Respiration-Deficient Astrocytes Survive As Glycolytic Cells In Vivo.

Neurons and glial cells exchange energy-rich metabolites and it has been suggested, originally based on in vitro data, that astrocytes provide lactate to glutamatergic synapses ("lactate shuttle"). Here, we have studied astrocytes that lack mitochondrial respiration in vitro and in vivo A novel mouse mutant (GLASTCreERT2::Cox10flox/flox) was generated, in which the administration of tamoxifen causes mutant astrocytes to fail in the assembly of mitochondrial cytochrome c oxidase (COX). Focusing on cerebellar Bergmann glia (BG) cells, which exhibit the highest rate of Cre-mediated recombination, we found a normal density of viable astrocytes even 1 year after tamoxifen-induced Cox10 gene targeting. Our data show that BG cells, and presumably all astrocytes, can survive by aerobic glycolysis for an extended period of time in the absence of glial pathology or unspecific signs of neurodegeneration.SIGNIFICANCE STATEMENT When astrocytes are placed into culture, they import glucose and release lactate, an energy-rich metabolite readily metabolized by neurons. This observation led to the "glia-to-neuron lactate shuttle hypothesis," but in vivo evidence for this hypothesis is weak. To study astroglial energy metabolism and the directionality of lactate flux, we generated conditional Cox10 mouse mutants lacking mitochondrial respiration in astrocytes, which forces these cells to survive by aerobic glycolysis. Here, we report that these mice are fully viable in the absence of any signs of glial or neuronal loss, suggesting that astrocytes are naturally glycolytic cells.

Glycolytic oligodendrocytes maintain myelin and long-term axonal integrity.

Oligodendrocytes, the myelin-forming glial cells of the central nervous system, maintain long-term axonal integrity. However, the underlying support mechanisms are not understood. Here we identify a metabolic component of axon-glia interactions by generating conditional Cox10 (protoheme IX farnesyltransferase) mutant mice, in which oligodendrocytes and Schwann cells fail to assemble stable mitochondrial cytochrome c oxidase (COX, also known as mitochondrial complex IV). In the peripheral nervous system, Cox10 conditional mutants exhibit severe neuropathy with dysmyelination, abnormal Remak bundles, muscle atrophy and paralysis. Notably, perturbing mitochondrial respiration did not cause glial cell death. In the adult central nervous system, we found no signs of demyelination, axonal degeneration or secondary inflammation. Unlike cultured oligodendrocytes, which are sensitive to COX inhibitors, post-myelination oligodendrocytes survive well in the absence of COX activity. More importantly, by in vivo magnetic resonance spectroscopy, brain lactate concentrations in mutants were increased compared with controls, but were detectable only in mice exposed to volatile anaesthetics. This indicates that aerobic glycolysis products derived from oligodendrocytes are rapidly metabolized within white matter tracts. Because myelinated axons can use lactate when energy-deprived, our findings suggest a model in which axon-glia metabolic coupling serves a physiological function.

Pharmacokinetics of concomitant cisplatin and paclitaxel administered by hyperthermic intraperitoneal chemotherapy to patients with peritoneal carcinomatosis from epithelial ovarian cancer.

BACKGROUND: Hyperthermic intraperitoneal chemotherapy (HIPEC) is advised as a treatment option for epithelial ovarian cancer (EOC) with peritoneal carcinomatosis. This study was designed to define the pharmacokinetics of cisplatin (CDDP) and paclitaxel (PTX) administered together during HIPEC. METHODS: Thirteen women with EOC underwent cytoreductive surgery (CRS) and HIPEC, with CDDP and PTX. Blood, peritoneal perfusate and tissue samples were harvested to determine drug exposure by high-performance liquid chromatography and matrix-assisted laser desorption ionization imaging mass spectrometry (IMS). RESULTS: The mean maximum concentrations of CDDP and PTX in perfusate were, respectively, 24.8±10.4 µg ml(-1) and 69.8±14.3 µg ml(-1); in plasma were 1.87±0.4 µg ml(-1) and 0.055±0.009 µg ml(-1). The mean concentrations of CDDP and PTX in peritoneum at the end of HIPEC were 23.3±8.0 µg g(-1) and 30.1±18.3 µg(-1)g(-1), respectively. The penetration of PTX into the peritoneal wall, determined by IMS, was about 0.5 mm. Grade 3-4 surgical complications were recorded in four patients, five patients presented grade 3 and two patients presented grade 4 hematological complications. CONCLUSIONS: HIPEC with CDDP and PTX after CRS is feasible with acceptable morbidity and has a favorable pharmacokinetic profile: high drug concentrations are achieved in peritoneal tissue with low systemic exposure. Larger studies are needed to demonstrate its efficacy in patients with microscopic postsurgical residual tumours in the peritoneal cavity.

