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PubMed1985

Decreased myocardial Na+K+ATPase activity in rats with reduced renal mass-saline hypertension.

Inhibition of cardiovascular Na,K-pump activity has been shown to promote an increase in the contractile activity of myocardial and vascular smooth muscle and a consequent rise in blood pressure (BP). It has also been shown that vascular Na,K-pump activity and myocardial Na+K+ATPase activity [the energy source for active sodium (Na) and potassium (K) transport] are decreased in rats with various forms of low renin hypertension including rats with reduced renal mass-saline (RRM-saline) hypertension. In the present study, left ventricular Na+K+ATPase activity from rats with RRM-saline hypertension was found to be decreased in membranes prepared by two independent methods: deoxycholate, sodium iodide (Nal)-treated microsomal fractions (method 1) and membranes prepared by the hypotonic, lithium bromide (LiBr) method (method 2). Relative to RRM normotensive control rats which drank distilled water, myocardial Na+K+ATPase activity from RRM-saline drinking rats was decreased by 18.2% in membranes prepared by method 1 and 33.6% in membranes prepared by method 2. The apparent affinities of Na+K+ATPase for K and for ouabain were unaltered relative to controls in membranes prepared from these hypertensive rats by method 1, and the sialic acid content and 5'-nucleotidase activity (two putative sarcolemmal markers) were unaltered in membranes from the hypertensive rats, prepared by methods 1 and 2 respectively. The Mg2+ATPase activity of membranes prepared by method 1 was increased in the RRM-saline hypertensive rats but because it was not increased in membranes prepared by method 2 the former observation does not appear to be of any pathophysiological importance. In other experiments, hypertension was reversed in RRM-saline hypertensive rats by restricting their salt intake (substitution of distilled water for drinking).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2006

Modelling number of services per conception of Japanese Black cattle by random regression.

Repeated records of number of services per conception (NSC) were collected on 607 Japanese Black cows. Data were analysed by random regression (RRM) and multiple trait (MTM) models, considering NSC in each parity as a separate trait. The chosen RRM included additive genetic and permanent environmental effects fitted with a third-order Legendre polynomials of parity. Heritabilities (h2) estimated by RRM decreased along the NSC trajectory from 0.15 in the first parity to 0.04 in the sixth parity and then increased up to 0.22 in the 10th parity. The corresponding estimates obtained by MTM ranged between 0.04 in parity 9 and 0.13 in parity 1. Permanent environmental proportions (p2) of the total phenotypic variance estimated by RRM showed similar pattern and magnitude to those of h2 estimated by the same method. On the contrary, the p2 estimated by MTM ranged between 0.04 in the first parity and 0.11 in the 10th parity. Additive genetic (r(G)), permanent environmental (r(P)) and phenotypic (r(PH)) correlations were also estimated. The values estimated by RRM between adjacent parities were higher than those of parities far apart. The corresponding values estimated by MTM were lower than those estimated by RRM with no certain trend. The results indicated that NSC in heifers is more heritable than NSC in cows with different parities. Reproductive traits are economically important traits and hence, they should be considered in breeding goals.

Analysis of Variance↗
PubMed2005

RNA-binding domain proteins in Kinetoplastids: a comparative analysis.

RNA-binding proteins are important in many aspects of RNA processing, function, and destruction. One class of such proteins contains the RNA recognition motif (RRM), which consists of about 90 amino acid residues, including the canonical RNP1 octapeptide: (K/R)G(F/Y)(G/A)FVX(F/Y). We used a variety of homology searches to classify all of the RRM proteins of the three kinetoplastids Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major. All three organisms have similar sets of RRM-containing protein orthologues, suggesting common posttranscriptional processing and regulatory pathways. Of the 75 RRM proteins identified in T. brucei, only 13 had clear homologues in other eukaryotes, although 8 more could be given putative functional assignments. A comparison with the 18 RRM proteins of the obligate intracellular parasite Encephalitozoon cuniculi revealed just 3 RRM proteins which appear to be conserved at the primary sequence level throughout eukaryotic evolution: poly(A) binding protein, the rRNA-processing protein MRD1, and the nuclear cap binding protein.

