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PubMed2002

Age-related changes in control of blood pressure and heart rate during sleep in the rat.

STUDY OBJECTIVES: The aim of this study was to determine age-related changes in the control of mean arterial pressure (MAP) and heart rate (HR) during sleep, and its relationship to the baroreflex in aging. DESIGN: MAP, HR, body temperature (TP), spontaneous activity (ACT), and sleeping/waking duration were monitored for 24 hours in groups of young (10-12 wk old) and old (23-24 mo old) rats. SETTING: The sleep laboratory at the University of Tokushima. PARTICIPANTS: Subjects were 8 young (10-12 wk old) and 7 old (23-24 mo old) Wistar rats. INTERVENTIONS: Reflex control of HR was evaluated by examining various pressure responses to an intravenous bolus injection of phenylephrine and sodium nitroprusside. MEASUREMENTS AND RESULTS: MAP and TP were recorded by a radiotelemetry system. HR was detected from the AP signal. ACT was counted by a photo-sensor system. In the case of old rats, the sensitivity of baroreflex control of HR was significantly depressed, and the spontaneous increase of MAP and HR during REM sleep and the MAP drop at the end of REM sleep were significantly enhanced. The old rats showed no large deterioration of the circadian profiles of MAP, HR, TP, and the amount of sleep. CONCLUSIONS: The baroreflex dysfunction is considered to appear in an early stage of the aging process, and to affect the control of MAP and HR during sleep.

Age Factors↗
PubMed2003

RNA-binding properties of HCF152, an Arabidopsis PPR protein involved in the processing of chloroplast RNA.

The nonphotosynthetic mutant of Arabidopsis hcf152 is impaired in the processing of the chloroplast polycistronic transcript, psbB-psbT-psbH-petB-petD, resulting in nonproduction of the essential photosynthetic cytochrome b6f complex. The nucleus-encoded HCF152gene was identified to encode a pentatricopeptide repeat (PPR) protein composed primarily of 12 PPR motifs, similar to other proteins of this family that were identified in mutants defected in chloroplast gene expression. To understand the molecular mechanism of how HCF152 modulates chloroplast gene expression, the molecular and biochemical properties should be revealed. To this end, HCF152 and several truncated versions were produced in bacteria and analyzed for RNA-binding and protein-protein interaction. It was found that two HCF152 polypeptides bind to form a homodimer, and that this binding is impaired by a single amino acid substitute near the carboxyl terminus, replacing leucine with proline. Recombinant HCF152 bound with higher affinity RNA molecules, resembling the petB exon-intron junctions, as well as several other molecules. The highest affinity was found to RNA composed of the poly(A) sequence. When truncated proteins composed of different numbers of PPR motifs were analyzed for RNA-binding, it was found that two PPR motifs were required for RNA-binding, but had very low affinity. The affinity to RNA increased significantly when proteins composed of more PPR motifs were analyzed, displaying the highest affinity with the full-length protein composed of 12 PPR motifs. Together, our data characterized the nuclear-encoded HCF152 to be a chloroplast RNA-binding protein that may be involved in the processing or stabilization of the petB transcript by binding to the exon-intron junctions.

Arabidopsis↗
PubMed2005

Processing of radial fast spin-echo data for obtaining T2 estimates from a single k-space data set.

Radially acquired fast spin-echo data can be processed to obtain T2-weighted images and a T2 map from a single k-space data set. The general approach is to use data at a specific TE (or narrow TE range) in the center of k-space and data at other TE values in the outer part of k-space. With this method high-resolution T2-weighted images and T2 maps are obtained in a time efficient manner. The mixing of TE data, however, introduces errors in the T2-weighted images and T2 maps that affect the accuracy of the T2 estimates. In this work, various k-space data processing methods for reconstructing T2-weighted images and T2 maps from a single radial fast spin-echo k-space data set are analyzed in terms of the accuracy of T2 estimates. The analysis is focused on the effect of image artifacts, object dependency, and noise on the T2 estimates. Results are presented in computer-generated phantoms and in vivo.

Algorithms↗
PubMed2005

HnRNP A3 genes and pseudogenes in the vertebrate genomes.

