Sigma 2 receptor expression levels in blood and bladder from healthy and bladder cancer cattle.

The expression of sigma-2 receptor (S2R) was assayed in blood and bladder samples from healthy cattle and in blood and bladder of cattle with deltapapillomavirus-associated urothelial tumors. Samples of bladder from cattle with neoplasia had significantly higher S2R than samples of bladder from healthy cattle (95% CI 0.31-0.82, P < 0.05). In addition, significantly higher S2R was detected in the blood of cattle with bladder cancer than blood from healthy cattle (95% CI 0.22-0.41, P < 0.05). The results provide evidence that increased expression of SR2 in blood could be useful as circulating biomarker for bladder cancer in cattle. PGRMC1 protein levels were also found to be increased in blood and bladder from cattle with cancer and increased expression of PGRMC1 transcripts was detected by quantitative real time PCR in samples from cattle neoplasia. Furthermore, electron microscopy revealed phagophores and numerous autophagosomes, ultrastructural hallmark of autophagy.

A systematic review on factors and consequences of parental distress as related to childhood cancer.

The literature including correlates of parental distress as related to childhood cancer is abundant. It is important to identify predictive factors and outcomes of this distress in parents. The objective of this review was to update previous syntheses on factors of distress and to identify outcomes of parents' distress in the recent literature (2007-2012). We performed a systematic review to identify all quantitative studies including measures of parental distress and associated factors during the study period. We found 56 eligible studies, of which 43 had a Low risk of bias (Cochrane guidelines). Forty-two reports included potential predictive factors. Significant relationships were found with clinical history of the child, sex of the parent, coping response and personal resources, pre-diagnosis family functioning, but not education/income or marital status. Twenty-five reports studied potential consequences of distress and focused on psychological adjustment in parents and children. Compared to past periods, a higher proportion of studies included fathers. Measures used to evaluate distress were also more homogeneous in certain domains of distress. This review underscores the need for appropriate methods for selecting participants and reporting results in future studies. Appropriate methods should be used to demonstrate causality between factors/consequences and distress.

Possible protective effects of kinins and converting enzyme inhibitors in cardiovascular tissues.

The main objective of this study was to determine if the components of the kallikrein-kinin system are released into the venous effluent from isolated perfused rat hearts. To assess the contribution of kinins and the vascular and cardioprotective effects of the ACE inhibitor ramipril, we determined the status of cardiac kallikrein (CKK), potent kinin-generating enzyme, in rats with right ventricular hypertrophy induced by chronic volume overload and left ventricular hypertrophy by aortic banding. CKK was measured as previously described (Nolly, H.L., Carbini, L., Carretero, O.A., Scicli, A.G., 1994). Kininogen by a modification of the technique of Dinitz and Carvalho (1963) and kinins were extracted with a Sep-Pak C18 cartridge and measured by RIA. CKK (169 +/- 9 pg Bk/30 min), kininogen (670 +/- 45 pg Bk/30 min) and immunoreactive kinins (62 +/- 10 pg Bk/30 min) were released into the perfusate. The release was almost constant over a 120 min period. Pretreatment with the protein synthesis inhibitor puromycin (10 mg i.p.) lowered the release of kallikrein (42 +/- 12 pg Bk/30 min, p < 0.001) and kininogen (128 +/- 56 pg Bk/30 min, p < 0.001). Addition of ramiprilat (10 micrograms/ml) increased kinin release from 54 +/- 18 to 204 +/- 76 pg Bk/30 min (p < 0.001). Aortic banding of rats increased their blood pressure (BP) (p < 0.001), relative heart weight (RHW) (p < 0.001) and CKK (p < 0.001). Ramipril treatment induced a reduction in BP (p < 0.05) and RHW (p < 0.005) while CKK remained elevated. Aortocaval shunts increased their ANF plasma levels (p < 0.05), RHW (p < 0.001) and CKK (p < 0.01). Ramipril treatment induced a reduction in RHW (p < 0.05), while CKK and ANF increased significantly (p < 0.05). The present data show that the components of the kallikrein-kinin system are continuously formed in the isolated rat heart and that ramipril reduces bradykinin breakdown with subsequent increase in bradykinin outflow. The experiments with aorta caval shunt and aortic banding show that cardiac tissues increase their kinin-generating activity and this was even higher in ramipril-treated animals. This may suggest that the actual level of kinins is finely tuned to the local metabolic demands. In this experimental model of cardiac hypertrophy. ACE inhibitors potentiate the actions of kinins and probably try to normalise endothelial cell function.

