Characterization of the biological conversion of naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene in direct micellar systems

The whole cell biological conversion of naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene by the E. coli JM109(pPS1778) recombinant strain carrying the naphthalene dioxygenase and regulatory genes cloned from Pseudomonas fluorescens N3 in micellar systems has been investigated using biochemical and chemico-physical techniques. Reverse and direct micellar systems have been tested. Non-ionic surfactants (Tween and Triton X series) were found not to inhibit either the growth of the bacteria and the expression of the hydroxylating dioxygenase enzyme in such systems and were utilized in order to speed up the naphthalene conversion by increasing its solubility and also its bioavailability. The phase behavior of the direct micellar system was characterized through light scattering and other chemico-physical techniques. Further addition of isopropyl-palmitate 1–2% v/v to the micellar systems resulted in an increase of the apparent substrate concentration in solution and particularly its bioavailability thus allowing faster catalytic conversions resulting in an increase in productivity for the process. Since the cis-dihydrodiols are acquiring considerable potential as chiral pool synthons in asymmetric synthesis for a variety of industrial processes, possible applications for efficient small and large-scale production of such compounds are discussed.     

Chiral arylpyrrolidinols: preparation and biological profile

The preparation and biological evaluation of a new class of arylpyrrolidinols is reported. The antinociceptive activity was evaluated in vivo with the hot plate test (HPT) and formalin test (FT), excluding any involvement on motor coordination with the rota-rod test (RRT). The nociceptive behavior in the late phase of FT (representative of chronic pain) suggests an involvement of the antiinflammatory process and it is clearly influenced by the stereochemical features, being the eutomer of phenylpyrrolidinols, the (2R,3S) enantiomer. Despite this, a specific mechanism of action is not yet clarified.     

Enzymatic synthesis of 12-ketoursodeoxycholic acid from dehydrocholic acid in a membrane reactor

Summary Dehydrocholic acid (3,7,12-trioxo-5-cholanic acid) (0.5% concentration) was completely and selectively reduced to 12-ketoursodeoxycholic acid (3, 7-dihydroxy-12-oxo- 5-cholanic acid) in a membrane reactor by means of 3-hydroxysteroid dehydrogenase and 7-hydroxysteroid dehydrogenase. Coenzyme regeneration was carried out with the glucose-glucose dehydrogenase system.     

Neonatal rat hepatocytes in primary tissue culture free from density-dependent regulation of growth

Summary Studies employing [³H]thymidine and radioautography as well as colchicine and Feulgen staining of DNA showed that up to 19-fold increases in the degree of cell crowding in vitro, i.e. from 1.45 to 27.55×10⁴ cells per specimen, did not change the rates of entry into DNA synthesis and mitosis of cultivated primary neonatal rat hepatocytes.     

Cognitive Modulation of Psychophysical,Respiratory and Autonomic Responses to Cold Pressor Test

In healthy subjects with high hypnotisability (highs) under hypnosis, subjectively effective suggestions for analgesia abolish the increases in blood pressure associated with cold pressor test (cpt) by reducing the peripheral vascular resistance. The aim of the present study was to investigate the effects of the suggestions of analgesia on the responses to cpt in healthy highs (n = 22) and in low hypnotisable participants (lows, n = 22) out of hypnosis. Cpt was administered without (CPT) and with suggestions for analgesia (CPT+AN). Psychophysical (pain intensity, pain threshold, cpt duration (time of immersion) and pain tolerance, defined as the difference between cpt duration and pain threshold), respiratory (amplitude and frequency) and autonomic variables (tonic skin conductance, mean RR interval (RR = 1/heart rate), blood pressure, skin blood flow) were studied. The suggestions for analgesia increased cpt duration and RR in both groups, but decreased pain intensity and enhanced pain threshold only in highs; in both groups they did not modulate systolic blood pressure, tonic skin conductance and skin blood flow; thus, increased parasympathetic activity appears responsible for the heart rate reduction induced by suggestions in both groups. In conclusion, our findings show that suggestions modulate pain experience differentially in highs and lows, and are partially effective also in lows. We hypothesize that the mechanisms responsible for the efficacy of suggestions in healthy lows may be involved also in their efficacy in chronic pain patients with low hypnotisability.     

Scintillometric determination of DNA repair in human cell lines: A critical appraisal

The ability of a variety of chemical and physical agents to stimulate DNA repair synthesis in human cell cultures was tested by a simplified scintillometric procedure, with the use of hydroxyurea (HU) to suppress DNA replicative synthesis. After incubation with [³H]thymidine, the radioactivity incorporated in to DNA was determined in controls (C) and treated (T) cultures and in the corresponding HU series (C_HU, T_HU). The ratios T_HU/C_HU and T_HU/T:C_HU/C, indicating absolute and relative increases of DNA radioactivity, were calculated. When both ratios were significantly higher than 1, they were taken as indices of DNA repair stimulation, whereas, no stimulation in inferred when both of them are ?1. The scintillometric estimate of DNA repair was always in agreement with the autoradiographic observations, so that the procedure adopted can be used as a rapid test for screening investigations.Agents giving a relative but no an absolute increase of DNA radioactivity are generally not inducers of repair synthesis as estimated by autoradiography. However, the same scintillometric results are also occasionally observed with DNA repair inducers, such as methyl methanesulphonate (MMS) and ethyl methanesulphonate (EMS), owing to alterations of thymidine pool radioactivity. These chemicals, besides affecting the levels of labelled precursors in the intracellular pool in the T series, differently modified the increase of pool radioactivity which is a regular effect of HU. With such chemicals, DNA repair synthesis can be detected only after normalization of th DNA radioactivity on the basis of pool alterations.The quantitative value of the autoradiographic estimate of DNA repair is also affected by the changes in the radioactivity of the thymidine pool although autoradiography retains its qualitative value.Dimethylnitrosamine, mitomycin C and potassium dichromate, described by other authors as inducers of DNA repair, also gave negative results by the scintillometric procedure after normalization of DNA radioactivities. However, in our hands, these agents were unable to stimulated repair synthesis, according to the results of autoradiography and isopynic centrifugation.The proposed scintillometric procedure is effective in indicating false negative inducers of DNA repair, not giving rise to false positives.     

