Development of diagnostic methods and study of the immunoreactivity of a mixture of recombinant core and E2 proteins fused to GST with control serum positive for hepatitis C

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Identification of unannotated exons of low abundance transcripts in Drosophila melanogaster and cloning of a new serine protease gene upregulated upon injury

Abstract Background The sequencing of the D.melanogaster genome revealed an unexpected small number of genes (~ 14,000) indicating that mechanisms acting on generation of transcript diversity must have played a major role in the evolution of complex metazoans. Among the most extensively used mechanisms that accounts for this diversity is alternative splicing. It is estimated that over 40% of Drosophila protein-coding genes contain one or more alternative exons. A recent transcription map of the Drosophila embryogenesis indicates that 30% of the transcribed regions are unannotated, and that 1/3 of this is estimated as missed or alternative exons of previously characterized protein-coding genes. Therefore, the identification of the variety of expressed transcripts depends on experimental data for its final validation and is continuously being performed using different approaches. We applied the Open Reading Frame Expressed Sequence Tags (ORESTES) methodology, which is capable of generating cDNA data from the central portion of rare transcripts, in order to investigate the presence of hitherto unnanotated regions of Drosophila transcriptome. Results Bioinformatic analysis of 1,303 Drosophila ORESTES clusters identified 68 sequences derived from unannotated regions in the current Drosophila genome version (4.3). Of these, a set of 38 was analysed by polyA+ northern blot hybridization, validating 17 (50%) new exons of low abundance transcripts. For one of these ESTs, we obtained the cDNA encompassing the complete coding sequence of a new serine protease, named SP212. The SP212 gene is part of a serine protease gene cluster located in the chromosome region 88A12-B1. This cluster includes the predicted genes CG9631, CG9649 and CG31326, which were previously identified as up-regulated after immune challenges in genomic-scale microarray analysis. In agreement with the proposal that this locus is co-regulated in response to microorganisms infection, we show here that SP212 is also up-regulated upon injury. Conclusion Using the ORESTES methodology we identified 17 novel exons from low abundance Drosophila transcripts, and through a PCR approach the complete CDS of one of these transcripts was defined. Our results show that the computational identification and manual inspection are not sufficient to annotate a genome in the absence of experimentally derived data.

Assessment of the role of transcript for GATA-4 as a marker of unfavorable outcome in human adrenocortical neoplasms

Abstract Background Malignant neoplasia of the adrenal cortex is usually associated with very poor prognosis. When adrenocortical neoplasms are diagnosed in the early stages, distinction between carcinoma and adenoma can be very difficult to accomplish, since there is yet no reliable marker to predict tumor recurrence or dissemination. GATA transcription factors play an essential role in the developmental control of cell fate, cell proliferation and differentiation, organ morphogenesis, and tissue-specific gene expression. Normal mouse adrenal cortex expresses GATA-6 while its malignant counterpart only expresses GATA-4. The goal of the present study was to assess whether this reciprocal change in the expression of GATA factors might be relevant for predicting the prognosis of human adrenocortical neoplasms. Since human adrenal cortices express luteinizing hormone (LH/hCG) receptor and the gonadotropins are known to up-regulate GATA-4 in gonadal tumor cell lines, we also studied the expression of LH/hCG receptor. Methods We conducted a study on 13 non-metastasizing (NM) and 10 metastasizing/recurrent (MR) tumors obtained from a group of twenty-two adult and pediatric patients. The expression of GATA-4, GATA-6, and LH/hCG receptor (LHR) in normal and tumoral human adrenal cortices was analysed using reverse transcriptase-polymerase chain reaction (RT-PCR) complemented by dot blot hybridization. Results Messenger RNA for GATA-6 was detected in normal adrenal tissue, as well as in the totality of NM and MR tumors. GATA-4, by its turn, was detected in normal adrenal tissue, in 11 out of 13 NM tumors, and in 9 of the 10 MR tumors, with larger amounts of mRNA found among those presenting aggressive clinical behavior. Transcripts for LH receptor were observed both in normal tissue and neoplasms. A more intense LHR transcript accumulation was observed on those tumors with better clinical outcome. Conclusion Our data suggest that the expression of GATA-6 in human adrenal cortex is not affected by tumorigenesis. GATA-4 expression is more abundant in MR tumors, while NM tumors express more intensely LHR. Further studies with larger cohorts are needed to test whether relative expression levels of LHR or GATA-4 might be used as prognosis predictors.

