The completion of the Mammalian Gene Collection (MGC).

Since its start, the Mammalian Gene Collection (MGC) has sought to provide at least one full-protein-coding sequence cDNA clone for every human and mouse gene with a RefSeq transcript, and at least 6200 rat genes. The MGC cloning effort initially relied on random expressed sequence tag screening of cDNA libraries. Here, we summarize our recent progress using directed RT-PCR cloning and DNA synthesis. The MGC now contains clones with the entire protein-coding sequence for 92% of human and 89% of mouse genes with curated RefSeq (NM-accession) transcripts, and for 97% of human and 96% of mouse genes with curated RefSeq transcripts that have one or more PubMed publications, in addition to clones for more than 6300 rat genes. These high-quality MGC clones and their sequences are accessible without restriction to researchers worldwide.

Nitric oxide upregulates expression of DNA-PKcs to protect cells from DNA-damaging anti-tumour agents.

Nitric-oxide synthase (NOS) activity has been detected in many human tumours, although its function is unclear. Here we show that exposure of cells to nitric oxide (NO) results in a 4-5-fold increase in expression of the DNA-dependent protein-kinase catalytic subunit (DNA-PKcs), one of the key enzymes involved in repairing double-stranded DNA breaks. This NO-mediated increase in enzymatically active DNA-PK not only protects cells from the toxic effects of NO, but also provides crossprotection against clinically important DNA-damaging agents, such as X-ray radiation, adriamycin, bleomycin and cisplatin. The NO-mediated increase in DNA-PKcs described here demonstrates the presence of a new and highly effective NO-mediated mechanism for DNA repair.

Microsomal CTP:choline phosphate cytidylyltransferase: kinetic mechanism of fatty acid stimulation.

Fatty acids are known to cause an increase in the incorporation of radioactive choline into phosphatidylcholine. A coincident increase in membrane cytidylyltransferase activity is well documented. The purpose of the present studies was to determine the direct effects of oleic acid on the kinetic properties of membrane cytidylyltransferase. An examination of the reaction characteristics of membrane cytidylyltransferase revealed that membranes from adult rat lung contained high CTPase activity. This activity prevented the determination of reaction velocities at low CTP concentrations. The CTPase activity was blocked by the addition of ADP or ATP to the reaction. The addition of 6.0 mM ADP to the assay mixture enabled us to determine the effect of oleate on the CTP Km. Oleate (122 microM) caused a significant decrease in CTP Km for microsomal cytidylyltransferase (0.99 mM to 0.33 mM) and H-Form cytidylyltransferase (1.04 mM to 0.27 mM). Oleate did not decrease the CTP Km for L-Form cytidylyltransferase. Oleate had no effect on the choline phosphate Km in microsomal, H-Form or L-Form cytidylyltransferase. Oleate also increased the Vmax for cytidylyltransferase. The increase was dependent upon the concentration of oleate with a maximal increase of 50-60% at 100-130 microM oleate. We conclude that oleate has a direct stimulatory effect on cytidylyltransferase when it is in the active form (membrane bound or H-Form lipoprotein complex). We suggest that the kinetic effects operate synergistically with other regulatory mechanisms such as translocation or conversion of inactive to active species. The direct effect of oleate on the cytidylyltransferase may be an important regulatory mechanism when CTP concentrations are limiting.

A case of Sweet syndrome associated with human granulocytic anaplasmosis.

BACKGROUND: Acute febrile neutrophilic dermatosis, or Sweet syndrome (SS), is a condition that is presumed to be triggered by infectious disease agents. We report a case of SS associated with human granulocytic anaplasmosis (HGA), which is of interest because Anaplasma phagocytophilum infects, multiplies in, and disrupts the function of neutrophils, the key infiltrating cell in SS. OBSERVATIONS: A patient with initial dermatologic manifestations of SS who did not respond to standard SS treatment was suspected to have concurrent HGA with the demonstration of leukopenia, thrombocytopenia, and elevated hepatic transaminase levels. The HGA diagnosis was established when morulae in neutrophils were observed on a peripheral blood smear, a finding confirmed by both serologic examination and polymerase chain reaction on the skin biopsy specimen used to establish the SS diagnosis. CONCLUSION: The significant involvement of neutrophils with both SS and HGA warrants a broader search for additional cases that may further define whether pathogenetic linkages could exist.

