Elevation of neuronal expression of NAIP reduces ischemic damage in the rat hippocampus.

We show here that transient forebrain ischemia selectively elevates levels of neuronal apoptosis inhibitory protein (NAIP) in rat neurons that are resistant to the injurious effects of this treatment. This observation suggests that increasing NAIP levels may confer protection against ischemic cell death. Consistent with this proposal, we demonstrate that two other treatments that increase neuronal NAIP levels, systemic administration of the bacterial alkaloid K252a and intracerebral injection of an adenovirus vector capable of overexpressing NAIP in vivo, reduce ischemic damage in the rat hippocampus. Taken together, these findings suggest that NAIP may play a key role in conferring resistance to ischemic damage and that treatments that elevate neuronal levels of this antiapoptotic protein may have utility in the treatment of stroke.

Identification of the transactivation domain of the human FHL3.

Four and a half LIM domain protein 3 (FHL3) has the transactivation and repressor activity, and plays important roles in regulating the expression levels of various genes. In this study, FHL3 was proved to possess the auto-activation ability when constructed into the pGBKT7 plasmid (a GALA DNA-binding domain (BD) cloning vector of the yeast two-hybrid system) and transformed into yeast Y190 cells. To determine the transactivation domain of FHL3, five mutants were constructed by sequentially deleting each LIM domain of FHL3 and then inserting them into the pGBKT7 plasmid. After being transformed into yeast Y190 cells, expression levels of the mutants were identified by Western blot analysis. The beta-galactosidase assay showed that the mutant without the fourth LIM domain (LIM4) lost the auto-activation ability. Further investigations on the mutants with deleted first or second zinc finger of LIM4 confirmed that the second zinc finger motif in LIM4 was responsible for the auto-activation of FHL3.

Neuroanatomical and pharmacological assessment of Fos expression induced in the rat brain by the phosphodiesterase-4 inhibitor 6-(4-pyridylmethyl)-8-(3-nitrophenyl) quinoline.

A major obstacle in the therapeutic development of phosphodiesterase-4 (PDE4) inhibitors is the production of adverse side effects such as nausea and vomiting. Immunohistochemical detection of Fos-like immunoreactivity (FLI) was used to address the neuroanatomical basis for the pharmacological actions of PDE4 inhibitors. The potent and selective PDE4 inhibitors 6-(4-pyridylmethyl)-8-(3-nitrophenyl) quinoline (PMNPQ) and rolipram elevated FLI in brain regions potentially relevant to the anti-depressant and emetic effects of PDE4 inhibition. PMNPQ and rolipram elevated FLI in the locus coeruleus, habenula, paraventricular nucleus of the thalamus, amygdala and nucleus accumbens, all structures with strong limbic connectivity implicated in arousal, memory and affective aspects of behaviour. Consistent with the emetic effects of PDE4 inhibitors such as PMNPQ and rolipram, these compounds elevated FLI in caudal brainstem nuclei such as the area postrema and nucleus of the solitary tract. Administration of the NK(1) antagonist RP 67580 prior to PMNPQ reversed increases in FLI produced by PMNPQ in these regions. RP 67580 did not, however, reduce PMNPQ-induced FLI in limbic structures. These findings suggest that PDE4 inhibitors produce emesis by increasing NK(1) receptor activation in the AP/NTS and implicate brain regions associated with reward and mood such as the amygdala, paraventricular nucleus of the thalamus, habenula and nucleus accumbens in the anti-depressant activity of such compounds.

Distribution of neuronal apoptosis inhibitory protein-like immunoreactivity in the rat central nervous system.

We have recently shown that spinal muscular atrophy (SMA), an autosomal recessive disorder characterized by motor neuron loss, is associated with deletion of a gene that encodes the neuronal apoptosis inhibitory protein (NAIP). In the present study, we have examined the distribution of NAIP-like immunoreactivity (NAIP-LI) in the rat central nervous system (CNS) by using an affinity-purified polyclonal antibody against NAIP. In the forebrain, immunoreactive neurons were detected in the cortex, the hippocampus (pyramidal cells, dentate granule cells, and interneurons), the striatum (cholinergic interneurons), the basal forebrain (ventral pallidum, medial septal nucleus, and diagonal band), the thalamus (lateral and ventral nuclei), the habenula, the globus pallidus, and the entopenduncular nucleus. In the midbrain, NAIP-LI was located primarily within neurons of the red nucleus, the substantia nigra pars compacta, the oculomotor nucleus, and the trochlear nucleus. In the brainstem, neurons containing NAIP-LI were observed in cranial nerve nuclei (trigeminal, facial, vestibular, cochlear, vagus, and hypoglossal nerves) and in relay nuclei (pontine, olivary, lateral reticular, cuneate, gracile nucleus, and locus coeruleus). In the cerebellum, NAIP-LI was found within both Purkinje and nuclear cells (interposed and lateral nuclei). Finally, within the spinal cord, NAIP-LI was detected in Clarke's column and in motor neurons. Taken together, these results indicate that NAIP-LI is distributed broadly in the CNS. However, high levels of NAIP-LI were restricted to those neuronal populations that have been reported to degenerate in SMA. This anatomical correspondence provides additional evidence for NAIP involvement in the neurodegeneration observed in acute SMA.