The hnRNP A/B type proteins are abundant nuclear factors that bind to Pol II transcripts and are involved in numerous RNA-related activities. To date most data on the hnRNP A/B family have been obtained with recombinant proteins and cell cultures. Further characterization can result from an examination of the impact of various modifications in intact functional loci; however, such characterization is hampered by the presence of numerous and widely dispersed hnRNP A/B-related sequences in the mammalian genome. We have found hnRNP A3, a poorly recognized member of the hnRNP A/B family, among candidate transcription factors that interact with the regulatory region of the Hoxc8 gene and screened the human and mouse genomes for genes that encode hnRNP A3. We demonstrate that the sequence reported previously as the human hnRNP A3 gene (Accession number S63912) and located on 10p11.1 belongs to a processed pseudogene of the functional intron-containing locus HNRPA3, which we have identified on 2q31.2. We have also identified its murine orthologs on mouse chromosome 2D and rat chromosome 3q23. Alternative splices were revealed at the N-terminus and in the middle of hnRNP A3. 14 and 28 additional loci in the human and mouse genome, respectively, were mapped and identified as hnRNP A3 processed pseudogenes. In addition, we have found and compared hnRNP A3 orthologous genes in Gallus gallus, Xenopus tropicalis, and Danio rerio. The present in silico analysis serves as a necessary step toward a further functional characterization of hnRNP A3.

Alternative Splicing↗
PubMed1995

Distribution and spatial geometry of dopamine interplexiform cells in the retina. II. External arborizations in the adult rat and monkey.

The morphology and distribution of dopaminergic interplexiform cells in adult rat and monkey retinas were analyzed to determine any correlation with the function of dopamine in the outer retinal layers. The retinas were processed as whole mounts for tyrosine hydroxylase immunohistochemistry. There was a network formed by the sclerally directed processes of interplexiform cells in the inner nuclear, outer plexiform, and outer nuclear layers running throughout the retina. Their density was higher in the superior retina than in the inferior retina of the rat and was especially high in the superior temporal quadrant. The external network in this quadrant was significantly less dense in the monkey than in the rat, as are the interplexiform cells. The somata of interplexiform and other dopaminergic cells were about the same size in both rats and monkeys. Computer-assisted reconstruction of external arborizations of individual cells showed that external processes lay very close to horizontal and photoreceptor cells and also to blood capillaries. Because they were long, thin, and highly varicose; branched at right angles; and often arose from an axon hillock, the external processes were identified as axons. Therefore, we define the dopaminergic interplexiform cells as multiaxonal neurons, with at least one outwardly directed axon that reaches the outer plexiform layer. The function of the network of external processes from the interplexiform dopaminergic cells is discussed in terms of modulating the release of dopamine to external layers.

Animals↗
PubMed1990

A mutant poliovirus containing a novel proteolytic cleavage site in VP3 is altered in viral maturation.

A six-amino-acid insertion containing a Q-G amino acid pair was introduced into the carboxy terminus of the capsid protein VP3 (between residues 236 and 237). Transfection of monkey cells with full-length poliovirus cDNA containing the insertion described above yields a mutant virus (Sel-1C-02) in which cleavage occurs almost entirely at the inserted Q-G amino acid pair instead of at the wild-type VP3-VP1 cleavage site. Mutant Sel-1C-02 is delayed in the kinetics of virus production at 39 degrees C and exhibits a defect in VP0 cleavage into VP2 and VP4 at 39 degrees C. Sucrose gradient analysis of HeLa cell extracts prepared from cells infected by Sel-1C-02 at 39 degrees C shows an accumulation of fast-sedimenting replication-packaging complexes and a significant amount of uncleaved VP0 present in fractions containing mature virions. Our data provide in vivo evidence for the importance of determinants other than the conserved amino acid pair (Q-G) for recognition and cleavage of the P1 precursor by proteinase 3CD and show that an alteration in the carboxy terminus of VP3 or the amino terminus of VP1 affects the process of viral maturation.

Amino Acid Sequence↗
PubMed1989

Developmental levels of processing in metaphor interpretation.

