Diabetes-related major lower limb amputation incidence is strongly related to diabetic foot service provision and improves with enhancement of services: peer review of the South-West of England.

AIMS: To investigate the relationship between high diabetes-related lower limb amputation incidence and foot care services in the South-West region of England. METHODS: The introduction of 10 key elements of foot care service provision in one area of the South-West resulted in stabilization of foot ulcer incidence and sustained reduction in amputation incidence from 2007. Services introduced included administrative support, standardized general practice foot screening, improved community podiatry staffing, hospital multidisciplinary foot clinics, effective care pathways, availability of an orthotist and audit. Peer reviews of the region's diabetes foot care services were undertaken to assess delivery of these service provisions and compare this with major amputation incidence in other regions with data provided by Yorkshire and Humber Public Health Observatory Hospital Episode Statistics. Recommendations were made to improve service provision. In 2015 changes in service provision and amputation incidence were reviewed. RESULTS: Initial reviews in 2013 showed that the 3-year diabetes-related major amputation incidence correlated inversely with adequate delivery of diabetes foot care services (P=0.0024, adjusted R2 =0.51). Repeat reviews in 2015 found that two or more foot care service improvements were reported by six diabetes foot care providers, with improvement in outcomes. The negative relationship between major amputation incidence and service provision remained strong both in the period 2012-2015 and in the year 2015 only (P ≤0.0012, adjusted R2 =0.56, and P= 0.0005, R2 =0.62, respectively). CONCLUSIONS: Major diabetes-related lower limb amputation incidence is significantly inversely correlated with foot care services provision. Introduction of more effective service provision resulted in significant reductions in major amputation incidence within 2 years. Failure to improve unsatisfactory service provision resulted in continued high amputation incidence.

Developmental program of murine erythroleukemia cells. Effect of the inhibition of protein synthesis.

The relationship between protein synthesis and commitment to terminal erythroid differentiation by dimethylsulfoxide-treated murine erythroleukemia (MEL) cells has been studied. Treatment with cycloheximide blocks the commitment of MEL cells. The effects of cycloheximide are completely reversible, however. Treatment of MEL cells before commitment delays commitment for a period of time equal to the length of inhibitor treatment. Puromycin exerts a similar effect on the commitment of MEL cells. These results indicate that there is a continuous requirement for protein synthesis before the commitment event.

Calcium regulates the commitment of murine erythroleukemia cells to terminal erythroid differentiation.

An alteration in the rate of calcium transport appears to be the rate-limiting event for the commitment of murine erythroleukemia (MEL) cells to initiate a program of terminal erythroid differentiation. The dimethyl sulfoxide (DMSO)-induced commitment of MEL cells to erythroid differentiation can be inhibited by treatment of cells with the calcium-chelating agent EGTA. Upon removal of EGTA, cells initiate commitment without the 12-h lag normally observed after treatment with DMSO alone. Treatment of cells with DMSO in the presence of calcium ionophore A23187 causes cells to initiate commitment from time zero with no lag. These results suggest that the lag is the time required for DMSO to alter the calcium transport properties of the cell.

Three differentially expressed Na,K-ATPase alpha subunit isoforms: structural and functional implications.

We have characterized cDNAs coding for three Na,K-ATPase alpha subunit isoforms from the rat, a species resistant to ouabain. Northern blot and S1-nuclease mapping analyses revealed that these alpha subunit mRNAs are expressed in a tissue-specific and developmentally regulated fashion. The mRNA for the alpha 1 isoform, approximately equal to 4.5 kb long, is expressed in all fetal and adult rat tissues examined. The alpha 2 mRNA, also approximately equal to 4.5 kb long, is expressed predominantly in brain and fetal heart. The alpha 3 cDNA detected two mRNA species: a approximately equal to 4.5 kb mRNA present in most tissues and a approximately equal to 6 kb mRNA, found only in fetal brain, adult brain, heart, and skeletal muscle. The deduced amino acid sequences of these isoforms are highly conserved. However, significant differences in codon usage and patterns of genomic DNA hybridization indicate that the alpha subunits are encoded by a multigene family. Structural analysis of the alpha subunits from rat and other species predicts a polytopic protein with seven membrane-spanning regions. Isoform diversity of the alpha subunit may provide a biochemical basis for Na,K-ATPase functional diversity.

Autonomic nervous system activity distinguishes among emotions.

