Expression of multiple larger-sized transcripts for several genes in oligodendrogliomas: potential markers for glioma subtype.

Astrocytomas and oligodendrogliomas are two brain tumors that follow different clinical courses. Although many of these tumors can be identified based on standard histopathological criteria, a significant percentage present notable problems in diagnosis. To identify markers that might prove useful in distinguishing glioma subtypes, we prepared and analyzed cDNA libraries for differential expression of genes in an astrocytoma (grade II), an oligodendroglioma (grade II), and a meningioma (benign). The tumor libraries were compared by sequencing randomly selected clones and tabulating the expression frequency of each gene. In addition to identifying several genes previously reported or expected to be differentially expressed among these tumors, several potential new brain tumor markers were identified and confirmed by Northern blot analysis of a panel of brain tumors. A surprising result of this analysis was the observation that several larger-sized transcripts for various genes were predominantly expressed in the oligodendroglioma tumors, when compared to the other brain tumors or in non-tumor gray matter. These findings are consistent with different pre-mRNA splicing patterns observed between oligodendrogliomas and astrocytomas. In support of this hypothesis, our screen revealed significantly higher levels of two hnRNP A1 transcripts in oligodendrogliomas. hnRNP A1 is a component of the spliceosome whose expression levels affect splice site selection in vivo. The preferential expression of larger-sized transcripts for several genes in oligodendrogliomas may be useful for distinguishing astrocytic and oligodendroglial gliomas.

Humoral autoreactivity to an alternatively spliced variant of ICA512/IA-2 in Type I diabetes.

AIMS/HYPOTHESIS: The receptor tyrosine phosphatase like-protein ICA512/IA-2 occurs as a proteolytically-processed 65,000 Mr type 1 transmembrane glycoprotein in beta cells and is a major autoantigen of Type I (insulin-dependent) diabetes mellitus. We investigated whether alternative splicing could affect humoral autoreactivity to the molecule. METHODS: Genomic and cDNA sequence analysis showed the presence of a ICA512 variant in islets and lymphoid tissues with an in-frame deletion of exon 13 which produces a secreted form lacking aa 557-629 including the transmembrane domain (aa 577 to 600). The alternatively spliced protein is detectable by western blotting in normal islets and translated into a protein that is processed to a series of soluble forms of 25,000-35,000 Mr Radioimmuno-precipitation assays for anti-ICA512 autoantibodies were developed with the widely used ICA512.bdc construct (which has exon 13 deleted) and a series of full-length and modified ICA512/IA-2 molecules. RESULTS: The assays showed that ICA512.bdc and ICA512604-979 gave the best discrimination between diabetic and control sera. With ICA512604-979 a somewhat greater proportion of patients expressing antibodies were detected than with ICA512.bdc in the groups studied (70.5 % vs 63.2 % of prediabetic/new-onset and 25.0 vs 13.9% in patients with diabetes > 20 years). Conversely, a small proportion (3 % recent-onset and 6% > 20 years) had antibodies to ICA512.bdc but not ICA512(604-979). CONCLUSION/INTERPRETATION: Important epitopes lie within the exon 13 region and others can be generated by the alternative splicing. As the deltaexon 13 variant is probably secreted by the beta cell, it could be recognized by the cellular and humoral arm of the immune system in the absence of cellular damage.

Clinical nutrition managers have access to sources of empowerment.

OBJECTIVE: To ascertain perceived access of dietitians to power in the workplace. DESIGN: The conceptual framework was Kanter's theory of organizational power. The Conditions for Work Effectiveness Questionnaire was used to measure perceived access to sources of power: information, support, resources, and opportunities. Demographic data were collected to identify factors that may enhance empowerment. SUBJECTS/SETTINGS: The questionnaire was sent to a random sample of 348 dietitians chosen from members of the Clinical Nutrition Management dietetic practice group of the American Dietetic Association. Blank questionnaires were returned by 99 (28.4%) people not working as clinical nutrition managers, which left 249 in the sample. STATISTICAL ANALYSES: Descriptive statistics were used to organize and summarize data. One-way analysis of variance and t tests were performed to identify differences in responses based on levels of education, work setting, and information technology skills. RESULTS: Usable questionnaires were received from 178 people (71.5%). On a 5-point scale, scores for access to information (mean +/- standard deviation [SD] = 3.8 +/- 0.7), opportunity (mean +/- SD = 3.6 +/- 0.7), support (mean +/- SD = 3.2 +/- 0.9), and resources (mean +/- SD = 3.1 +/- 0.8) demonstrated that clinical nutrition managers perceived themselves as having substantial access to sources of empowerment. Those having higher levels of education, working in larger hospitals, having better-developed information technology skills, and using information technology more frequently had statistically significant higher empowerment scores (P = < or = .05) than contrasting groups in each category. APPLICATIONS/CONCLUSION: Clinical nutrition managers are empowered and able to assume leadership roles in today's health care settings. Their power may be enhanced by asserting more pressure to gain greater access to sources of power: support, information, resources, and opportunities.