We outline a theory of metaphor interpretation. The theory posits varying levels of semantic processing and formalizes them in terms of kinds of semantic-mapping operators that transform properties of the metaphoric vehicle (i.e., predicate) into properties of the metaphoric topic (i.e., subject). We used cognitive-developmental theory to estimate the mental-processing complexity of the various mapping operators, and thereby to predict the timing of their emergence in childhood, and to construct a measure of the metaphoric-processing levels. Metaphor interpretations collected from children (aged 6-12 years) and adults were coded and scored according to the processing levels. In two separate developmental studies, processing score increased with age in a predictable way. Growth in a mental-attentional resource accounted for much of the developmental variance in metaphor interpretation. Possible moderating effects of knowledge and context are discussed.

Child↗
PubMed2005

Delayed neutrophil apoptosis attenuated by melatonin in human acute pancreatitis.

OBJECTIVES: This study evaluated the expression of neutrophil apoptosis and the effects of melatonin at different concentrations on delayed neutrophil apoptosis in different severities of acute pancreatitis in patients. METHODS: The study population was comprised of 10 patients with severe acute pancreatitis (SAP) and 10 with mild acute pancreatitis (MAP). A total of 10 mL of blood was drawn 24 hours after the onset of the clinical disease for isolation and incubation of the human neutrophils with 4 different concentrations of melatonin. Neutrophil apoptosis activity, CD18 expression, and respiratory burst activity were assessed with flow cytometry 12 hours after incubation. Another group of neutrophils from a healthy control group was used (n = 6) for comparison. RESULTS: Neutrophil apoptosis in patients with SAP is delayed compared with that of patients with MAP. Neutrophils from patients with SAP or MAP are functionally activated. Melatonin at concentrations of 10(-8), 10(-7), or 10(-6) M reverses the delayed process and enhances apoptosis activity in neutrophils in patients with MAP. Melatonin at concentrations of 10(-7) and 10(-6) M reverses the delayed process and increases apoptosis activity in neutrophils in patients with SAP. Neutrophils from patients with SAP and MAP showed significantly increased CD18 expression and respiratory burst activity. Melatonin at concentrations of 10(-7) or 10(-6) M reverses CD18 expression and respiratory burst activity in neutrophils in patients with SAP. CONCLUSIONS: This study highlights the importance of neutrophil apoptosis in patients with SAP and raises the possibility of a therapeutic strategy. Study data show that melatonin promotes neutrophil apoptosis in human acute pancreatitis.

Acute Disease↗
PubMed2004

Localization of the motor and speech zones of the cerebral cortex by functional magnetic resonance tomography.

Functional magnetic resonance tomography provides a non-invasive method for mapping the cerebral cortex. The aim of the present work was to assess the potential and suitability of this method in a series of brain disorders. Studies were performed on 32 volunteers (mean age 37.8 +/- 20.9 years) and 16 patients with tumors of the cerebral cortex (mean age 36.2 +/- 24.2 years). Initial functional images were processed by statistical methods. Computed activation maps were superimposed on anatomical images. In 89% of cases, functional magnetic resonance tomography allowed the motor cortex and Broca's area to be localized; in almost 69%, the method impinged on the therapeutic tactics used in patients with cerebral cortex lesions. Thus, functional magnetic resonance tomography can be used in clinical conditions to obtain information not yielded by other diagnostic methods and which can be used to plan the neurosurgical treatment of patients with supratentorial brain tumors with maximum preservation of cerebral cortex function. Assessments of the state of the motor and speech areas by this tomographic method has potential applications in neurosurgery and neurophysiology.

Adolescent↗
PubMed1991

Phosphorylation of the avian retrovirus integration protein and proteolytic processing of its carboxyl terminus.