Efecto cardio y vasculoprotector de las kininas y los inhibidores de la enzima de conversión / Cardiovascular protective effect of kinins and converting enzyme inhibitors

ANTECEDENTES: Las kininas son oligopéptidos que generados localmente en corazón y vasos sanguíneos estimulan la producción endotelial de factores relajantes (prostaciclina, óxido nítrico y factor endotelial hiperpolarizante). El nivel de kininas depende de: a) la tasa de producción por kalikreínas y b) la tasa de destrucción por kininasas, entre las que se encuentra la enzima de conversión de angiotensina (CE). Los inhibidores de la enzima de conversión (CEI) empleados con notable éxito en el tratamiento de la hipertensión arterial y la insuficiencia cardíaca actuarían en parte prolongando la vida media del péptido. El objetivo de este trabajo es examinar: a) si los componentes del sistema kalikreína-kininógeno-kininas (SKKK) se liberan al perfusado en corazones aislados y perfundidos, b) la influencia de ramiprilat sobre la tasa de secreción de kininas y c) la acción biológica de las kininas en una preparación de vasos coronarios aislados. MATERIAL Y METODO: Los corazones de Ratas Wistar (300 ñ 30 gr) se aislaron y perfundieron en forma retrógrada con buffer Krebs-Henseleit equilibrado con 95 por ciento de oxígeno y 5 por ciento de anhídrido carbónico a un flujo constante (5 ml/min) mediante una bomba de perfusión Gilson. Kalikreína se midió por su capacidad de generar kininas; kininógeno, por una modificación de un método previamente descripto; y las kininas extraídas con un Set-Pack Cartridge, se dosaron por RIA. RESULTADOS: Los componentes del SKKK se forman y liberan continuamente en la preparación de corazón aislado (hasta 120 minutos). El pretratamiento con puromicina, un inhibidor de la síntesis proteica, descendió significativamente el release de kalikreína y kininógeno, confirmando la síntesis ex novo en el propio tejido cardíaco. El agregado del CEI, ramiprilat, incrementó significativamente el nivel de kininas en el perfusado. Kalikreína, per se, actuando sobre su sustrato en la propia pared del vaso libera kininas que relajan los vasos coronarios. Las kininas generadas permanentemente en la pared vascular en pequeñas cantidades se ponen de manifiesto en presencia de ramiprilat, relajando los vasos coronarios. El agregado de HOE 140, un inhibidor selectivo B2 de las kininas, bloquea el efecto relajante, confirmando que tanto la acción de las kalikreínas liberadas localmente como el efecto de los CEIs es mediado por kininas endógenas...

Efecto cardio y vasculoprotector de las kininas y los inhibidores de la enzima de conversión / Cardiovascular protective effect of kinins and converting enzyme inhibitors