Genes expressed in the mouse pituitary corticotrope AtT-20/D-16v tumor cell line.

The pituitary corticotrope AtT-20 stable cell line has been used as a model system to study peptide secretion, glucocorticoid regulation, and several other processes. In order to better understand this model cell line, a phage cDNA library was generated from AtT-20/D-16v cell mRNA and cDNA sequences were obtained for 317 clones representing 203 known genes and 48 novel cDNAs. The sequencing results revealed the prevalence of the mouse leukemia virus in this cell line and also identified a number of putatively secreted molecules that were not previously recognized as being secreted from AtT-20/D-16v cells or pituitary corticotropes. Nine completely novel cDNAs and 39 cDNAs homologous to known ESTs were also identified. A listing of other genes known to be expressed in AtT-20/D-16v cells is also provided.

Induction of integral membrane PAM expression in AtT-20 cells alters the storage and trafficking of POMC and PC1.

Peptidylglycine alpha-amidating monooxygenase (PAM) is an essential enzyme that catalyzes the COOH-terminal amidation of many neuroendocrine peptides. The bifunctional PAM protein contains an NH2-terminal monooxygenase (PHM) domain followed by a lyase (PAL) domain and a transmembrane domain. The cytosolic tail of PAM interacts with proteins that can affect cytoskeletal organization. A reverse tetracycline-regulated inducible expression system was used to construct an AtT-20 corticotrope cell line capable of inducible PAM-1 expression. Upon induction, cells displayed a time- and dose-dependent increase in enzyme activity, PAM mRNA, and protein. Induction of increased PAM-1 expression produced graded changes in PAM-1 metabolism. Increased expression of PAM-1 also caused decreased immunofluorescent staining for ACTH, a product of proopiomelanocortin (POMC), and prohormone convertase 1 (PC1) in granules at the tips of processes. Expression of PAM-1 resulted in decreased ACTH and PHM secretion in response to secretagogue stimulation, and decreased cleavage of PC1, POMC, and PAM. Increased expression of a soluble form of PAM did not alter POMC and PC1 localization and metabolism. Using the inducible cell line model, we show that expression of integral membrane PAM alters the organization of the actin cytoskeleton. Altered cytoskeletal organization may then influence the trafficking and cleavage of lumenal proteins and eliminate the ability of AtT-20 cells to secrete ACTH in response to a secretagogue.

Patients report positive nutrition counseling outcomes.

OBJECTIVE: Assess outcomes of patient nutrition counseling. DESIGN: A descriptive study based on the results of a telephone interview performed 2 to 8 weeks after counseling. SUBJECTS/SETTING: Subjects were 400 adult patients referred for nutrition counseling at 2 academic health centers. Of these, 274 patients received nutrition counseling during hospitalization and 126 as outpatients. STATISTICAL ANALYSIS: Descriptive statistics were used to summarize data and the Mann-Whitney U statistic and logistic regressions were used to test significant differences (P < .05) between inpatient and outpatient counseling outcomes. RESULTS: Most patients (83%) gave a partial or full description of their diet modifications and 79% had a moderate or good understanding of their diet. Most patients reported that the dietitian's advice was suited to their special needs (88%) and that they knew what to eat (83%). A majority (62%) had made dietary changes, but 17% said they had had trouble changing their diets as suggested. After talking with a dietitian, 57% felt better emotionally, 37% felt better physically, 64% felt in control of their condition, and 43% noticed improved health indicators. Initial analysis indicated that outpatients reported better outcomes than inpatients; further analysis showed that these differences could be attributed to younger ages among the outpatient sample. APPLICATIONS/CONCLUSIONS: Patient nutrition counseling has positive outcomes. Therefore, key counseling points should be introduced or reinforced in inpatient settings, in conjunction with multiple-session protocols during the pre- and/or posthospitalization continuum of care. Dietitians, managers, administrators, and credentialing agencies should work together to secure and promote the necessary physical, personnel, and financial resources to make this happen.

