Signal transducer and activator of transcription 3 is a transcriptional factor regulating the gene expression of SALL4.

Both signal transducer and activator of transcription 3 (STAT3) and SALL4 are important in maintaining the pluripotent and self-renewal state of embryonic stem cells. We hypothesized that STAT3, a latent transcriptional factor, may regulate the gene expression of SALL4. In support of this hypothesis, DNA sequence analysis of the SALL4 gene promoter revealed four putative STAT3-binding sites. Using a SALL4-luciferase reporter gene assay, we found that modulation of the STAT3 activity significantly up-regulated the luciferase activity. By chromatin immunoprecipitation, the segment of the SALL4 promoter showing the highest affinity to STAT3 was localized to -366 to -163, in which there was only one putative STAT3 binding site starting at -199. Site-directed mutagenesis of all four putative STAT3-binding sites in the SALL4 promoter significantly reduced its responsiveness to STAT3, although the most dramatic effect was seen at the binding site starting at -199. We further tested the functional relationship between STAT3 and SALL4 using MDA-MB-231, a breast cell line carrying constitutive SALL4 expression and STAT3 activity. Down-regulation of the STAT3 activity using a dominant-negative construct resulted in a significant decrease in the expression of SALL4. To conclude, our data suggest that STAT3 and SALL4 probably cooperate in both physiological and pathological states.

Multidrug resistance proteins MRP3, MRP1, and MRP2 in lung cancer: correlation of protein levels with drug response and messenger RNA levels.

Previously (L. C. Young et al., Clin. Cancer Res., 5: 673-680, 1999), we found, in a panel of 23 lung cancer cell lines that had not been selected for in vitro drug resistance, that the mRNA levels of MRP3 and MRP1, two members of the ATP-binding cassette superfamily of transport proteins, correlated with resistance to doxorubicin, vincristine, VP-16, and cis-diamminedicholoroplatinum(II). To extend these studies, we measured multidrug resistance protein (MRP)1, MRP2, and MRP3 protein levels in a panel of 30 lung cancer cell lines that included the original 23 cell lines as well as an additional 7 unselected lung cancer cell lines. In the case of MRP3, a polyclonal antibody was developed that was found to be a sensitive reagent for the detection of MRP3 by Western blot analysis. We found good agreement in the original 23 cell lines between the cognate mRNA and protein levels for MRP1, MRP2, and, especially, MRP3 (r, 0.852), supporting the use of semiquantitative PCR to predict MRP1, MRP2, and MRP3 protein levels in patient samples. There were also strong correlations between the mRNA and protein levels of MRP3 and MRP1, which suggested that these genes might be expressed in a coordinate manner. MRP3, MRP1, and MRP2 protein levels were higher in the non-small cell lung cancer (NSCLC) than in the SCLC cell lines and, in addition, MRP3 and MRP2 were detected almost exclusively in the NSCLC cell lines. Finally, we found that both MRP3 and MRP1, but not MRP2, protein levels correlated with decreased sensitivity of these lung cancer cell lines to doxorubicin, VCR, VP-16, and cis-diamminedicholoroplatinum(II). These findings are consistent with our hypothesis that both MRP3 and MRP1 are components of the multifactorial multidrug resistance phenotype of lung cancer and that MRP3 contributes to the intrinsic resistance of NSCLC cells.

Expression of the MRP and MDR1 multidrug resistance genes in small cell lung cancer.

Acquired multidrug resistance is a major obstacle to a cure for small cell lung cancer (SCLC). Overexpression of the MDR1 gene occurs infrequently in multidrug-resistant SCLC cell lines. The multidrug resistance protein (MRP) can confer multidrug resistance, but its role in clinically acquired drug resistance is unknown. The purpose of this study was to measure expression of MRP and MDR1 mRNA in cell lines and clinical samples from SCLC patients and to correlate the results with drug sensitivity profiles. Twenty-three SCLC cell lines and 10 tumor samples from SCLC patients were examined. Samples expressing MRP and MDR1 were identified by reverse transcription-PCR, and levels of MRP mRNA in the cell lines were measured by quantitative reverse transcription-PCR. One of 23 cell lines (4%) expressed MDR1 mRNA, whereas MRP expression was detected in 19 of 23 cell lines (83%). There was a significant correlation between doxorubicin resistance and MRP expression levels (r = 0.422; P = 0.045). Of the 10 clinical samples, 3 expressed only MRP, 2 expressed only MDR1, and 4 expressed both drug resistance genes. In summary, MRP is frequently expressed in clinical samples and cell lines from SCLC patients, and the levels correlate with doxorubicin resistance in unselected SCLC cell lines. Expression of MDR1 can be detected in clinical samples of SCLC but is rarely found in cell lines from drug-resistant patients. These multidrug resistance proteins may contribute to the multifactorial problem of clinically acquired drug resistance in SCLC.

