Simultaneous wireless and high-resolution detection of nucleus accumbens shell ethanol concentrations and free motion of rats upon voluntary ethanol intake.

Highly sensitive detection of ethanol concentrations in discrete brain regions of rats voluntarily accessing ethanol, with high temporal resolution, would represent a source of greatly desirable data in studies devoted to understanding the kinetics of the neurobiological basis of ethanol's ability to impact behavior. In the present study, we present a series of experiments aiming to validate and apply an original high-tech implantable device, consisting of the coupling, for the first time, of an amperometric biosensor for brain ethanol detection, with a sensor for detecting the microvibrations of the animal. This device allows the real-time comparison between the ethanol intake, its cerebral concentrations, and their effect on the motion when the animal is in the condition of voluntary drinking. To this end, we assessed in vitro the efficiency of three different biosensor designs loading diverse alcohol oxidase enzymes (AOx) obtained from three different AOx-donor strains: Hansenula polymorpha, Candida boidinii, and Pichia pastoris. In vitro data disclosed that the devices loading H. polymorpha and C. boidinii were similarly efficient (respectively, linear region slope [LRS]: 1.98 ± 0.07 and 1.38 ± 0.04 nA/mM) but significantly less than the P. pastoris-loaded one (LRS: 7.57 ± 0.12 nA/mM). The in vivo results indicate that this last biosensor design detected the rise of ethanol in the nucleus accumbens shell (AcbSh) after 15 minutes of voluntary 10% ethanol solution intake. At the same time, the microvibration sensor detected a significant increase in the rat's motion signal. Notably, both the biosensor and microvibration sensor described similar and parallel time-dependent U-shaped curves, thus providing a highly sensitive and time-locked high-resolution detection of the neurochemical and behavioral kinetics upon voluntary ethanol intake. The results overall indicate that such a dual telemetry unit represents a powerful device which, implanted in different brain areas, may boost further investigations on the neurobiological mechanisms that underlie ethanol-induced motor activity and reward.

Beta caryophyllene and caryophyllene oxide, isolated from Aegle marmelos, as the potent anti-inflammatory agents against lymphoma and neuroblastoma cells.

Aegle marmelos (Indian Bael) is a tree which belongs to the family of Rutaceae. It holds a prominent position in both Indian medicine and Indian culture. We have screened various fractions of Aegle marmelos extracts for their anticancer properties using in vitro cell models. Gas chromatography-Mass spectrometry (GC-MS) was employed to analyze the biomolecules present in the Aegle marmelos extract. Jurkat and human neuroblastoma (IMR-32) cells were treated with different concentrations of the fractionated Aegle marmelos extracts. Flow cytometric analysis revealed that optimal concentration (50 µg/ml) of beta caryophyllene and caryophyllene oxide fractions of Aegle marmelos extract can induce apoptosis in Jurkat cell line. cDNA expression profiling of pro-apoptotic and anti-apoptotic genes was carried out using real time PCR (RT-PCR). Down-regulation of anti-apoptotic genes (bcl-2, mdm2, cox2 and cmyb) and up-regulation of pro-apoptotic genes (bax, bak1, caspase-8, caspase-9 and ATM) in Jurkat and IMR-32 cells treated with the beta caryophyllene and caryophyllene oxide fractions of Aegle marmelos extract revealed the insights of the downstream apoptotic mechanism. Furthermore, in-silico approach was employed to understand the upstream target involved in the induction of apoptosis by the beta caryophyllene and caryophyllene oxide fractions of Aegle marmelos extract. Herein, we report that beta caryophyllene and caryophyllene oxide isolated from Aegle marmelos can act as potent anti-inflammatory agents and modulators of a newly established therapeutic target, 15-lipoxygenase (15-LOX). Beta caryophyllene and caryophyllene oxide can induce apoptosis in lymphoma and neuroblastoma cells via modulation of 15-LOX (up-stream target) followed by the down-regulation of anti-apoptotic and up-regulation of pro-apoptotic genes.

Reliability of hamilton-norwood classification.

