Identification and cloning of a mobile transposon fromAspergillus niger var.awamori

Aspergillus niger var.awamori contains multiple copies of a transposable element, Vader. This element was detected as a 437-bp insertion in four independently isolated spontaneous mutants of theniaD (nitrate reductase) gene. The Vader element is present in approximately 15 copies in bothA. niger var.awamori andA. niger. A single copy of Vader was detected from only one of the two laboratory strains ofA. nidulans which were also examined. Insertion of the Vader element into theniaD gene ofA. niger var.awamori caused a 2-bp duplication (TA) of the target sequence. The Vader element is flanked by a 44-bp inverted repeat. The genetic stabilities of the inserted Vader elements atniaD were examined by studying reversion frequencies resulting in colonies able to grow on nitrate as a sole nitrogen source. MutantsniaD392 andniaD436 reverted at a frequency of 9x10^-3 and 4x10^-2, respectively. Two of the mutants,niaD587 andniaD410, reverted at a lower frequency of 6x10^-4.     

Tacrolimus and mycophenolate mofetil after nonmyeloablative matched-sibling donor allogeneic stem-cell transplantations conditioned with fludarabine and low-dose total body irradiation.

We evaluated tacrolimus/mycophenolate mofetil (MMF) for graft-versus-host disease (GVHD) prophylaxis after a nonmyeloablative stem cell transplantation (NST) from a matched sibling donor (MSD). Thirty-two patients (median age, 57 years) with advanced hematologic malignancies, who were poor candidates for a conventional myeloablative transplantation, received fludarabine (30 mg/m(2), day -4 to day -2), total-body irradiation (TBI) (200 cGy, day 0), infusion of donor peripheral blood progenitor cells (day 0), oral tacrolimus 0.06 mg/kg twice daily (from day 3), and oral MMF at 15 mg/kg twice daily (days 0-+27). Tacrolimus was tapered from day +100 to day +180 in those patients with indolent malignancies (n = 25), and from day +35 to day +56 in those with aggressive tumors (n = 7). Regimen toxicities and myelosuppression were mild, allowing 75% of patients to have entirely outpatient transplantations. One patient (3%) experienced a nonfatal graft rejection. Rates of grades II-IV and III-IV acute GVHD were 15.6% and 3%, respectively. Acute GVHD was diagnosed at median day +78 (range, days +31-+84). Extensive chronic GVHD was observed in 10 of 24 evaluable patients (41.6%) at a median onset of day +198 (range, days +128-+277), either spontaneously (n = 5) or elicited after tumor progression (n = 5). Five patients experienced transplantation-related mortality (TRM) (15.6%) from either acute GVHD-related multiorgan failure (MOF) (n = 3) or infectious complications (n = 2). At median follow-up of 19 months (range, 2-41 months), the overall survival, progression-free survival, and disease-free survival rates are 62.5%, 50%, and 40%, respectively. In conclusion, the use of tacrolimus/MMF after MSD NST is associated with encouraging rates of GVHD control.     

CD3 antigen-mediated calcium signals and protein kinase C activation are higher in CD45R0+ than in CD45RA+ human T lymphocyte subsets

T lymphocytes may be separated into subsets according to their expression of CD45 isoforms. The CD45R0⁺ T cell subset has been reported to proliferate in response to recall antigen and to mitogenic mAb to a much greater extent than the CD45RA⁺ subset. This difference could be due to more efficient coupling of the T cell antigen receptor complex to mitogenic signaling pathways. To investigate this possibility, CD3 antigen-induced calcium signals, diacylglycerol (DAG) production and protein kinase C (PKC) activation levels were compared in CD45RA⁺ and CD45R0⁺ human T lymphocyte subsets derived from peripheral blood. The mean CD3-induced rise in intracellular calcium was 80% greater in CD45R0⁺ than in CD45RA⁺ cells. Basal DAG levels in CD45R0⁺ cells were found to be, on average, 60% higher than in CD45RA⁺ cells (p = 0.002), but the CD3-induced production of DAG over background was not different in the two subsets (p = 0.4). Basal PKC activity, and CD3-induced PKC activation levels over background, were found to be 50% and 140% higher, respectively, in CD45R0⁺ cells than in CD45RA⁺ cells (p = 0.015 and 0.023). The CD45R0⁺ subset contained a higher proportion of cells expressing activation markers, such as CD25, CD71 and major histocompatibility complex class II, when compared to the CD45RA⁺ subset. Our results suggest that the elevated basal DAG levels observed in the CD45R0⁺ subset may reflect the recent activation of these cells. Both the higher basal DAG and CD3-induced elevation in intracellular calcium observed in the CD45R0⁺ cells may contribute to the greater PKC activation signals triggered by CD3 mAb in this subset. These findings elucidate the greater response of CD45R0⁺ T cells to mitogenic stimuli compared to CD45RA⁺ cells.     