ANTECEDENTES: Las kininas son oligopéptidos que generados localmente en corazón y vasos sanguíneos estimulan la producción endotelial de factores relajantes (prostaciclina, óxido nítrico y factor endotelial hiperpolarizante). El nivel de kininas depende de: a) la tasa de producción por kalikreínas y b) la tasa de destrucción por kininasas, entre las que se encuentra la enzima de conversión de angiotensina (CE). Los inhibidores de la enzima de conversión (CEI) empleados con notable éxito en el tratamiento de la hipertensión arterial y la insuficiencia cardíaca actuarían en parte prolongando la vida media del péptido. El objetivo de este trabajo es examinar: a) si los componentes del sistema kalikreína-kininógeno-kininas (SKKK) se liberan al perfusado en corazones aislados y perfundidos, b) la influencia de ramiprilat sobre la tasa de secreción de kininas y c) la acción biológica de las kininas en una preparación de vasos coronarios aislados. MATERIAL Y METODO: Los corazones de Ratas Wistar (300 ñ 30 gr) se aislaron y perfundieron en forma retrógrada con buffer Krebs-Henseleit equilibrado con 95 por ciento de oxígeno y 5 por ciento de anhídrido carbónico a un flujo constante (5 ml/min) mediante una bomba de perfusión Gilson. Kalikreína se midió por su capacidad de generar kininas; kininógeno, por una modificación de un método previamente descripto; y las kininas extraídas con un Set-Pack Cartridge, se dosaron por RIA. RESULTADOS: Los componentes del SKKK se forman y liberan continuamente en la preparación de corazón aislado (hasta 120 minutos). El pretratamiento con puromicina, un inhibidor de la síntesis proteica, descendió significativamente el release de kalikreína y kininógeno, confirmando la síntesis ex novo en el propio tejido cardíaco. El agregado del CEI, ramiprilat, incrementó significativamente el nivel de kininas en el perfusado. Kalikreína, per se, actuando sobre su sustrato en la propia pared del vaso libera kininas que relajan los vasos coronarios. Las kininas generadas permanentemente en la pared vascular en pequeñas cantidades se ponen de manifiesto en presencia de ramiprilat, relajando los vasos coronarios. El agregado de HOE 140, un inhibidor selectivo B2 de las kininas, bloquea el efecto relajante, confirmando que tanto la acción de las kalikreínas liberadas localmente como el efecto de los CEIs es mediado por kininas endógenas... (AU)

Ethanol production by a mixed culture of flocculent strains of Zymomonas mobilis and Saccharomyces sp.

Pure and mixed cultures of Zymomonas mobilis and Saccharomyces sp. were tested for the production of ethanol using sucrose as the carbon source. Both strains, isolated from spontaneously fermenting sugar-cane juice, are flocculent and alcohol-tolerant. The best results were obtained using a mixed culture, with a yield of 0.5 g ethanol/g sugar consumed and a volumetric productivity of 1.5 g ethanol l-1 h-1. No levan was produced even if a sucrose-based medium was used.

Construction of an integrative shuttle vector for Zymomonas mobilis.

An integrative shuttle vector, pZMOCP1, was constructed by ligating EcoRV digests of the plasmid cloning vector pBluescript and pZMP1, a cryptic plasmid of Zymomonas mobilis PROIMI A1. The 7.2-kb plasmid pZMOCP1 replicated in Escherichia coli and could also be transferred from this host by electroporation to Z. mobilis ATCC 29191. The transformants were selected by ampicillin resistance. The integrative characteristic was detected by hybridization in situ. The vector was stably maintained in Z. mobilis after 200 generations without selective pressure.

Multiple amino acids determine the DNA binding specificity of the Msx-1 homeodomain.

This study investigates the sequence features that contribute to the differential DNA binding properties of two divergent homeodomains, Msx-1 and HoxA3. We show that these homeodomains have overlapping, but nonidentical, DNA binding site preferences. We defined the amino acid residues that contribute to the observed differences in DNA binding specificity by producing a series of mutated polypeptides in which selected residues in Msx-1 were replaced with the corresponding ones in HoxA3. These analyses show that the DNA binding specificity of Msx-1 versus HoxA3 results from the cumulative action of multiple residues in all segments of the homeodomain (i.e., the N-terminal arm and helices I, II, and III). Therefore, substitutions of residues in both helix III and the N-terminal arm (but not in either segment alone) produced an Msx-1 polypeptide whose binding site preference was indistinguishable from that of HoxA3. Residues in helices I and II also influence DNA binding activity; these oppositely charged residues (e.g., lysine 19 and glutamate 30) may mediate ionic interactions between helices I and II which stabilize DNA binding by Msx-1. These findings demonstrate a critical interplay between residues in each homeodomain segment for appropriate conformation of the protein-DNA complex.

Transcriptional repression by Msx-1 does not require homeodomain DNA-binding sites.