Evaluation of gingival microcirculation in patients undergoing fixed orthodontic treatment: a pilot study.

AIM: Among the many biological effects which occur during orthodontic movement, we decided to investigate gingival microcirculation. The aim of the study was to evaluate the biological microvascular response to the application of orthodontic force in vivo. MATERIALS AND METHODS: Forty patients (case group) between 9-22 years of age (average± DS 12±3.01) were selected for the study (M/F ratio: 20/20). They needed fixed orthodontic treatment due to several types of malocclusion. Forty healthy subjects (control group) were also recruited (M/F ratio 20/20; average age 12 years ± 4.01; Mean±SD =10.04±1.7). A videocapillaroscopic examination was performed on each patient on the buccal alveolar mucosa at the pre- treatment time (t0), 1 month after the beginning of the treatment (t1), after 2 months (t2), after 6 months (t3), and after 12 months (t4). RESULTS: Capillary density increases significantly from t0 to t1. Between t1 to t2, t2 to t3 the density underwent another increase. Between t3 and t4 (69.22 ± 3.63) the density showed no increase. In the control group no statistical differences were observed. CONCLUSION: Videocapillaroscopy allows the in vivo evaluation and quantification of the microcirculatory changes consequent to the application of orthodontic force, managing to detect subclinical changes in angiogenesis. In fact, the study revealed an increase in the density of the capillaries which is directly proportionate to the application time of the orthodontic device, i.e. the exogenous mechanical force. This research offers new perspectives for the future of monitoring fixed orthodontic therapy.

Personal exposure to particulate matter is associated with worse health perception in adult asthma.

BACKGROUND: Epidemiological studies have shown positive associations between particulate matter (PM) air pollution and short-term mortality and morbidity for asthma. The hypothesis that lung inflammation is responsible for these effects has been tested in panel and controlled exposure studies in asthmatic adults, with inconsistent results. OBJECTIVES: We investigated whether personal exposure to PM10 and PM2.5 were related to changes in the clinical course of asthma and to lung inflammatory responses in adult asthmatics. METHODS: A cohort of 32 asthmatic patients was followed for 2 years. Asthma control test (ACT) and St George's Respiratory Questionnaire (SGRQ) scores, forced expired volume in the first second (FEV1), exhaled nitric oxide (Fe(NO)), and pH of exhaled breath condensate (EBC) were determined on 6 occasions during different seasons. Personal exposure to PM was measured for 24 hours prior to clinical assessments. RESULTS: A 10 microg/m3 increase in PM10 personal exposure was associated with an increase in SGRQ scores (regression coefficient beta = 0.22; 95% confidence interval [CI], -0.005 to 4.451; P =.055) and with a decrease in ACT scores (beta = -0.022; 95% CI, -0.045 to 0.001; P = .060), whereas no associations were found between PM10 and FEV1, Fe(NO), or EBC pH. A positive association was detected between Fe(NO) and outdoor O3 (P = .042) and SO2 (P = .042) concentrations in the subgroup of nonsmoking asthmatics. CONCLUSIONS: We concluded that increments in personal exposure to PM10 are associated with a decrease in asthma control and health-related quality of life. However, this study does not provide evidence that 24-hour exposures to PM are associated with short-term changes in lung function or inflammatory responses of the lung.

Effects of recombinant protein misfolding and aggregation on bacterial membranes.