Recombinant vaccinia-PSA (PROSTVAC) can induce a prostate-specific immune response in androgen-modulated human prostate cancer.

OBJECTIVES: Prostate cancer recurrence, evidenced by rising prostate-specific antigen (PSA) levels after radical prostatectomy, is an increasingly prevalent clinical problem in need of new treatment options. Preclinical studies have suggested that for tumors in general, settings of minimal cancer volume may be uniquely suitable for recombinant vaccine therapy targeting tumor-associated antigens. A clinical study was undertaken to evaluate the safety and biologic effects of vaccinia-PSA (PROSTVAC) administered to subjects with postprostatectomy recurrence of prostate cancer and to assess the feasibility of interrupted androgen deprivation as a tool for modulating expression of the vaccine target antigen, as well as detecting vaccine bioactivity in vivo. METHODS: A limited Phase I clinical trial was conducted to evaluate the safety and biologic effects of vaccinia-PSA administered in 6 patients with androgen-modulated recurrence of prostate cancer after radical prostatectomy. End points included toxicity, serum PSA rise related to serum testosterone restoration, and immunologic effects measured by Western blot analysis for anti-PSA antibody induction. RESULTS: Toxicity was minimal, and dose-limiting toxicity was not observed. Noteworthy variability in time required for testosterone restoration (after interruption of androgen deprivation therapy) was observed. One subject showed continued undetectable serum PSA (less than 0.2 ng/mL) for over 8 months after testosterone restoration, an interval longer than those reported in previous androgen deprivation interruption studies. Primary anti-PSA IgG antibody activity was induced after vaccinia-PSA immunization in 1 subject, although such antibodies were detectable in several subjects at baseline. CONCLUSIONS: Interrupted androgen deprivation may be a useful tool for modulating prostate cancer bioactivity in clinical trials developing novel biologic therapies. Immune responses against PSA may be present among some patients with prostate cancer at baseline and may be induced in others through vaccinia-PSA immunization.

Onycholysis associated with paclitaxel.

Chemotherapeutic agents are known to cause a myriad of cutaneous side effects that the dermatologist is often called upon to identify and treat. The taxoid drug paclitaxel is commonly used in oncology. To date, there have been few adverse dermatologic effects reported secondary to paclitaxel use. This is in contrast to the related drug docetaxel. We report a case in which paclitaxel caused onycholysis and nail loss in a patient being treated for lung cancer. To our knowledge, this finding has not previously been reported in the American dermatologic literature, though it has been reported in association with docetaxel use. It is important for clinicians to recognize that onycholysis can be associated with paclitaxel. Prompt recognition may prevent the unnecessary use of antibiotics or antifungal medications. Discontinuation of paclitaxel chemotherapy generally is not required, and regrowth of nails can be expected following completion of therapy.

Targeted discovery of novel human exons by comparative genomics.

A complete and accurate set of human protein-coding gene annotations is perhaps the single most important resource for genomic research after the human-genome sequence itself, yet the major gene catalogs remain incomplete and imperfect. Here we describe a genome-wide effort, carried out as part of the Mammalian Gene Collection (MGC) project, to identify human genes not yet in the gene catalogs. Our approach was to produce gene predictions by algorithms that rely on comparative sequence data but do not require direct cDNA evidence, then to test predicted novel genes by RT-PCR. We have identified 734 novel gene fragments (NGFs) containing 2188 exons with, at most, weak prior cDNA support. These NGFs correspond to an estimated 563 distinct genes, of which >160 are completely absent from the major gene catalogs, while hundreds of others represent significant extensions of known genes. The NGFs appear to be predominantly protein-coding genes rather than noncoding RNAs, unlike novel transcribed sequences identified by technologies such as tiling arrays and CAGE. They tend to be expressed at low levels and in a tissue-specific manner, and they are enriched for roles in motor activity, cell adhesion, connective tissue, and central nervous system development. Our results demonstrate that many important genes and gene fragments have been missed by traditional approaches to gene discovery but can be identified by their evolutionary signatures using comparative sequence data. However, they suggest that hundreds-not thousands-of protein-coding genes are completely missing from the current gene catalogs.