Expression of multidrug resistance protein-related genes in lung cancer: correlation with drug response.

Recently, cDNAs have been identified that encode four human proteins (MRP2-5) with structural similarity to the multidrug resistance protein (MRP). Preliminary studies have shown that levels of mRNAs encoding MRP2, MRP3, and MRP5, are increased in some drug-selected cell lines, but the correlation of MRP2-5 mRNA levels with drug resistance has not been examined. Using a collection of small cell lung cancer (SCLC) and non-SCLC patient samples and unselected cell lines established from patients at various stages of treatment, we examined the expression of MRP2, MRP3, MRP4, and MRP5, as well as MDR1 and MRP, by PCR. The levels of individual mRNAs were correlated with the sensitivity of these cell lines to doxorubicin (DOX), vincristine, VP-16, and cis-diamminedichloroplatinum(II), as determined by a modified MTT assay. Using both SCLC and non-SCLC cell lines, we confirmed the previously observed correlation of MRP mRNA levels with resistance to DOX (B. G. Campling et al., Clin. Cancer Res., 3:115-122, 1997) and found a strong correlation of MRP3 mRNA levels with resistance of the cell lines to DOX. In addition, the mRNA levels of both MRP and MRP3 correlated with resistance of the cell lines to vincristine, VP-16, and cis-diamminedichloroplatinum(II). These findings are consistent with the suggestion that MRP3, like MRP, may contribute to the drug resistance phenotype of lung cancer cells.

On the site by which alpha-dendrotoxin binds to voltage-dependent potassium channels: site-directed mutagenesis reveals that the lysine triplet 28-30 is not essential for binding.

We constructed a synthetic gene encoding the published amino acid sequence of DTx from Dendroaspis angusticeps, a ligand of voltage-dependent postassium channels that facilitates neurotransmitter release. We expressed it in Escherichia coli as a fusion protein secreted in the culture medium. The recombinant DTx was generated in vitro by chemical treatment and recovered as two isoforms. One of them (rDTx), like the venom toxin, has an N-terminal pyroglutamate whereas the other (rQDTx) has a free N-terminal glutamine. Chromatographic differences between rDTx and natural DTx led us to re-examine the amino acid sequence of natural DTx. In contrast to what was previously published, position 12 was an Asp and not Asn. Despite this difference, rDTx and DTx had similar toxicity in mice and binding affinity to synaptosomes, suggesting that residue 12 is not important for DTx function. Nor is the N-terminal residue implicated in DTx function since rDTx and rQDTx also had similar biological activities. We also synthesized and expressed a mutant of the DTx gene in which the lysine triplet 28-30 was changed into Ala-Ala-Gly. The two resulting recombinant isoforms exhibited only small decreases in biological activity, excluding the possibility that the positively charged lysine triplet 28-30 of DTx is directly involved in the toxin functional site.

Novel tacrine analogues for potential use against Alzheimer's disease: potent and selective acetylcholinesterase inhibitors and 5-HT uptake inhibitors.

Several novel analogues of tacrine have been synthesized and tested for their ability to inhibit acetylcholinesterase, butyrylcholinesterase, and neuronal uptake of 5-HT (serotonin) and noradrenaline. Changes in the size of the carbocyclic ring of tacrine produced modest potency against cholinesterase enzymes. Addition of a fourth ring resulted in compounds with marked selectivity for acetylcholinesterase (AChE) over butyrylcholinesterase (BChE): e.g. 6-amino-4,5-benzo-5H-cyclopenta[1,2-b]-quinoline (14a) had an IC50 of 0.35 microM against AChE and 3.1 microM against BChE. Some tetracyclic compounds are 100-400 times more active than tacrine as inhibitors of neuronal uptake of serotonin, in particular 13-amino-6,7-dihydro-5H-benzo-[3,4]cyclohepta[1,2-b]quinoline (18), which had an IC50 of 20 nM. These compounds would be expected to facilitate both cholinergic and monoaminergic transmission. They should be worth investigating in models of memory impairment.

