Fast method for the determination of short-chain-length polyhydroxyalkanoates (scl-PHAs) in bacterial samples by In Vial-Thermolysis (IVT).

A new method based on the GC-MS analysis of thermolysis products obtained by treating bacterial samples at a high temperature (above 270°C) has been developed. This method, here named "In-Vial-Thermolysis" (IVT), allowed for the simultaneous determination of short-chain-length polyhydroxyalkanoates (scl-PHA) content and composition. The method was applied to both single strains and microbial mixed cultures (MMC) fed with different carbon sources. The IVT procedure provided similar analytical performances compared to previous Py-GC-MS and Py-GC-FID methods, suggesting a similar application for PHA quantitation in bacterial cells. Results from the IVT procedure and the traditional methanolysis method were compared; the correlation between the two datasets was fit for the purpose, giving a R2 of 0.975. In search of further simplification, the rationale of IVT was exploited for the development of a "field method" based on the titration of thermolyzed samples with sodium hydrogen carbonate to quantify PHA inside bacterial cells. The accuracy of the IVT method was fit for the purpose. These results lead to the possibility for the on-line measurement of PHA productivity. Moreover, they allow for the fast and inexpensive quantification/characterization of PHA for biotechnological process control, as well as investigation over various bacterial communities and/or feeding strategies.

Cation-Selective Channel Regulated by Anions According to Their Hofmeister Ranking.

Specificity of small ions, the Hofmeister ranking, is long-known and has many applications including medicine. Yet it evades consistent theoretical description. Here we study the effect of Hofmeister anions on gramicidin A channels in lipid membranes. Counterintuitively, we find that conductance of this perfectly cation-selective channel increases about two-fold in the H2 PO4-

A therapeutic dose of FK506 does not affect collagen turnover pathways in healthy human gingival fibroblasts.

We previously demonstrated that a high dose of tacrolimus (1 mumol/L) induced expression of matrix metalloproteinase (MMP) proteins in human cultured gingival fibroblasts, suggesting a molecular mechanism maintaining gingival collagen homeostasis in tacrolimus-treated patients. Herein we have analyzed whether the effect on collagen turnover might be influenced by a therapeutic tacrolimus dose. Human gingival fibroblasts were incubated for 72 hours with 10 nmol/L, 100 nmol/L, and 1 mumol/L tacrolimus, or left untreated (CT). Collagen type I and III (COL-I, COL-III), lysyl hydroxylase 2b (LH2b), MMP-1 and -2, tissue inhibitor of MMP-1 and transforming growth factor-beta1 (TGF-beta1) mRNA levels were assayed by reverse-transcriptase polymerase chain reaction, collagen protein levels by dot blot, and MMP activity by sodium dodecyl sulfate zymography. Tacrolimus did not affect COL-I, COL-III, or MMP gene expression, while LH2b and TGF-beta1 tended to be down-regulated after 1 mumol/L FK506. Conversely, protein levels of MMP-1 (P = NS) and MMP-2 (P < .05 vs CT, 10 nmol/L, 100 nmol/L) were up-regulated after 1 mumol/L tacrolimus. Our findings confirmed that a high dose of tacrolimus does not induce interstitial collagen overexpression by gingival fibroblasts and induces up-regulation of MMPs protein levels. Interestingly, at doses corresponding to whole blood trough levels, tacrolimus did not exert any evident effect on collagen turnover pathways, suggesting that tacrolimus is likely to not affect collagen homeostasis in the gingival connective tissue compartment of FK506-immunosuppressed subjects. This effect did not seem to be dose-dependent.

Effect of a topical treatment in organotypic culture of human breast skin after exposure to gamma-rays.