Glucose is the major component controlling irregular spike activity after feeding in primates.

The aim of this study was to examine the caloric components responsible for the postprandial increase in small intestinal electrical spike activity. Monopolar electrodes were surgically placed on the jejunum and on the ileum of 8 cynomolgus monkeys 2 wk before study. Myoelectric activity was measured after a 14-h fast, and was compared with activity seen either after feeding or after intragastric injection of test solutions [MCT oil (100 kcal), Crisco oil (100 kcal), glucose (40 kcal), glucose polymer (100 kcal), or an equivalent volume of saline control]. Data were analyzed by a spike train analysis program to provide activity front cycle length, quantitative measurements of the activity, front spike activity, and frequency of irregular spike activity before and after feeding. Oral feeding of a predominantly carbohydrate banana meal or an intragastric solution resulted in a significant delay in periodic activity fronts and a significant increase in jejunal postprandial spike activity over fasted irregular spike activity. Increases in postprandial spike activity appeared to parallel increases in the serum glucose concentration. These studies demonstrate that phasic fasting spike activity occurs in the primate small intestine and that this activity is disrupted by oral feeding. Carbohydrates were responsible for the increased postprandial spike activity and for the disruption of regular spike activity after meals. A local effect of the carbohydrate is the most likely explanation. With carbohydrate or fat solutions, changes can occur in postprandial spike activity independent of the delay in periodic activity front activity. The increase in postprandial spike activity and the postprandial delay in periodic activity front activity appear to be controlled through separate mechanisms.

Migrating action-potential complex activity in absence of fluid production is produced by B subunit of cholera enterotoxin.

The myoelectric response and fluid output from in vivo rabbit ileal loops injected with B subunits of purified cholera enterotoxin are compared with the response to the purified cholera holotoxin. Migrating action-potential complex (MAPC) frequency is similar after injection of purified cholera toxin or B subunits. In contrast, fluid output after B-subunit injection is significantly (P less than 0.05) less than fluid output after purified cholera toxin injection. Specific antiserum to the holotoxin incubated with holotoxin at equivalence significantly decreased both MAPC activity (P less than 0.05) and fluid output (P less than 0.001) from the purified toxin. Purified cholera toxin and B subunits, modified by reaction with 2-nitrophenylsulfonyl chloride to produce monomeric B fractions with decreased GM1 ganglioside-binding properties, produced significantly (P less than 0.05) less fluid output and MAPC activity. Binding of the toxin or B subunits in the aggregated form is essential for the expression of MAPC activity or fluid output. These results suggest that the B subunit of cholera toxin produces MAPC activity in the rabbit ileum in the absence of fluid production. Furthermore, previous assumptions that MAPC activity is linked to fluid secretion should be reconsidered. It appears that the gut responses to cholera toxin, fluid production, or MAPC activity are produced by separate mechanisms.

Antitumor efficacy of tumor-antigen-encoding recombinant poxvirus immunization in Dunning rat prostate cancer: implications for clinical genetic vaccine development.