This study investigates the transcriptional properties of Msx-1, a murine homeodomain protein which has been proposed to play a key role in regulating the differentiation and/or proliferation state of specific cell populations during embryogenesis. We show, using basal and activated transcription templates, that Msx-1 is a potent repressor of transcription and can function through both TATA-containing and TATA-less promoters. Moreover, repression in vivo and in vitro occurs in the absence of DNA-binding sites for the Msx-1 homeodomain. Utilizing a series of truncated Msx-1 polypeptides, we show that multiple regions of Msx-1 contribute to repression, and these are rich in alanine, glycine, and proline residues. When fused to a heterologous DNA-binding domain, both N- and C-terminal regions of Msx-1 retain repressor function, which is dependent upon the presence of the heterologous DNA-binding site. Moreover, a polypeptide consisting of the full-length Msx-1 fused to a heterologous DNA-binding domain is a more potent repressor than either the N- or C-terminal regions alone, and this fusion retains the ability to repress transcription in the absence of the heterologous DNA site. We further show that Msx-1 represses transcription in vitro in a purified reconstituted assay system and interacts with protein complexes composed of TBP and TFIIA (DA) and TBP, TFIIA, and TFIIB (DAB) in gel retardation assays, suggesting that the mechanism of repression is mediated through interaction(s) with a component(s) of the core transcription complex. We speculate that the repressor function of Msx-1 is critical for its proposed role in embryogenesis as a regulator of cellular differentiation.

Comparison of heated water-filled mattress and space-heated room with infant incubator in providing warmth to low birthweight newborns.

BACKGROUND: Prevention of excessive heat loss is fundamental to survival of low birthweight (LBW) newborns. The use of infant incubators (INC) is beyond the resources of developing countries, and the space-heated room (SHR) has been the only feasible means of providing thermal protection to LBW newborns. Recently a thermostatically controlled, heated, water-filled mattress (HWM) has been developed as a potentially simpler and affordable alternative. METHODS: In a neonatal care ward of a referral hospital in Addis Ababa, 62 < 1 week old newborns, weighing 1000-1999 g, who were well enough to breathe comfortably in room air and tolerate oral feeds, were randomly allocated to INC, HWM or SHR and followed for 3 weeks. The level of cold stress as assessed by core-to-skin temperature gradient and the rate of weight gain were the main outcome measures. RESULTS: The level of cold stress was lowest in the INC, intermediate in the HWM and highest in the SHR. Relative to the INC group, the HWM group exhibited a modest increase in the occurrence of clinically important hyperthermic or hypothermic deviations in core temperature (rate ratio (RR) = 2.3; 95% CI: 0.9, 5.6), and the SHR displayed a definite increase (RR = 4.0; 95% CI: 1.7, 9.3). During the first week, the rate of weight gain was highest in the INC group (3.6 g/kg/day), lowest in the SHR group (-2.3 g/kg/day, P < 0.05 versus INC) and intermediate in the HWM group (1.6 g/kg/day, P > 0.1 versus INC). CONCLUSION: Care in the SHR produced clinically significant thermal stresses and was associated with deficient early neonatal growth, but the use of HWM may constitute a feasible and clinically acceptable alternative in providing warmth to LBW newborns during the neonatal period.

Design of a "minimAl" homeodomain: the N-terminal arm modulates DNA binding affinity and stabilizes homeodomain structure.

This report investigates the sequence specificity requirements for homeodomain structure and DNA binding activity by the design and synthesis of a "minimAl" homeodomain (for minimalist design and alanine scanning mutagenesis) which contains the consensus residues and in which all nonconsensus residues have been replaced with alanine. The murine homeodomain Msx served as the prototype for the minimAl homeodomain, Ala-Msx. We show that Ala-Msx binds to DNA specifically, albeit with lower affinity than Msx. A derivative of the minimAl homeodomain, Ala-Msx(NT), which contains a native rather than an alanine-substituted N-terminal arm, has similar DNA binding affinity as Msx. We show that the native N-terminal arm stabilizes the tertiary structure of the minimAl homeodomain. Although Ala-Msx resembles a molten-globule protein, the structure of Ala-Msx(NT) is similar to Msx. The requirement for an intact N-terminal arm is not unique to the minimAl homeodomain, since the N-terminal arm also promotes high-affinity binding activity and appropriate tertiary structure of Msx. Therefore, the homeodomain "scaffold" consists of consensus residues, which are sufficient for DNA recognition, and nonconsensus residues in the N-terminal arm, which are required for optimal DNA binding affinity and appropriate tertiary structure. MinimAl design provides a powerful strategy to probe homeodomain structure and function. This approach should be of general utility to study the sequence specificity requirements for structure and function of other DNA-binding domains.