The expression of recombinant proteins is known to induce a metabolic rearrangement in the host cell. We used aggregation-sensitive model systems to study the effects elicited in Escherichia coli cells by the aggregation of recombinant glutathione-S-transferase and its fusion with the green fluorescent protein that, according to the expression conditions, accumulate intracellularly as soluble protein, or soluble and insoluble aggregates. We show that the folding state of the recombinant protein and the complexity of the intracellular aggregates critically affect the cell response. Specifically, protein misfolding and aggregation induce changes in specific host proteins involved in lipid metabolism and oxidative stress, a reduction in the membrane permeability, as well as a rearrangement of its lipid composition. The temporal evolution of the host cell response and that of the aggregation process pointed out that the misfolded protein and soluble aggregates are responsible for the membrane modifications and the changes in the host protein levels. Interestingly, native recombinant protein and large insoluble aggregates do not seem to activate stress markers and membrane rearrangements.

Carbon monoxide pollution is associated with decreased lung function in asthmatic adults.

The aim of the present study was to test the effects of exposure to air pollutants on lung function. A panel of 19 adult asthmatics living in Padua (Italy) was followed for five 30-day periods during 2 yrs consecutively (1,492 morning and 1,434 evening measures analysed). Peak expiratory flow (PEF) and forced expiratory volume in 1 s (FEV(1)) were measured using a pocket electronic meter. Daily levels of air pollutants and meteorological variables were collected at outdoor city monitoring sites. Significant inverse associations were observed between morning and evening PEF and carbon monoxide level (p = 0.01-0.03), without clear differences between lags (0-3 days). An increment of 1 mg.m(-3) CO was associated with a PEF variation ranging -2.6- -2.8%. All effect estimates on PEF for CO remained significant and even increased after controlling for particles with a 50% cut-off aerodynamic diameter of 10 microm (PM(10)), nitrogen dioxide and sulphur dioxide in single and multi-pollutant models. A similar trend was observed for FEV(1), but the associations were nonsignificant. A nonsignificant inverse relationship between evening PEF and SO(2) was also detected. PEF and FEV(1) were not related to PM(10) and NO(2) concentrations. The present results indicate that, in this panel of adult asthmatics, the worsening of lung function is associated with exposure to gaseous pollutants and occurs at levels of CO and SO(2) lower than current European standards.

[Asbestos-related lung cancer]. / Il tumore polmonare correlato all'asbesto.

Lung cancer is the leading cause of tumour death and a large percentage of it is associated with tobacco smoking. Epidemiology has shown that asbestos cumulative exposures increase the risk of lung cancer to a variable extent, depending on the manufacturing process and the specific job. The risk appears relatively small (< or = 2) and is detectable after massive exposures only. Clinical diagnosis of asbestos-related lung cancer is based upon medical history (exposures > 25 ff.ml years double the risk), possible lung fibrosis and counts of asbestos bodies and fibers in bronchoalveolar lavage and lung tissues. Pleural plaques do not correlate with the cumulative exposures that are associated with lung cancer. The multiplicative interaction between smoke and asbestos is only detectable when the risk associated with asbestos exposure is increased, i.e. after high exposures.

[Academic occupational medicine]. / La medicina del lavoro accademica.

This assay analyses the sorry state of occupational medicine, particularly in Italian Academy, and discusses the opportunities for its revitalization. Contrary to its past history, occupational medicine is only witnessing the ongoing extraordinary revolution in biomedical sciences and taking no advantage from it. The main reason for this academic decline may be due, paradoxically, to its success. The change of paradigm, from clinical medicine to preventive activities was relatively quick, missing a clear understanding of their differences in backgrounds, methods and objectives. Moreover, the spread of different disciplines across occupational medicine has led to an impoverish role of biomedical sciences and to diminished medical skills of occupational physicians. The wide range of opportunities offered by translational medicine gives to the discipline unprecedented chances of revitalization.

Expression of cloned Saccharomyces diastaticus glucoamylase under natural and inducible promoters.

Any one of three homologous genes - STA1, STA2 and STA3 - encoding glucoamylase isozymes I, II and III respectively, allows the Saccharomyces species to utilize starch as a sole carbon source. We show in this paper that glucoamylase II production can be increased 4-fold over the level produced by STA2 strains, by using a two-step fermentation and a yeast strain transformed with a high-copy-number plasmid carrying the STA2 gene. The accumulation of anomalous STA2 mRNA species, mainly differing at their 5' ends, and saturation of step(s) in the secretory pathway appear to be among the major factors limiting glucoamylase expression in synthetic media.

Homology-derived three-dimensional structure prediction of Candida cylindracea lipase.