BACKGROUND: Hamilton-Norwood scale (HNS) has been largely used to assess clinically the severity of androgenetic alopecia (AGA), especially for therapeutical trials and even to establish its association with important diseases such as ischemic heart disease and prostate cancer. OBJECTIVE: To study HNS reproducibility in the hands of dermatologists and dermatology residents. MATERIALS AND METHODS: Seven dermatologists and 16 residents in dermatology classified 43 photographs of male heads with different degrees of AGA. In a second study, 8 appraisers (3 dermatologists and 5 residents in dermatology) examined 56 pictures with the same procedure and repeated the observation 3 months later. In the first study, the inter-rater agreement was estimated by calculating an intra-class correlation coefficient (ICC). In the second study, for intra-rater repeatability, each rater's scores from session 1 were paired with his/her scores for the same subjects in session 2, and the ordinary least products linear regression was calculated. RESULTS: In the first study, the concordance of appraisers was unsatisfactory (ICC = 0.63-0.68)]. In the second study, repeatability was poor, without any significant difference between dermatologists and dermatology residents. COMMENT: Reliability of HNS is unsatisfactory even in the hands of expert appraisers. To obtain better reliability, the number of classes should be reduced, but with such reduction HNS would be usable to classify patients only in a broad way.

Synthesis, platelet adhesion and cytotoxicity studies of new glycerophosphoryl-containing polyurethanes.

In this work we synthesized new MDI -based poly(ether)urethanes (PEUs) with phospholipid-like residue as chain extender. Polymers were prepared by a conventional two-step solution polymerization procedure using 4,4' diphenylmethanediisocyanate (MDI) and poly(1,4- butanediol) with 1000 as molecular weight to form prepolymers which were successively polymerized with 1 glycerophosphorylcholine (1-GPC), 2-glycerophosphorylcholine (2-GPC) or glycerophosphorylserine (GPS) as chain extenders. Two reference polymers bearing 1,4-butandiol (BD) have been also synthesized. The polymers obtained were characterized by Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC) and modulated scanning calorimetry (MDSC). The biocompatibility of synthesized segmented polyurethanes was then investigated by platelet-rich plasma contact studies and related scanning electron microscopy (SEM) photographs for blood compatibility and cytotoxicity assay (MTT test) on material elution to assess the effect of any toxic leachables on cellular viability. Three polymers among all have given very satisfactory results suggesting to investigate more deeply their possible use in biomedical devices.

A novel human homologue of the SH3BGR gene encodes a small protein similar to Glutaredoxin 1 of Escherichia coli.

Glutaredoxins (GRXs) are ubiquitous GSH-dependent oxidoreductases, which catalyze the reduction of protein-glutathionyl-mixed disulfides and are considered to play an important role in the enzymatic regulation of redox-sensitive proteins. In this paper, we describe the identification and characterization of a new human homologue of the SH3BGR gene, named SH3BGRL3 (SH3 domain binding glutamic acid-rich protein like 3). SH3BGRL3 is widely expressed and codes for a highly conserved small protein, which shows a significant similarity to Glutaredoxin 1 (GRX1) of Escherichia coli and is predicted to belong to the Thioredoxin Superfamily. However, the SH3BGRL3 protein lacks both the conserved cysteine residues, which characterize the enzymatic active site of GRX. This structural feature raises the possibility that SH3BGRL3 could function as an endogenous modulator of GRX biological activity. EGFP-SH3BGRL3 fusion protein expressed in COS-7 cells localizes both to the nucleus and to the cytoplasm. The SH3BGRL3 gene was mapped to chromosome 1p34.3-35.

Effects of fluvastatin treatment on red blood cell Na+ transport systems in hypercholesterolemic subjects.