The integration protein (IN) of the Prague A strain of Rous sarcoma virus (RSV) was analyzed by high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three polypeptides of similar proportions and molecular mass (32 kDa) were immunoprecipitated by an antiserum directed against the first 10 amino acids of the amino terminus of IN. However, the faster-migrating nonphosphorylated polypeptide was not immunoprecipitated by two different polyclonal antisera directed against the last 11 amino acids of the carboxyl terminus of IN. These results suggest that the faster-migrating species was proteolytically processed at its carboxyl terminus. RSV IN is phosphorylated on an S residue located five amino acids from its carboxyl terminus. Two different missense mutations at this S residue resulted in the isolation of slow-growing viable mutants whose phenotypes were stable. Each mutation at residue 282 eliminated both major phosphorylated-Ser-containing tryptic peptides observed with wild-type IN. An S----F mutation resulted in the conversion of all IN polypeptides to one species that was not precipitable by carboxyl-terminal antisera, suggesting that this amino acid transition promoted proteolysis at the carboxyl terminus. An S----D mutation resulted in the recovery of one major (greater than 95%) slower-migrating polypeptide that was immunoprecipitated by carboxyl-terminal antisera, suggesting that this negatively charged D residue (similar to phosphorylated Ser) inhibited proteolysis. Modification of the S residue at amino acid 262 to R had no apparent effect on the proteolytic processing or phosphorylation of IN.

Amino Acid Sequence↗
PubMed2006

Crystallographic protein model-building on the web.

UNLABELLED: X-ray crystallography is the most widely used method to determine the 3D structure of protein molecules. One of the most difficult steps in protein crystallography is model-building, which consists of constructing a backbone and then amino acid side chains into an electron density map. Interpretation of electron density maps represents a major bottleneck in protein structure determination pipelines, and thus, automated techniques to interpret maps can greatly improve the throughput. We have developed WebTex, a simple and yet powerful web interface to TEXTAL, a program that automates this process of fitting atoms into electron density maps. TEXTAL can also be downloaded for local installation. AVAILABILITY: Web interface, downloadable binaries and documentation at http://textal.tamu.edu

Algorithms↗
PubMed1987

The tRNA-tufB operon transcription termination and processing upstream from tufB.

Two genes, tufA and tufB, located at 73 and 88 minutes of the Escherichia coli linkage map, code for the polypeptide chain elongation factor EF-Tu. tufB is transcribed with four upstream tRNA genes, thrU, tyrU, glyT and thrT, into a cotranscript of approximately 1800 nucleotides. Here we show that in vivo processing yields a 1320 nucleotide transcript of tufB. S1 nuclease fine mapping reveals that the processing site is located in the intergenic region at about 72 to 74 nucleotides upstream from the initiation codon of the tufB cistron. A deletion in the cloned tRNA-tufB operon, encompassing the 3' half of thrU, the complete tyrU, glyT, thrT genes and ten nucleotides of the intergenic region, causes a threefold increase of the rate of plasmid tufB transcription, a fourfold increase of plasmid-borne tufB RNA and a twofold increase of plasmid-borne EF-TuB. We conclude that the deletion has eliminated a transcription termination site probably located after the thrT gene. Termination at this site uncouples tRNA synthesis from tufB transcription.

Chromosome Mapping↗
PubMed2000

The MAP kinase inhibitors, PD098059, UO126 and SB203580, inhibit IL-1beta-dependent PGE(2) release via mechanistically distinct processes.

1. In common with human bronchial epithelial cells, pulmonary A549 cells release prostaglandin (PG) E(2) in response to pro-inflammatory cytokines. We have therefore used these cells to examine the effect of the selective mitogen activated protein (MAP) kinase inhibitors; PD098059, a mitogen activated and extracellular regulated kinase kinase (MEK) 1 inhibitor, UO126, a dual MEK1 & MEK2 inhibitor, and SB203580, a p38 MAP kinase inhibitor in the IL-1beta-dependent release of PGE(2). 2. Following IL-1beta treatment the extracellular regulated kinases (ERKs) and the p38 MAP kinases were rapidly phosphorylated. 3. PD09059, UO126 and SB203580 prevented IL-1beta-induced PGE(2) release at doses that correlated closely with published IC(50) values. Small or partial effects at the relevant doses were observed on induction of cyclo-oxygenase (COX) activity or COX-2 protein suggesting that the primary effects were at the level of arachidonate availability. 4. Neither PD098059 nor SB203580 showed any effect on IL-1beta-induced arachidonate release. We therefore speculate that the MEK1/ERK and p38 kinase cascades play a role in the functional coupling of arachidonate release to COX-2. 5. In contrast, UO126 was highly effective at inhibiting IL-1beta-dependent arachidonate release, implicating MEK2 in the activation of the PLA(2) that is involved in IL-1beta-dependent PGE(2) release. 6. We conclude that the MEK1, MEK2 and p38 MAP kinase inhibitors, PD098059, UO126 and SB203580, are highly potent in respect of inflammatory PG release. Finally, we conclude that these inhibitors act via mechanistically distinct processes, which may have anti-inflammatory benefits.