Mechanical and biochemical responses to endothelin-1 and endothelin-3 in bovine bronchial smooth muscle.

1. In this study, mechanical responses to endothelin-1 and endothelin-3 were examined in bovine bronchial smooth muscle. In addition, the involvement of phosphatidylinositol 4,5-bisphosphate hydrolysis (PIP2) in the responses to these peptides was assessed by measurement of inositol (1,4,5) trisphosphate (I(1,4,5)P3) production using a specific mass assay. 2. ET-1 evoked contractions of bovine bronchi which were concentration-dependent and initiated at between 10(-9) M and 10(-8) M. ET-1-evoked responses were unaffected by slight elevation of tone with potassium chloride (3 x 10(-2) M), methacholine (10(-6) M) or U46619 (10(-7) M). 3. Contractions to ET-1 were not altered by pre-incubation with atropine (10(-5) M), indomethacin (10(-5) M), nifedipine (10(-5) M), phosphoramidon (3.67 x 10(-5) M) or by removal of the epithelium. 4. ET-3 evoked small contractions which were not concentration-dependent. In the presence of phosphoramidon (3.67 x 10(-5) M) however, concentration-dependent contractions were obtained to ET-3 which were unaffected by atropine (10(-5) M) or by removal of the epithelium, but were significantly attenuated by indomethacin (10(-5) M). Nifedipine (10(-5) M) virtually abolished this response. 5. Both ET-1 and ET-3 (in the presence of phosphoramidon)-evoked contractions were significantly enhanced by the presence of the phorbol ester phorbol 12,13-dibutyrate (10(-8) M). Neither ET-1-, nor ET-3-mediated responses were antagonized by the protein kinase C (PKC) inhibitor, Ro 31-8220 (3 x 10(-9) - 3 x 10(-8) M). 6. ET-1 (3 x 10(-7) M) evoked a biphasic rise in levels of I(1,4,5)P3 which was unaltered by preincubation with atropine, whilst ET-3 (10(-10) - 3 x 10(-7) M) failed to alter levels of I(1,4,5)P3 at any time point examined, even in the presence of phosphoramidon (3.67 x 10(-5) M). 7. These results suggest that, in bovine bronchial smooth muscle, ET-l does not evoke contraction via cyclo-oxygenase metabolites, does not evoke release of the neurotransmitter substance acetylcholine, or require calcium influx via dihydropyridine-sensitive channels. ET-1 evokes 1(1,4,5)P3 production, but stimulation of protein kinase C may not be critical for the associated contraction. In contrast,ET-3-evoked contractions are partly mediated by cyclo-oxygenase metabolites. ET-3 does not stimulate PIP2 hydrolysis, nor activate PKC, but may, either directly or as a requirement of intermediates released in response to ET-3, rely upon extracellular calcium.

Mechanical and biochemical responses to endothelin-1 and endothelin-3 in human bronchi.

In human bronchi, contractions to endothelin-1 were unaltered by atropine (10(-5) M), indomethacin (10(-6) M), nifedipine (10(-5) M), or phosphoramidon (3.67 x 10(-5) M). Endothelin-3-evoked contractions were markedly enhanced by phosphoramidon (3.67 x 10(-5) M), unaffected by atropine (10(-5) M), and attenuated by indomethacin (10(-6) M) or nifedipine (10(-5) M). Phorbol 12,13-dibutyrate (PDB, 10(-8) M) enhanced both endothelin-1- and endothelin-3 (plus phosphoramidon)-evoked responses, an effect abolished by Ro 31-8220 (3 x 10(-8) M). Contractions to endothelin-1 or endothelin-3 alone were unaltered by staurosporine 10(-8)-3 x 10(-7) M) or Ro 31-8220 (3 x 10(-9)-3 x 10(-8) M). Endothelin-1 (3 x 10(-7) M), but not endothelin-3 (10(-10)-3 x 10(-7) M), evoked a rise in levels of inositol (1,4,5) trisphosphate (Ins(1,4,5)P3). These results suggest that endothelin-1 does not act via cyclo-oxygenase metabolites nor require Ca2+ influx via dihydropyridine-sensitive channels. It evokes Ins(1,4,5)P3 production, but does not rely upon protein kinase C activation for contraction. Endothelin-3-evoked contractions are partly mediated by cyclo-oxygenase metabolites. Endothelin-3 does not stimulate phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis, nor utilise protein kinase C to produce contraction, but its actions may rely upon extracellular Ca2+.