Untersuchungen zur Chemosensitivität von testikulären Tumorzellen gegenüber Cisplatin

Metastasierender Krebs ist bei Erwachsenen in der Regel nicht heilbar. Eine Ausnahme stellen testikuläre Keimzelltumoren (TKZT) dar, da über 75 % der Patienten mit fortgeschrittenen metastasierenden TKZT mit einer auf Cisplatin basierenden Kombinations-Chemotherapie geheilt werden können. Zelllinien, die aus TKZT isoliert wurden, behalten diese Cisplatin-Sensitivität in vitro bei. Somit spiegeln Testistumorzelllinien die klinische Situation wider und sind deswegen ein gutes Modellsystem um zu untersuchen, welche Faktoren der Cisplatin-Sensitivität zugrunde liegen. Die Ursachen der Cisplatin-Sensitivität in Testistumoren sind nicht bekannt. Es wurde bereits gezeigt, dass Testistumorzellen eine geringe Kapazität für die Entfernung von Cisplatin-induzierten DNA-Platinierungen aufweisen. Dieser Defekt in der DNA-Reparatur könnte ein Faktor für die beobachtete Cisplatin-Sensitivität sein. Cisplatin induziert sowohl Intrastrang-Vernetzungen als auch Interstrang-Vernetzungen (ICLs). Die Bildung und Reparatur der Cisplatin-induzierten Intrastrang-Vernetzungen wurde mittels DNA-Slot-Blot, die Bildung und Entfernung von Interstrang-Vernetzungen wurde mithilfe des Comet-Assays untersucht. In der vorliegenden Arbeit wurde gezeigt, dass die Reparatur von Intrastrang-Vernetzungen in Testis- und Blasentumorzelllinien vergleichbar ist. Somit sind Testistumorzellen in diesem Reparaturweg nicht beeinträchtigt. Im Unterschied dazu zeigte sich, dass Testistumorzellen die ICLs nicht oder nur mit einer reduzierten Kapazität entfernen können.Da die ICL-Reparatur über die Bildung von DNA-Doppelstrangbrüchen (DSB) mit anschließender DSB-Reparatur verläuft, wurde die Kinetik der DSB-Reparatur anhand der Immundetektion der Histon-Variante γH2AX, die zur Visualisierung von DSB verwendet wird, verfolgt. γH2AX Foci wurden nach Behandlung mit Cisplatin in Testistumorzellen und Blasentumorzellen gebildet. Anders als in Blasentumorzellen blieb der Prozentsatz an γH2AX-positiven Zellen in Testistumorzellen bestehen. Offensichtlich konnten die Testistumorzellen die Cisplatin-induzierten ICLs nicht korrekt prozessieren, was dazu führte, dass γH2AX Foci persistierten. Da unreparierte DNA-Läsionen eine DNA-schadensabhängige Antwort einleiten können, wurde die Aktivierung der Hauptfaktoren dieser Signalwege untersucht. In den Testistumorzellen zeigte sich eine Erhöhung der p53 Proteinmenge nach Cisplatin-Behandlung. Des Weiteren wurde die durch Cisplatin induzierte Aktivierung von ATM/ATR, Chk1/Chk2, Bax und Noxa in Testis- und Blasentumorzellen vergleichend untersucht. Es wurde bereits gezeigt, dass der Reparaturfaktor ERCC1-XPF in Testistumorzelllinien reduziert vorliegt. Um eine mögliche Rolle von ERCC1-XPF für die Reparatur-Defizienz der ICLs und Cisplatin-Sensitivität in Testistumorzellen zu analysieren, wurde ERCC1-XPF in der Testistumorenzelllinie 833K mithilfe eines Expressionsvektors überexprimiert, und der Einfluss von ERCC1-XPF auf ICL-Reparatur sowie Cisplatin-Sensitivität wurde ermittelt. Überexpression von ERCC1-XPF führte zur Reparatur der ICLs in 833K-Zellen und verminderte die Cisplatinsensitivität. Somit scheint die Cisplatinsensitivität der Testistumorzellen, zumindest zum Teil, auf einer verminderten ICL-Reparatur zu beruhen. Des Weiteren wurde in „proof of principle“ Experimenten ERCC1-XPF in der Cisplatin-resistenten Blasentumorzelllinie MGH-U1 mittels siRNA herunterreguliert, und die Auswirkung der Herunterregulation auf die ICL-Reparatur und die Cisplatinsensitivität wurde geprüft. RNA-Interferenz-vermittelte Herunterregulierung von ERCC1-XPF reduzierte die Prozessierung der Cisplatin-induzierten ICLs und verstärkte die Cisplatinsensitivität in MGH-U1 Zellen. Somit wurde in dieser Arbeit zum ersten Mal gezeigt, dass die Testistumorzellen in Vergleich zu Blasentumorzellen in der Reparatur von ICLs defizient sind, wobei die verminderte ICL-Reparatur auf die geringe Expression von ERCC1-XPF zurückgeführt werden konnte. Diese ICL-Reparatur-Defizienz könnte, zumindest zu einem Teil, für die Sensitivität der Testistumoren gegenüber Cisplatin verantwortlich sein.

