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.

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.

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.

High-dose cytosine arabinoside as the initial treatment of poor-risk patients with acute nonlymphocytic leukemia: a Leukemia Intergroup Study.

Sixty-seven patients with newly diagnosed acute nonlymphocytic leukemia (ANLL) who were considered to be poor candidates for treatment with cytosine arabinoside (ara-C)/anthracycline antibiotic therapy were treated with high-dose ara-C (HDara-C) remission induction therapy. Thirty-four of the 67 patients had a hematologic disorder before developing acute leukemia or had a history of exposure to marrow toxins, 23 patients were greater than 70 years old, and 10 patients had medical problems that were felt to be a contraindication to therapy with an anthracycline antibiotic. Forty-two percent of patients entered complete remission (CR), whereas 22% failed to enter remission because of persistent leukemia. Treatment was associated with substantial toxicity varying from nausea and vomiting to irreversible cerebellar toxicity. Thirty-four percent of patients died during therapy. Poor performance status, a low serum albumin, and a low platelet count were associated with death during remission induction therapy, whereas a high pretherapy leukemic cell mass and a large number of residual leukemic cells in the marrow after six days of therapy were associated with treatment failure due to persistent leukemia.

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.

Limited efficacy of a four-day course of high-dose cytosine arabinoside in the treatment of poor-risk patients with acute nonlymphocytic leukemia.

High-dose cytosine arabinoside therapy was administered to 29 patients with poor-prognosis acute nonlymphocytic leukemia. In an attempt to reduce toxicity, therapy was divided into an initial 4-day course of therapy, followed by a 3-day course of the marrow aspirate examined 1 week after the end of treatment contained substantial numbers of leukemic cells. Of the 29 patients, 5 entered complete remission, 2 of them after the initial 4-day course of therapy. The toxicity of split-course therapy was the same as that of conventional 6-day high-dose cytosine arabinoside therapy. This study demonstrates that the modification of high-dose cytosine arabinoside therapy used in this study failed to reduce toxicity and produced a lower remission rate than that obtained with the 6-day course of therapy.

Relationship between plasma adriamycin levels and the outcome of remission induction therapy for acute nonlymphocytic leukemia.

Plasma adriamycin and adriamycinol levels were measured in 45 patients with acute nonlymphocytic leukemia 3 h after the drug was administered. A wide range of levels as found. Plasma levels increased after the administration of each of the three daily doses of the drug. High plasma levels were associated with both death during remission induction therapy and, for patients who entered remission, long remissions.

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.

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.

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.

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.

Study of bound water of poly-adenine using high frequency dielectric measurements.

The high frequency dielectric constant of poly-adenine (poly-A) was measured between 1 MHz and 1 GHz. The purpose of these experiments was to investigate the state of water molecules that are bound to the charged groups of the poly-A molecule. Analysis of the data using the Maxwell's mixture equation revealed the dielectric constant of bound water higher than we expected. Using Onsager's internal field in Debye's equation, we calculated the dielectric constant of water in the vicinity of a charged ion. The result of this computation demonstrates that the dielectric constant of bound water is much smaller than the normal value only in the immediate proximity of charged ions (within 2 A). The dielectric constant increases rapidly to the normal value as the distance increases from 2 to 4 A. This observation indicates that charged sites of polyions have only short range interactions with the surrounding water molecules. However, this conclusion pertains only to rotary diffusion of bound water since dielectric measurement is unable to detect translational diffusion.

[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.

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.

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.