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Soluble vascular cell adhesion molecule-1 and E-selectin levels in relation to vascular risk factors and to E-selectin genotype in the first degree relatives of NIDDM patients and in NIDDM patients.

To investigate the metabolic and genetic associations of levels of soluble adhesion molecules, plasma levels of soluble E-selectin and vascular cell adhesion molecule-1 were measured in 60 non-insulin-dependent diabetes mellitus (NIDDM) patients, 60 first-degree relatives of NIDDM patients and 60 control subjects, none of whom displayed clinical features of vascular disease. In addition, E-selectin A561C genotype, coding for a serine to arginine change, was determined. E-selectin levels were elevated in the patient group; 57 [52-63] (mean [95% confidence intervals]) ng/ml, compared with both relatives; 44 [39-50] ng/ml p = 0.001 and controls 39.5 [36-43] ng/ml p = 0.0001. E-selectin levels correlated with triglycerides, tissue-plasminogen activator and plasminogen activator inhibitor-1 activity in all groups. Levels of E-selectin were related to E-selectin genotype, being higher in subjects possessing the arginine allele (51.4 vs 44.5 ng/ml p < 0.05). E-selectin levels were higher in males than females in controls (female 35 [32-39] vs male 45 [40-51] ng/ml p = 0.004), and NIDDM relatives (female 38 [33-44] vs male 52 [45-61] ng/ml p = 0.004) but not in NIDDM patients where levels were similar (female 58 [49-69] vs male 56 [50-62] ng/ml, ns). There was no difference in soluble vascular cell adhesion molecule-1 levels between the three groups (control 640 [598-686] ng/ml, NIDDM relatives 634 [593-678] ng/ml and NIDDM patients 664 [608-725] ng/ml). In controls and patients vascular cell adhesion molecule-1 levels correlated with von Willebrand factor (vWF). The results indicate that levels of E-selectin relate to vascular risk factors in control subjects, NIDDM relatives and NIDDM patients.

Adult↗

A maximum likelihood method for detecting functional divergence at individual codon sites, with application to gene family evolution.

The tailoring of existing genetic systems to new uses is called genetic co-option. Mechanisms of genetic co-option have been difficult to study because of difficulties in identifying functionally important changes. One way to study genetic co-option in protein-coding genes is to identify those amino acid sites that have experienced changes in selective pressure following a genetic co-option event. In this paper we present a maximum likelihood method useful for measuring divergent selective pressures and identifying the amino acid sites affected by divergent selection. The method is based on a codon model of evolution and uses the nonsynonymous-to-synonymous rate ratio (omega) as a measure of selection on the protein, with omega = 1, < 1, and > 1 indicating neutral evolution, purifying selection, and positive selection, respectively. The model allows variation in omega among sites, with a fraction of sites evolving under divergent selective pressures. Divergent selection is indicated by different omega's between clades, such as between paralogous clades of a gene family. We applied the codon model to duplication followed by functional divergence of (i) the epsilon and gamma globin genes and (ii) the eosinophil cationic protein (ECP) and eosinophil-derived neurotoxin (EDN) genes. In both cases likelihood ratio tests suggested the presence of sites evolving under divergent selective pressures. Results of the epsilon and gamma globin analysis suggested that divergent selective pressures might be a consequence of a weakened relationship between fetal hemoglobin and 2,3-diphosphoglycerate. We suggest that empirical Bayesian identification of sites evolving under divergent selective pressures, combined with structural and functional information, can provide a valuable framework for identifying and studying mechanisms of genetic co-option. Limitations of the new method are discussed.

Amino Acids↗

[Analysis program for quantitative detection of chromosome aberrations using comparative genomic hybridization].

Comparative genomic hybridization (CGH) is a molecular cytogenetic method for the detection of chromosomal imbalances between a tumor and a normal genome. In order to produce quantitative and reproducible results, we developed an image analysis program that allows the detection and mapping of the genetic alterations. The result is represented as a CGH sum karyogram in which the genetic changes are documented as color coded-chromosomes. The aim of our investigation is to correlate the genotype with the phenotype on the basis of CGH sum karyograms and thus to achieve a genetic characterization that will supplement the morphological tumor description.

Cell Transformation, Neoplastic↗

Application of universal primers for identification of Foot-and-mouth disease virus and Swine vesicular disease virus by PCR and PCR-ELISA.