Emotion-specific activity in the autonomic nervous system was generated by constructing facial prototypes of emotion muscle by muscle and by reliving past emotional experiences. The autonomic activity produced distinguished not only between positive and negative emotions, but also among negative emotions. This finding challenges emotion theories that have proposed autonomic activity to be undifferentiated or that have failed to address the implications of autonomic differentiation in emotion.

Ouabain resistance conferred by expression of the cDNA for a murine Na+, K+-ATPase alpha subunit.

The molecular basis for the marked difference between primate and rodent cells in sensitivity to the cardiac glycoside ouabain has been established by genetic techniques. A complementary DNA encoding the entire alpha 1 subunit of the mouse Na+- and K+-dependent adenosine triphosphatase (ATPase) was inserted into the expression vector pSV2. This engineered DNA molecule confers resistance against 10(-4) M ouabain to monkey CV-1 cells. Deletion of sequences encoding the carboxyl terminus of the alpha 1 subunit abolish the activity of the complementary DNA. The ability to assay the biological activity of this ATPase in a transfection protocol permits the application of molecular genetic techniques to the analysis of structure-function relationships for the enzyme that establishes the internal Na+/K+ environment of most animal cells. The full-length alpha 1 subunit complementary DNA will also be useful as a dominant selectable marker for somatic cell genetic studies utilizing ouabain-sensitive cells.

Dual signaling regulated by calcyon, a D1 dopamine receptor interacting protein.

The synergistic response of cells to the stimulation of multiple receptors has been ascribed to receptor cross talk; however, the specific molecules that mediate the resultant signal amplification have not been defined. Here a 24-kilodalton single transmembrane protein, designated calcyon, we functionally characterize that interacts with the D1 dopamine receptor. Calcyon localizes to dendritic spines of D1 receptor-expressing pyramidal cells in prefrontal cortex. These studies delineate a mechanism of Gq- and Gs-coupled heterotrimeric GTP-binding protein-coupled receptor cross talk by which D1 receptors can shift effector coupling to stimulate robust intracellular calcium (Ca2+i) release as a result of interaction with calcyon. The role of calcyon in potentiating Ca2+-dependent signaling should provide insight into the D1 receptor-modulated cognitive functions of prefrontal cortex.

D4 Dopamine receptor genes of zebrafish and effects of the antipsychotic clozapine on larval swimming behaviour.

Zebrafish, a model developmental genetic organism, is being increasingly used in behavioural studies. We have initiated studies designed to evaluate the response of zebrafish to antipsychotic drugs. This study focuses on characterization of zebrafish D4 dopamine receptors (D4Rs) and the response of larval zebrafish to the atypical antipsychotic clozapine. The D4R is of interest because of its high affinity for clozapine, while interest in clozapine stems from its effectiveness in reducing symptoms in acutely psychotic, treatment-resistant schizophrenic patients. By mining the zebrafish genomic database, we identified three distinct D4R genes, drd4a, drd4b and drd4c, and generated full-length open reading frames encoding each of the three D4Rs by reverse transcription-polymerase chain reaction. Gene mapping studies showed that each D4R gene mapped to a distinct chromosomal location in the zebrafish genome, and each gene exhibited a unique expression profile during embryogenesis. When administered to larval zebrafish, clozapine produced a rapid and profound effect on locomotor activity. The effect of clozapine was dose-dependent, resulted in hypoactivity and was prevented by the D4-selective agonist ABT-724. Our data suggest that the inhibitory effect of clozapine on the locomotor activity of larval zebrafish may be mediated through D4Rs.

Identification of a region within the Na,K-ATPase alpha subunit that contributes to differential ouabain sensitivity.

To analyze determinants within the Na,K-ATPase alpha subunit that contribute to differential ouabain sensitivity, we constructed and expressed a panel of chimeric cDNA molecules between ouabain-resistant and ouabain-sensitive alpha subunit cDNAs. When introduced into ouabain-sensitive monkey CV-1 cells, ouabain-resistant rat alpha 1 subunit cDNA and chimeras in which the 5' end of ouabain-sensitive human alpha 1 or rat alpha 2 subunit cDNA was replaced by the 5' end of rat alpha 1 subunit cDNA conferred resistance to 100 microM ouabain. Monkey cells transfected with the reciprocal chimeras were unable to survive selection in 1 microM ouabain. Rat alpha 2 subunit cDNA and a chimera in which the 5' end of rat alpha 1 subunit cDNA was replaced by the 5' end of rat alpha 2 subunit cDNA conferred resistance to 0.5 microM ouabain. These results suggest that determinants of ouabain resistance reside within the amino-terminal portions of the rat alpha 1 and alpha 2 subunits. Expression of chimeric alpha subunit cDNAs should prove useful for elucidating the structural basis of Na,K-ATPase function.