Purification of a 5-HT uptake inhibitor from the venom of Cerastes vipera.

A protein that inhibits the re-uptake of 5-hydroxytryptamine into rat brain synaptosomes was isolated from the venom of the Sahara sand viper (Cerastes vipera) by gel filtration and reverse phase chromatography. It has a molecular weight of 13,739 Da and an IC50 of about 50 nM for blocking uptake of 3H-5-HT into rat brain synaptosomes. It also augmented the responses to 5-HT in a smooth muscle preparation. It has phospholipase A2 activity, but it has no lytic activity as measured by its inability to release lactate dehydrogenase from rat brain synaptosomes. Determination of the N-terminal sequence revealed a similarity with a phospholipase A2 previously isolated from Cerastes cerastes venom.

Some pharmacological studies on the effects of Cerastes vipera (Sahara sand viper) snake venom.

The effects of the venom of the Sahara sand viper (Cerastes vipera) were studied on isolated chick biventer cervicis, isolated rat atria and vas deferens preparations, and on the electrocardiogram of anaesthetized rats. Effects on 3H-noradrenaline uptake were studied using rat brain synaptosomes. At 50 micrograms/ml and 100 micrograms/ml, the venom caused a transient increase in rate and force of contractions of the rat atria followed by an irreversible depression. These effects were not prevented by atenolol, atropine or a combination of the two. In the presence of 25 microM lignocaine, the effects of venom on rat atria were reversible by washing. At 100 micrograms/ml, the venom transiently increased responses of vas deferens preparations to indirect stimulation, but had little effect on responses to noradrenaline, KCl, and ATP. In the presence of an alpha 1-adrenoceptor antagonist (prazosin) or a P2-purinergic receptor antagonist (suramin), the venom still significantly increased twitch height and responses to noradrenaline but not to KCl or ATP. The effect of the venom did not change after exposure to a combination of prazosin, suramin and tetrodotoxin. The venom (100 micrograms/ml) significantly decreased twitches to indirect and direct stimulation in chick biventer cervicis preparations. Responses to exogenously applied acetylcholine, carbachol and KCl were also decreased. Venom blocked the synaptosomal uptake of 3H-noradrenaline (IC50 = 5 micrograms/ml), and caused severe bradycardia in vivo. Some of the direct effects on muscle preparations are possibly due to the venom's phospholipase A2 activity.

Physicians' attitudes and behaviors regarding hepatitis B immunization.

BACKGROUND: Hepatitis B virus (HBV) infections are a serious health problem in the United States, where approximately 18,000 cases are reported each year to the Centers for Disease Control. Even among health care providers, reported vaccination rates have ranged from 17% to 68%. The purpose of this study was to determine the important factors influencing physicians in an academic medical center to receive the HBV vaccination since the introduction of recombinant yeast-derived vaccines. METHODS: The 1282 house staff and attending physicians in a university medical center were surveyed regarding their HBV vaccination history. The characteristics of vaccinated and nonvaccinated physicians and their attitudes and concerns regarding vaccination were compared. RESULTS: Of the 813 physicians who responded, 54.0% had been vaccinated. Vaccination rates varied with level of training, from 91.9% among first- and second-year residents to 32.2% among attending physicians. Although physicians in specialties at higher risk for infection were more likely to have been vaccinated, only 40.0% of pathologists and 51.9% of obstetrician-gynecologists reported having been vaccinated. Using multivariate analysis, we found important demographic predictors of HBV vaccination included physician sex and years since graduation, as well as level of training and specialty. Physicians who had been offered the vaccine were more likely to have been vaccinated. CONCLUSIONS: These results show that many physicians in an academic medical center, particularly those at an early stage of their training, have received HBV vaccination. Our results suggest that programs offering hepatitis B vaccinations to physicians can be effective in reducing this group's risk of hepatitis B infections. Special efforts may be necessary to reach physicians who have completed their training.