A Multilocus Genetic Study in a Cohort of Italian SLE Patients Confirms the Association with STAT4 Gene and Describes a New Association with HCP5 Gene

Background

Systemic lupus erythematosus (SLE) is an autoimmune disease with complex pathogenesis in which genes and environmental factors are involved. We aimed at analyzing previously identified loci associated with SLE or with other autoimmune and/or inflammatory disorders (STAT4, IL10, IL23R, IRAK1, PSORS1C1, HCP5, MIR146a, PTPN2, ERAP1, ATG16L1, IRGM) in a sample of Italian SLE patients in order to verify or confirm their possible involvement and relative contribution in the disease.

Materials and methods

Two hundred thirty-nine consecutive SLE patients and 278 matched healthy controls were enrolled. Study protocol included complete physical examination, and clinical and laboratory data collection. Nineteen polymorphisms were genotyped by allelic discrimination assays. A case-control association study and a genotype-phenotype correlation were performed.

Results

STAT4 was the most associated gene [P = 3×10⁻⁷, OR = 2.13 (95% CI: 1.59–2.85)]. IL10 confirmed its association with SLE [rs3024505: P = 0.02, OR = 1.52 (95% CI: 1.07–2.16)]. We describe a novel significant association between HCP5 locus and SLE susceptibility [rs3099844: P = 0.01, OR = 2.06 (95% CI: 1.18–3.6)]. The genotype/phenotype correlation analysis showed several associations including a higher risk to develop pericarditis with STAT4, and an association between HCP5 rs3099844 and anti-Ro/SSA antibodies.

Conclusions

STAT4 and IL10 confirm their association with SLE. We found that some SNPs in PSORS1C1, ATG16L1, IL23R, PTPN2 and MIR146a genes can determine particular disease phenotypes. HCP5 rs3099844 is associated with SLE and with anti-Ro/SSA. This polymorphism has been previously found associated with cardiac manifestations of SLE, a condition related with anti-Ro/SSA antibodies. Thus, our results may provide new insights into SLE pathogenesis.     

Environmental Enrichment Extends Photoreceptor Survival and Visual Function in a Mouse Model of Retinitis Pigmentosa

Slow, progressive rod degeneration followed by cone death leading to blindness is the pathological signature of all forms of human retinitis pigmentosa (RP). Therapeutic schemes based on intraocular delivery of neuroprotective agents prolong the lifetime of photoreceptors and have reached the stage of clinical trial. The success of these approaches depends upon optimization of chronic supply and appropriate combination of factors. Environmental enrichment (EE), a novel neuroprotective strategy based on enhanced motor, sensory and social stimulation, has already been shown to exert beneficial effects in animal models of various disorders of the CNS, including Alzheimer and Huntington disease. Here we report the results of prolonged exposure of rd10 mice, a mutant strain undergoing progressive photoreceptor degeneration mimicking human RP, to such an enriched environment from birth. By means of microscopy of retinal tissue, electrophysiological recordings, visual behaviour assessment and molecular analysis, we show that EE considerably preserves retinal morphology and physiology as well as visual perception over time in rd10 mutant mice. We find that protective effects of EE are accompanied by increased expression of retinal mRNAs for CNTF and mTOR, both factors known as instrumental to photoreceptor survival. Compared to other rescue approaches used in similar animal models, EE is highly effective, minimally invasive and results into a long-lasting retinal protection. These results open novel perspectives of research pointing to environmental strategies as useful tools to extend photoreceptor survival.     

A hormonal role for endogenous opiate alkaloids: vascular tissues

The distribution of morphine-containing cells in the central nervous system, adrenal gland, and its presence in blood may serve to demonstrate that this signal molecule can act as a hormone besides its role in cell-to-cell signaling within the brain. This speculative review is the result of a literature evaluation with an emphasis on studies from our laboratory. Opioid peptides and opiate alkaloids have been found to influence cardiac and vascular function. They have also been reported to promote ischemic preconditioning protection in the heart. Given the presence of morphine and the novel mu(3) opiate receptor on vascular endothelial cells, including cardiac and vascular endothelial cells in the median eminence, it would appear that endogenous opiate alkaloids are involved in modulating cardiac function, possible at the hormonal level. This peripheral target tissue, via nitric oxide coupling to mu opiate receptors, may serve to down regulate the excitability of this tissue given the heart's high performance state as compared to that of the saphenous vein, a passive resistance conduit. With this in mind, morphine and other endogenous opiate alkaloids may function as a hormone.