We propose a structural model of Candida cylindracea lipase (CCL) based on the reported X-ray structure of the highly homologous Geotrichum candidum lipase (GCL). The network of interactions around the active site, the salt and disulfide bridge pattern is conserved in the proposed structure. Functional, structural and evolutionary aspects of the peculiar usage of CTG codons by C. cylindracea ATCC 14830 are discussed.

Cloning and nucleotide sequences of two lipase genes from Candida cylindracea.

Two lipase-encoding genes (LIP1 and LIP2) have been isolated from a SacI genomic library of the yeast Candida cylindracea and their nucleotide sequences have been determined. Comparison with the sequence of a cDNA ruled out the presence of introns in the two genes. Both ORFs encode for mature proteins of 534 residues with putative signal peptides of 15 and 14 amino acids, respectively. When compared with other lipase sequences, the two C. cylindracea lipases showed homology only with the Geotrichum candidum lipase, whereas they shared a significant similarity with several esterases.

Design and realization of a tailor-made enzyme to modify the molecular recognition of 2-arylpropionic esters by Candida rugosa lipase.

Within a research project aimed at probing the substrate specificity and the enantioselectivity of Candida rugosa lipase (CRL), computer modeling studies of the interactions between CRL and methyl (+/-)-2-(3-benzoylphenyl)propionate (Ketoprofen methyl ester) have been carried out in order to identify which amino acids are essential to the enzyme/substrate interaction. Different binding models of the substrate enantiomers to the active site of CRL were investigated by applying a computational protocol based on molecular docking, conformational analysis, and energy minimization procedures. The structural models of the computer generated complexes between CRL and the substrates enabled us to propose that Phe344 and Phe345, in addition to the residues constituting the catalytic triad and the oxyanion hole, are the amino acids mainly involved in the enzyme-ligand interactions. To test the importance of these residues for the enzymatic activity, site-directed mutagenesis of the selected amino acids has been performed, and the mutated enzymes have been evaluated for their conversion and selectivity capabilities toward different substrates. The experimental results obtained in these biotransformation reactions indicate that Phe344 and especially Phe345 influence CRL activity, supporting the findings of our theoretical simulations.

Evolutionary origin of nonuniversal CUGSer codon in some Candida species as inferred from a molecular phylogeny.

CUG, a universal leucine codon, has been reported to be read as serine in various yeast species belonging to the genus Candida. To gain a deeper insight into the origin of this deviation from the universal genetic code, we carried out a phylogenetic analysis based on the small-subunit ribosomal RNA genes from some Candida and other related Hemiascomycetes. Furthermore, we determined the phylogenetic relationships between the tRNA(Ser)CAG, responsible for the translation of CUG, from some Candida species and the other serine and leucine isoacceptor tRNAs in C. cylindracea. We demonstrate that the group of Candida showing the genetic code deviation is monophyletic and that this deviation could have originated more than 150 million years ago. We also describe how phylogenetic analysis can be used for genetic code predictions.

Mutants provide evidence of the importance of glycosydic chains in the activation of lipase 1 from Candida rugosa.

Sequence analysis of Candida rugosa lipase 1 (LIP1) predicts the presence of three N-linked glycosylation sites at asparagine 291, 314, 351. To investigate the relevance of sugar chains in the activation and stabilization of LIP1, we directed site mutagenesis to replace the above mentioned asparagine with glutamine residues. Comparison of the activity of mutants with that of the wild-type (wt) lipase indicates that both 314 and 351 Asn to Gln substitutions influence, although at a different extent, the enzyme activity both in hydrolysis and esterification reactions, but they do not alter the enzyme water activity profiles in organic solvents or temperature stability. Introduction of Gln to replace Asn351 is likely to disrupt a stabilizing interaction between the sugar chain and residues of the inner side of the lid in the enzyme active conformation. The effect of deglycosylation at position 314 is more difficult to explain and might suggest a more general role of the sugar moiety for the structural stability of lipase 1. Conversely, Asn291Gln substitution does not affect the lipolytic or the esterase activity of the mutant that behaves essentially as the wt enzyme. This observation supports the hypothesis that changes in activity of Asn314Gln and Asn351Gln mutants are specifically due to deglycosylation.

Design, total synthesis, and functional overexpression of the Candida rugosa lip1 gene coding for a major industrial lipase.