Two approaches for simultaneous identification of both Foot-and-mouth disease virus (FMDV) and Swine vesicular disease virus (SVDV) are described: (1) a single-step reverse transcription-PCR with three primers and (2) a PCR-ELISA assay with two universal primers for genome amplification and two virus-specific probes for identification. These methods are based on the use of 3D gene universal PCR primers, the structure of which was optimized and refined due to the close relationship between the two viruses belonging to different genera of the Picornaviridae family. In procedure (1), a three-primer PCR containing one universal antisense primer and two virus-specific primers was shown to differentiate between FMDV and SVDV in one reaction, due to the different length of the amplified DNA fragments (600 and 340 base pairs, respectively). In procedure (2), the two viruses were identified by PCR-ELISA, i.e. PCR for the 3D gene followed by two parallel hybridizations with FMDV and SVDV-specific probes in microplate wells and ELISA detection. The application of universal primers could halve the number of PCR experiments in both cases, as compared to the usual virus-specific PCR procedures. Also, we investigated the 3D gene structure of several SVDV strains isolated at different times. No essential changes were detected in the regions coding for conserved motifs of the RNA-dependent RNA polymerase recognized by our universal primers. The multi-primer PCR was successfully tested on 38 FMDV and 15 SVDV strains, and the PCR-ELISA on 32 FMDV and 16 SVDV strains including clinical material from disease cases.

Amino Acid Motifs↗

Dynamics of gene expression for microtubule-associated protein MAP1B, embryonic alpha-tubulin and late neural beta-tubulin mRNAs in the hippocampus of aged rats.

In the present study we characterized the developmental changes in the prevalence of mRNA coding for microtubule-associated protein, MAP1B, embryonic alpha-tubulin and late neural beta-tubulin in rat hippocampus and forebrain from 1 to 720 days of age using RNA gel-blot analysis. We find that (i) the microtubule-associated protein, MAP1B, signal was relatively abundant at early postnatal stages when compared with mature animals. The hybridization signal in the 24-month-old rats was was approximately 1.7 times that observed in 6-month-old rats. (ii) Embryonic alpha-tubulin and late neural beta-tubulin were differentially regulated during rat brain development. This regulation is characterized by a dramatic decrease in the amount of alpha-tubulin after day-1 and a coincident increase in the production of late neural beta-tubulin. Both messages became stabilized at moderate levels during the subsequent developmental stages. However, the averaged signal for beta-tubulin was then approximately 1.8-fold increased in 24- vs. 6-month-old rats. These results are consistent with hypothesis of an age-associated increase in reactive synaptogenesis where the healthy neurons sprout new connections to compensate for neuronal loss occurring in neighboring neurons.

Aging↗

Analysis of ECG data, for data compression.

A number of papers on the subject of data reduction techniques applied to ECG Data have recently been published; however, the authors found that most of these articles did not consider quantization techniques, which can be effectively applied to ECG data without any complex parameter extraction procedures. In this paper the authors have looked at the effects of quantization on ECG data and techniques of reducing the amount of data needed to represent these signals. Basically, 3 data reduction techniques, linear prediction using differential pulse code modulation, spectral analysis and slope change detection are investigated and a relative assessment of their performance is presented. This analysis revealed that a slope change detection, as applied to prefiltered data, can be used to represent ECG data at a rate of 2 bits/sample, while maintaining the mean squared error and peak error below 1% and 5% respectively. This technique therefore gives an effective 3 to 1 reduction over the original sampled data, since it was found that the original data could be quantized to 6 bits without significant loss of waveform information.

Electrocardiography↗

Ovarian atrial natriuretic peptide during the rat estrous cycle.

The changes in ovarian levels of immunoreactive atrial natriuretic peptide (irANP) and arginine vasopressin (irAVP) were observed during the estrous cycle of rat. We also demonstrated the synthesis of ovarian ANP. In adult 4-day cycling rats, ovarian level of irANP was found to be the highest on proestrus and was to be the lowest on diestrus. Ovarian irANP level inversely correlated with ovarian level of irAVP. On reverse-phase HPLC, two distinct peaks of ovarian irANP, high and low molecular weight forms, existed in the each stage of the estrous cycle. However, no significant changes in plasma and atrial concentrations of ANP were observed during the cycle. The rat ovary contained mRNA coding for ANP. These data showing the synchronized cyclic change of ovarian irANP and irAVP with the estrous cycle suggest that the ovary locally synthesizes ANP and ovarian ANP may play regulatory roles on the follicular fluid dynamics.

Analysis of Variance↗

Is there a constancy for velocity?