Administrators' perceptions of nutrition services in home health care agencies.

OBJECTIVE: Ascertain views of home care administrators regarding the need for nutrition services in home care, current status and gaps in dietitian services, and obstacles toward expansion. DESIGN: Mailed survey. SUBJECTS: Members of the National Association for Home Care residing in California, New York, Pennsylvania, Ohio, and Texas. Of the 1,992 questionnaires sent, 402 (20.1%) were completed and used for data analysis. STATISTICAL ANALYSES PERFORMED: Descriptive statistics. RESULTS: Respondents gave high importance to nutrition (6.2 on a 7-point scale), and 39.6% estimated that more than half of their patients were malnourished. Most respondents reported using dietitian services, either as consultants (45.5%) or full-time (5.8%) or part-time (11.9%) employees. Nurses (85.5%) were listed most often as providers of nutrition services; also listed were dietitians (70.6%), pharmacists (17.2%), and physicians (11.2%). More than half of the respondents did not have, but would like to have, a dietitian serve as a nutrition consultant, prepare nutrition care plans, and provide nutrition counseling for patients and caregivers. Primary deterrents noted were lack of reimbursement by third-party payers and lack of physician request. Dietitian services ranked as having the highest value were patient education and counseling, medical nutrition therapy for specific conditions, development of nutrition care plans, staff in-service training, and nutrition assessments. Most home care administrators expected dietitian hours, services, and staff to increase during the next 5 years. APPLICATIONS: The growing home health care market is fertile ground for dietitians. To expand their role in home care, dietitians can position nutrition services as being vital to cost-effective, high-quality care; augment their skills in consultation, training, nutrition support, and outcomes research; strengthen collaborative ties with key home health care professionals; and advocate for Medicare coverage of medical nutrition therapy.

A novel neuroendocrine intracellular signaling pathway.

Expression of many components of the secretory pathway in peptidergic neuroendocrine cells is precisely controlled in response to secretagogues. Regulated endocrine-specific protein (RESP18) was identified as a dopamine-regulated intermediate pituitary transcript. Although the amino acid sequence of RESP18 initially suggested that it might be a novel preprohormone, its widespread expression in peptide-producing neurons and endocrine cells and its localization to the lumen of the endoplasmic reticulum suggested that it subserves a unique function. Subtractive hybridization of a pituitary corticotrope AtT-20 cell line engineered for inducible RESP18 expression demonstrated a RESP18-dependent induction of several transcripts. Regulation of RESP18 expression in vitro and in vivo was accompanied by changes in the same transcripts. Several cDNAs encoding transcripts up-regulated by RESP18 were analyzed by DNA sequencing, searching the GenBank databases for homologous proteins, and Northern blotting. One novel clone showed a tissue distribution nearly identical to that of RESP18. One clone was identical to rat LIMK2, a protein kinase containing modular protein-protein interaction LIM (lin-11, isl-1, mec-3) domains. Another clone was similar to monomeric bacterial isocitrate dehydrogenases. Like the unfolded protein response, these data demonstrate a novel signaling pathway from the secretory pathway lumen to the nucleus. RESP18 acts as a lumicrine peptide (an intracellular luminal autocrine hormone) inducing this pathway.

Dietetics and foodservice personnel are ready for team problem solving.

OBJECTIVE: To determine the readiness of dietetics and foodservice personnel for contributing to team problem solving. DESIGN: A descriptive, correlation study in which a five-part questionnaire was designed to collect data. SUBJECTS: Dietetics and foodservice personnel (n = 632) in eight hospital settings; 321 subjects (51%) volunteered to participate. STATISTICAL ANALYSES PERFORMED: Statistical data to summarize demographic information and two-way analysis of variance with a Scheffe post hoc analysis was used to investigate differences between dietetics and foodservice personnel on each of the subscales. RESULTS: In general, all respondents were somewhat oriented toward group problem solving and were confident in their problem-solving skills. Problem-solving expertise within the organization decreased for the respondents as the problem moved from their direct work areas to the organization level. Dietitians displayed higher self-efficacy in contributing to problem-solving teams than did foodservice personnel, indicating a 75% chance that they would contribute whereas all other respondents indicated a 50% chance of contributing. APPLICATIONS: The results of this study generate optimism for involving all dietetics and foodservice personnel in team problem solving. However, training activities are needed for both foodservice personnel and dietetics professionals.

