusion protein led to greater prodrug cytotoxicity than that produced by TK alone in the Renca cell line, which is sensitive to bystander killing. Combination gene transfer of this CKI with TK therefore sustained the synthesis of TK by genetically modified cells to enhance the susceptibility of bystander cells to prodrug cytotoxicity and increased the efficacy of this gene transfer approach.