Human observers are unable to use disparity information to transform the angular velocity signal into a precise object-based code. The Weber fraction for discriminating changes in objective velocity (cm/sec) is about twice the Weber fraction for discriminating changes in angular velocity (deg/sec), and is substantially higher than predicted from a combination of the errors in judging disparity and angular velocity. By comparison, judgments of the distance traversed by the moving target show excellent size constancy. The discrimination of changes in objective size (cm) is as precise as the discrimination of changes in angular size (deg). The angular velocity signal is useful without transformation into an object-centered signal; it guides eye and body movements, and is the basis of motion parallax judgments. The need to retain this angular signal may explain why there is no efficient mechanism for velocity constancy.

Discrimination, Psychological↗

Impairment of intracellular antiviral defense with age: age-dependent changes in expression of interferon-induced and double-stranded RNA-activated 2-5A synthetase in rat.

The 2',5'-oligoadenylate (2-5A) system is involved in the defense of mammalian cells against virus infection. In a previous study [25], we demonstrated that the activities of the enzymes which synthesize and degrade 2-5A [2-5A synthetase (2-5OAS) and 2',3'-exoribonuclease] and of the enzyme that is activated by 2-5A (ribonuclease L) change during aging and development in different tissues of rat. The age-dependent decrease in 2-5OAS activity and increase in 2-5A nuclease activity results in a decrease in the cellular 2-5A content, suggesting that the efficiency of the antiviral 2-5A system is impaired in aged rats. Here we determined the age-dependent changes in the level of mRNA coding for the class I isoenzyme of 2-5OAS (M(r) 40-46 kDa) in rat liver and brain using a cDNA which was recently cloned from rat hippocampus. We found that the decrease in 2-5OAS activity is accompanied by a decrease in the level of 2-5OAS mRNA; in old animals (32-33 months old), the amount of 2-5OAS mRNA was reduced to 20-30% compared to young adult (2-3 months old) (100%) and middle-aged adult animals (12 months old) (110-120%). In addition, Western-blotting experiments revealed that the amount of class I 2-5OAS capable of binding to its activator, poly(I).poly(C), is also diminished in the livers and brains of old rats compared to those of young adult and middle-aged adult animals.

2',5'-Oligoadenylate Synthetase↗

Production of the antigen and the antibody of the JC virus major capsid protein VP1.

The DNA of the major capsid protein VP1 of the human polyomavirus JC virus (JCV), Taiwan-3 strain, was generated from the urine of an autoimmune disease patient by polymerase chain reaction (PRC). The VP1 DNA was cloned into a prokaryotic expression vector, pGEX-4T-1, for expression in E. coli. The nucleotide sequences and the deduced amino acid sequences were determined and compared with the JC virus prototype, Mad-1. Thirty nucleotides were different between these two strains. Six of the altered nucleotides affected amino acid coding and ten of them caused changes in endonuclease recognition sites. The recombinant VPI protein was purified and used to raise monospecific antiserum in rabbit. Recombinant JCV VP1 protein and its monospecific antiserum are important clinical reagents and could possibly be developed as a subunit vaccine and as a serological diagnostic antigen in the future. In addition, the region between amino acid residues 40 and 80 of JCV VP1 is predicted to be an antigenic epitope on the basis of its hydropathy plot and comparison with the VP1 sequences of SV40 and BK virus.

Amino Acid Sequence↗

Hormonal regulation of translatable mRNA of glucose-6-phosphate dehydrogenase in primary cultures of adult rat hepatocytes.

The quantity of translatable mRNA of glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ 1-oxidoreductase, EC 1.1.1.49) in primary cultures of adult rat hepatocytes subjected to different hormonal conditions was determined with a reticulocyte-lysate, cell-free system. The level of glucose-6-phosphate dehydrogenase mRNA was about 5-fold higher in the presence of insulin than in its absence. This increase of glucose-6-phosphate dehydrogenase mRNA reached a maximum 12 h after the addition of insulin. The maximum level of induction of glucose-6-phosphate dehydrogenase mRNA required 10(-8) M insulin. Glucagon and triiodothyronine had no effect on the glucose-6-phosphate dehydrogenase mRNA level. The increase of glucose-6-phosphate dehydrogenase activity correlated with the increase in level of mRNA of this enzyme. This suggests that the changes in glucose-6-phosphate dehydrogenase activity in response to the above hormonal changes are primarily due to changes in the amount of mRNA coding for this enzyme.

Animals↗

Stabilization of a protein-tyrosine phosphatase mRNA upon mitogenic stimulation of T-lymphocytes.