Short-term effects of systemic antibiotics during periodontal healing

OBJECTIVES: To investigate the short-term effects of nonsurgical therapy (scaling and root planing, SRP) on the subgingival microbiota in chronic (CP) and aggressive (AP) periodontal disease. METHOD AND MATERIALS: Ninety-seven CP and AP subjects underwent full-mouth SRP on 2 consecutive days. AP patients were randomly assigned to either receive systemic metronidazole plus amoxicillin (AP+AB) or were treated mechanically alone (AP). Pathogens were identified with 16S rRNA oligodeoxynucleotide probes and dot-blot hybridization before and at days 2, 3, 4, 7, 10, and 21 of healing. CP subjects were treated by scaling and root planing along with placebo tablets. RESULTS: Initially, AP cell counts were 69.9- (Porphyromonas gingivalis), 10.2- (Aggregatibacter actinomycetemcomitans), 5.7- (Tannerella forsythia), and 3.3-fold (Prevotella intermedia) enhanced compared to CP cell counts. Following SRP, immediate elimination occurred in single individuals of all three treatment groups at day 2. After SRP plus antibiotic therapy (AP+AB), the prevalence scores dropped beyond the levels of AP and CP, beginning at day 7, and remained low until day 21 (P =or< .05). Clinical healing statistically benefited from SRP with no differences among the three treatment groups. CONCLUSION: Nonsurgical therapy resulted in both a suppression and early elimination of single taxa immediately after completion of active treatment. Systemic antibiotics significantly accelerate the suppression of the periodontal microflora, but have limited effect on the elimination of target isolates during healing.

Patient characteristics but not virulence factors discriminate between asymptomatic and symptomatic E. coli bacteriuria in the hospital

Background Escherichia coli is a common cause of asymptomatic and symptomatic bacteriuria in hospitalized patients. Asymptomatic bacteriuria (ASB) is frequently treated with antibiotics without a clear indication. Our goal was to determine patient and pathogen factors suggestive of ASB. Methods We conducted a 12-month prospective cohort study of adult inpatients with E. coli bacteriuria seen at a tertiary care hospital in St. Louis, Missouri, USA. Urine cultures were taken at the discretion of treating physicians. Bacterial isolates were tested for 14 putative virulence genes using high-throughput dot-blot hybridization. Results The median age of the 287 study patients was 65 (19–101) years; 78% were female. Seventy percent had community-acquired bacteriuria. One-hundred ten (38.3%) patients had ASB and 177 (61.7%) had symptomatic urinary tract infection (sUTI). Asymptomatic patients were more likely than symptomatic patients to have congestive heart failure (p = 0.03), a history of myocardial infarction (p = 0.01), chronic pulmonary disease (p = 0.045), peripheral vascular disease (p = 0.04), and dementia (p = 0.03). Patients with sUTI were more likely to be neutropenic at the time of bacteriuria (p = 0.046). Chronic pulmonary disease [OR 2.1 (95% CI 1.04, 4.1)] and dementia [OR 2.4 (95% CI 1.02, 5.8)] were independent predictors for asymptomatic bacteriuria. Absence of pyuria was not predictive of ASB. None of the individual virulence genes tested were associated with ASB nor was the total number of genes. Conclusions Asymptomatic E. coli bacteriuria in hospitalized patients was frequent and more common in patients with dementia and chronic pulmonary disease. Bacterial virulence factors could not discriminate symptomatic from asymptomatic bacteriurias. Asymptomatic E. coli bacteriuria cannot be predicted by virulence screening.