Arachidonic Acid↗
PubMed2003

Neural resources for processing language and environmental sounds: evidence from aphasia.

Although aphasia is often characterized as a selective impairment in language function, left hemisphere lesions may cause impairments in semantic processing of auditory information, not only in verbal but also in nonverbal domains. We assessed the 'online' relationship between verbal and nonverbal auditory processing by examining the ability of 30 left hemisphere-damaged aphasic patients to match environmental sounds and linguistic phrases to corresponding pictures. The verbal and nonverbal task components were matched carefully through a norming study; 21 age-matched controls and five right hemisphere-damaged patients were also tested to provide further reference points. We found that, while the aphasic groups were impaired relative to normal controls, they were impaired to the same extent in both domains, with accuracy and reaction time for verbal and nonverbal trials revealing unusually high correlations (r = 0.74 for accuracy, r = 0.95 for reaction time). Severely aphasic patients tended to perform worse in both domains, but lesion size did not correlate with performance. Lesion overlay analysis indicated that damage to posterior regions in the left middle and superior temporal gyri and to the inferior parietal lobe was a predictor of deficits in processing for both speech and environmental sounds. The lesion mapping and further statistical assessments reliably revealed a posterior superior temporal region (Wernicke's area, traditionally considered a language-specific region) as being differentially more important for processing nonverbal sounds compared with verbal sounds. These results suggest that, in most cases, processing of meaningful verbal and nonverbal auditory information break down together in stroke and that subsequent recovery of function applies to both domains. This suggests that language shares neural resources with those used for processing information in other domains.

Adult↗
PubMed1978

Membrane proteins synthesized but not processed by isolated maize chloroplasts.

One-dimensional maps of proteolytic fragments generated by digestion with Staphylococcus aureus protease in sodium dodecyl sulfate (SDS) were used to identify three polypeptides synthesized by isolated Zea mays chloroplasts. This technique does not depend upon proper incorporation of the newly synthesized polypeptides into a more complex structure for their identification. The only preliminary purification required is electrophoretic separation on SDS-polyacrylamide gels. The pattern of radioactive fragments from labeled proteins which co-migrate with the alpha and beta subunits of chloroplast coupling factor (CF1) corresponds precisely to the pattern of stainable fragments derived from subunits of the purified enzyme. A 34,500-dalton protein is the major membrane-associated product of protein synthesis by isolated maize chloroplasts. From the similarity in the fragments formed by digestion with S. aureus protease, it appears that this radioactive protein is probably a precursor of a 32,000-dalton protein which is a component of the thylakoid. The alpha and beta subunits of CF1 newly synthesized by isolated chloroplasts are not fully extractable by procedures which normally solubilize the enzyme from membranes. The 34,500-dalton protein is not processed to the 32,000-dalton form in any great amount by isolated chloroplasts. A 19,000-dalton fragment of the 32,000-dalton protein is protected from digestion when thylakoids are treated with proteases, while the newly synthesized 34,500-dalton protein is fully susceptible. The isolated chloroplast does not appear to be able to fully integrate these newly made proteins into the membrane structure.

Cell Membrane↗
PubMed1995

Improved recombinant tandem expression of translation initiation factor IF2 in RNASE E deficient E. coli cells.