Evidence that dysregulated DNA mismatch repair characterizes human nonmelanoma skin cancer.

BACKGROUND: In addition to an established role in the repair of postreplicative DNA errors, DNA mismatch repair (MMR) proteins also contribute to cellular responses to exogenous DNA damage. Previously, we have shown that Msh2-null mice display increased sensitivity to ultraviolet (UV) B-induced tumorigenesis, but squamous cell carcinomas (SCC) generated are microsatellite stable, suggesting a role for MMR other than postreplicative repair in UV-induced cutaneous tumour formation. OBJECTIVES: We questioned whether there was evidence of MMR dysfunction in human SCC, thus validating the mouse models of MMR-dependent UVB-induced skin cancer. METHODS: Using tissue microarrays we examined both nuclear and cytoplasmic levels of MMR proteins MSH2, MSH6, MSH3, MLH1 and PMS2 in more than 200 cases of cutaneous SCC and basal cell carcinoma (BCC). RESULTS: We found that subsets of these 10 MMR protein measures were increased in nonmelanoma skin cancer (NMSC) compared with normal epidermal samples; this was particularly true of SCC. In fact, based on post hoc tests and MMR protein distribution patterns, BCC was distinct from SCC. With the exception of nuclear MSH2, the BCC had lower levels of identified MMR protein measures than SCC. We believe this to be important because not only is SCC more aggressive than BCC, but evidence suggests that these two NMSC subtypes arise through different molecular pathways. CONCLUSIONS: In combination with previously established roles for MMR proteins in response to UVB-induced DNA damage, our data point towards an expanded perspective of the importance of MMR proteins in the suppression of UVB-induced tumorigenesis and, potentially, tumour behaviour.

Summer outbreak of respiratory disease in an Australian prison due to an influenza A/Fujian/411/2002(H3N2)-like virus.

An outbreak of influenza A occurred in a prison system in New South Wales, Australia in January 2003 during the southern hemisphere summer. This report documents only the third confirmed outbreak of influenza in a prison environment. The outbreak investigation included case ascertainment, state-wide surveillance, a case-control study and interventions to limit the outbreak such as infection control, quarantine, cohorting of cases, and the use of antiviral medication for prophylaxis. A total of 37 clinical cases were identified. Influenza A virus was detected in 11 of the 22 respiratory tract specimens collected. The virus was typed as an influenza A/Fujian/411/2002 (H3N2)-like virus. This strain subsequently became the predominant virus strain during the northern hemisphere winter and the following 2003 Australian southern hemisphere winter influenza season.

Changing epidemiology of hepatitis A in the 1990s in Sydney, Australia.

Surveillance of hepatitis A in residents of Eastern Sydney Health Area identified substantial epidemics in homosexual males in 1991-2 with a peak rate of 520 per 100,000 recorded in males aged 25-29 years, and again in 1995-6, with a peak rate of 405 per 100,000 per year in males aged 30-34 years. During 1994-5 an epidemic was detected among disadvantaged youth associated with injecting drug use; peak rates of 200 per 100,000 per year were reported in males aged 25-29 years and of 64 per 100,000 per year among females aged 20-24 years. The epidemiology of hepatitis A in these inner suburbs of Sydney is characterized by very few childhood cases and recurrent epidemics among homosexual men. Identified risk groups need to be targeted with appropriate messages regarding the importance of hygiene and vaccination in preventing hepatitis A. However, poor access to health services among disadvantaged youth and a constant influx of young homosexual males into these inner suburbs present major challenges to hepatitis A control.

Outbreak of hepatitis A among homosexual men in Sydney.