Herd-specific strains of Mycoplasma bovis in outbreaks of mycoplasmal mastitis and pneumonia

Mycoplasma bovis causes severe economic losses in livestock production, particularly on the Northern American continent and more recently also in continental Europe. The aim of the current study was to evaluate whether the recently emerging outbreaks were due to a particular clone or strain of M. bovis or whether these outbreaks are due to multiple infectious strains of M. bovis. The study is based on the analysis M. bovis isolated from cattle of herds with outbreaks of mycoplasmal mastitis or pneumonia from geographically non related parts of Switzerland. M. bovis isolates were typed by insertion sequence (IS) element analysis based upon ISMbov1 and ISMbov2 southern-blot hybridization. We observed a strong divergence of M. bovis strains among affected herds which mostly were herd specific. This argues against the assumption that a recent infiltration of a particular clone of M. bovis is the cause of the perilous emerging outbreaks. The study suggests that transmission occurs from animal to animal most probably via milk.

Mechanisms of protein regulation in rat skeletal muscle during resistance exercise and training

The cellular mechanisms through which adult rat skeletal muscle protein is regulated during resistance exercise and training was investigated. A model of non-voluntary resistance exercise was described which involves the electrically-stimulated contraction of the lower leg muscles of anesthetized rats against a weighted pulley-bar. Muscle protein synthesis rates were measured by in vivo constant infusion of $\sp3$H-leucine following a single bout of resistance exercise. Specific messenger RNA levels were determined by dot-blot hybridization analysis using $\sp{32}$P-labelled DNA probes after a single bout and multiple bouts of phasic training. The effects of phasic training on increasing skeletal muscle mass was assessed. Between 12 and 36 hours following a single resistance exercise bout (24-192 contractions), total mixed and myofibril protein synthesis rates were significantly increase (32%-65%) after concentric (gastrocnemius m.) and eccentric (tibialis anterior m.) contractions. Eccentric contractions had greater effects on myofibril synthesis with more prolonged increases in synthesis rates. Lower numbers of eccentric than concentric contractions were required to increase synthesis. Cellular RNA was increased after exercise but the relative levels of skeletal $\alpha$-actin and cytochrome c mRNAs were unchanged. Since increases in synthesis rates exceeded increases in RNA, post-transcriptional mechanisms may be primarily responsible for increased protein synthesis after a resistance exercise bout. After 10-22 weeks of phasic eccentric resistance training, muscle enlargement (16%-30%) was produced in the tibialis anterior m. after all training paradigms examined. In contrast, gastrocnemius m. enlargement after phasic concentric training occurred after moderate (24/bout) but not after high (192/bout) repetition training. The absence of muscle growth in the gastrocnemius m. after high repetition training despite increased synthesis rates after the initial bout and RNA and possibly mRNA accumulation during training suggests a role for post-translational mechanisms (protein degradation) in the control of muscle growth in the gastrocnemius m. It is concluded that muscle protein during resistance exercise and training is regulated at several cellular levels. The particular response may be influenced by the exercise intensity and duration, the training frequency and the type of contractile work (eccentric vs. concentric) performed. ^