Hox proteins have different affinities for a consensus DNA site that correlate with the positions of their genes on the hox cluster.

The hox genes, members of a family of essential developmental regulators, have the intriguing property that their expression in the developing murine embryo is colinear with their chromosomal organization. Members of the hox gene family share a conserved DNA binding domain, termed the homeodomain, which mediates interactions of Hox proteins with DNA regulatory elements in the transcriptional control regions of target genes. In this study, we characterized the DNA binding properties of five representative members of the Hox family: HoxA5, HoxB4, HoxA7, HoxC8, and HoxB1. To facilitate a comparative analysis of their DNA binding properties, we produced the homeodomain regions of these Hox proteins in Escherichia coli and obtained highly purified polypeptides. We showed that these Hox proteins interact in vitro with a common consensus DNA site that contains the motif (C/G)TAATTG. We further showed that the Hox proteins recognize the consensus DNA site in vivo, as determined by their ability to activate transcription through this site in transient transfection assays. Although they interact optimally with the consensus DNA site, the Hox proteins exhibit subtle, but distinct, preferences for DNA sites that contain variations of the nucleotides within the consensus motif. In addition to their modest differences in DNA binding specificities, the Hox proteins also vary in their relative affinities for DNA. Intriguingly, their relative affinities correlate with the positions of their respective genes on the hox cluster. These findings suggest that subtle differences in DNA binding specificity combined with differences in DNA binding affinity constitute features of the "Hox code" that contribute to the selective functions of Hox proteins during murine embryogenesis.

DNA affinity cleaving analysis of homeodomain-DNA interaction: identification of homeodomain consensus sites in genomic DNA.

We have incorporated the DNA-cleaving moiety o-phenanthroline-copper at amino acid 10 of the Msx-1 homeodomain, and we have analyzed site-specific DNA cleavage by the resulting Msx-1 derivative. We show that amino acid 10 of the Msx-1 homeodomain is close to the 5' end of the consensus DNA site 5'-(C/G)TAATTG-3' in the Msx-1-DNA complex. Our results indicate that the orientation of the Msx-1 homeodomain relative to DNA is analogous to the orientation of the engrailed and Antennapedia homeodomains. We show further that DNA affinity cleaving permits identification of consensus DNA sites for Msx-1 in kilobase DNA substrates. The specificity of the approach enabled us to identify an Msx-1 consensus DNA site within the transcriptional control region of the developmental regulatory gene Wnt-1. We propose that incorporation of o-phenanthroline-copper at amino acid 10 of a homeodomain may provide a generalizable strategy to determine the orientation of a homeodomain relative to DNA and to identify homeodomain consensus DNA sites in genomic DNA.

Phosphorylation of the c-Fos transrepression domain by mitogen-activated protein kinase and 90-kDa ribosomal S6 kinase.

Phosphorylation of the C terminus of c-Fos has been implicated in serum response element-mediated repression of c-fos transcription after its induction by serum growth factors. The growth-regulated enzymes responsible for this phosphorylation in early G1 phase of the cell cycle and the sites of phosphorylation have not been identified. We now provide evidence that two growth-regulated, nucleus- and cytoplasm-localized protein kinases, 90-kDa ribosomal S6 kinase (RSK) and mitogen-activated protein kinase (MAP kinase), contribute to the serum-induced phosphorylation of c-Fos. The major phosphopeptides derived from biosynthetically labeled c-Fos correspond to phosphopeptides generated after phosphorylation of c-Fos in vitro with both RSK and MAP kinase. The phosphorylation sites identified for RSK (Ser-362) and MAP kinase (Ser-374) are in the transrepression domain. Cooperative phosphorylation at these sites by both enzymes was observed in vitro and reflected in vivo by the predominance of the peptide phosphorylated on both sites, as opposed to singly phosphorylated peptides. This study suggests a role for nuclear RSK and MAP kinase in modulating newly synthesized c-Fos phosphorylation and downstream signaling.

Dimerization and DNA binding alter phosphorylation of Fos and Jun.