The early radiation of epidermal reactions can lead to healing of the lesion or radiation necrosis. There is no general agreement for either the prevention and/or treatment of radiation skin response, also as little is known about the immediate phases of this phenomenon. We investigated the early effects exerted by Healing and Wound Emulsion (HWE) on human skin response after ionizing radiation. Epidermal morphology, Heat Shock Protein (HSP) 70, and Transforming Growth Factor-beta1 (TGF-beta1) gene expression were investigated in organotypic human skin cultures undergoing a double dose of gamma-rays (2 Gy). HSP70 gene expression tended to be induced in the HWE group 6 hours after cream administration and was significantly up-regulated after 48 hours, when epidermal morphological alterations were evident. TGF-beta1 seems not affected in cream treated samples. HWE may stimulate skin to mount an early defensive response against damage induced by gamma rays.

[The outpatient care in the Latium region (Italy) in 2001]. / L'assistenza specialistica ambulatoriale nella regione Lazio nell'anno 2001.

The Region of Latium has been operating an Outpatient Care Information System (SIAS) since 1997 to monitor the supply of outpatient care in a territory with a population of over five million. The present work has the aim of describing the outpatient care in the region, in terms of number of facilities involved by category (public and private, operating in the regional public health system) and volume of procedures rendered to residents in 2001. Of the 971 outpatient facilities operating in hospitals and elsewhere--37% state managed and 67% private--distributed in a non-uniform manner throughout the region, the majority is concentrated in the city of Rome, which by itself accounts for 49% of its total amount of facilities, and in a lesser measure in the other provincial capitals (Viterbo, Rieti, Frosinone, Latina). In 2001, 71 million procedures were performed, comprising 17 million prescriptions, for an economic value of over 400 million Euros. The three specialties of greatest use were Lab Analysis, Physical Therapy and Rehabilitation, and Radiology, making up 88% of the total outpatient procedures performed within the precinct of the regional health service, in respective measures of 57%, 27%, and 4%. It is noted that the public facilities are prevalently polyspecialistic while a great number of private facilities are monospecialistic and perform procedures almost exclusively (96%) in the three specialties of greatest use. The other specialties which receive notable use are Cardiology, Eye Care, Orthopedics and Neurology. In general, the greater the number of facilities there are in either the public or private sector, the greater the level of activity in terms of procedures performed, with the exception of the area of Physical Therapy and Rehabilitation where the correlation is inversely proportioned; in fact, for this specialty the public facilities, which are represented in a much greater number throughout the region, supply only 7% of the volume of activity.

Protective effects of delapril combined with indapamide or hydrochlorothiazide in spontaneously hypertensive stroke-prone rats: a comparative dose-response analysis.

In previous articles, we have shown that the combination of the angiotensin-converting enzyme (ACE) inhibitor delapril (12 mg/kg/day) and the diuretic indapamide (1 mg/kg/ day) was able to prolong the life span significantly in salt-loaded stroke-prone spontaneously hypertensive rats (SHRsp). Because this finding was partly dependent on the antagonism of salt-loading effects by pharmacologic induction of diuresis, which prevented any increase in blood pressure values, we decided to evaluate whether lower doses of the combination could be equally protective without changing the progression of hypertension. Thus, we studied several treatments with progressively lower doses of delapril (6, 3, or 1.5 mg/kg/day) combined with indapamide (0.5, 0.25, or 0.125 mg/kg/day) or hydrochlorothiazide (2.5, 1.25, or 0.625 mg/kg/day) in salt-loaded SHRsp. Salt-loaded untreated animals were considered to be the control group. In agreement with previous experiments, control rats reached 50% mortality approximately 7 weeks after the beginning of salt loading. The combination of delapril and hydrochlorothiazide at the two lowest doses was not able to delay animal death significantly, whereas treatment with delapril and indapamide at the lowest dose was effective (50% survival rate, 15 weeks). The groups treated with the highest dose of delapril and hydrochlorothiazide or with the intermediate or highest dose of delapril and indapamide did not reach 50% mortality by the end of the experiment, at 44 weeks of treatment (i.e., when animals reached age 1 year). Only the highest delapril and indapamide doses were able to increase diuresis, but for a relatively short period. None of the treatments was able to lower or control blood pressure levels adequately. Therefore, blood pressure levels by themselves were not predictive of rat mortality. In contrast, the maximal value of proteinuria in the weeks preceding death was inversely correlated with the survival time. In conclusion, this study shows that low doses of an ACE inhibitor in combination with a diuretic can be effectively protective in a model of severe hypertension, independent of any change in blood pressure levels.