One potential use for prostate-cancer-associated genes discovered through ongoing genetics studies entails the construction of virus- or plasmid-based recombinant vector vaccines encoding these new tumor-associated antigens (TAA) to induce TAA-specific immune responses for the prevention or therapy of prostate cancer. Clinical trials evaluating prototypes of such recombinant vaccines are under way. TAA-encoding recombinant vector vaccines, however, have not previously been evaluated in a prostate-cancer animal model. For assessment of the potential susceptibility of prostate cancer to genetic immunization strategies using TAA-encoding recombinant vectors, the antitumor efficacy of a model recombinant viral vector encoding a TAA was evaluated in rat Dunning prostate cancer. Recombinant vaccinia was chosen as a prototype virus vector encoding a TAA for these studies, and beta-galactosidase was chosen as a model target TAA. Dunning AT-2 cells were transduced with a retroviral vector to express beta-galactosidase, and the susceptibility of tumorigenic AT-2-lacZ cells to immunization with vaccinia-lacZ was measured using protection studies in Copenhagen and nu/nu rats. Stably transduced AT-2-lacZ cells expressing beta-galactosidase as measured by enzymatic substrate-based assays were found to retain their tumorigenicity in vivo despite abundant expression of rat major histocompatibility complex (MHC) class I. Immunization with model TAA-encoding recombinant vaccinia-lacZ conferred significant protection against subsequent growth of AT-2-lacZ cells in vivo (P = 0.01); however, the efficacy of such immunization was markedly dependent on the volume of tumor challenge. The antitumor efficacy of TAA-encoding recombinant vaccinia immunization was abrogated in nu/nu rats, suggesting a T-cell-dependent mechanism of activity. These studies suggest that prostate cancer may be a suitable target for immunization strategies using TAA-encoding recombinant vectors. Such immunization strategies may be more effective in settings of minimal cancer burden.

Multiple phase variation in haemolytic, adhesive and antigenic properties of Streptococcus gordonii.

Streptococcus gordonii gave rise to beta-haemolytic variants (Bhp+ for beta-haemolysin production) at frequencies of 10(-4)-10(-3) on agar medium containing washed horse erythrocytes. Bhp+ variants reverted to the wild-type alpha-haemolytic phenotype (Bhp-) at the same frequencies. There was a significant probability (> or = 0.1) that phase variation in Bhp and phase variation in the previously described Spp (sucrose promoted phenotype) would occur concomitantly, but there was no correlation between these phenotypes. There was evidence also of independent phase variation in adhesion to saliva-coated hydroxyapatite (Asp for adhesion to salivary pellicles), in lactose-sensitive coaggregation (Cls for coaggregation, lactose-sensitive) and in the concentrations of particular cell surface antigens (Cap for cell antigen profile) in strains that had undergone phase changes in Spp and/or Bhp. Phase variation in all these phenotypes were transitions between high and low levels of activity and each appeared to occur as an independent event. Significant associations (P << 0.0001 by contingency table analysis) between particular phenotypes such as Bhp and Asp and between Asp, Cls and Cap phenotypes, however, were apparent. The results suggest that S. gordonii cells become predisposed to phase variation and that the resulting independent phenotypic changes may give rise to phenotypically diverse streptococcal populations able to accommodate rapid and transient environmental changes in the mouth.

Early castration reduces prostatic carcinogenesis in transgenic mice.

OBJECTIVES: To test the hypothesis that transgenic mouse models of prostate cancer could be useful for testing chemoprevention strategies by evaluating the effects of early castration on prostate carcinogenesis in TRAMP mice. Human prostate cancer, unlike other cancers, requires androgens for oncogenesis yet acquires partial androgen independence in the castrated milieu. This paradigm is the basis for an ongoing clinical trial using selective androgen deprivation for prostate cancer chemoprevention. However, preclinical correlates for hormonal prevention or other chemoprevention strategies of prostate cancer have not previously been demonstrated in autochthonous models of prostate carcinogenesis. METHODS: Magnetic resonance imaging was used to longitudinally measure prostate growth in castrated and noncastrated TRAMP mice, and mice were prospectively examined for the onset of advanced, palpable prostate cancer. Modulation of androgen-responsive oncogene expression, as well as oncogene expression in refractory cancers, was evaluated by Western blot. RESULTS: Early castration significantly reduced prostate tumor growth as measured by magnetic resonance imaging and improved cancer-free survival. Prevention of prostate cancer development in these mice was associated with durable suppression of androgen-responsive oncogene expression (T-antigen expression not detectable by Western blot); prostate cancers refractory to the hormonal prevention strategy demonstrated androgen-independent oncogene expression. CONCLUSIONS: These findings suggest that carcinogenesis related to androgen-responsive oncogene expression can be prevented in some cases by hormonal manipulation and that transgenic TRAMP mice are useful for the preclinical evaluation of hormonal and possibly other strategies of prostate cancer chemoprevention.

