Natriuretic peptide guanylyl cyclase receptors in the kidney of the Japanese eel, Anguilla japonica.

Natriuretic peptides are linked to osmoregulation, cardiovascular and volume regulation in fishes. The peptides bind to two guanylyl-cyclase-linked receptors, natriuretic peptide receptor-A (NPR-A) and NPR-B, to elicit their effects. Atrial natriuretic peptide (ANP) binds principally to NPR-A, whereas C-type natriuretic peptide (CNP) binds to NPR-B. The teleost kidney has an important role in the maintenance of fluid and electrolyte balance; therefore, the location of NPR-A and NPR-B in the kidney could provide insights into the functions of natriuretic peptides. This study used homologous, affinity purified, polyclonal antibodies to NPR-A and NPR-B to determine their location in the kidney of the Japanese eel, Anguilla japonica. Kidneys from freshwater and seawater acclimated animals were fixed overnight in 4% paraformaldehyde before being paraffin-embedded and immunostained. NPR-A immunoreactivity was found on the apical membrane of proximal tubule 1 and the vascular endothelium including the glomerular capillaries. In contrast, NPR-B immunoreactivity was located on the smooth muscle of blood vessels including the glomerular afferent and efferent arterioles, and on smooth muscle tissue surrounding the collecting ducts. No difference in the distribution of NPR-A and NPR-B was observed between freshwater and seawater kidneys. Immunoreactivity was not observed in any tissue in which the antibodies had been preabsorbed. In addition, there was no difference in NPR-A and NPR-B mRNA expression between freshwater-acclimated and seawater-acclimated eels. These results suggest that, although utilizing the same second messenger system, ANP and CNP act on different targets within the kidney and presumably elicit different effects.

Altered cell cycle distribution, hyperplasia, and inhibited differentiation in Arabidopsis caused by the D-type cyclin CYCD3.

CYCD3;1 expression in Arabidopsis is associated with proliferating tissues such as meristems and developing leaves but not with differentiated tissues. Constitutive overexpression of CYCD3;1 increases CYCD3;1-associated kinase activity and reduces the proportion of cells in the G1-phase of the cell cycle. Moreover, CYCD3;1 overexpression leads to striking alterations in development. Leaf architecture in overexpressing plants is altered radically, with a failure to develop distinct spongy and palisade mesophyll layers. Associated with this, we observe hyperproliferation of leaf cells; in particular, the epidermis consists of large numbers of small, incompletely differentiated polygonal cells. Endoreduplication, a marker for differentiated cells that have exited from the mitotic cell cycle, is inhibited strongly in CYCD3;1-overexpressing plants. Transcript analysis reveals an activation of putative compensatory mechanisms upon CYCD3;1 overexpression or subsequent cell cycle activation. These results demonstrate that cell cycle exit in the G1-phase is required for normal cellular differentiation processes during plant development and suggest a critical role for CYCD3 in the switch from cell proliferation to the final stages of differentiation.

Cell cycle regulation of cyclin-dependent kinases in tobacco cultivar Bright Yellow-2 cells.

Plants possess two major classes of cyclin-dependent kinases (CDK) with cyclin-binding motifs PSTAIRE (CDK-a) and PPTA/TLRE (CDK-b). Tobacco (Nicotiana tabacum L. cv Bright Yellow-2) cells are the most highly synchronizable plant culture, but no detailed analysis of CDK activities has been reported in this system. Here we describe isolation of new PPTALRE CDKs (Nicta;CdkB1) from Bright Yellow-2 cells and present detailed analysis of the mRNA, protein and kinase activity levels of CdkB1, and the PSTAIRE CDKA during the growth and cell cycles. CdkA and CdkB1 transcripts are more abundant in exponential than in stationary phase cells, but the two genes show strikingly different regulation during the cell cycle. CdkA mRNA and protein accumulate during G1 in cells re-entering the cell cycle, and immunoprecipitated histone H1 kinase activity increases at the G1/S boundary. Aphidicolin synchronized cells show the highest CDKA-associated histone H1 kinase activity during S-G2 phases, although CdkA mRNA and protein levels are not significantly regulated. In contrast, CdkB1 transcripts are present at very low levels until S phase and CDKB1 protein and kinase activity is almost undetectable in G1. CdkB1 mRNA accumulates through S until M phase and its associated kinase activity peaks at the G2/M boundary, confirming that transcription of PPTALRE CDKs is cell cycle regulated. We suggest that CDKA kinase activity likely plays roles at the G1/S phase boundary, during S phase, and at the G2/M phase transition, and that CDKB1 kinase activity is present only at G2/M.