This study was performed to ascertain the effects of short-term cholesterol-lowering therapy with fluvastatin on red blood cells Na+ transport systems. Forty familial hypercholesterolemic subjects (FH; 19 men and 21 women) without hypertension or cardiovascular disease were given a placebo for 4 weeks, and then randomized in two groups. Twenty (fluvastatin group) were given fluvastatin (40 mg/day), and the other 20 (placebo group) continued placebo administration. After the placebo period and after 4 and 12 weeks of placebo or fluvastatin treatment, we measured Na+/K+ pump activity, Na+/K+ cotransport (Na+/K+ Ct), Na+/Li+ countertransport (Na+/Li+ Cnt), passive Na+ permeability (Na+PP), and internal Na+ content (Na+i). The same parameters were measured in 23 control subjects (C) with normal cholesterolemic values, who were matched for sex and age. FH had higher Na+/Li+ Cnt values than C (193.2 +/- 59.4 vs. 139.8 +/- 48.7 microM cells/h; p < 0.01), an increase in Na(+)PP (0.034 +/- 0.012/h vs. 0.018 +/- 0.004/h; p < 0.001), and higher Na(+)i (7.5 +/- 1.5 vs. 6.2 +/- 0.9 mM cells; p < 0.001). In hypercholesterolemic subjects, Na(+)i values were correlated with cholesterol (total and LDL) and apo B levels, whereas an inverse correlation was found for HDL-c and apo AI levels. Reduced total and LDL cholesterol and apo B levels after fluvastatin treatment caused a decrease in both Na(+)/Li(+) Cnt (from 186.1 +/- 60.5 to 125.1 +/- 34.0 microM cells/h; p < 0.001) and Na(+) PP (from 0.035 +/- 0.013/h to 0.02 +/- 0.016/h; p < 0.01), and an increase in Na+/K+ pump activity (from 1,549.0 +/- 507.7 to 1,894.2 +/- 536.2 microM cells/h; p < 0.04), with a significant reduction in the internal Na+ content (from 7.5 +/- 1.6 to 5.8 +/- 2.4 mM cells; p < 0.001). Our findings show that hypercholesterolemia affects red blood cell Na+ transport systems, with an increase in Na+/Li+Cnt, Na+PP, and the internal Na+ content. Cholesterol-lowering treatment with fluvastatin influences Na+ transport systems and reduces the internal Na+ content. This might also be responsible for the greater vascular reactivity observed in hypercholesterolemic patients, and its amelioration after a reduction in cholesterol levels.

Crystallization and preliminary X-ray diffraction studies of phospholipase D from Streptomyces sp.

Crystals of purified phospholipase D (E.C. 3.1.4.4) from Streptomyces sp. strain PMF have been grown under two different crystallization conditions using vapour diffusion. Both conditions gave monoclinic crystals in space group P2(1). The unit-cell parameters were a = 57.28, b = 57.42, c = 68.70 A, beta = 93.17 degrees. The crystals diffract at 110 K to a resolution beyond 1.4 A using synchrotron radiation.

Can functional regions of proteins be predicted from their coding sequences? The case study of G-protein coupled receptors.

A filter based on a set of unsupervised neural networks trained with a winner-take-all strategy discloses signals along the coding sequences of G-protein coupled receptors. By comparing with the existing experimental data it appears that these signals correlate with putative functional domains of the proteins. After protein alignment within subfamilies, signals cluster in protein regions which, according to the presently available experimental results, are described as possible functional domains of the folded proteins. The mapping procedure reveals characteristic regions in the coding sequences common and/or characteristic of the receptor subtype. This is particularly noticeable for the third cytoplasmic loop, which is likely to be involved in the molecular coupling of all the subfamilies with G-proteins. The results indicate that our mapping can highlight intrinsic representative features of the coding sequences which, in the case of G-protein coupled receptors, are characteristic of protein functional regions and suggest a possible application of the filter for predicting functional determinants in proteins starting from the coding sequence.

Identification and characterization of a new human gene encoding a small protein with high homology to the proline-rich region of the SH3BGR gene.

As part of an effort to identify genes potentially involved in the Down Syndrome pathogenesis, in this paper we report the identification and characterization of a new human gene (named SH3BGRL), which shows a high homology to the SH3BGR gene, previously mapped to the Down Syndrome region of chromosome 21. The SH3BGRL gene encodes for a small protein of 114 amino acids, sharing 60% identity and 84% conservation on the amino acid level with the middle, proline-rich region of the SH3BGR gene and containing a similar SH3 (Scr homology 3) binding motif. The SH3BGRL and the proline-rich region of SH3BGR proteins appear to be highly conserved, sharing 95 and 98% identity, respectively, with the mouse homologues. A 1.9 kb transcript of the SH3BGRL gene has been found in all the tissues examined, in contrast with the expression pattern of the SH3BGR gene which is transcribed only in heart and skeletal muscle. The SH3BGR gene and its homologue, SH3BGRL, could be members of a new family of genes containing a highly conserved proline-rich functional domain. The SH3BGRL gene has been mapped by fluorescent in situ hybridization to Chromosome Xq13.3.