Fos and Jun form dimeric complexes that bind to activator protein 1 (AP-1) DNA sequences and regulate gene expression. The levels of expression and activities of these proteins are regulated by a variety of extracellular stimuli. They are thought to function in nuclear signal transduction processes in many different cell types. The role of Fos and Jun in gene transcription is complex and may be regulated in several ways including association with different dimerization partners, interactions with other transcriptional factors, effects on DNA topology, and reduction/oxidation of a conserved cysteine residue in the DNA-binding domain. In addition, phosphorylation has been suggested to control the activity of Fos and Jun. Here we show that phosphorylation of Fos and Jun by several protein kinases is affected by dimerization and binding to DNA. Jun homodimers are phosphorylated efficiently by casein kinase II, whereas Fos-Jun heterodimers are not. DNA binding also reduces phosphorylation of Jun by casein kinase II, p34cdc2 (cdc2) kinase, and protein kinase C. Phosphorylation of Fos by cAMP-dependent protein kinase and cdc2 is relatively insensitive to dimerization and DNA binding, whereas phosphorylation of Fos and Jun by DNA-dependent protein kinase is dramatically stimulated by binding to the AP-1 site. These results imply that different protein kinases can distinguish among Fos and Jun proteins in the form of monomers, homodimers, and heterodimers and between DNA-bound and non-DNA-bound proteins. Thus, potentially, these different states of Fos and Jun can be recognized and regulated independently by phosphorylation.

The dental health of adults in an integrated urban development in Addis Ababa, Ethiopia.

Two hundred and forty-three Ethiopian adults (18 years-old and over) were examined for caries, periodontal disease, malocclusions and enamel opacities. These adults were the parents of children cared for by an independent charitable organisation, the Ethiopian Gemini Trust. The prevalence of dental caries was generally low with a mean DMFT for the sample of 2.7 (+/- 0.2) and a mean DMFS of 6.7 (+/- 0.6), although one adult had a DMFS of 62. A high proportion of the adults (83.5 per cent) had calculus, but only 2 per cent had deep pocketing. Twenty-three per cent of the adults had a malocclusion and for 6 per cent of these this was moderate to severe. The most prevalent enamel defects were hypoplasias and diffuse opacities with 22 per cent of adults having one or more index teeth affected. Access to dental services was virtually non-existent as judged by the clinical status of these adults.

"Killer" canine removal and its sequelae in Addis Ababa.

Children and adolescents, aged 2 to 18 years, from 300 poor families in Addis Ababa were examined to determine the prevalence of the traditional practice of primary canine tooth removal. Fifteen percent of the primary canine teeth were found to have been affected, and 7% of the permanent canines had been damaged by this practice. A questionnaire to a subset of 40 families revealed some of the reasons that this procedure is still carried out, in spite of the considerable associated morbidity.

Nucleotides flanking a conserved TAAT core dictate the DNA binding specificity of three murine homeodomain proteins.

Murine homeobox genes play a fundamental role in directing embryogenesis by controlling gene expression during development. The homeobox encodes a DNA binding domain (the homeodomain) which presumably mediates interactions of homeodomain proteins with specific DNA sites in the control regions of target genes. However, the bases for these selective DNA-protein interactions are not well defined. In this report, we have characterized the DNA binding specificities of three murine homeodomain proteins, Hox 7.1, Hox 1.5, and En-1. We have identified optimal DNA binding sites for each of these proteins by using a random oligonucleotide selection strategy. Comparison of the sequences of the selected binding sites predicted a common consensus site that contained the motif (C/G)TAATTG. The TAAT core was essential for DNA binding activity, and the nucleotides flanking this core directed binding specificity. Whereas variations in the nucleotides flanking the 5' side of the TAAT core produced modest alterations in binding activity for all three proteins, perturbations of the nucleotides directly 3' of the core distinguished the binding specificity of Hox 1.5 from those of Hox 7.1 and En-1. These differences in binding activity reflected differences in the dissociation rates rather than the equilibrium constants of the protein-DNA complexes. Differences in DNA binding specificities observed in vitro may contribute to selective interactions of homeodomain proteins with potential binding sites in the control regions of target genes.