The expression of a non-receptor type protein-tyrosine phosphatase (the T-cell phosphatase or PTP-S) which shows homology with basic domains of Fos and Jun, was investigated upon mitogenic stimulation of rat splenic T lymphocytes. As studied by Northern blot analysis of total cellular RNA, mitogenic stimulation of T lymphocytes by concanavalin A resulted in an increase in the level of PTP-S mRNA; there was little or no change in the level of mRNA coding for PTP-1 (which is also a non-receptor type tyrosine phosphatase). Maximum increase of about 3-fold in the level of PTP-S mRNA occurred after 72 h of mitogenic stimulation. Mitogenic stimulation did not increase the level of PTP-S transcripts in the nucleus. The half-life of PTP-S mRNA in unstimulated lymphocytes was about 25 min which increased to 5 h after mitogenic stimulation. An inhibitor of protein synthesis, cycloheximide, increased the level of PTP-S transcripts by 6-fold in control lymphocytes but did not increase the level of PTP-1 transcripts. Treatment with cycloheximide increased the half-life of PTP-S transcripts in resting lymphocytes. The PTP-S gene product was identified as a 42 kDa polypeptide by immunoblotting. The level of PTP-S gene product increased upon mitogenic stimulation of lymphocytes by Con A and reached a maximum after 72 h, as determined by immunoblotting. These results suggest that post-transcriptional regulation of mRNA stability is an important factor in controlling the level of this phosphatase mRNA during mitogenic stimulation of T-lymphocytes.

Animals↗

A physical map of the chromosomal region determining O-antigen biosynthesis in Vibrio cholerae O1.

We have previously described the cosmid cloning of the genes determining the biosynthesis of the Inaba and Ogawa O-antigens of the lipopolysaccharides of Vibrio cholerae O1 (Manning et al., 1986). By Southern hybridization analysis of chromosomal and cosmid DNA, and heteroduplex analysis between the clones we have been able to precisely define the region of contiguous chromosomal DNA in the vicinity of the O-antigen-encoding region. These data and comparison of end points of clones and of deletion derivatives demonstrate that at least 16 kb of a 19-kb SstI fragment is required to encode O-antigen biosynthesis. Expression of O-antigen is independent of the orientation of this SstI fragment with respect to cloning vectors suggesting that its regulatory region has been cloned intact. No detectable differences were observed in the restriction patterns of the Inaba and Ogawa coding regions implying that only minor changes are involved when serotype conversion (Inaba to Ogawa or vice versa) occurs. Bhaskaran [Ind. J. Med. Res. 47 (1959) 253-260] originally defined this region associated with O-antigen biosynthesis oag; however, to be consistent with other organisms [Hitchcock et al., J. Bacteriol. 166 (1986) 699-705], it is suggested this be changed to rfb.

Antigens, Bacterial↗

Evidence that V+ fibronectin, GFAP and S100 beta mRNAs are increased in the hippocampus of aged rats.

In the present study, using RNA gel-blot analysis, we characterized the developmental changes in the prevalence of mRNA coding for fibronectin (FN), glial fibrillary acidic protein (GFAP), neurotrophic protein S100 beta, and beta-actin mRNA in rat hippocampus and forebrain from 1 to 720 days of age. We found that the FN and mRNA containing the V segment (FN-V) was relatively abundant at early postnatal stages, but very few transcripts were detected in adult rats. However, the hybridization signal for the juvenile FN-V mRNA was up to approximately 8-fold increased in some but not all 24-versus 6-month-old rats. Also, GFAP and S100 beta transcripts were faintly expressed at an early developmental stage, then the level of expression steadily increased starting with day 21. The greatest increase averages approximately 1.8-fold for GFAP in 24-month-old rats, and approximately 1.5-fold for S100 beta in 15-month-old versus 6-month-old rats. As all these messages are localized primarily in astrocytes, we conclude that (a) astrocyte might play an active role in aging hippocampus and (b) an increase in S100 beta and GFAP mRNA expression may precede that for FN-V mRNA expression in a hypothetical pathway of molecular events underlying neurodegeneration in the hippocampus of old rats. We also note the considerable variability among the 24-month-old rats, suggesting that aging is an individual process.

Actins↗

Prior pain induces heightened motor responses during clustered care in preterm infants in the NICU.