Longitudinal cohort analysis of lethal prostate cancer progression in transgenic mice.

PURPOSE: Human prostate cancer is variably lethal, shows heterogeneous progression, and exhibits a spectrum of histopathology. Traditional rodent models of prostate cancer lack these characteristics. An alternative, autochthonous model of prostate cancer consists of transgenic mice which develop prostate cancer due to prostatic expression of SV40 T antigen. Lethal progression of such cancers in individual mice has not been previously characterized. Studies were undertaken to characterize the longitudinal progression of prostate cancers in these transgenic mice. METHODS: A prospective longitudinal cohort study was undertaken to characterize prostate cancer volume, progression, lethality, and histological heterogeneity in a transgenic mouse model of prostatic adenocarcinoma. Fifty-one transgenic mice were followed prospectively to determine the age at onset of palpable tumor and age at cancer-related death. Tumor volume was followed longitudinally by magnetic resonance imaging (MRI) in a subset of these mice and lethal cancers were evaluated by histopathology. RESULTS: Primary tumors became palpable at 10-38 weeks of age. Palpable tumors always preceded lethal progression. Cancer death followed 2-9 weeks later, and age at cancer death varied from 24 to 39 weeks of age. The histopathological changes were heterogeneous. Primary tumors were detectable by MRI before they became detectable by palpation. MRI showed that, analogous to human prostate cancers, volume of early stage primary tumors did not necessarily predict age at cancer death. CONCLUSION: Prostate cancer in transgenic mice mimics heterogeneic tumor progression in human prostate cancer, providing a uniquely relevant pre-clinical model. Tumor detection by MRI and palpation are valid surrogate measures of tumor progression in this model.

The effect of outpatient preoperative evaluation of hospital inpatients on cancellation of surgery and length of hospital stay.

UNLABELLED: To evaluate the possible effects of outpatient preoperative evaluation (OPE) for new surgical patients who will be inpatients, we conducted an observational study at a university hospital in The Netherlands. Various outcomes before and after the introduction of an OPE clinic were compared. The study population comprised all 21,553 elective adult inpatients operated on between January 1, 1997 and December 31, 1999. Cardiac surgery, obstetric and pediatric patients, and patients operated on in same-day surgery were excluded. The main outcome measures were surgical cases canceled for medical reasons, rate of same-day admissions (who were expected to increase), and length of hospital stay. After introduction of OPE, the rate of cancellations for medical reasons decreased from 2.0% to 0.9% (adjusted odds ratio 0.7, 95% CI, 0.5--0.9). The rate of same-day admissions increased from 5.3% before to 7.7% after OPE introduction (adjusted odds ratio 1.2, 95% CI, 1.01--1.39), and the total hospital length of stay (in days) significantly decreased by a factor of 0.92 (0.90--0.94), which was partly the result of a reduction in preoperative admission time. We concluded that, although smaller than anticipated, the use of OPE for potential inpatients leads to a significant reduction of cancelled cases and of length of admission. Further increase of these benefits from OPE requires changes in institutional policy, such as forcing surgical departments to increase their number of same-day admissions. IMPLICATIONS: An observational study was conducted to compare various outcomes before and after the introduction of outpatient preoperative evaluation (OPE). Although smaller than anticipated, OPE for potential inpatients leads to a significant reduction of canceled cases and of length of admission.

Epidemiologia molecular do gênero arcobacter / Molecular epidemiology of genus arcobacter

Pertencente à família Campylobacteriaceae, os bacilos gram-negativos curvos Arcobacter apresentam quatro subtipos: A. cryaerophylos, A. butzleri, A. nitrofrigilis e A. skirrowii. Estudos de DNA e rRNA foram usados para evidenciar as diferenças genotípicas entre estas e as dos gêneros Campylobacter e Helicobacter. Tais bactérias foram encontradas em carcaças de suínos, bovinos e aves, bem como em esgotos, sedimentos de pântanos salgados e humanos com enterites, o que sugere que possam ser patógenos humanos transmitidos principalmente através de água não tratada e carnes pouco cozidas