Ouabain-resistant mutants of the rat Na,K-ATPase alpha 2 isoform identified by using an episomal expression vector.

Site-directed mutagenesis was used to identify residues responsible for the greater than 1,000-fold difference in ouabain sensitivity between the rat Na,K-ATPase alpha 1 and alpha 2 isoforms. A series of mutagenized cDNAs was constructed that replaced residues of the rat alpha 2 subunit with the corresponding residues from the rat alpha 1 subunit. These cDNAs were cloned into a mammalian episomal expression vector (EBOpLPP) and expressed in ouabain-sensitive primate cells. Either of two single substitutions introduced into the rat alpha 2 subunit cDNA (Leu-111----Arg or Asn-122----Asp) conferred partial resistance (approximately 10 microM ouabain) upon transformed cells. This resistance was intermediate between the levels conferred by the rat alpha 1 cDNA (approximately 500 microM ouabain) and the rat alpha 2 cDNA (approximately 0.2 microM ouabain). A double substitution of the rat alpha 2 cDNA (Leu-111----Arg and Asn-122----Asp) conferred a resistance level equivalent to that obtained with rat alpha 1. These results demonstrate that the residues responsible for isoform-specific differences in ouabain sensitivity are located at the end of the H1-H2 extracellular domain. The combination of site-directed mutagenesis and episomal expression provides a useful system for the selection and analysis of mutants.

Rat-brain Na,K-ATPase beta-chain gene: primary structure, tissue-specific expression, and amplification in ouabain-resistant HeLa C+ cells.

We deduced the complete amino acid sequence of the rat brain Na,K-ATPase beta-subunit from cDNA. The rat brain beta-subunit exhibits a high degree of primary sequence and secondary structural homology with the human and Torpedo beta-subunit polypeptides. Analysis of rat tissue RNA reveals that the beta-subunit gene encodes four separate mRNA species which are expressed in a tissue-specific fashion. In ouabain-resistant HeLa C+ cells, beta-subunit DNA sequences are amplified (approximately 20-fold) and beta-subunit mRNAs are overproduced relative to levels in parental HeLa cells. These results suggest that the beta-subunit plays an important role in Na,K-ATPase structure-function and in the mechanism underlying cellular resistance to the cardiac glycosides.

Amplification of DNA sequences coding for the Na,K-ATPase alpha-subunit in ouabain-resistant C+ cells.

We have studied the mechanism of cellular resistance to cardiac glycosides in C+ cells. C+ cells were resistant to ouabain and overproduced plasma membrane-bound Na,K-ATPase relative to parental HeLa cells. Overexpression of Na,K-ATPase in C+ cells correlated with increased ATPase mRNA levels and amplification (approximately 100 times) of the ATPase gene. Growth of C+ cells in ouabain-free medium resulted in a marked decline in ATPase mRNA and DNA levels. However, when cells were reexposed to ouabain, they proliferated and ATPase mRNA and DNA sequences were reamplified. Restriction analysis of C+ and other human DNA samples revealed the occurrence of rearrangements in the region of the Na,K-ATPase gene in C+ cells. Furthermore, C+ cells expressed an ATPase mRNA species not found in HeLa cells. These results suggest that amplification of the gene coding for Na,K-ATPase results in overproduction of Na,K-ATPase polypeptides. Amplification of the ATPase gene or the expression of new ATPase mRNA sequences or both may also be responsible for acquisition of the ouabain-resistant phenotype.

Chromosome-mediated transfer of the murine Na,K-ATPase alpha subunit confers ouabain resistance.

We transferred murine NIH 3T3 metaphase chromosomes into monkey CV-1 cells to investigate the different ouabain sensitivities of rodent and primate cells. In 16 ouabain-resistant transferents, the mouse Na,K-ATPase alpha 1 subunit gene was detected, suggesting that structural differences between the rodent and primate alpha 1 subunits determine the different ouabain sensitivities.

Small cell carcinoma presenting as an extrapulmonary neoplasm: sites of origin and response to chemotherapy.