Identification and characterization of a new human cDNA from chromosome 21q22.3 encoding a basic nuclear protein.

Congenital heart disease (CHD) affects over 40% of Down syndrome (DS) patients. The region proposed to contain the gene(s) for DS CHD has been restricted to 21q22.2-22.3, from D21S55 to MX1. The identification and functional characterization of the genes mapping to this region is a necessary step to understand the pathogenesis of CHD in DS. In an effort to contribute to the construction of a transcriptional map of the DS CHD region we have performed direct cDNA selection using a YAC contig that maps between ETS2 and D21S15 and cDNAs synthesised from fetal heart structures. Here we describe the identification and characterization of a new gene, WRB, that maps to 21q22.3 between ACTL5 and HMG 14 and appears to be widely expressed in adult and fetal tissues. The new gene encodes a basic protein of unknown function containing a tryptophan-rich carboxyl-terminal region and a potential nuclear localization signal. Immunofluorescence analysis shows a predominant localization in the cell nucleus. The understanding of the biological function of the protein product should clarify the potential role of WRB in the pathogenesis of DS CHD.

Cloning a new human gene from chromosome 21q22.3 encoding a glutamic acid-rich protein expressed in heart and skeletal muscle.

The identification and functional characterization of genes on chromosome 21 is a necessary step to understand the pathogenesis of the various phenotypic anomalies that affect Down syndrome patients. Using direct cDNA selection we have identified a new gene, SH3BGR, that maps to 21q22.3, proximal to HMG14, and is differentially expressed in heart and skeletal muscle. SH3BGR encodes a novel protein that is characterized by the presence of a proline-rich region containing the consensus sequence for a SH3-binding domain and by an acidic carboxyl-terminal region containing a glutamic acid-rich domain predicted to assume a coiled coil. The presence of two functional domains involved in protein-protein interactions suggests that SH3BGR could be part of a multimeric complex. Its overexpression might alter specific functions of muscular tissue and therefore take part in the pathophysiology of muscular hypotonia in Down syndrome.

Self-organizing neural maps of the coding sequences of G-protein-coupled receptors reveal local domains associated with potentially functional determinants in the proteins.

Mapping of the coding sequences of the best characterized subfamilies of G-protein-coupled receptors is performed with unsupervised neural networks based on a winner-take-all strategy. High order features therefrom extracted originate signals along the aligned protein sequences of the different subfamilies. These plots reveal characteristic domains common and/or characteristic of the receptor subfamily. By comparison with the existing experimental results, it is obtained that most of the regions signalled by clustering overlap with possible functional regions in the folded proteins. This is particularly noticeable for the third cytoplasmic loop, which is likely to be involved in the molecular coupling with the G-proteins. The results suggest that functional regions in proteins may be characterized by intrinsic representative features in the coding sequences which can be enlighted by high order mapping.

Hamming-Clustering method for signals prediction in 5' and 3' regions of eukaryotic genes.

MOTIVATION: Gene expression is regulated by different kinds of short nucleotide domains. These features can either activate or terminate the transcription process. To predict the signal sites in the 5' and 3' gene regions we applied the Hamming-Clustering network (HC) to the TATA box, to the transcription initiation site and to the poly(A) signal determination in DNA sequences. This approach employs a technique deriving from the synthesis of digital networks in order to generate prototypes, or rules, which can be directly analysed or used for the construction of a final neural network. RESULTS: More than 1000 poly-A signals have been extracted from EMBL database rel. 42 and used to build the training and the test set. A full set of the eukaryotic genes (1252 entry) from the Eukaryotic Promoter Database (EPD rel. 42) have been used for the TATA-box signal and transcription network approach. The results show the applicability of the Hamming-Clustering method to functional signal prediction.

Evidence for an essential lysyl residue in phospholipase D from Streptomyces sp. by modification with diethyl pyrocarbonate and pyridoxal 5-phosphate.