Expression of RESP18 in peptidergic and catecholaminergic neurons.

We examined the expression of regulated endocrine-specific protein of 18-kD (RESP18) in selected peptidergic and catecholaminergic neurons of adult rat brain. In the hypothalamic paraventricular, supraoptic, and accessory nuclei, RESP18 mRNA was highly expressed in neurons immunostained for oxytocin and vasopressin. RESP18 mRNA was also highly expressed in paraventricular nucleus neurons immunostained for corticotropin-releasing hormone, thyrotropin-releasing hormone, and somatostatin. RESP18 mRNA was expressed in POMC cells of the arcuate nucleus, in neuropeptide Y cells of the dorsal tegmental nucleus, lateral reticular nucleus, and hippocampus, and in brainstem catecholaminergic neurons. RESP18 mRNA expression was high in all paraventricular and arcuate neurons, but RESP18 protein was detectable in the perikarya of a subset of these neurons, suggesting an important post-transcriptional component to the regulation of RESP18 expression. RESP18 antisera immunostained perikarya but not axon fibers or terminals. Sub-cellular fractionation of homogenates of several hypothalamic nuclei identified RESP18 protein in fractions enriched in endoplasmic reticulum. The presence of 22- and 24-kD RESP18 isoforms in the neural lobe of the pituitary indicated that some RESP18 protein exited the endoplasmic reticulum. The post-transcriptional regulation of RESP18 expression and localization of RESP18 protein primarily to the endoplasmic reticulum suggests that RESP18 plays a regulatory role in peptidergic neurons.

Therapists' anxiety and attitudes toward computerized documentation in the clinical setting.

Many hospitals are converting to electronic records and allied health professionals are required to modify their traditional documentation practices to accommodate this new technology. This paper discusses a study conducted to determine the computer anxiety and attitudes of physical, occupational, and speech therapists in a large urban teaching hospital before and after the implementation of a computerized documentation system. Fifty-three therapists surveyed with a preinstallation questionnaire reported mild computer anxiety and generally good attitudes about the planned computer system. A greater amount of previous computer use and better self-related computer skills were consistent with less computer anxiety. Seven of the original sample became the first to use the computer system. After their six month trial period, surveys revealed a reduction in computer anxieties. Manual time logs completed before and after the system implementation revealed a significant decrease in total documentation time when using computers.

The expression of regulated endocrine-specific protein of 18 kDa in peptidergic cells of rat peripheral endocrine tissues and in blood.

We examined the cellular localization of regulated endocrine-specific protein of 18 kDa (RESP18) and mRNA in peripheral endocrine tissues. In situ hybridization and immunocytochemistry identified RESP18 mRNA in most cells of the anterior and intermediate pituitary, with RESP18 protein apparent in many anterior pituitary cells but very few intermediate pituitary cells. In the adrenal medulla and superior cervical ganglion, RESP18 mRNA co-localized with dopamine beta-mono-oxygenase and neuropeptide Y. In the thyroid, RESP18 mRNA was localized to C-cells. RESP18 mRNA was expressed in most of the cells of the pancreatic islets, co-localizing with insulin, glucagon, and somatostatin. No RESP18 mRNA or protein was detected in the adrenal cortex, ovary, neural lobe of the pituitary, parathyroid, exocrine pancreas, thyroid follicular cells, placenta, mammary tissue, liver, lung, or atria. As in the intermediate lobe of the pituitary, high levels of RESP18 mRNA in the pancreatic islets and adrenal medulla did not always correlate with immunodetectable RESP protein, suggesting that post-transcriptional mechanisms are important in controlling RESP18 expression. Western blot analyses identified 18 kDa RESP and higher molecular weight isoforms of RESP in most tissues and in plasma. Subcellular fractionation of the anterior pituitary identified 18 kDa RESP18 in fractions enriched in endoplasmic reticulum and secretory granules, with the higher molecular weight isoforms of RESP18 concentrated in fractions enriched in secretory granules. The broad neuroendocrine distribution of RESP18 suggests that it subserves an important function in the secretory pathway that is common to the production of many secreted peptides.