Analysis of the Soehner-Dmochowski strain of Moloney murine sarcoma virus

The Soehner-Dmochowski strain of murine sarcoma virus (MuSV-SD) was derived from a bone tumor of a New Zealand Black (NZB) rat infected with the Moloney strain of MuSV, which carries the gene encoding the v-mos protein. Serial passage of cell-free tumor extracts both decreased the latent period and resulted in osteosarcomas. Cells from a late passage tumor were established in culture, cell-free extracts frozen, and later inoculated into newborn NZB rats. One of the resulting bone tumors was established in culture and clonal cell lines derived, of which S4 was selected for the present study. The objectives of the study were two-fold: an examination of the genetic organization of MuSV-SD, and an examination of the biochemical characteristics of the viral proteins, since this is an acutely transforming virus which may yield insights into the mechanism of transformation caused by the v-mos protein. Blot hybridization of digested S4 genomic DNA reveals three candidate MuSV-SD integrated viral DNAs. The largest of these, MuSV-SD-6.5, was cloned from an S4 cosmid library, and the complete MuSV-SD-mos sequence was determined. The predicted amino acid sequence of the v-mos protein was compared to that of MuSV-124 and Ht-1, which show a 96.5% and 97.1% similarity, respectively. To characterize the MuSV-SD-mos protein further, immunochemical assays were performed using anti-mos antisera. The immunoblot analysis and immunoprecipitation assays demonstrated that similar levels of the v-mos protein were present in cells chronically infected with either MuSV-SD or MuSV-124; however, the immune complex kinase assay revealed greatly reduced in vitro serine kinase activity of the MuSV-SD-mos protein compared to that of MuSV-124. Sequence analysis demonstrated that the serine at amino acid residue 358 of the MuSV-SD-mos protein, like that of MuSV-Ht-1, had been mutated to a glycine. Mutations of this serine residue have been shown to affect the detectable in vitro kinase activity, however, v-mos proteins containing this mutation still retain transforming properties. Therefore, although the characteristic in vitro kinase activity of the MuSV-SD-mos protein has not been demonstrated, it is clear that this virus is a potent transforming agent. ^

DNA probes for the detection of Fasciola hepatica in snails

Fasciola hepatica, also called the large liver fluke, is a trematode which can infect most mammals. Monitoring the infection rate of snails, which function as intermediate hosts and harbour larval stages of F. hepatica, is an important component of epidemiological studies on fascioliasis. For this purpose, DNA probes were generated which can be used for the detection of F. hepatica larvae in snails. Four highly repetitive DNA fragments were cloned in a plasmid vector and tested by Southern blot hybridization to the DNA of various trematodes for specificity and sensitivity. The probes Fhr-I, Fhr-II and Fhr-III hybridized only to F. hepatica DNA. Fhr-IV contained ribosomal RNA gene sequences and cross-hybridize with the DNA from various other trematode species. Squash blot analysis showed that the different probes were able to detect the parasite larvae in trematode-infected snails even as isolated single larvae. No signals were obtained in squash blots of uninfected snails. Probes Fhr-I, Fhr-II and Fhr-III are thus useful specific tools for studying the epidemiology of fascioliasis. The probe Fhr-IV, because of its broader spectrum, can be used to detect the larvae of a wide range of trematode species of waterbirds, which are the causative agents of swimmer's itch.

STUDIES INTO THE MOLECULAR MECHANISMS REGULATING MUSCULAR ATROPHY RESULTING FROM HINDLIMB IMMOBILIZATION IN THE RAT (PROTEIN, SYNTHESIS, RNA)

The purpose of the work performed in this dissertation was to examine some of the possible regulatory mechanisms involved in the initiation of muscular atrophy during periods of decreased muscle utilization resulting from hindlimb immobilization in the rat. A 37% decrease in the rate of total muscle protein synthesis which has been observed to occur in the first 6 h of immobilization contributes significantly to the observed loss of protein during immobilization.^ The rates of cytochrome c and actin synthesis were determined in adult rat red vastus lateralis and gastrocnemius muscles, respectively, by the constant infusion and incorporation of ('3)H-tyrosine into protein. The fractional synthesis rates of both actin and cytochrome c were significantly decreased (P < 0.05) in the 6th h of hindlimb immobilization.^ RHA was extracted from adult rat gastrocnemius muscle by modification of the phenol: chloroform: SDS extraction procedures commonly used for preparation of RNA for hybridization analysis from other mammalian tissues. RNA content of rat gastrocnemius muscle, as determined by this method of extraction and its subsequent quantification by UV absorbance and orcinol assay, was significantly greater than the RNA content previously determined for adult rat gastrocnemius by other commonly employed methods.^ RNA extracted by this method from gastrocnemius muscles of control and 6h immobilized rats was subjected to "dot blot" hybridization to ('32)P-labelled probe from plasmid p749, containing a cDNA sequence complementary to (alpha)-actin mRNA and from rat skeletal muscle. (alpha)-Actin specific mRNA content as estimated by this procedure is not significantly decreased in rat gastrocnemius following 6h or hindlimb immobilization. However, (alpha)-actin specific mRNA content is significantly decreased (P < 0.05) in adult rat gastrocnemius (alpha)-actin specific mRNA is not decreased in adult rat gastrocnemius muscle following 6h of immobilization, a time when actin synthesis is significantly decreased, it is concluded that a change in (alpha)-actin specific mRNA content is not the initiating event responsible for the early decrease in actin synthesis observed in the 6th h of immobilization. ^