Regulation of both gene expression and protein stability provides genetically assisted target evaluation (GATE) for microbial target validation.

The attempt to develop novel antibiotics, active against organisms resistant to current therapies, has led researchers to seek and explore new drug targets. The rapid sequencing and analysis of entire microbial genomes has identified large numbers of genes that may be sufficiently different from their human counterparts to be exploited as targets for antimicrobial treatment. As a first step, the importance of the various putative targets for microbial growth and survival must be assessed. Emerging validation technologies are becoming increasingly sophisticated and, in certain cases, allow prioritisation of the best targets. In this paper, genetically assisted target evaluation (GATE) is introduced as a versatile target validation technology. GATE concomitantly manipulates both synthesis and stability of the targeted protein using copper ions as an effector. This technology allows rapid quantitation of the lethal consequences of inactivation of targeted gene products in Saccharomyces cerevisiae. Additional tools can then be applied to extend these results into pathogenic organisms, such as Candida albicans.

The Arabidopsis D-type cyclins CycD2 and CycD3 both interact in vivo with the PSTAIRE cyclin-dependent kinase Cdc2a but are differentially controlled.

D-type cyclins (CycD) play key roles in linking the Arabidopsis cell cycle to extracellular and developmental signals, but little is known of their regulation at the post-transcriptional level or of their cyclin-dependent kinase (CDK) partners. Using new antisera to CycD2 and CycD3, we demonstrate that the CDK partner of these Arabidopsis cyclins is the PSTAIRE-containing CDK Cdc2a. Previous analysis has shown that transcript levels of CycD2 and CycD3 are regulated in response to sucrose levels and that both their mRNA levels and kinase activity are induced with different kinetics during the G(1) phase of cells reentering the division cycle from quiescence. Here we analyze the protein levels and kinase activity of CycD2 and CycD3. We show that CycD3 protein and kinase activity parallel the abundance of its mRNA and that CycD3 protein is rapidly lost from cells in stationary phase or following sucrose removal. In contrast to both CycD3 and the regulation of its own mRNA levels, CycD2 protein is present at constant levels. CycD2 kinase activity is regulated by sequestration of CycD2 protein in a form inaccessible to immunoprecipitation and probably not complexed to Cdc2a.

Cyclin D control of growth rate in plants.

The mechanisms by which plants modulate their growth rate in response to environmental and developmental conditions are unknown, but are presumed to involve specialized regions called meristems where cell division is concentrated. The possible role of cell division in influencing meristem activity and overall plant growth rate is controversial, with a prevailing view that cell division is secondary to higher order meristem controls. Here we show that a reduction in the length of the cell-cycle G1 phase and faster cell cycling occur when the rate of cell division in transgenic tobacco plants is increased by the plant D-type cyclin CycD2 (ref. 8). The plants have normal cell and meristem sizes, but elevated overall growth rates, an increased rate of leaf initiation and accelerated development in all stages from seedling to maturity. We conclude that cell division is a principal determinant of meristem activity and overall growth rate, and propose that modulation of plant growth rate is achieved through regulation of G1.

Sugar control of the plant cell cycle: differential regulation of Arabidopsis D-type cyclin gene expression.