Stage-specific expression of RESP18 in the testes.

RESP18 (regulated endocrine-specific protein of 18 KD) is an endoplasmic reticulum (ER) protein that was identified by coordinate dopaminergic regulation with pro-opiomelanocortin in the rat neurointermediate pituitary. Many attributes of RESP18 suggest an important function in neuroendocrine cells. Several neuropeptides, growth factors, and enzymes involved in biosynthesis of classical chemical neurotransmitters, have been identified in germ cells, Sertoli cells, and spermatozoa. In this study, screening of reproductive tissues revealed high levels of RESP18 protein and mRNA in the testes but not in ovaries or epididymis. The testes and sperm expressed 18-KD RESP18 and a unique 19-KD isoform. To better understand RESP18 expression in the testes, we have examined the stages of the cycle of the seminiferous epithelium by immunohistochemistry and Western blot analyses. Immunohistochemical analysis showed that RESP18 protein was expressed exclusively in spermatocytes and maturing spermatids. RESP18 protein was expressed at high levels in Step 1-8 round spermatids, in which the PC4 prohormone convertase, nerve growth factor, and proenkephalin are also expressed. Western blots, Northern blots, and indirect immunofluorescence staining demonstrated RESP18 expression in sperm.

Analysis of wild-type and mutant aspartate aminotransferases using integrated rate equations.

A general integrated rate equation was fit to reaction progress curves catalyzed by wild-type E. coli aspartate aminotransferase and the site-specific mutant enzymes, H193Q and Y70F. A nonlinear step-regression code, revised for this study selected from all kinetic constants in a general integrated rate equation for all unbranched enzyme mechanisms with stoichiometries upto two substrates and two products including terms for substrate inhibitions and that of an exogenous inhibitor. For each aspartate aminotransferase enzyme studied only kinetic constants consistent with a substituted enzyme mechanism were found statistically significant, thus the enzyme mechanism and sources of inhibition were determined objectively by statistics. The kinetic constants for wild-type and Y70F aspartate aminotransferase were similar to those previously reported indicating the validity of the integrated rate equation analysis. Minor changes in kinetic constants were observed for the H193Q mutant enzyme suggesting that the catalytic effects of the electrostatic hydrogen bonding network extending from the pyridine nitrogen of the cofactor through Asp-222, His-189 ends prior to His-193.

Production of radiolabeled neuropeptide precursors by in vitro transcription and translation.

Bioactive peptide hormones and neurotransmitters are required for neuroendocrine regulation of cellular functions. Importantly, proteolytic processing of inactive neuropeptide precursors is required to generate physiologically active peptide hormones and neurotransmitters. Studies of the processing enzymes require authentic neuropeptide precursors as substrates, rather than peptide substrates. This study demonstrates an efficient method to general 35S-precursors from cloned cDNAs by in vitro transcription and translation. In vitro transcription of neuropeptide cDNAs with SP6 RNA polymerase generates large amounts (micrograms) of corresponding RNAs. Subsequent in vitro translation of RNAs with wheat germ extract and 35S-methionine generates large quantities of 35S-precursors (10-25 million cpm 35S-precursor protein per reaction) with high specific radioactivity. The radiolabeled precursor substrates offer a reliable, sensitive and accurate method for detecting the proteolytic activity. Importantly, specific detection of the primary proenkephalin processing activity in chromaffin granules by 35S-enkephalin precursor as substrate, but not by peptide methylcoumarinamide (MCA) substrates, illustrates the significance of using full-length precursor to detect appropriate processing enzymes. This study demonstrates that efficient production of radiolabeled neuropeptide precursors by in vitro transcription and translation will be useful for in vitro assays of relevant processing proteases.

Dietitians in home care: a survey of current practice.