Changes in nicotinic acetylcholine receptor subunit mRNAs and nicotinic binding in spontaneously hypertensive stroke prone rats.

We have studied nicotinic acetylcholine receptors in spontaneously hypertensive 'stroke prone' (SHsp) rats. We found a significant decrease in 125I-alpha-bungarotoxin binding and alpha7 subunit mRNA levels in cortical areas of the SHsp rats with respect to Wistar Kyoto (WKy) normotensive rats. Antihypertensive drug treatment counteracted these changes in cerebral cortex but not in hippocampus. No significant change was instead found in [3H]-epibatidine binding and alpha4 and beta2 subunit mRNA levels. SHsp rats showed decreased latency at the active avoidance test and transiently increased threshold at both hot-plate and tail-flick tests in comparison with WKy rats. None of these behavioral parameters was correlated with 125I-alpha-bungarotoxin binding in cortical areas. In conclusion, present data show a preferential impairment of alpha-bungarotoxin sensitive nAChRs in SHsp rats.

The emergence of the volume transmission concept.

Interneuronal communication in the central nervous system (CNS) have always been of basic importance for theories on the cerebral morphofunctional architecture. Our group has proposed that intercellular communication in the brain can be grouped into 2 broad classes based on some general features of the transmission: wiring (WT) and volume (VT) transmission. WT occurs via a relatively constrained cellular chain (wire), while VT consists of 3-dimensional diffusion of signals in the extracellular fluid (ECF) for distances larger than the synaptic cleft. Both morphological and functional evidence indicates that dopamine (DA) synapses in striatum are 'open' synapses, i.e., synapses which favor diffusion of the transmitter into the surrounding ECF and observations are compatible with the view that DA varicosities can synthesize, store and release DA for VT. The DAergic mesostriatal transmission has, therefore, been examined by several groups to give experimental support to VT. Moreover, due to its minor structural requirements, VT may become prevalent under some pathological conditions, e. g. Parkinson's disease. In animal models of DAergic pathway degeneration, it has been shown that a compensatory activation of surviving DA terminals may lead to a preferential potentiation of VT. WT and VT favor different and complementary types of computation. VT is markedly slower and less safe than WT, but has minor spatial constraints and allows the reach of a large number of targets. Models of neuronal systems integrating classical neuronal circuits and diffusible signals begin to show how WT and VT may interact in the neural tissue.

Synaptosomal iron-dependent lipid peroxidation inhibition after subarachnoid hemorrhage by lazaroid in vivo treatment.

The production of oxygen-free radicals and their subsequent peroxidative action on membrane unsaturated fatty acids could be enhanced after subarachnoid hemorrhage (SAH). We have studied the effects of the in vivo pharmacological treatment with a lazaroid (U78517F) after experimental SAH, on lipid peroxidative patterns in cortical synaptosomal preparations. U78517F is a lipid-soluble antioxidant with a potent action to inhibit iron-dependent lipid peroxidation. Experimental SAH was induced in anesthetized rats by slow injection of 0.3 mL of autologous arterial blood into cisterna magna. The hemorrhagic animals were treated with 5 mg/kg iv of U78517F immediately after surgical operation. The animals were sacrificed 1 d after the hemorrhage and the thiobarbituric acid reactive material (TBAR) was assayed in basal conditions and after 1, 3, 5, 10, and 20 min of incubation at 37 degrees C with a pro-oxidant mixture on three different rat groups: sham-operated (0.3 mL of mock cerebrospinal fluid (CSF) into cisterna magna), hemorrhagic (0.3 mL of autologous arterial blood into cisterna magna), and hemorrhagic-treated. The hemorrhagic event did not influence the membrane lipoperoxidation levels in basal conditions, whereas peroxidative stimulation in vitro caused significant increases in hemorrhagic animals compared to the sham-operated, and in hemorrhagic-treated animals, the synaptosomal TBARs were similar to controls. The pharmacological treatment showed its effectiveness only following incubations with pro-oxidants; therefore, U78517F seems to be protective for membranes in case of severe lipid peroxidative stress.