Amino Acid Motifs↗
PubMed2006

Evaluation of MGIT 960-based antimicrobial testing and determination of critical concentrations of first- and second-line antimicrobial drugs with drug-resistant clinical strains of Mycobacterium tuberculosis.

The objectives of this study were to (i) compare agreement of the MGIT 960 system for first-line drugs with a methodology (the resistance ratio method [RRM]) that had been used in clinical trials, relating drug susceptibility to clinical outcome; (ii) compare the performance of the MGIT 960, RRM, and microtiter plate assay (MPA) methodologies for second-line drug testing; and (iii) define critical concentrations for ciprofloxacin and moxifloxacin for liquid-culture-based testing. The large collection of clinical isolates of Mycobacterium tuberculosis (n = 247) used included 176 (71%) multidrug-resistant isolates. The results for MGIT 960 and the RRM for rifampin and isoniazid (n = 200) were in excellent (99 to 100%) agreement for all strains. For streptomycin, 97% of the results at the critical concentration and 92% at high concentration, and for pyrazinamide 92% of results overall, were concordant, but for ethambutol, fewer than 85% (65% for the critical concentration and 84% for the high concentration) of the MGIT-based results were concordant with those for the RRM. The MGIT 960, RRM, and MPA assays (n = 133) correlated well for most second-line drugs tested. For susceptibility to ofloxacin, the MGIT 960 and MPA results were in full agreement. The amikacin and rifabutin results obtained by MGIT 960 agreed with the RRM results in 131 (99%) cases, and for capreomycin, they agreed for 129 of 133 isolates tested (97%). For prothionamide testing, only a limited number of drug-resistant isolates were available for testing and drawing definitive conclusions. We propose critical concentrations of 1.0 microg/ml and 0.125 microg/ml for ciprofloxacin and moxifloxacin, respectively, for liquid-culture-based testing.

Antitubercular Agents↗
PubMed1991

Structural alterations of microvascular smooth muscle cells in reduced renal mass hypertension.

Loss of microvessels (anatomic rarefaction) occurs in chronic reduced renal mass (RRM) hypertension and is mediated via structural degeneration of vascular smooth muscle (VSM) and endothelial cells. The purpose of the present study was to determine if structural changes occur in VSM cells of the microvessels that remain in the tissue of rats with chronic RRM hypertension. Samples of cremaster muscles were taken from normotensive control rats and rats with acute (3-7 days) and chronic (14-28 days) RRM hypertension (75% reduction in kidney mass with 4% NaCl loading). The samples were fixed in situ and processed for light and electron microscopy. Ultrastructural morphology of VSM cells in terminal arterioles of control animals was normal. Although VSM morphology in many microvessels of RRM hypertensive rats was also normal, some vessels exhibited structural changes that were not present in arterioles of the normotensive animals. The most striking change was the appearance of more extensive dense bodies anchoring the contractile filaments around the outer membrane of the cells. Extreme vasoconstriction was observed in some arterioles of RRM rats as long as 2 weeks after salt loading. Focal areas of VSM cell proliferation were evident. Many of the changes occurring in RRM were detected as early as 1 week after the onset of hypertension. These observations suggest that renal mass reduction-salt loading hypertension is associated with early structural and functional changes in the VSM cells.

Animals↗
PubMed2006

The solution structure of REF2-I reveals interdomain interactions and regions involved in binding mRNA export factors and RNA.

The RNA binding and export factor (REF) family of mRNA export adaptors are found in several nuclear protein complexes including the spliceosome, TREX, and exon junction complexes. They bind RNA, interact with the helicase UAP56/DDX39, and are thought to bridge the interaction between the export factor TAP/NXF1 and mRNA. REF2-I consists of three domains, with the RNA recognition motif (RRM) domain positioned in the middle. Here we dissect the interdomain interactions of REF2-I and present the solution structure of a functionally competent double domain (NM; residues 1-155). The N-terminal domain comprises a transient helix (N-helix) linked to the RRM by a flexible arm that includes an Arg-rich region. The N-helix, which is required for REF2-I function in vivo, overlaps the highly conserved REF-N motif and, together with the adjacent Arg-rich region, interacts transiently with the RRM. RNA interacts with REF2-I through arginine-rich regions in its N- and C-terminal domains, but we show that it also interacts weakly with the RRM. The mode of interaction is unusual for an RRM since it involves loops L1 and L5. NMR signal mapping and biochemical analysis with NM indicate that DDX39 and TAP interact with both the N and RRM domains of REF2-I and show that binding of these proteins and RNA will favor an open conformation for the two domains. The proximity of the RNA, TAP, and DDX39 binding sites on REF2-I suggests their binding may be mutually exclusive, which would lead to successive ligand binding events in the course of mRNA export.