BACKGROUND: Acute pain is a significant stressor for preterm infants in neonatal intensive care units (NICU); however, little is known about the effects of acute pain on subsequent motor responses during clusters of tactile handling. AIMS: (1) To compare facial, body and heart rate reactivity in preterm infants at 32 weeks gestational age (GA) during routine care-giving tasks following a rest period (RCC: diapering, measuring abdominal girth and axillary temperature, mouth care) with their responses to Clustered Care following blood collection (PCC). (2) To examine how GA at birth affects patterns of stress and self-regulatory behaviors during RCC and PCC. STUDY DESIGN: Within-group crossover design (random order). SUBJECTS: Preterm infants, N=54 (mean GA at birth 29.3 +/- 2.2 weeks; mean birth weight 1257 +/- 423 g) were assessed at 32 weeks GA in the NICU. OUTCOME MEASURES: The Newborn Developmental Care and Assessment Program (NIDCAP) and Neonatal Facial Coding System (NFCS) were coded from continuous bedside video recordings. Changes in mean heart rate (HR) were computed using custom physiologic software. RESULTS: All infants had heightened facial, body and HR responses when CC followed a painful procedure compared to when they had not been handled prior to CC. Infants born at earlier GA (<30 weeks) had equal numbers of stress cues during RCC and PCC, but dampened self-regulatory behaviors during PCC. CONCLUSION: Prior pain induces heightened biobehavioral reactivity in preterm infants during subsequent tactile procedures. In addition, clustering care is particularly stressful for infants born at earlier GA.

Cross-Over Studies↗

Arrhythmogenic right ventricular cardiomyopathy due to a novel plakophilin 2 mutation: wide spectrum of disease in mutation carriers within a family.

BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a familial disease, with male preponderance, characterized by progressive fibrofatty replacement of the right ventricle and ventricular arrhythmias. Mutations in plakophilin-2 (PKP2), a desmosomal protein, have been reported to underlie familial ARVC. We report a novel ARVC PKP2 mutation and present the clinical findings in three female mutation carriers. METHODS: We sequenced PKP2 from genomic DNA isolated from peripheral blood lymphocytes in a female proband who presented with cardiac arrest and in her four first-degree relatives. Clinical testing and diagnosis of ARVC was based on International Task Force criteria. RESULTS: The proband was diagnosed with ARVC due to right ventricular enlargement and regional hypokinesis, along with repolarization abnormalities and frequent ventricular ectopy. A novel 28 bp insertion in exon 11 of the PKP2 gene was found which causes a frameshift in the coding region. This results in a change in the amino acid sequence of the protein with a premature stop codon at position 740. Of the four relatives, only the mother and younger sister were identified as mutation carriers. The mother was phenotypically normal, while the younger sister has repolarization abnormalities and frequent ventricular ectopy. CONCLUSIONS: We report a novel PKP2 mutation that causes familial ARVC. All mutation carriers in this kindred group were women, and the family showed incomplete penetrance and variable expression of ARVC. Premature truncation of the plakophilin-2 protein appears to be the predominant mechanism whereby PKP2 mutations elicit the ARVC phenotype.

Adolescent↗

Left ventricular wall motion analysis using real-time three-dimensional ultrasound.

This study tested the ability of real-time 3-D (RT 3-D) echocardiography to detect and delineate regions of abnormal contraction (akinesia or dyskinesia) in a canine model of regional myocardial injury and to develop methods to simplify injury assessments. Closed chest RT 3-D scans were obtained and regional left ventricular (LV) contractile function was assessed in nine animals at baseline and after myocardial cryoinjury with a 1-cm cryoprobe. Evaluation of contractile function was based on radial shortening of LV chamber cross-sections at multiple levels. Radial length changes were analyzed using color-coded circumferential maps of the LV. Seven sets of motion maps demonstrated new areas of poorly contracting myocardium in the cryoinjured region relative to baseline. Two sets of data were excluded due to insufficient LV visualization. Motion maps derived from RT 3-D echo have the ability to detect and localize regions of abnormal LV wall motion.

Animals↗

Differential clustering of Mycoplasma mycoides subsp. mycoides SC strains by PCR-REA of the bgl locus.

In order to develop a specific tool differentiating the African field strains of Mycoplasma mycoides subsp. mycoides SC from other potentially less virulent strains, including the vaccine strains, we have developed a PCR followed by a restriction enzyme analysis (PCR-REA). This approach also differentiates the African field strains from the Australian strains and the type strain PG1. The genomic marker detected by the PCR-REA is based on a single nucleotide change in the bgl gene that codes for 6-phospho-beta-glucosidase (Bgl), an enzyme that is involved in sugar metabolism.

Africa↗