Eight (4%) of 203 consecutive prospectively staged and treated patients with small cell carcinoma (SCC) had no evidence of pulmonary or mediastinal tumor on chest roentgenogram or at fiberoptic bronchoscopy at the time of diagnosis. There were two distinct clinical presentations in these SCC patients with exclusively extrapulmonary tumors. Four had discrete localized extrapulmonary neoplasms, presumably originating in these sites. In the other 4 cases with either regional or widely metastatic disease, no obvious primary tumor could be documented in the lungs or elsewhere. One complete and two partial responses to chemotherapy (duration 6 to greater than 11 mo) occurred in 6 evaluable patients. Two remaining patients were inevaluable for response because they received adjuvant chemotherapy after irradiation or excision of the primary tumor and are free of disease at 15 and 28 months. Results document two clinicopathologic entities of extrapulmonary SCC, more firmly establish that it can be responsive to chemotherapy, and encourage systemic therapy as part of initial treatment planning.

Dynamic in vivo interactions among Myc network members.

Members of the Myc oncoprotein network (c-Myc, Max, and Mad) play important roles in proliferation, differentiation, and apoptosis. We expressed chimeric green fluorescent protein (GFP) fusions of c-Myc, Max, and three Mad proteins in fibroblasts. Individually, c-Myc and Mad proteins localized in subnuclear speckles, whereas Max assumed a homogeneous nuclear pattern. These distributions were co-dominant and dynamic, however, as each protein assumed the pattern of its heterodimeric partner when the latter was co-expressed at a higher level. Deletion mapping of two Mad members, Mad1 and Mxi1, demonstrated that the domains responsible for nuclear localization and speckling are separable. A non-speckling Mxi1 mutant was also less effective as a transcriptional repressor than wild-type Mxi1. c-Myc nuclear speckles were distinct from SC-35 domains involved in mRNA processing. However, in the presence of co-expressed Max, c-Myc, but not Mad, co-localized to a subset of SC-35 loci. These results show that Myc network proteins comprise dynamic subnuclear structures and behave co-dominantly when co-expressed with their normal heterodimerization partners. In addition, c-Myc-Max heterodimers, but not Max-Mad heterodimers, localize to foci actively engaged in pre-mRNA transcription/processing. These findings suggest novel means by which Myc network members promote transcriptional activation or repression.

Glucocorticoid modulation of transformed cell proliferation is oncogene specific and correlates with effects on c-myc levels.

In the presence of the glucocorticoid hormone dexamethasone, bovine papillomavirus-1 (BPV-1)-transformed C127 mouse fibroblasts assume a flattened morphology and reach a saturation density of only 50% of that attained without hormone. This phenotypic reversion of transformation is dependent on the continued presence of dexamethasone and occurs with concentrations as low as 1 nM. Dexamethasone also suppresses the growth of the parental C127 cells as well as that of cells transformed by polyoma middle-T. In contrast, the growth of C127 cells transformed by the oncogenes v-H-ras, v-mos, or v-fes is inhibited by low concentrations of dexamethasone (1 nM) and stimulated by higher concentrations (0.1-1 microM), possibly due to dexamethasone-induced transcription from the viral long terminal repeat promoters as is shown for v-H-ras. On the other hand, inhibition of BPV-transformed cell line growth by dexamethasone does not appear to be related to hormone effects on BPV-1 oncogene transcription. Indeed, in several cases, dexamethasone increases the steady state transcript levels of the BPV-1 oncogenes, E5 and E6-E7, while suppressing cellular proliferation. Dexamethasone also rapidly reduces the steady state levels of c-myc in the BPV-transformed cells but has less effect on c-myc expression in the ras-transformed cells. These results demonstrate that the growth-promoting actions of the papillomavirus transforming genes, but not those of several retroviral oncogenes, may be overcome by dexamethasone, which appears to act by down-regulation of c-myc expression.

Effect of thyroid hormone on the abundance of Na,K-adenosine triphosphatase alpha-subunit messenger ribonucleic acid.

The effects of thyroid hormone on Na,K-ATPase alpha-subunit mRNA (mRNA alpha) content and Na,K-ATPase activity were measured in renal cortex, heart, and cerebrum of hypothyroid rats 24 and 72 h after injection of diluent or T3. Use of a cDNA probe complementary to rat brain mRNA alpha in Northern blot analysis revealed a single 26-27 S band in RNA isolated from these three tissues regardless of thyroid status. Tissue mRNA alpha content was estimated by dot blot analysis of whole cell extracts and isolated total RNA. Injection of T3 augmented mRNA alpha content by 2.1- to 2.5-fold in kidney cortex and myocardium at 24 h. After three daily injections of T3, the increases in mRNA alpha were evident despite a global increase in RNA content associated with hypertrophy of these target tissues. Furthermore, the increases in abundance of mRNA alpha after 72 h of T3 treatment correlated with enhancement of Na,K-ATPase activity. In contrast, both mRNA alpha and enzyme activity were invariant in the cerebrum. These data suggest that T3-induced augmentation of Na,K-ATPase activity is mediated, at least in part, by increased mRNA alpha content in target tissues.