Amino Acid Sequence↗
PubMed2000

Telementored laparoscopic cholecystectomy: a pilot study.

Few laparoscopic surgical experts exist relative to the number of surgeons needing training in laparoscopic surgical techniques. This study tested application of telemedicine technology in the mentoring of surgeons during laparoscopic cholecystectomy. Our Surgical Telementoring Suite provided real-time audio and video telecommunication to the operating room. Data points for telementored laparoscopic cholecystectomy (TLC, n = 6) were compared to age and sex-matched controls having standard laparoscopic cholecystectomy (SLC, n = 6) with mentors physically present in the operating room. TLC data were also compared between cases performed with a staff surgeon and resident as mentorees (SRM, n = 3), versus two residents as mentorees (RRM, n = 3). Data were analyzed with chi-square testing. The level of statistical significance was set at p < 0.05. No major operative complications occurred in either group (p > 0.05). Total operative times were similar (92.2 +/- 18.4 minutes SLC vs. 94.7 +/- 25.3 minutes TLC, p > 0.05). Additional data compared between SRM and RRM groups included time to establishment of a pneumoperitoneum of 12-15 mm Hg (7.0 +/- 6.1 minutes SRM vs. 6.7 +/- 2.9 minutes RRM), time to placement of all four trocars (13.0 +/- 3.6 minutes SRM vs. 10.3 +/- 3.1 minutes RRM, time to isolation and proximal clipping of the cystic duct (38.0 +/- 12.1 minutes SRM vs. 55.7 +/- 29.0 minutes RRM), and time to removal of the gallbladder (77.3 +/- 25.4 minutes vs. 77.7 +/- 27.5 minutes RRM). For all data points, p > 0.05. We conclude that telementoring is a safe, effective method for teaching the techniques of LC. This is true for operating teams composed of surgical residents, with or without staff surgeons present.

Adolescent↗
PubMed1996

Application of random-effects regression models in relapse research.

This article describes and illustrates use of random-effects regression models (RRM) in relapse research. RRM are useful in longitudinal analysis of relapse data since they allow for the presence of missing data, time-varying or invariant covariates, and subjects measured at different timepoints. Thus, RRM can deal with "unbalanced" longitudinal relapse data, where a sample of subjects are not all measured at each and every timepoint. Also, recent work has extended RRM to handle dichotomous and ordinal outcomes, which are common in relapse research. Two examples are presented from a smoking cessation study to illustrate analysis using RRM. The first illustrates use of a random-effects ordinal logistic regression model, examining longitudinal changes in smoking status, treating status as an ordinal outcome. The second example focuses on changes in motivation scores prior to and following a first relapse to smoking. This latter example illustrates how RRM can be used to examine predictors and consequences of relapse, where relapse can occur at any study timepoint.

Alcoholism↗
PubMed1996

Prognostic significance of radial margins of clearance in rectal cancer.

A retrospective review of 325 patients was undertaken to analyse whether involvement of the radial resection margin (RRM) could predict locally recurrent disease or distant metastases in patients who had curative surgery for rectal or rectosigmoid cancer. Information on the RRM was available in 253 patients. The RRM was involved in 31 (12 per cent). Nine of these 31 patients developed local recurrence (29 per cent), while only 17 local recurrences were diagnosed in 217 patients (8 per cent) without involvement of the RRM (P < 0.01). At 2 years the overall local recurrence rate was 10 per cent. Distant metastases were diagnosed in 46 patients (18 per cent) and RRM involvement was identified as a prognostic factor depending on lymph node involvement (N stage) (P = 0.02). Local recurrence and some distant metastases result from microscopically incomplete resection. Assessment of the radial depth of tumour invasion by careful histological examination of x791p4ecimen may be used for selection of patients for adjuvant radiotherapy and/or chemotherapy.