Presence of antibodies to heat stress proteins and its possible significance in workers exposed to high temperature and carbon monoxide.

Antibodies to the ubiquitous group of stress proteins known as heat shock proteins (Hsps) have been found to be associated with a number of diseases in humans. Hsps are known to be induced by certain xenobiotics, some of which are common in the working environment. The biological significance of the presence of such autoantibodies is presently unclear. In the present study, we used immunoblotting to investigate the presence of antibodies against the different stress proteins, Hsp27, Hsp60, Hsp71, Hsc (heat shock cognate) 73 and Hsp89 alpha and beta in groups of workers exposed to high temperature or carbon monoxide. These data were related to a detailed clinical evaluation and to various laboratory measurements including electrocardiogram (ECG), B echogram, white blood cell counts and typing, the activity of alanine aminotransferase (ALT), acid phosphatase (ACP) and alkaline phosphatase (ALP) and lymphocyte DNA damage. Antibodies to Hsp27 and Hsp71 were found more frequently in the high temperature and carbon monoxide-exposed groups than in controls (P < 0.05). The carbon monoxide-exposed group showed the highest incidence of anti-Hsp antibodies. Anti-Hsp60 antibodies were only detected in workers exposed to high temperature or carbon monoxide. The percentage of workers with abnormal ECG, B echogram changes and displaying hepatitis B antigen (HBsAg) was higher in the carbon monoxide group than in the control group (P < 0.05). There was a significant increase in the activity of ALT in the high temperature and carbon monoxide groups and in the activities of ACP and ALP in the carbon monoxide group (P < 0.05). The extent of DNA damage measured in lymphocytes was higher in workers from the high temperature and carbon monoxide-exposed groups. We suggest that the increased frequency of antibodies to Hsps is the result of these damages of the release of denatured Hsps and of a decrease in the phagocytic ability of macrophages in these workers. The data gathered in the present study show a statistical relation between the occurrence of antibodies against Hsps and the frequency of health problems in workers and suggest a potential role for the antibodies as useful biomarkers to assess whether workers are experiencing environmental stress.

Effect of chemoradiotherapy and neoadjuvant chemoradiotherapy in resectable pancreatic cancer: a systematic review and meta-analysis.

OBJECTIVE: Controversy remains existed whether chemoradiotherapy (CRT), especially neoadjuvant chemoradiotherapy (neoadjuvant CRT) achieves a significant benefit in resectable pancreatic cancer (PC) treatment. In this meta-analysis, we aimed to clarify the benefits of CRT and neoadjuvant CRT in resectable PC. METHODS: Eligible trials were identified from MEDLINE, EMBASE, Cochrane center, China National Knowledge Internet and Wanfang database since their inception to July 31, 2013. Only patients with resectable PC, who underwent tumor resection and received CRT and/or neoadjuvant CRT, were enrolled. The treatment outcomes were overall survival (OS) and progression-free survival (PFS). Hazard ratio (HR) with a 95% confidence interval (CI) was used to measure the pooled effect according to a fixed-effects model. The statistical heterogeneity between trials was detected by χ(2) and I (2) test. Sensitivity analyses were also carried out. RESULTS: A total of 28 studies were identified as relevant, but only 17 studies with a total of 3,088 patients were included in the comparison between CRT versus non-CRT, and a total number of three studies with 189 patients included in the comparison between neoadjuvant CRT versus postoperative CRT. The comparison between CRT and non-CRT showed that the overall pooled HR for death was 0.96 (95% CI 0.89-1.03; P = 0.28). The HR for progress was 0.83 (95% CI 0.68-1.03, P = 0.09). Comparison between neoadjuvant CRT and adjuvant CRT revealed a pooled HR of 0.93 (95% CI 0.69-1.25; P = 0.62). CONCLUSIONS: This meta-analysis showed that CRT showed no significant effect on OS and PFS when compared to non-CRT. Neoadjuvant CRT showed no significant effect over postoperative adjuvant CRT.

Sequence dependence of energy transfer in DNA oligonucleotides.