Diethyl pyrocarbonate inactivated phospholipase D from Streptomyces PMF with second-order rate constants of 0.7 M-1 s-1 at pH 6.1 or 222 M-1 s-1 at pH 8.3 and 25 degrees C, and modified 5 His residues per enzyme molecule. The His residues, however, were not essential for activity because: (a) the second-order rate constants for reaction of diethyl pyrocarbonate with the His residues of the enzyme, which were 1.4 M-1 s-1 at pH 6.1 or 7.2 M-1 s-1 at pH 8.3 and 25 degrees C, differed, both at low and high pH values, from the inactivation rates, and (b) the reversal of His modification by hydroxylamine was not accompanied by recovery of activity. As demonstrated by dinitrophenylation experiments carried out on the treated enzyme, diethyl pyrocarbonate also modified up to 20 Lys residues per enzyme molecule. Other amino acid residues and the conformation and hydrodynamic volume of the enzyme were not modified. The involvement of a Lys residue in enzyme activity was confirmed through experiments with pyridoxal 5-phosphate which inactivated phospholipase D, after NaBH4 reduction, with a second-order rate constant of 3.5 M-1 s-1 at pH 8.5 and 15 degrees C. The inactivation took place with concomitant modification of 4 Lys residues, only one of which was found to be essential using the kinetic method of Tsou (Tsou, C.-L. (1962) Sci. Sin. 11, 1535-1538). Dicaproyl phosphatidylcholine markedly protected the enzyme against inactivation by DEP or PLP, and this strongly suggests that the essential Lys residue is located in or near the substrate binding site.

Purification and properties of two phospholipases D from Streptomyces sp.

Two enzymes with phospholipase D activity were purified from Streptomyces strains (PMF and PM43) by column chromatography on Fractogel TSK CM-650(S), Sephadex G-100 and Fractogel EMD DEAE-650(M). The purified preparations were found to be homogeneous by SDS-PAGE, capillary electrophoresis and analytical gel filtration. The molecular masses, assessed by MALDI-MS spectrometry, were 53.864 kDa for PMF and 54.147 kDa for PM43. The isoelectric point was 9.1 for both enzymes. The enzymes were most active at around 60 degrees C and stable between pH 4 and 9 and below 50 degrees C. The pH optima were between 4 and 6 for PMF and between 6 and 7 for PM43. Both phospholipases displayed high transphosphatidylation activity but PMF was more selective than PM43.

Comparison of two methods for the recognition of guanine and cytosine rich regions on human gene sequences.

The authors compared two methods to recognise cytosine and guanine rich zones on 19 DNA sequences. The computer method based on an artificial neural network algorithm indicated presence of guanine and cytosine rich regions also in genes not previously known to contain CpG islands.

Potentially functional regions of nucleic acids recognized by a Kohonen's self-organizing map.

Computer recognition of short functional sites on DNA, such as promoter regions or intron-exon boundaries, has recently attracted much interest. In this paper we have focused our attention on the automatic recognition of relevant features of human nucleic acid sequences by means of an unsupervised artificial neural network model. Sixty messenger RNA and 31 genomic DNA sequences were analysed. The results showed that in mRNA, the minimal similarity 60 base pattern was guanine- and cytosine-rich and located in most sequences in a range of 250 bases from either the middle point of the signal peptide coding region or from the start of the coding region. On DNA sequences a region defined by a cluster of minimal similarity patterns was present in many of the analysed genes. This zone may be related to alternative splicing and DNA methylation.

[Computer program recognition of a cDNA sequence specifying signal peptides]. / Riconoscimento mediante programma computazionale delle sequenze di cDNA che specificano i peptidi segnale.

An application of a computational analysis of cDNA sequences is presented in this paper. The goal is the identification of functional domains on sequence data. The results show the capability of this technique to identify a zone of DNA associated with the signal peptide coding region, whose biological function at DNA or RNA level is still unknown.

Identification of a new motif on nucleic acid sequence data using Kohonen's self-organizing map.

Here we present a performance test of a Kohonen features map applied to the fast extraction of uncommon sequences from the coding region of the human insulin receptor gene. We used a network with 30 neurons and with a variable input window. The program was aimed at detecting unique or uncommon DNA regions present in crude sequence data and was able to automatically detect the signal peptide coding regions of a set of human insulin receptor gene data. The testing of this program with HSIRPR cDNA release (EMBL data bank) indicated the presence of unique features in the signal peptide coding region. On the basis of our results this program can automatically detect 'singularity' from crude sequencing data and it does not require knowledge of the features to be found.