Disease-Free Survival↗
PubMed1993

Functional analysis of pre-mRNA splicing factor SF2/ASF structural domains.

Human pre-mRNA splicing factor SF2/ASF has an activity required for general splicing in vitro and promotes utilization of proximal alternative 5' splice sites in a concentration-dependent manner by opposing hnRNP A1. We introduced selected mutations in the N-terminal RNA recognition motif (RRM) and the C-terminal Arg/Ser (RS) domain of SF2/ASF, and assayed the resulting recombinant proteins for constitutive and alternative splicing in vitro and for binding to pre-mRNA and mRNA. Mutants inactive in constitutive splicing can affect alternative splice site selection, demonstrating that these activities involve distinct molecular interactions. Specific protein-RNA contact mediated by Phe56 and Phe58 in the RNP-1 submotif of the SF2/ASF RRM are essential for constitutive splicing, although they are not required for RRM-mediated binding to pre-mRNA. The RS domain is also required for constitutive splicing activity and both Arg and Ser residues are important. Analysis of domain deletion mutants demonstrated strong synergy between the RRM and a central degenerate RRM repeat in binding to RNA. These two domains are sufficient for alternative splicing activity in the absence of an RS domain.

Alternative Splicing↗
PubMed2004

Personal health behaviors in women who have undergone risk-reducing mastectomy.

BACKGROUND: Risk-reducing mastectomy (RRM) remains an effective yet controversial modality for the primary prevention of breast cancer. Is the choice of RRM consistent with a generally proactive healthy lifestyle? METHODS: The National Prophylactic Mastectomy Registry is a volunteer database comprised of 460 women who have undergone bilateral RRM. Each patient received a questionnaire drawn from the Centers for Disease Control Behavioral Risk Factor Surveillance System. Their responses were compared with gender-specific national data from the Centers for Disease Control. RESULTS: Women in the RRM group were statistically more likely to engage in risk-reducing personal health behavior including not smoking, exercising, maintaining their health, and taking advantage of screening programs. CONCLUSIONS: Women in the registry who underwent RRM practiced a more "healthy" lifestyle than gender-matched controls. Therefore, the decision to have prophylactic surgery may have been part of a proactive approach toward their overall physical well-being.

Adult↗
PubMed2003

Urgent adolescent psychiatric consultation: from the accident and emergency department to inpatient adolescent psychiatry.

The Rapid Response Model (RRM) provides psychiatric services to children and adolescents seen at the Accident and Emergency (A&E) department or at the Urgent Consultation Clinic of the Child and Adolescent Psychiatry Division the next day. In a naturally occurring experiment, the RRM was introduced, withdrawn and restarted. When RRM was withdrawn at one site, it was implemented at another. The RRM reduced nighttime Emergency Consultations and inpatient admissions from A&E, while it increased daytime consultations and daytime admissions. The RRM provided timely, organized emergency psychiatric services. A&E staff expressed satisfaction with the service.

Adolescent↗
PubMed1997

Crystal structure of human UP1, the domain of hnRNP A1 that contains two RNA-recognition motifs.

BACKGROUND: Heterogeneous nuclear ribonucleoprotein (hnRNP) A1 is one of the most abundant core proteins of hnRNP complexes in metazoan nuclei. It behaves as a global regulator of alternative pre-mRNA splicing by antagonizing the activities of several serine/arginine-rich splicing factors (SR proteins), resulting in the activation of distal alternative 5' splice sites and skipping of optional exons. Purified hnRNP A1 has nucleic acid annealing activity. The protein also shuttles continuously between the nucleus and the cytoplasm, a process mediated by signals within its C-terminal glycine-rich domain. The N-terminal region of human hnRNP A1, termed unwinding protein 1 (UP1), contains two RNA-recognition motifs (RRMs), RRM1 and RRM2. Understanding the structural elements by which hnRNP A1 interacts with RNA will have broad implications for studies of RNA processing. RESULTS: The crystal structure of UP1 has been determined to 1.9 A resolution. Each RRM independently adopts the characteristic RRM fold, consisting of a four-stranded antiparallel beta-pleated sheet and two alpha helices packed on one side of the beta sheet. The two RRMs are antiparallel and held in close contact, mainly by two Arg-Asp ion pairs. As a result, the two four-stranded beta sheets are brought together to form an extended RNA-binding surface. A segment of the linker connecting the two RRMs is flexible in the absence of bound RNA, but the general location of the linker suggests that it can make direct contacts with RNA. Comparison with other RRM structures indicates that a short 310 helix, found immediately N-terminal to the first beta strand in RRM1, may interact with RNA directly. CONCLUSIONS: The RRM is one of the most common and best characterized RNA-binding motifs. In certain cases, one RRM is sufficient for sequence-specific and high affinity RNA binding; but in other cases, synergy between several RRMs within a single protein is required. This study shows how two RRMs are organized in a single polypeptide. The two independently folded RRMs in UP1 are held together in a fixed geometry, enabling the two RRMs to function as a single entity in binding RNA, and so explaining the synergy between the RRMs. The UP1 structure also suggests that residues which lie outside of the RRMs can make potentially important interactions with RNA.