The sequence, temperature, concentration, and solvent dependence of singlet energy transfer from normal DNA bases to the 2-aminopurine base in synthesized DNA oligomers were investigated by optical spectroscopy. Transfer was shown directly by a variable fluorescence excitation band at 260-280 nm. Adenine (A) is the most efficient energy donor by an order of magnitude. Stacks of A adjacent to 2AP act as an antenna for 2AP excitation. An interposed G, C, or T base between A and 2AP effectively blocks transfer from A to 2AP. Base stacking facilitates transfer, while base pairing reduces energy transfer slightly. The efficiency is differentially temperature dependent in single- and double-stranded oligomers and is highest below 0 degrees C in samples measured. An efficiency transition occurs well below the melting transition of a double-stranded decamer. The transfer efficiency in the duplex decamer d(CTGA[2AP]TTCAG)(2) is moderately dependent on the sample and salt concentration and is solvent dependent. Transfer at physiological temperature over more than a few bases is improbable, except along consecutive A's, indicating that singlet energy transfer is not a major factor in the localization of UV damage in DNA. These results have features in common with recently observed electron transfer from 2AP to G in oligonucleotides.

[DNA methylation of monohalogenated methanes of F344 rats]. / DNA-Methylierung von monohalogenierten Methanen in F-344 Ratten.

Monohalogenated methanes (methyl chloride, methyl bromide and methyl iodide) are mutagenic and carcinogenic. The possible mechanism of these effects, DNA methylation, was studied. DNA adducts from organs of F344 rats exposed to these chemicals were separated and identified with high performance liquid chromatography (HPLC) and gas-chromatography/mass-spectrometry (GC/MS). DNA adducts, 7-methylguanine (7-MeG) and O6-Methylguanine(0(6)-MeG), incorporation of 14C into de novo synthesis of nucleobases could be observed in enzymatic DNA hydrolysates by HPLC and determination of the radioactivity in the fractions. The formation of DNA adducts in the studied organs was only quantitatively different. The formation of O6-MeG was further proved by analysing the acidic hydrolysates using HPLC with non-radioactive O6-MeG as internal standard. 7-MeG and 3-MeA were identified with GC/MS analysis.

The combined effects of high temperature and carbon monoxide on heat stress response.

In this study, we have examined the effects of exposure to high temperature, carbon monoxide or a combination of both conditions in a model system, the rat and in industrial workers. In the rat liver, HSP70 mRNA and HSP70 synthesis were measured by dot hybridization and western blot. The results showed that after a heat stress HSP70 mRNA and its product, HSP70 increased significantly and there was a synergism in the combined effects of high temperature and carbon monoxide exposure on the induction of HSP70 mRNA and HSP70 synthesis. Heat played a major role in this induction. The presence of antibodies to human HSP27, HSP60, HSP70, HSC73, HSP89 alpha and beta in workers exposed to heat, carbon monoxide was also measured by western blot using purified HSPs as antigens. Plasma free amino acids were measured in the same group of workers. The incidence of antibodies to HSP27 and HSP70 was significantly higher in the workers working in an environment with extreme heat, and high carbon monoxide emission than in a control group. The carbon monoxide exposed group showed the highest incidence of antibodies to HSPs. Although our previous results indicated that workers had an insufficient protein intake, plasma free amino acids tended to increase, especially in methionine and tryptophan two kinds of amino acids which are absent from the main stress protein, HSP70. These results suggest that the major problems that these workers may face are how to facilitate the use of plasma free amino acids and reduce the inhibition of synthesis of normal proteins when they are exposed to occupational harmful factors. These results also add new information on the measurement of HSPs as a potential biomonitor to assess whether organisms are experiencing metabolic stress within their environment.

A study of primary- and re-infection with hepatitis C virus in blood transfusion recipients.

A nested polymerase chain reaction was used to assess viraemia in blood transfusion recipients with no serological evidence of hepatitis C virus (HCV) infection (naive recipients) and in recipients with prior or existing HCV infection (infected recipients), who were transfused with HCV-positive blood. In 10 hepatitis cases in naive recipients, defined as primary infection, nine showed clinical hepatitis, and one was sub-clinical; the time between transfusion and elevation of alanine aminotransferase (ALT) levels was 15-60 days (37.9 +/- 13.9). All 10 naive recipients showed abnormal ALT, and 10/10 and 7/10 were persistently positive for anti-HCV and HCV-RNA, respectively, for more than 1 year. Similarly, in five cases in previously infected recipients, defined as re-infection, 4/5 showed clinical hepatitis, the time to elevation of ALT was 30-46 days (34.8 +/- 6.4), and 5/5 and 3/5 were persistently positive for anti-HCV and HCV-RNA, respectively, for more than 1 year. All five infected recipients showed abnormal ALT. In conclusion, there was no significant difference (P = 0.05) in the frequency of the markers of infection resulting from primary or re-infection with HCV, suggesting that primary infection fails to induce a protective immune response.