STRUCTURAL ANALYSIS OF THE TRANSCRIPTION PRODUCTS OF A TEMPERATURE-SENSITIVE PHENOTYPIC MUTANT OF MOLONEY SARCOMA VIRUS--MUSV TS110

Cells infected with a temperature sensitive phenotypic mutant of Moloney sarcoma virus (MuSVts110) exhibit a transformed phenotype at 33('(DEGREES)) and synthesize two virus specific proteins, p85('gag-mos), a gag-mos fusion protein and p58('gag), a truncated gag precursor protein (the gag gene codes for viral structural proteins and mos is the MuSV transforming gene). At 39('(DEGREES)) only p58('gag) is synthesized and the morphology of the cells is similar to uninfected NRK parental cells. Two MuSVts110 specific RNAs are made in MuSVts110-infected cells, one of 4.0 kb in length, the other of 3.5 kb. Previous work indicated that each of these RNAs arose by a single central deletion of parental MuSV genetic material, and that p58('gag) was made by the 4.0 kb RNA and p85('gag-mos) from the 3.5 kb RNA. The objective of my dissertation research was to map precisely the deletion boundaries of both of the MuSVts110 RNAs, and to determine the proper reading frame across both deletion borders. This work succeeded in arriving at the following conclusions: (a) Using S-1 nuclease analysis and primer extension sequencing, it was found that the 4.0 kb MuSVts110 RNA arose by a 1488 base deletion of 5.2 kb parental MuSV genomic RNA. This deletion resulted in an out of frame fusion of the gag and mos genes that resulted in the formation of a "stop" codon which causes termination of translation just beyond the c-terminus of the gag region. Thus, this RNA can only be translated into the truncated gag protein p58('gag). (b) S-1 analysis of RNA from cells cultivated at different temperatures demonstrated that the 4.0 kb RNA was synthesized at all temperatures but that synthesis of the 3.5 kb RNA was temperature sensitive. These observations supported the data derived from blot hybridization experiments the interpretation of which argued for the existence of a single provirus in MuSVts110 infected cells, and hence only a single primary transcript (the 4.0 kb RNA). (c) Analyses similar to those described in (a) above showed that the 3.5 kb RNA was derived from the 4.0 kb MuSVts110 RNA by a further deletion of 431 bases, fusing the gag and mos genes into a continuous reading frame capable of directing synthesis of the p85('gag-mos) protein. These sequence data and the presence of only one MuSVts110-specific provirus, indicate that a splice mechanism is employed to generate the 3.5 kb RNA since the gag and mos genes are observed to be fused in frame in this RNA. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI ^

Cigarette smoking and cervical intraepithelial neoplasia among colposcopy patients

Epidemiologic and biochemical evidence suggest that smoking is an independent risk factor for cervical neoplasia; however, only two studies have adjusted by the potential confounding effect of human papillomavirus (HPV). To determine the association between self-reported current cigarette smoking and cervical intraepithelial neoplasia (CIN), we conducted a case-control study that controlled for HPV infection and other reported risk factors. The medical records of all new patients referred to the University of Texas M. D. Anderson Cancer Center (UTMDACC) Colposcopy Clinic were reviewed. The study population (n = 564) consisted of all white, black, and Hispanic non-pregnant women who were residents of Texas, and had no history of treatment for cervical neoplasia. Cases (n = 313) included women diagnosed at the UTMDACC with CIN; while controls (n = 251) included those patients diagnosed at the colposcopy clinic as non-CIN (negative 47%, inflammation or atypia 25%, and koilocytosis 27%). Diagnosis was based on a colposcopically directed biopsy in 95% of the subjects, and all subjects were tested for HPV by dot blot hybridization. The crude odds ratio for cigarette smoking and CIN was 1.37 (95% CI 0.97-1.95); however, after adjusting for HPV, age, education, race, number of sexual partners, and age at first sexual intercourse, the odds ratio decreased to 0.91 (95% CI 0.61-1.41). A higher crude odds ratio was observed with CIN 3 (OR = 1.75, 95% CI 1.08-2.83), but this effect also disappeared after adjustment (OR = 1.06, 95% CI 0.57-1.96). Similar results were observed when controlling only for HPV: OR = 1.11 (95% CI 0.77-1.59) for CIN combined and 1.25 (95% CI 0.76-2.08) for CIN 3. These findings suggest that cigarette smoking is not an independent risk factor for CIN in this population, and that HPV may be an important confounding factor for this association. ^