Superoxide dismutase activity in cisternal cerebrospinal fluid after aneurysmal subarachnoid haemorrhage.

It has been recognised that the level of superoxide dismutase (SOD) significantly increases in CSF as the result of cerebral ischaemic damage. The aim of this study was to correlate the CSF levels of SOD enzymatic activity to the patterns of subarachnoid haemorrhage with regards to ischaemic complications due to vasospasm. A series of 78 patients operated on for intracranial aneurysms was studied; all patients were monitored with serial TCD measurements every second day after SAH. CSF samples were obtained at surgery by cisternal puncture of the subarachnoid cistern nearest to the aneurysm. SOD activity was assayed spectrophotometrically. Mean cisternal CSF level of SOD in 12 control cases (12.99 +/- 2.33 U/ml) is significantly higher (p < 0.01) than in 26 patients operated on between day 1 and 3 from last SAH episode (4.44 +/- 0.7 U/ml) and in 40 patients treated by delayed surgery (7.64 +/- 0.92 U/ml). In 13 patients presenting neurological deterioration related to arterial vasospasm mean cisternal SOD level was 12.23 +/- 1.86 U/ml; in 27 cases without vasospasm mean level was 5.43 +/- 0.7 U/ml (p < 001). The present results suggest that (a) cisternal CSF levels of SOD significantly decreases after SAH, probably in relation to an impaired synthesis in the brain compartment and that (b) a substantial elevation of SOD levels is evident in patients suffering ischaemic complications vasospasm-related. Biochemical events in the brain compartment could influence the expression and release of anti-oxidant enzymes in CSF after SAH.

Protective effects of delapril, indapamide and their combination chronically administered to stroke-prone spontaneously hypertensive rats fed a high-sodium diet.

1. Stroke-prone spontaneously hypertensive rats (SHRsp) have been used widely to test agents putatively capable of vascular protection. These animals present an accelerated time course of hypertension and a reduced life-span. When fed a high-sodium diet from the eighth week of life, a further acceleration in blood pressure increase is obtained, and rats start to die after 5 weeks of diet as a consequence of cerebral haemorrhage. In this model, angiotensin-converting enzyme (ACE) inhibitors were repeatedly proved to prevent vascular lesions and death. Notably, this effect was independent of any hypotensive effect. On the contrary, diuretics were shown not to be equally effective. A combination of ACE inhibitors and diuretics, although known to have synergistic effects in the therapy of hypertension, has never previously been tested. 2. Our aim was to study the effects of long-term treatment with the ACE inhibitor delapril (12 mg day-1 kg-1), the thiazide-like diuretic indapamide (1 mg day-1 kg-1), and their combination (12 and 1 mg day-1 kg-1 respectively), on the survival of SHRsp rats fed a high-sodium diet from the eighth week of life onwards. The effects of the treatments on blood pressure, body weight, food and fluid intake, diuresis, proteinuria and the appearance of lesion signs and death were assessed weekly. When control rats reached 50% mortality, they were killed, together with some drug-treated rats, to compare lesions in brain and kidney. The other drug-treated rats continued treatments until 50% mortality was reached in two treatment groups. 3. All drug treatments were able to delay death significantly when compared with control rats, which reached 50% mortality after 6 weeks of salt loading. This event was preceded by a highly significant increase in proteinuria, diuresis and fluid intake that took place 3 weeks after the increase in blood pressure over the initial range. In delapril- or indapamide-treated SHRsp these changes were never seen, even when animals started to die. In the combination-treated group, a significant increase (P < 0.01) in fluid intake and diuresis, but not proteinuria, was observed from the third week of treatment onwards. 4. Treatment with delapril or indapamide did not block the progressive increase in blood pressure as observed in control animals. However, the increase in blood pressure was markedly retarded with respect to control rats. At variance with this, in combination-treated animals blood pressure levels were maintained until the end of the experiment within the 99% confidence interval initially observed in control animals. 5. Infarctual and haemorrhagic cerebral lesions were observed in 38% of control rats; no lesions were noted in brains of age-matched rats receiving a drug treatment. Kidneys from control animals presented major degenerative lesions of glomeruli and arteries, characterized by fibrinoid necrosis. This condition was absent in drug-treated animals, which presented minor signs of ischaemic lesion. Heart hypertrophy, when heart weight was expressed as a percentage of body weight, was similar in saline-, delapril- or indapamide-treated rats. At variance with this, in combination-treated animals the heart weight to body weight ratio was significantly (P < 0.01) lower than in the other groups. 6. In conclusion, the diuretic indapamide showed similar protective effects as the ACE inhibitor delapril on acute vascular lesions and survival of SHRsp. Moreover, their combination synergized in preventing heart hypertrophy consequent to longterm hypertension. This results is probably related to the enhanced diuresis and the better control of blood pressure levels selectively found in combination-treated animals.