In most plants, sucrose is the major transported carbon source. Carbon source availability in the form of sucrose is likely to be a major determinant of cell division, and mechanisms must exist for sensing sugar levels and mediating appropriate control of the cell cycle. We show that sugar availability plays a major role during the G(1) phase by controlling the expression of CycD cyclins in Arabidopsis. CycD2 mRNA levels increase within 30 min of the addition of sucrose; CycD3 is induced after 4 h. This corresponds to induction of CycD2 expression early in G(1) and CycD3 expression in late G(1) near the S-phase boundary. CycD2 and CycD3 induction is independent both of progression to a specific point in the cell cycle and of protein synthesis. Protein kinase activity of CycD2- and CycD3-containing cyclin-dependent kinases is consistent with the observed regulation of their mRNA levels. CycD2 and CycD3 therefore act as direct mediators of the presence of sugar in cell cycle commitment. CycD3, but not CycD2, expression responds to hormones, for which we show that the presence of sugars is required. Finally, protein phosphatases are shown to be involved in regulating CycD2 and CycD3 induction. We propose that control of CycD2 and CycD3 by sucrose forms part of cell cycle control in response to cellular carbohydrate status.

Paxillin association in vitro with integrin cytoplasmic domain peptides.

Short cytoplasmic domains of integrin heterodimers are crucial for transduction of signals generated by adhesion of cells to the extracellular matrix. Here, we describe the use of peptides mimicking the intracellular tails of integrin alpha5beta1 to assay in vitro associations with cytoskeletal proteins. Our results suggest that the focal adhesion protein, paxillin, may interact directly with the intracellular region of the integrin beta1 subunit. Paxillin is known to form stable complexes with several signaling molecules, including focal adhesion kinase. Physical interaction between paxillin and the beta1 cytoplasmic domain suggests a model in which paxillin may function as a key intermediary in integrin-mediated signal transduction.

Diagnosis of malignant hyperthermia: a comparison of the in vitro contracture test with the molecular genetic diagnosis in a large pedigree.

Malignant hyperthermia (MH) is an inherited skeletal muscle disorder and is one of the major causes of death resulting from anaesthesia. MH is currently diagnosed by the in vitro contracture test performed on a muscle biopsy. Genetic linkage analysis on an Irish MH pedigree showed that when the thresholds for the standardised European protocol for MHS diagnosis was applied, linkage between the MHS phenotype and the RYR1 locus was excluded. When we raised the threshold values for assignment of MHS status and assumed MHN diagnosis in subjects where this threshold was not attained, tight linkage between MHS and RYR1 markers was observed, suggesting that MHS is linked to the RYR1 locus in this pedigree. Confirmation of these results was borne out by the fact that all of the MHS patients in the pedigree exceeding the raised threshold carried the known MHS Gly341Arg RYR1 mutation. The results obtained could be explained (1) by false positive diagnosis of MHS in the recombinant subjects, (2) by the presence of a mutation in a predisposing gene other than RYR1, or (3) by the presence of mild subclinical myopathies. The implications of these results for heterogeneity studies is discussed.

Characterization of biphenyl catabolic genes of gram-positive polychlorinated biphenyl degrader Rhodococcus sp. strain RHA1.

Rhodococcus sp. strain RHA1 is a gram-positive polychlorinated biphenyl (PCB) degrader which can degrade 10 ppm of PCB48 (equivalent to Aroclor1248), including tri-, tetra-, and pentachlorobiphenyls, in a few days. We isolated the 7.6-kb EcoRI-BamHI fragment carrying the biphenyl catabolic genes of RHA1 and determined their nucleotide sequence. On the basis of deduced amino acid sequence homology, we identified six bph genes, bphA1A2A3A4, bphB, and bphC, that are responsible for the initial three steps of biphenyl degradation. The order of bph genes in RHA1 is bphA1A2A3A4-bphC-bphB. This gene order differs from that of other PCB degraders reported previously. The amino acid sequences deduced from the RHA1 bph genes have a higher degree of homology with the tod genes from Pseudomonas putida F1 (49 to 79%) than with the bph genes of Pseudomonas sp. strains KF707 and KKS102 (30 to 65%). In Escherichia coli, bphA gene activity was not observed even when expression vectors were used. The activities of bphB and bphC, however, were confirmed by observing the transformation of biphenyl to a meta-cleavage compound with the aid of benzene dioxygenase activity that complemented the bphA gene activity (S. Irie, S. Doi, T. Yorifuji, M. Takagi, and K. Yano, J. Bacteriol. 169:5174-5179, 1987). The expected products of the cloned bph genes, except bphA3, were observed in E. coli in an in vitro transcription-translation system. Insertion mutations of bphA1 and bphC of Rhodococcus sp. strain RHA1 were constructed by gene replacement with cloned gene fragments.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship of anatomic disease to appropriateness ratings of coronary angiography.