OBJECTIVE: To determine current practice patterns of dietitians who work in home health care. DESIGN: A mail survey was sent and a reminder postcard was mailed 2 weeks later. SUBJECTS: Registered dietitians (1,305) who returned a postcard indicating employment in home care. A total of 660 questionnaires (50.6%) were returned; of these, 252 were usable for data analysis. STATISTICAL ANALYSES PERFORMED: Descriptive statistics for summary data and a paired t test to compare level of importance and level of expertise needed for nutrition care skills. RESULTS: The typical respondent was a consultant, relatively new to home care, working fewer than 10 hours a week, and paid an hourly wage (mean=$61.45/hour). Most agencies did not bill separately for nutrition services but 28.2% received third-party payment for services. Primary diagnoses of home care patients were diabetes and cancer. Many dietitians worked with patients receiving enteral tube feedings (55%), total parenteral nutrition (29%), or both (17%), but most made fewer than two visits per week to these patients. Areas of skill rated highest in level of importance for dietitians working in home care were patient counseling, caregiver education, documentation, and dietary history. These plus the ability to develop a care plan received highest expertise ratings. Disparity between skill importance and personal expertise was greatest for activities in the clinical and administrative categories. Most respondents expected growth and role expansion for dietitians in home care, but lack of reimbursement was the greatest deterrent. APPLICATIONS: To expand their role in the home care field, dietitians need to develop additional skills, improve outcome documentation, diversify roles, and create new relationships with home care providers and case managers.

The processing proteases prohormone thiol protease, PC1/3 and PC2, and 70-kDa aspartic proteinase show preferences among proenkephalin, proneuropeptide Y, and proopiomelanocortin substrates.

Proteases of cysteine, aspartic, and subtilisin classes have been indicated as candidate prohormone processing enzymes. The chromaffin granule proenkephalin processing proteases have been characterized as the novel cysteine protease prohormone thiol protease (PTP), a 70-kDa aspartic proteinase, and the subtilisin-like PC1/3 and PC2 enzymes. The goal of this study was to assess whether these processing proteases possess preference(s) for prohormone substrates. The recombinant prohormones proenkephalin, proneuropeptide Y (pro-NPY), and proopiomelanocortin (POMC) were expressed in Escherichia coli using the T7 expression system and purified for in vitro processing studies. Results indicated that the chromaffin granule processing proteases possess selectivity for particular prohormones. PTP preferred proenkephalin, with good cleavage of pro-NPY and slow processing of POMC. In contrast, the 70-kDa aspartic proteinase cleaved POMC most readily, with cleavage of proenkephalin and some processing of pro-NPY. PC1/3 and PC2 preferred POMC among the prohormones tested. Importantly, these results indicate that prohormone selectivity of processing proteases may be an important factor in predicting the primary and rate-limiting protease(s) required for processing a particular prohormone.

Proenkephalin-processing enzymes in chromaffin granules: model for neuropeptide biosynthesis.

Our discovery of precursor preference of processing enzymes indicates possible development of future drugs that target specific proteases uniquely associated associated with processing of a particular prohormone. For example, selective processing of PE by the PTP suggests that future evaluation of modulation of PTP through central nervous system drug reagents may modify the endogenous analgesic effects of the enkephalins. With respect to blood pressure, neuropeptide Y (NPY) that is released from sympathetic nerve terminals is a strong vasoconstrictor. Our finding that only PTP (not PC1/3, PC2, or the aspartic proteinase) possesses the ability to convert pro-NPY to NPY suggests that investigation of inhibitors of peripheral PTP in blood pressure regulation should be initiated. Overall, elucidation of the proteolytic components required in prohormone processing will provide insights into the molecular mechanisms of human disease.

Expression of recombinant pro-neuropeptide Y, proopiomelanocortin, and proenkephalin: relative processing by 'prohormone thiol protease' (PTP).

The preference of the 'prohormone thiol protease' (PTP), a candidate prohormone processing enzyme, for different peptide precursors was assessed in vitro with recombinant prohormones near estimated in vivo levels. Pro-neuropeptide Y (pro-NPY), proopiomelanocortin (POMC), and proenkephalin (PE) were expressed at high levels in E. coli. Purification of prohormones utilized a combination of DEAE-Sepharose, Mono Q, and preparative electrophoresis. PTP cleaved PE most readily, and also cleaved pro-NPY. The processing of POMC by PTP was minimal. These results demonstrate PTP's preference for certain prohormone substrates.