Antioxidant enzymatic system and free radicals pathway in two different human cancer cell lines.

Resistance to oxidative damage is an important feature of cancer cells. Cellular anti-radical enzymes, lipid peroxidation, glutathione pathway, capability to produce ROS, and cells' susceptibility to H2O2 and menadione toxicity, were analyzed in DND-1A and HeLa cancer cell lines. SOD and GSHPx activities were higher in DND-1A than in HeLa cells. Lipid peroxidation was the same in both cell lines, while menadione stimulation of ROS production was tenfold higher in HeLa cells. Total and reduced, but not oxidized, glutathione levels, were tenfold smaller in HeLa cells. H2O2 proved fatal to HeLa cells after 12 hours' incubation, while it was ineffective on DND-1A; DND-1A cells were more sensitive to menadione toxicity than HeLa cells. The two lines behaved differently in response to the above treatments. These observations might be important in designing more specific cancer treatments.

Oxidative stress after acute and chronic application of beta-amyloid fragment 25-35 in cortical cultures.

The aim of this work was to investigate whether free radical reactions play a role in beta-amyloid neurotoxicity. Rat cortical neurons were exposed acutely (24 h) or chronically (3, 7 days) to beta-amyloid biologically active fragment beta 25-35 (50 microM). In these conditions, where only the longest exposure induced neuronal death, superoxide dismutase activity was increased after acute exposure but no change was detected after chronic treatments, whereas a different pattern was observed for glutathione peroxidase. In the basal condition, there was an eight-fold increase in dichlorofluoroscein, used as peroxide production marker, in neuronal cells after 7 days treatment with beta 25-35. Moreover, the intracellular peroxide production induced by Fe2+/ascorbate stimulation was amplified by beta 25-35, increasingly up to 7 days of exposure, by which time the dichlorofluoroscein-stimulated levels were 33 times higher than in controls. In conclusion, our results show that oxidative stress and free radical production are linked to beta 25-35 exposure and may contribute to neurodegenerative events associated with beta-amyloid deposits in Alzheimer's disease.

Oxidative events in neuronal and glial cell-enriched fractions of rat cerebral cortex.

The aim of this work was to investigate how neurons and glial cells separated from rat brain cortex respond to "in vitro" oxidative stress induced by incubation of the cellular fractions in the presence of prooxidant mixtures; in addition, the endogenous enzymatic antioxidant capacity of the purified fractions was investigated. Neuronal and glial cell-enriched fractions were obtained from rat cerebral cortex following passages of the tissue through meshes and centrifugations. The following parameters were evaluated: antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSHPx), and glucose-6-phosphate dehydrogenase (G6PDH); lipid peroxidation products (TBARS) prior to (basal) and after (iron-stimulated) incubation with a mixture of iron and ascorbic acid; intracellular production of reactive oxygen species (ROS) using a fluorescent probe, dichlorofluorescin-diacetate, in basal, iron-stimulated, and menadione stimulated conditions. SOD and GSHPx activities showed no significant changes between neurons and glia, whereas CAT and G6PDH activities were found to be significantly lower in glia than in neurons. TBARS levels were significantly lower in the glial fraction than in neurons, both in basal and iron-stimulated conditions. ROS production showed no differences between neurons and glia in both basal and menadione-stimulated conditions. Iron-stimulation produced a marked increase in ROS production, limited to the neuronal fraction, with the glial values being similar to the basal ones. Our conclusion is that glia and neurons isolated from rat cerebral cortex show a similar pattern of the most important antioxidant enzymes and of their basal ROS production, whereas glia is more resistant in "oxidative stress" conditions.