The prokaryotic translation initiation factor IF2 exists in a varying number of nested forms in different species. In E. coli three natural forms exist, IF2 alpha, IF2 beta and IF2 gamma differing only in the N-terminal: IF2 beta and IF2 gamma lack 158 and 165 amino acid residues, respectively, as compared to IF2 alpha. We have earlier shown that the smaller forms of IF2 are not the result of a specific proteolysis of IF2 alpha, but produced from individual translation initiation sites in the mRNA. However it has not been known whether the expression in E. coli of IF2 beta and IF2 gamma is dependent on or related to a posttranscriptional processing of the polycistronic nusA operon, containing infB, the gene for IF2. Here we have used S1 mapping to study the existence of such mRNA processing in the region between the initiation sites for IF2 alpha and IF2 beta/IF2 gamma. The results show a Ribonuclease E cleavage site at position +200 in the infB mRNA between the translation initiation sites. However, studies of the overexpression of the different forms of IF2 show that the relative expression of IF2 alpha and IF2 beta/IF2 gamma is independent of RNase E activity. Thus E. coli exhibits a true tandem translation of intact infB mRNA with multiple in-frame translation initiation sites resulting in gene products of different sizes. An additional observation is a significant increase in the level of overexpression of IF2 in cells devoid of RNase E activity. We conclude that due to lack of RNase E activity, the amount of plasmid-transcribed infB mRNA available for translation is accumulated, resulting in an elevated amount of recombinant IF2. This observation may have a more general application within the field of recombinant protein production and expression efficiency.

Bacterial Proteins↗
PubMed2006

Sensitive detection of Mycobacterium avium subsp. paratuberculosis in bovine semen by real-time PCR.

AIMS: To develop a fast and sensitive protocol for detection of Mycobacterium avium subsp. paratuberculosis (MAP) in bovine semen and to make a critical evaluation of the analytical sensitivity. METHODS AND RESULTS: Processed semen was spiked with known amounts of MAP. Semen from different bulls as well as semen of different dilutions was tested. The samples were treated with lysing agents and beadbeating and the DNA was extracted with phenol and chloroform. Real-time PCR with a fluorescent probe targeting the insertion element IS900 detected as few as 10 organisms per sample of 100 mul semen. PCR-inhibition was monitored by inclusion of an internal control. Pre-treatment with immunomagnetic separation was also evaluated, but was not shown to improve the overall sensitivity. CONCLUSIONS: Real-time PCR is a sensitive method for detection of MAP in bovine semen. Lysis by mechanical disruption followed by phenol and chloroform extraction efficiently isolated DNA and removed PCR-inhibitors. SIGNIFICANCE AND IMPACT OF THE STUDY: The high sensitivity of the applied method allows reliable testing of bovine semen used for artificial insemination to prevent the spread of Johne's disease, caused by MAP.

Animals↗
PubMed1999

Neural mechanisms for processing binocular information II. Complex cells.

Complex cells in the striate cortex exhibit extensive spatiotemporal nonlinearities, presumably due to a convergence of various subunits. Because these subunits essentially determine many aspects of a complex cell receptive field (RF), such as tuning for orientation, spatial frequency, and binocular disparity, examination of the RF properties of subunits is important for understanding functional roles of complex cells. Although monocular aspects of these subunits have been studied, little is known about their binocular properties. Using a sophisticated RF mapping technique that employs binary m-sequences, we have examined binocular interactions exhibited by complex cells in the cat's striate cortex and the binocular RF properties of their underlying functional subunits. We find that binocular interaction RFs of complex cells exhibit subregions that are elongated along the frontoparallel axis at different binocular disparities. Therefore responses of complex cells are largely independent of monocular stimulus position or phase as long as the binocular disparity of the stimulus is kept constant. The binocular interaction RF is well described by a sum of binocular interaction RFs of underlying functional subunits, which exhibit simple cell-like RFs and a preference for different monocular phases but the same binocular disparity. For more than half of the complex cells examined, subunits of each cell are consistent with the characteristics specified by an energy model, with respect to the number of subunits as well as relationships between the subunit properties. Subunits exhibit RF binocular disparities that are largely consistent with a phase mechanism for encoding binocular disparity. These results indicate that binocular interactions of complex cells are derived from simple cell-like subunits, which exhibit multiplicative binocular interactions. Therefore binocular interactions of complex cells are also multiplicative. This suggests that complex cells compute something analogous to an interocular cross-correlation of images for a local region of visual space. The result of this computation can be used for solving the stereo correspondence problem.

Animals↗