Amino Acid Sequence↗
PubMed1999

Functional analysis of SNF, the Drosophila U1A/U2B" homolog: identification of dispensable and indispensable motifs for both snRNP assembly and function in vivo.

In Drosophila, the spliceosomal protein SNF fulfills the functions of two vertebrate proteins, U1 snRNP-UlA and U2 snRNP-U2B". The structure and sequence of SNF, U1A, and U2B" are nearly identical with two RNA recognition motifs (RRM) separated by a short linker region, yet they have different RNA-binding properties: U1A binds U1 snRNA, U2B" binds U2 snRNA, and SNF binds both snRNAs. Structure/function studies on the human proteins have identified motifs in the N-terminal RRM that are critical for RNA-binding specificity but have failed to identify a function for the C-terminal RRM. Interestingly, SNF is chimeric in these motifs, suggesting a basis for its dual specificity. Here, we test the importance of these motifs by introducing site-directed mutations in the snf coding region and examining the effects of these mutations on assembly into the snRNP and on snf function in vivo. We found that an N-terminal RRM mutant protein predicted to eliminate RNA binding still assembles into snRNPs and is capable of rescuing snf's lethal phenotype only if the normally dispensable C-terminal RRM is present. We also found that the mixed motif in the "RNA-specificity" domain is necessary for SNF's dual function whereas the mixed motif in the U2A'-protein-binding region is not. Finally, we demonstrate that animals carrying a snf mutation that converts SNF from a bifunctional protein to a U1 snRNP-specific protein are viable. This unexpected result suggests that SNF's presence within the U2 snRNP is not essential for splicing.

Amino Acid Sequence↗
PubMed2001

Structural basis for recognition of AU-rich element RNA by the HuD protein.

Hu proteins bind to adenosine-uridine (AU)-rich elements (AREs) in the 3' untranslated regions of many short-lived mRNAs, thereby stabilizing them. Here we report the crystal structures of the first two RNA recognition motif (RRM) domains of the HuD protein in complex with an 11-nucleotide fragment of a class I ARE (the c-fos ARE; to 1.8 A), and with an 11-nucleotide fragment of a class II ARE (the tumor necrosis factor alpha ARE; to 2.3 A). These structures reveal a consensus RNA recognition sequence that suggests a preference for pyrimidine-rich sequences and a requirement for a central uracil residue in the clustered AUUUA repeats found in class II AREs. Comparison to structures of other RRM domain-nucleic acid complexes reveals two base recognition pockets in all the structures that interact with bases using residues in conserved ribonucleoprotein motifs and at the C-terminal ends of RRM domains. Different conformations of nucleic acid can be bound by RRM domains by using different combinations of base recognition pockets and multiple RRM domains.

AT Rich Sequence↗
PubMed2005

Prospective comparison of cardiopulmonary events during minilaparoscopy and colonoscopy under conscious sedation.