Changes in non-synaptosomal and synaptosomal mitochondrial membrane-linked enzymatic activities after transient cerebral ischemia.

Non-synaptosomal and synaptosomal mitochondrial membrane-linked enzymatic activities, NADH-cytochrome c reductase rotenone insensitive (marker of the outer membrane) and cytochrome oxidase (marker of the inner membrane), were measured in rat brain hippocampus and striatum immediately after and 1, 4 and 7 days following the induction of complete transient ischemia (15 min) by the four vessel occlusion method. Furthermore citrate synthetase activity was measured with and without Triton X-100 in order to qualitatively evaluate the membrane permeability. Non-synaptosomal mitochondrial membranes showed reduction of both activities only in the late reperfusion phase: NADH-CCRRi decreased in striatal mitochondria after 4-7 days and only after 7 days in the hippocampus. COX activity decreased only in striatal mitochondria 7 days after ischemia. Non-synaptosomal mitochondrial membrane permeability did not show changes. Synaptosomal mitochondria showed a decrease of NADH-CCRRi only at 7 days of reperfusion both in hippocampus and striatum, while COX activity decreased only during ischemia and returned to normal levels in the following days in the two areas considered. In summary, free mitochondria showed insensitiveness to ischemia but they resulted damaged in the late reperfusion phase, while mitochondria from the synaptic terminal showed ischemic damage, partially restored during reperfusion. The striatal mitochondria showed a major susceptibility to ischemia/reperfusion damage, showing changes earlier than the hippocampal ones.

Experimental subarachnoid hemorrhage: events related to anti-oxidant enzymatic systems and eicosanoid peroxide enhancement.

Experimental and clinical studies have emphasized the role of free radicals in the pathogenesis of vasospasm and neurological dysfunction after subarachnoid hemorrhage (SAH). Increases in both enzymatic (arachidonic acid cascade and eicosanoid peroxide production) and non-enzymatic (thiobarbituric acid reactive substances production) lipid peroxidation were found, pointing out the key role of arachidonic acid cascade in impairing membrane functionality in the post-hemorrhage brain. The aim of this work is to investigate whether a correlation exists between time-dependent modifications of eicosanoid peroxide production ("ex vivo" release of leukotriene C4 = LTC4) and antioxidant enzymatic systems in the brain after experimental subarachnoid hemorrhage in the rat. The release of the LTC4 is significantly enhanced at 1, 6 and 48 hours after SAH induction. Cu-Zn superoxide dismutase (SOD) activity is significantly reduced at 6 and 48 hours after SAH induction; Mn-SOD activity is significantly affected at 1, 6 and 48 hours after the hemorrhage. GSH-Px activity is significantly reduced only in the late phase (48 hours) after SAH. The linear regression of statistical analysis, performed to investigate any possible relationship among time-dependent modifications shows that the "ex vivo" release of LTC4 is significantly related to the decreasing trend of MnSOD activity (p < 0.001). The present results suggest that after SAH, a deficit in endogenous anti-oxidant defenses may play a role in making the brain more susceptible to lipid peroxidative events.

Cellular and molecular events of ischemic brain damage.

Many events occurring during and after cerebral ischemia are well known, but they are not known enough to fully elucidate the mechanisms of brain damage. Energy failure alone cannot explain the functional damage occurring during the reperfusion phase, and, at present, four features of the ischemic and postischemic brain are the focus of interest: development of acidosis, calcium overload, free radical formation, and nitric oxide overproduction. It is likely that these events, perhaps with other less known ones, contribute altogether to the occurrence of irreversible damage. An understanding of them might lead to the development of a drug, or a drug cocktail, able to counteract ischemic lesions.