BACKGROUND: At Harvard Community Health Plan (HCHP), Brookline, Mass, a mixed-model health maintenance organization (HMO), coronary angiography is performed at numerous community and tertiary-level teaching hospitals. OBJECTIVE: To determine the appropriateness of coronary angiography within HCHP according to RAND (1992) criteria and to examine the relationship between the appropriateness rating and (1) the clinical indication for catheterization and (2) the extent of anatomic disease. METHOD: A retrospective, randomized hospital medical record review of 292 patients enrolled in HCHP who underwent coronary angiography in 1992, stratified by four distinct HCHP subgroups. RESULTS: Of the coronary angiographies reviewed, 78% were rated appropriate, 16% uncertain, and only 6% inappropriate across the entire sample. Ratings were comparable in all subdivisions of HCHP despite an incidence rate of catheterization in one of the three HMO divisions that was 60% and 40% higher than in the other two divisions. The lowest appropriateness ratings were for Asymptomatic patients (43%) and those with Chest Pain of Uncertain Origin (35%) (capital letters refer to the RAND clinical indication criteria mentioned above). A rating of necessity was not a better discriminator of anatomic disease than a rating of appropriateness alone: 82% and 84%, respectively, were found to have disease by angiography. CONCLUSION: The low HCHP rate of inappropriateness for coronary angiography is comparable with the RAND 1992 New York State data. This finding, coupled with marked differences in the incidence rate of this procedure among the HCHP divisions, is consistent with either major differences in the sickness of the HMO's sub-populations or, more likely, a lack of specificity of the RAND criteria for coronary angiography.

Peptide ligands for integrin alpha v beta 3 selected from random phage display libraries.

The integrin alpha v beta 3 binds promiscuously to cell-adhesive proteins: vitronectin, fibronectin, and several others containing the RGD motif. We have explored molecular recognition by alpha v beta 3 through selection of ligands from large random libraries of peptides displayed on phage. Ligands bound by alpha beta 3 consisted primarily of RGD peptides; however, these peptides showed considerable heterogeneity with respect to the identities of amino acids flanking RGD. The tolerance of alpha v beta 3 for RGD peptides of diverse composition is consistent with its role in vivo as a versatile receptor for RGD-containing extracellular matrix proteins. Peptide ligands for alpha v beta 3 also included a novel binding sequence, identical to a tetrapeptide found in vitronectin, which is a candidate for a synergistic site in this adhesive protein that may act in concert with RGD to promote molecular recognition.

Patient reports about ambulatory care.

To improve, managers need information on the process of care and patient satisfaction. The study described in this article validates a survey for assessing the process of care and satisfaction with ambulatory care visits and illustrates how this information can be used to estimate the impact of different visit processes on patient satisfaction.

Mutation screening of the RYR1 gene in malignant hyperthermia: detection of a novel Tyr to Ser mutation in a pedigree with associated central cores.