BACKGROUND AND STUDY AIMS: Cardiorespiratory parameters were examined throughout diagnostic minilaparoscopy procedures. The same parameters were analyzed during colonoscopy, and the data were compared. PATIENTS AND METHODS: Sixty-five consecutive unselected patients undergoing minilaparoscopy (group 1: ASA I, n = 34; group 2: ASA II/III, n = 31) and 61 consecutive unselected patients undergoing colonoscopy (group 3: ASA I, n = 31; group 4: ASA II/III, n = 30) were included. Oxygen saturation (Sao (2)), heart rate (HR) and mean arterial pressure (RRm) were measured continuously, and 12-lead electrocardiography (ECG) recordings were made at specific times during each procedure. RESULTS: Minor differences were observed, particularly after premedication, probably due to different dosage regimens and timing in the two examination techniques. After premedication, testing for differences from baseline values showed a minor decrease in Sao (2) and RRm in the minilaparoscopy groups in comparison with the colonoscopy groups (median Sao (2), group 1: 99.9 % +/- 0 vs. group 3 : 100 % -1, P = 0.0078; median RRm, group 1: 99.5 - 4 mm Hg vs. group 3 : 96 -16 mm Hg, P = 0.046, and median RRm, group 2 : 110 + 1 mm Hg vs. group 4 : 101 -13.5 mm Hg, P = 0.0007). HR increased in minilaparoscopy in comparison with colonoscopy (median HR: group 2 : 77 + 4 beats/min vs. group 4 : 75.5 +/- 0 beats/min; P = 0.01). Comparison of defined relevant pathological changes in Sao (2), RRm, HR, and ECG showed no significant differences. DISCUSSION: These data indicate that diagnostic minilaparoscopy under conscious sedation is only associated with limited risk in patients with compensated cardiopulmonary diseases. This is probably due to the low insufflation pressure used.

Blood Pressure↗
PubMed2004

Domain architectures and characterization of an RNA-binding protein, TLS.

Translocated in liposarcoma (TLS) is an important protein component of the heterogeneous nuclear ribonucleoprotein complex involved in the splicing of pre-mRNA and the export of fully processed mRNA to the cytoplasm. We examined the domain organization of human TLS by a combined approach using limited proteolysis, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, circular dichroism, inductively coupled plasma atomic emission spectroscopy, and NMR spectroscopy. We found that the RNA recognition motif (RRM) and zinc finger-like domains exclusively form protease-resistant core structures within the isolated TLS protein fragments, while the remaining regions, including the Arg-Gly-Gly repeats, appear to be completely unstructured. Thus, TLS contains the unstructured N-terminal half followed by the RRM and zinc finger-like domains, which are connected to each other by a flexible linker. We also carried out NMR analyses to obtain more detailed insights into the individual RRM and zinc finger-like domains. The 113Cd NMR analysis of the zinc finger-like domain verified that zinc is coordinated with four cysteines in the C4 type scheme. We also investigated the interaction of each domain with an oligo-RNA containing the GGUG sequence, which appears to be critical for the TLS function in splicing. The backbone amide NMR chemical shift perturbation analyses indicated that the zinc finger domain binds GGUG-containing RNA with a dissociation constant of about 1.0 x 10(-5) m, whereas the RRM domain showed no observable interaction with this RNA. This surprising result implies that the zinc finger domain plays a more predominant role in RNA recognition than the RRM domain.

Amino Acid Sequence↗
PubMed2006

Structure of eIF3b RNA recognition motif and its interaction with eIF3j: structural insights into the recruitment of eIF3b to the 40 S ribosomal subunit.

Mammalian eIF3 is a 700-kDa multiprotein complex essential for initiation of protein synthesis in eukaryotic cells. It consists of 13 subunits (eIF3a to -m), among which eIF3b serves as a major scaffolding protein. Here we report the solution structure of the N-terminal RNA recognition motif of human eIF3b (eIF3b-RRM) determined by NMR spectroscopy. The structure reveals a noncanonical RRM with a negatively charged surface in the beta-sheet area contradictory with potential RNA binding activity. Instead, eIF3j, which is required for stable 40 S ribosome binding of the eIF3 complex, specifically binds to the rear alpha-helices of the eIF3b-RRM, opposite to its beta-sheet surface. Moreover, we identify that an N-terminal 69-amino acid peptide of eIF3j is sufficient for binding to eIF3b-RRM and that this interaction is essential for eIF3b-RRM recruitment to the 40 S ribosomal subunit. Our results provide the first structure of an important subdomain of a core eIF3 subunit and detailed insights into protein-protein interactions between two eIF3 subunits required for stable eIF3 recruitment to the 40 S subunit.

Amino Acid Motifs↗