Fatal pneumonia in an adult due to respiratory syncytial virus.

Respiratory syncytial virus (RSV) infections in adults are generally mild, and no fatalities in uninstitutionalized adults have been reported. To our knowledge, we document herein the first case of fatal RSV pneumonia in a previously healthy elderly woman living at home, in whom complement fixation titers against RSV rose from less than 1:8 to reach 1:256 at death. Cytoplasmic inclusions characteristic of RSV were detected at autopsy.

Co-localization and polarized distribution of Na,K-ATPase alpha 3 and beta 2 subunits in photoreceptor cells.

Na,K-ATPase plays a central role in the visual sensitivity of photoreceptors by driving the dark current of vision. The alpha 3 and beta 2 isoforms of Na,K-ATPase were previously shown to be the major alpha and beta subunit mRNAs expressed in photoreceptors. Here we compared the distribution of beta-subunits of the enzyme in the retina and kidney, using electron microscopic immunocytochemistry with specific antibodies against alpha 3, beta 1, and beta 2 isoforms as well as with an antibody (Ax2) that binds to alpha 2 and/or alpha 3 isoforms. Both the alpha 3 and beta 2 isoforms were localized to photoreceptor inner segments at highest labeling density between the base of the connecting cilium and the outer limiting membrane (OLM). Quantitative analysis of Ax2 antibody binding to alpha 3 revealed a significant decrease in labeling density below the OLM and above the base of the connecting cilium. Although the beta 2-subunit has been reported to have adhesive functions in glial cells in cerebellum, we detected beta 2 in the photoreceptor, a cell of neural origin, but not in the Mueller cell, the glial cell of the retina. Moreover, anti-beta 2 antibodies bound maximally to portions of photoreceptor cells not involved in cell-cell contact.

A program to immunize hospitalized preschool-aged children: evaluation and impact.

OBJECTIVE: The Standards for Pediatric Immunization Practices suggest that hospitalization be viewed as an opportunity to vaccinate children. The purpose of the present study is 1) to determine the immunization status of an urban population of hospitalized preschool-aged children, 2) to study the impact of an immunization program designed to vaccinate hospitalized 0 to 2-year-old children who are underimmunized at admission, and 3) to make immunization a routine part of care for the hospitalized child. METHODS: Prospective evaluation of the immunization status of hospitalized 0 to 2-year-old residents of Philadelphia admitted to an urban children's hospital was performed. With verification of the child's immunization record through the primary care provider (PCP), needed immunizations were given and records were forwarded to notify the PCP. Educational information was provided to families and health care providers. MAIN OUTCOME MEASURE: The percentage of children fully immunized on admission compared with the percentage at the time of discharge. Results. Two thousand three hundred twenty-nine children from 0 to 2 years of age were hospitalized during the 22-month study period. Immunization records were verified in 86% (2006), requiring an average of 1.5 phone calls to the PCP. The mean patient age was 10 months. Average hospital length of stay was 4 days. On admission, 49% (980) of the 2006 study patients were fully immunized. The remaining 51% (1026) were eligible for vaccination. Immunizations were delayed greater than or equal to 2 months in 18% (355) of the children. Neither type of health care insurance nor site of primary care affected the immunization status of those evaluated at the time of admission. Sixty-six percent (N = 674) of eligible patients received at least one vaccination before hospital discharge. Medical contraindications accounted for only 4% of the reasons eligible patients were not immunized. Of the 2006 children evaluated, the percentage of those fully vaccinated for age increased significantly from 44% on admission to 70% on discharge. CONCLUSION: As a result of this program, there was a significant improvement in vaccination percentage at the time of hospital discharge in this group of urban preschool-aged children. The development of an immunization program to vaccinate hospitalized preschool children is an opportunity to immunize in the urban setting where there is a high prevalence of underimmunization. In addition, it provides an opening for educational programs for families, nurses, and housestaff and linkage to the community PCPs.