The ryanodine receptor gene (RYR1) has been shown to be mutated in a small number of malignant hyperthermia (MH) pedigrees. Missense mutations in this gene have also been identified in two families with central core disease (CCD), a rare myopathy closely associated with MH. In an effort to identify other RYR1 mutations responsible for MH and CCD, we used a SSCP approach to screen the RYR1 gene for mutations in a family exhibiting susceptibility to MH (MHS) where some of the MHS individuals display core regions in their muscle. Sequence analysis of a unique aberrant SSCP has allowed us to identify a point mutation cosegregating with MHS in the described family. The mutation changes a conserved tyrosine residue at position 522 to a serine residue. This mutation is positioned relatively close to five of the six MHS/CCD mutations known to date and provides further evidence that MHS/CCD mutations may cluster in the amino terminal region of the RYR1 protein.

Assessing the needs of women and clinicians for the management of menopause in an HMO.

OBJECTIVE: To determine the perceived needs of perimenopausal women regarding the management of menopause and the resource needs of the clinicians who treat them. SETTING: A large staff and group network model health maintenance organization (HMO) in New England. PARTICIPANTS: A random sample of 790 perimenopausal women aged 45-60 years who were members of the HMO in 1991, and a random sample of 180 clinicians in internal medicine, family practice, and obstetrics/gynecology practicing in the HMO during 1991. METHOD: Mailed surveys of women and clinicians were designed to assess possible needs and attitudes that could lead to the improvement of care for menopausal women. The chi-square test was used to determine differences in perceived needs and satisfaction levels among women with differences in self-reported menopausal status. The Kruskal-Wallis one-way analysis of variance and the Mann-Whitney U test were used in the clinician survey to test for differences among specialties and between genders. RESULTS: The key findings include that: 1) most (81%) of the women wanted to see a woman clinician, 2) many (50%) were interested in a menopause support group, 3) 30% reported that their care for menopause had been fair to poor, 4) only 55% of the primary care specialists (including internal medicine and family practice) reported high confidence in their abilities to treat menopause, compared with 68% of the obstetric/gynecology clinicians, and 5) 56% of the clinicians surveyed said that support from the HMO to their practices for the treatment of menopause was fair to poor. CONCLUSIONS: There is an opportunity for better care for perimenopausal women as reported by two sources, HMO clinicians and members. To provide this care, clinicians may need explicit guidelines as well as administrative supports such as educational materials and specialty access. Since the capability for menopausal care from clinicians in obstetrics/gynecology is perceived to be higher than that from primary care clinicians, an opportunity for cross-specialty collaboration and training may exist.

Detection of a novel common mutation in the ryanodine receptor gene in malignant hyperthermia: implications for diagnosis and heterogeneity studies.

Malignant hyperthermia (MH) is a potentially fatal autosomal dominant disorder of skeletal muscle and is triggered in susceptible people by all commonly used inhalational anaesthetics. To date, the ryanodine receptor gene (RYR1) has been shown to be mutated in a small number of malignant hyperthermia susceptible (MHS) cases. To determine if a common RYR1 mutation exists that might account for a significant number of MHS cases, we have investigated the RYR1 gene in unrelated patients for the presence of new mutations by the single-stranded conformation polymorphism method and have identified a novel Gly341Arg mutation which accounts for approximately 10% of Caucasian MHS cases. The implications of this common mutation in MHS diagnosis and heterogeneity studies are discussed.

Mutations in the ryanodine receptor gene in central core disease and malignant hyperthermia.

Central core disease (CCD) of muscle is an inherited myopathy which is closely associated with malignant hyperthermia (MH) in humans. CCD has recently been shown to be tightly linked to the ryanodine receptor gene (RYR1) and mutations in this gene are known to be present in MH. Mutation screening of RYR1 has led to the identification of two previously undescribed mutations in different CCD pedigrees. One of these mutations was also detected in an unrelated MH pedigree whose members are asymptomatic of CCD. The data suggest a model to explain how a single mutation may result in two apparently distinct clinical phenotypes.

Strategic planning applied to quality in asthma management for children.

This strategic plan translates the HCHP vision statement into a working plan for one major clinical condition--asthma in children. It is a working plan for clinicians and managers across specialties and levels. The results of the projects will improve in a measurable way significant clinical practice and outcomes, in keeping with the FY 1993 strategic goals.