Gestational exposure to ethanol suppresses msx2 expression in developing mouse embryos

Ethanol acts as a teratogen in developing fetuses causing abnormalities of the brain, heart, craniofacial bones, and limb skeletal elements. To assess whether some teratogenic actions of ethanol might occur via dysregulation of msx2 expression, we examined msx2 expression in developing mouse embryos exposed to ethanol on embryonic day (E) 8 of gestation and subjected to whole mount in situ hybridization on E11–11.5 using a riboprobe for mouse msx2. Control mice exhibited expression of msx2 in developing brain, the developing limb buds and apical ectodermal ridge, the lateral and nasal processes, olfactory pit, palatal shelf of the maxilla, the eye, the lens of the eye, otic vesicle, prevertebral bodies (notochord), and endocardial cushion. Embryos exposed to ethanol in utero were significantly smaller than their normal counterparts and did not exhibit expression of msx2 in any structures. Similarly, msx2 expression, as determined by reverse transcription–PCR and Northern blot hybridization, was reduced ≈40–50% in fetal mouse calvarial osteoblastic cells exposed to 1% ethanol for 48 hr while alkaline phosphatase was increased by 2-fold and bone morphogenetic protein showed essentially no change. Transcriptional activity of the msx2 promoter was specifically suppressed by alcohol in MC3T3-E1 osteoblasts. Taken together, these data demonstrate that fetal alcohol exposure decreases msx2 expression, a known regulator of osteoblast and myoblast differentiation, and suggest that one of the “putative” mechanisms for fetal alcohol syndrome is the inhibition of msx2 expression during key developmental periods leading to developmental retardation, altered craniofacial morphogenesis, and cardiac defects.

Three members of a novel small gene-family from Arabidopsis thaliana able to complement functionally an Escherichia coli mutant defective in PAPS reductase activity encode proteins with a thioredoxin-like domain and “APS reductase” activity

Three different cDNAs, Prh-19, Prh-26, and Prh-43 [3′-phosphoadenosine-5′-phosphosulfate (PAPS) reductase homolog], have been isolated by complementation of an Escherichia coli cysH mutant, defective in PAPS reductase activity, to prototrophy with an Arabidopsis thaliana cDNA library in the expression vector λYES. Sequence analysis of the cDNAs revealed continuous open reading frames encoding polypeptides of 465, 458, and 453 amino acids, with calculated molecular masses of 51.3, 50.5, and 50.4 kDa, respectively, that have strong homology with fungal, yeast, and bacterial PAPS reductases. However, unlike microbial PAPS reductases, each PRH protein has an N-terminal extension, characteristic of a plastid transit peptide, and a C-terminal extension that has amino acid and deduced three-dimensional homology to thioredoxin proteins. Adenosine 5′-phosphosulfate (APS) was shown to be a much more efficient substrate than PAPS when the activity of the PRH proteins was tested by their ability to convert 35S-labeled substrate to acid-volatile 35S-sulfite. We speculate that the thioredoxin-like domain is involved in catalytic function, and that the PRH proteins may function as novel “APS reductase” enzymes. Southern hybridization analysis showed the presence of a small multigene family in the Arabidopsis genome. RNA blot hybridization with gene-specific probes revealed for each gene the presence of a transcript of ≈1.85 kb in leaves, stems, and roots that increased on sulfate starvation. To our knowledge, this is the first report of the cloning and characterization of plant genes that encode proteins with APS reductase activity and supports the suggestion that APS can be utilized directly, without activation to PAPS, as an intermediary substrate in reductive sulfate assimilation.