Melatonin secretion in the Mashona mole-rat, Cryptomys darlingi--influence of light on rhythmicity

The hormone melatonin is synthesised and secreted from the pineal gland in darkness and triggers the daily and seasonal timing of various physiological and behavioural processes. The Mashona mole-rat, Cryptomys darlingi, lives in subterranean burrows that are completely sealed and is therefore rarely, if ever, exposed to light under natural conditions. Hence, this species is of particular interest for studies on rhythms of melatonin secretion. We investigated how plasma melatonin concentrations of the Mashona mole-rat responded to exposure to a long-term standard photoperiod of 12 h light, 12 h dark (12:12 LD), constant light (LL) and constant dark (DD). In addition, we examined whether plasma melatonin concentration was coupled to locomotor activity. Mashona mole-rats displayed rhythms of plasma melatonin concentration that appeared entrained to the standard LD photoperiod, suggesting that the mole-rat is capable of perceiving and entraining to this photic zeitgeber. Furthermore, under chronic constant lighting conditions (DD, LL), circadian rhythms in plasma melatonin concentration were observed, suggesting the possible existence of an endogenous rhythm. Light suppressed melatonin secretion, but constant light did not abolish the rhythm of plasma melatonin concentration. Between active and non-active animals, no difference in plasma melatonin concentration was found for any of the sequential photoperiods (LD1 DD, LD2, LL), tentatively suggesting that the rhythm of melatonin secretion is uncoupled from that of locomotor activity.     

Characterization of the hamster FancG/Xrcc9 gene and mutations in CHO UV40 and NM3

The human FANCG/XRCC9 gene, which is defective in Fanconi anemia complementation group G (FA-G) cells, was first cloned by genetic complementation of the mitomycin C (MMC) sensitivity of CHO mutant UV40. The CHO NM3 mutant was subsequently assigned to the same complementation group. The parental AA8 CHO cells are hemizygous at the FancG locus, and we identified frameshift mutations that result in N-terminal truncations of the protein in both UV40 and NM3. Hypersensitivity to DNA cross-linking agents, such as MMC, typically characterizes FA cells. By introducing the native CHO FancG gene into mutant NM3, we demonstrate that hamster FancG fully corrects the 3-fold sensitivity to methyl methanesulfonate (MMS) as well as the 10-fold sensitivity to MMC, whereas resistance to ionizing radiation did not increase appreciably. In contrast, hamster cDNA transformants showed incomplete correction for both MMC and MMS sensitivity. The constitutively expressed FancG protein is present in the cytoplasmic, nuclear and chromatin fractions. FancG protein levels and subcellular localization do not change appreciably as a function of cell cycle position. Our results are consistent with roles of FancG in both the nuclear and cytoplasmic compartments to maintain genomic stability in response to various genotoxic agents.     

The NAD(P)H:quinone oxidoreductase I C609T polymorphism modifies the risk of Barrett esophagus and esophageal adenocarcinoma.

PURPOSE: The role of genetic susceptibility to esophageal adenocarcinoma and its precursor lesion Barrett esophagus has not been fully elucidated. This study investigated the effect of polymorphisms in the manganese superoxide dismutase (MnSOD) and NAD(P)H:quinone oxidoreductase 1 (NQO1) genes in modulating the risk of developing Barrett esophagus or esophageal adenocarcinoma. METHODS: A total of 584 patients (146 esophagitis, 200 Barrett esophagus, 144 esophageal adenocarcinoma, and 94 controls) were genotyped for the MnSOD C14T and NQO1 C609T polymorphisms using polymerase chain reaction and restriction fragment length polymorphism analysis. RESULTS: The NQO1 TT genotype was less common in Barrett esophagus (2.0%) and esophageal adenocarcinoma (1.4%) patients, compared with both esophagitis patients (7.6%) and controls (5.4%). After adjustment for sex, age, body mass index, reflux symptoms, and smoking status, patients with the homozygous TT genotype had a 4.5-fold decreased risk of developing Barrett esophagus (odds ratio = 0.22, 95% confidence interval = 0.07-0.76, P = 0.01) and a 6.2-fold decreased risk of esophageal adenocarcinoma (odds ratio = 0.16, 95% confidence intervals = 0.03-0.94, P = 0.04) compared with individuals with the TC and CC genotypes. No significant differences between groups were observed for the MnSOD polymorphism (P = 0.289). CONCLUSIONS: Overall, the results of this study suggest that the NQO1 TT genotype may offer protection from reflux complications such as Barrett esophagus and esophageal adenocarcinoma.     

Sequence Analysis Demonstrates that VP6, NSP1 and NSP4 Genes of Indian Neonatal Rotavirus Strain 116E are of Human Origin

We have sequenced the genes encoding the inner capsid protein VP6 and the nonstructural proteins NSP1 and NSP4 of the Indian neonatal serotype P8[11]G9 human/bovine reassortant candidate vaccine rotavirus strain 116E. These three genes share a high degree of sequence and deduced amino acid homology with human prototype strain Wa. Our results confirm and extend those of previous RNA-RNA hybridization studies which suggested that these genes are of human origin, and will facilitate examination of the host immune response to 116E induced by natural infection and vaccination. This revised version was published online in July 2006 with corrections to the Cover Date.     