OBJECTIVES: This study examined an outbreak of hepatitis A in eastern Sydney during 1991-1992. METHODS: Data were based on cases of hepatitis A in eastern Sydney residents reported from January 1991 to October 1992. RESULTS: Five hundred seventy cases of hepatitis A were reported. Of these, 515 (90%) occurred in male patients, of whom 330 were reported to be homosexual or bisexual. The highest attack rate (71/10,000) occurred in men aged 20 to 29 years of age. CONCLUSIONS: This outbreak affected large numbers of young homosexual men living in the inner eastern suburbs of Sydney. Hepatitis A vaccination should be considered for all susceptible homosexual men. Further research into the use of hepatitis A vaccine as an outbreak control measure is also recommended.

Difficulties in clinical diagnosis of measles: proposal for modified clinical case definition.

OBJECTIVE: To examine the accuracy of clinical diagnosis of measles and to develop an improved measles clinical case definition. DESIGN: Case survey. SETTING: Eastern Sydney, December 1990 to August 1993. SUBJECTS: All cases of measles notified to the Eastern Sydney Public Health Unit without or before serological confirmation. OUTCOME MEASURES: Demographic and clinical details, measles- and rubella-specific IgM and measles complement fixation titres in acute and convalescent (when available) sera and epidemiological links with confirmed measles cases. RESULTS: Of 49 subjects reported and with complete follow-up, 24 were confirmed with measles, four with rubella and 21 had no definite diagnosis. Clinical diagnosis of measles had a false positive rate of 51%. Subjects with confirmed measles were significantly more likely to have a cough (23/24) than those with no definite diagnosis (15/21; P = 0.03) and to be febrile on the day of rash onset (23/24 versus 10/21; P = 0.001). The Centers for Disease Control definition had a sensitivity of 92% but specificity of only 24%. A modified case definition of rash, cough and fever present at onset of rash had equal sensitivity but greater specificity (57%). CONCLUSIONS: As measles is no longer common in Australia, clinical diagnosis is unreliable. When a public health response is needed, cases should be confirmed by serological tests or, if not available, we propose use of our modified clinical case definition.

Association of mastopoiesis with haemopoietic tissues in the neonatal rat.

Mast cells in the newborn rat occur in harmopoietic foci of liver and spleen, but disappear from those foci as extramedullary harmopoiesis ceases during the initial postnatal month. At the same time, mast cells increasingly populate bone marrow and connective tissues of heart, lung, stomach and portal tract of liver.

Modulation of the effect of atrial natriuretic peptide in human and bovine bronchi by phosphoramidon.

1. We have previously shown that atrial natriuretic peptide causes bronchodilatation and reduces bronchial reactivity when administered intravenously or by inhalation to asthmatic patients. We wished to determine the direct effect of exogenously applied atrial natriuretic peptide on isolated airway and the role of proteases important in atrial natriuretic peptide degradation in other organ systems. 2. The ability of atrial natriuretic peptide (alpha-human atrial natriuretic peptide 28-amino acid) to relax precontracted tissues and to protect against methacholine-induced contraction was studied in human and bovine tissue. The role of neutral endopeptidase-24.11 and other proteases in regulating the effect of atrial natriuretic peptide on bronchial smooth muscle was also examined by studying the influence of phosphoramidon, a protease inhibitor, whose actions include the inhibition of neutral endopeptidase-24.11, and the protease inhibitors leupeptin, aprotinin and soybean trypsin inhibitor on the airway response to atrial natriuretic peptide. 3. In human and bovine tissue atrial natriuretic peptide (10(-6) mol/l) caused a slight relaxation of methacholine-contracted tissue [mean (SEM) percentage inhibition of contraction of 13.2 (3.02)% and 9.41 (2.63)% respectively] and evoked a significant rightward shift of the cumulative concentration-response curve to methacholine [pD2 5.15 (0.23) and 4.85 (0.1) compared with control values of 6.14 (0.1) and 5.85 (0.16), respectively]. 4. Phosphoramidon potentiated atrial natriuretic peptide-induced relaxation of methacholine-induced tone and the ability of atrial natriuretic peptide to protect against methacholine-induced contraction. The combination of leupeptine, aprotinin and soybean trypsin inhibitor did not significantly alter the bronchial response to atrial natriuretic peptide in either human or bovine tissues.(ABSTRACT TRUNCATED AT 250 WORDS)