The dimorphic yeast Candida rugosa has an unusual codon usage that hampers the functional expression of genes derived from this yeast in a conventional heterologous host. Commercial samples of C. rugosa lipase (CRL) are widely used in industry, but contain several different isoforms encoded by the lip gene family, among which the isoform encoded by the gene lip1 is the most prominent. In a first laborious attempt, the lip1 gene was systematically modified by site-directed mutagenesis to gain functional expression in Saccharomyces cerevisiae. As alternative approach, the gene (1647 bp) was completely synthesized with an optimized nucleotide sequence in terms of heterologous expression in yeast and simplified genetic manipulation. The synthetic gene was functionally expressed in both hosts S. cerevisiae and Pichia pastoris, and the effect of heterologous leader sequences on expression and secretion was investigated. In particular, using P. pastoris cells, the synthetic gene was functionally overexpressed, allowing for the first time to produce recombinant Lipl of high purity at a level of 150 U/mL culture medium. The physicochemical and catalytic properties of the recombinant lipase were compared with those of a commercial, nonrecombinant C. rugosa lipase preparation containing lipase isoforms.

Cloning and analysis of Candida cylindracea lipase sequences.

Lipases (Lip) hydrolyze triglycerides into fatty acids and glycerol. Lip produced by the yeast Candida cylindracea are encoded by multiple genomic sequences. We report the molecular cloning and characterization of three genes from this family. They encode putative mature 57-kDa proteins of 534 amino acids (aa). To date, five Lip-encoding genomic sequences from C. cylindracea have been characterized in our laboratory. The five deduced aa sequences share an overall homology of 80%. These sequences have been aligned with each other and with those of homologous enzymes, the Lip from the mould Geotrichum candidum and the acetylcholinesterase from Torpedo californica, whose three-dimensional structures have been solved by X-ray analysis. The C. cylindracea Lip appear to have a structural organization similar to that described for both enzymes.

Organophosphate polyneuropathy: pathogenesis and prevention.

Organophosphorus-induced delayed polyneuropathy (OPIDP) is initiated by the phosphorylation of a protein neurotoxic esterase (NTE) in the nervous system. A second step, the "aging" of the phosphoryl-enzyme complex, is required to produce the toxic effect. The experimental evidence for this molecular target and the importance of the aging process are reviewed. The catalytic activity of NTE has been used to develop an in vitro screening test that may distinguish the organophosphorus compounds (OPs) that cause neuropathy from those that do not, thereby providing a means for prevention of OPIDP. Moreover, a biochemical screening test, the determination of NTE activity in blood lymphocytes, may predict the development of OPIDP after acute or chronic exposure to OPs, and requires evaluation by carefully designed studies of occupational exposure to OPs.

Human serum "A"-esterases. Hydrolysis of O,O-dimethyl-2,2-dichlorovinyl phosphate.

Some characteristics of the hydrolysis of O,O-dimethyl-2,2 dichlorovinyl phosphate (DDVP) by human serum are reported and compared with the hydrolysis of O,O-diethyl-4-nitrophenyl phosphate (paraoxon) which is a substrate for Paraoxonase, a known "A"-esterase of human serum. When incubated with human serum, DDVP was losing its inhibitory power toward acetylcholinesterase (AChE). The loss of DDVP followed first order kinetics and was proportional to serum dilution. The disappearance of DDVP after incubation with human serum was not due to protein binding. Apparent Km and Vm for the hydrolysis of DDVP were 7.1 mM and 143 nmol.min-1.ml-1. The pH sensitivity, EDTA inhibitory and Ca2+ requirements of DDVP-ase were similar to those of Paraoxonase. DDVP inhibited the Paraoxonase activity and paraoxon inhibited the DDVP-ase activity. Ca2+, Ag+ and Hg2+ were better inhibitors of the Paraoxonase than the DDVP-ase. The rate of heat inactivation was also different; at 55 degrees Paraoxonase inactivated almost completely within 10 min, while DDVP-ase lost only about 10% activity over 1 hr. Consequently, DDVP-ase and Paraoxonase can be differentiated by means of heat sensitivity. The DDVP-ase was normally distributed in a population of 60 individuals, while Paraoxonase is known to show a marked polymorphism.