Inhibition of TCR-mediated shedding of L-selectin (CD62L) on human and mouse CD4+ T cells by metalloproteinase inhibition: analysis of the regulation of Th1/Th2 function

The generation of a productive primary immune response is dependent on the ability of na?ve T lymphocytes to recirculate through peripheral lymph organs to encounter specific antigen. The process of na?ve CD4(+) T cell entry into lymph nodes correlates with cell surface expression of L-selectin (CD62L), which mediates early tethering and rolling events to endothelium prior to entry. Here, we demonstrate that surface expression of CD62L enhances CD4(+) T cell activation in vitro. The synthetic hydroxamate metalloproteinase inhibitor (BB-3103), specifically inhibits activation-induced shedding of CD62L from CD4(+) T cells by TCR cross-linking and lowers proliferation in part by reducing rapid tyrosine phosphorylation of zeta-associated protein 70 kDa (ZAP-70) and by increasing cytosolic free Ca(2+) concentration mobilization. BB-3103 also inhibited the proliferative response of both murine CD4(+) Th1 and Th2 subsets in vitro but the inhibitory effects were sustained only in Th2-type cells. Similarly, BB-3103 mediated prolonged inhibition of allergen-dependent peripheral T cell proliferation in atopic dermatitis patients but not in healthy controls. Analysis of CD62L expression on murine CD4(+) T cell subsets revealed that surface expression was maintained on Th1 cells but not Th2 cells. The differential effects of BB-3103 on primed effector CD4(+) T cells may provide new insights into generating therapeutic agents capable of redressing the Th2/Th1 imbalance in allergic diseases.     

The application of operative ultrasound immediately following carotid endarterectomy

One hundred and seventy-five carotid bifurcations have been scanned using intraoperative ultrasonic imaging. The technique is simple, reproducible, rapid, and reliable. Intimal flaps, clamp stenoses, residual plaques, and platelet accumulations were detected by this method. Most technical defects occurred in the external carotid artery (12%), and it is recommended that significant lesions remaining in this vessel should be removed. Defects in the internal carotid artery and common carotid artery were much less common (7%) and most of these were quite small, not requiring reopening. In fact, only 2% required reopening. In 1% of patients there were defects detected that led to stroke, which would have been removed on present criteria. Although there were other causes of stroke, technical error remains an immediately reversible source of postoperative stroke that can be avoided by the use of operative ultrasonic imaging. Long-term minor defects, as followed by noninvasive tests, rarely became significant and bore no relationship to the development of restenosis.

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Resumen Ciento setenta y cinco bifurcaciones carotídeas han sido estudiadas intraoperatoriamente al cierre de la arteriotomía para endarterectomía y una vez que los clamps y asas habían sido retirados y el flujo restaurado, mediante la imagenología de ultrasonido. La técnica es simple, reproducible, rápida, y confiable. Pliegues (flaps) de la íntima, estenosis por clamps, placas residuales, y acumulaciones plaquetarias fueron detectadas por este método. La mayoría de los defectos de técnica operatoria se presentaron en la carótida externa (12%) y por ello se recomienda que lesiones de significación que queden presentes en este vaso deben ser removidas. Los defectos en la arteria carótida interna y en la carótida primitiva fueron mucho menos frecuentes (7%) y su mayoría fueron mínimos y no requirieron reapertura del vaso. En 1% de los pacientes se presentaron defectos que dieron lugar a accidente cerebral los cuales, bajo criterios actuales, habrían sido removidos. Aunque hubo otras causas de accidente cerebral, los errores técnicos siguen siendo una fuente reversible inmediata de accidente cerebral postoperatorio que puede ser evitado con el uso de imagenología de ultrasonido intraoperatoria. Los defectos menores estudiados a largo plazo por medios no invasivos muy rara vez probaron ser de significación y no aparecieron relacionados con el desarrollo de reestenosis.

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Résumé Cent soixante-quinze fourches carotidiennes ont été soumises à une échographie per-opératoire. La méthode d'exploration est simple, aisée à répéter et fiable. Les lambeaux d'intima, les sténoses secondaires au clampage, les plaques laissées en place, les accumulations de plaquettes peuvent être détectées par cette exploration. La majorité des impairs opératoires s'observent au niveau de la carotide externe (12% des cas) et imposent la correction des lésions patentes. Les malfaçons au niveau de la carotide interne et de la carotide primitive sont moins fréquentes (7% des cas). Les lésions sont souvent peu importantes, leur correction ne s'imposant que dans 2% des cas. Dans 1% des cas, ces lésions artérielles opératoires aboutissent à une lésion cérébrale qui aurait pu être évitée. En effet, bien que d'autres causes de lésion cérébrale existent, les altérations imputables à l'acte chirurgical sont réversibles dès lors qu'elles sont mises en évidence par l'échographie opératoire. Les lésions mineures persistantes qui sont décelées par les tests non-invasifs ne sont que rarement à l'origine d'une sténose artérielle post-opératoire.