Insulin receptor substrate-1 pleckstrin homology and phosphotyrosine-binding domains are both involved in plasma membrane targeting.

The localization of insulin receptor substrate (IRS) molecules may be responsible for the differential biological activities of insulin and other peptides such as platelet-derived growth factor. The subcellular localization of IRS-1 is controversial, with some reports suggesting association with the cytoskeleton and other studies reporting membrane localization. In this study, we used immunofluorescence microscopy to define the localization of IRS-1. In the basal state, recombinant IRS-1 was localized predominantly in the cytoplasm. In response to insulin, recombinant IRS-1 translocated to the plasma membrane. We have also studied the localization of green fluorescent protein (GFP) fusion proteins. Unlike native IRS-1, a fusion protein containing GFP plus full-length IRS-1 appeared to localize in inclusion bodies. In contrast, when GFP was fused to the N terminus of IRS-1 (i.e. the pleckstrin homology and phosphotyrosine-binding domains), this fusion protein was targeted to the plasma membrane. Mutations of phosphoinositide-binding sites in both the pleckstrin homology and phosphotyrosine-binding domains significantly reduced the ability of Myc-tagged IRS-1 to translocate to the plasma membrane following insulin stimulation. However, these mutations did not cause a statistically significant impairment of tyrosine phosphorylation in response to insulin. This raises the possibility that IRS-1 tyrosine phosphorylation may occur prior to plasma membrane translocation.

Only a small fraction of purified hepatitis C RNA-dependent RNA polymerase is catalytically competent: implications for viral replication and in vitro assays.

The enzymatic activity of a C-terminally truncated form of the RNA-dependent RNA polymerase, termed NS5B(Delta21), of the hepatitis C virus (strain BK) has been investigated using both homopolymeric and heteropolymeric RNA templates. Incorporation of nucleotides into a heteropolymeric RNA template as catalyzed by NS5B(Delta21) is characterized by biphasic reaction time courses. At high concentrations of nucleoside triphosphate in reactions allowing a preincubation of NS5B(Delta21) and RNA template, an initial rapid phase of the reaction is followed by a slower linear phase. The amplitude of the first phase of the reaction varies directly with the concentration of the enzyme in the reaction. It is shown here that full-length copies of the template are produced during the first phase of the reaction. Our results reveal that NS5B(Delta21) is processive but only a small fraction, less than 1%, of the purified enzyme present participates productively in the reaction. Most importantly, the turnover number for the hepatitis C NS5B(Delta21) is comparable to those observed for other polymerases such as the HIV-1 reverse transcriptase. The combined results reconcile in part the apparent discrepancy of the low, observed specific activity of the purified enzyme and the rapid generation of HCV in vivo.

Characterization of Drosophila insulin receptor substrate.

Insulin receptor substrate (IRS) proteins are phosphorylated by multiple tyrosine kinases, including the insulin receptor. Phosphorylated IRS proteins bind to SH2 domain-containing proteins, thereby triggering downstream signaling pathways. The Drosophila insulin receptor (dIR) C-terminal extension contains potential binding sites for signaling molecules, suggesting that dIR might not require an IRS protein to accomplish its signaling functions. However, we obtained a cDNA encoding Drosophila IRS (dIRS), and we demonstrated expression of dIRS in a Drosophila cell line. Like mammalian IRS proteins, the N-terminal portion of dIRS contains a pleckstrin homology domain and a phosphotyrosine binding domain that binds to phosphotyrosine residues in both human and Drosophila insulin receptors. When coexpressed with dIRS in COS-7 cells, a chimeric receptor (the extracellular domain of human IR fused to the cytoplasmic domain of dIR) mediated insulin-stimulated tyrosine phosphorylation of dIRS. Mutating the juxtamembrane NPXY motif markedly reduced the ability of the receptor to phosphorylate dIRS. In contrast, the NPXY motifs in the C-terminal extension of dIR were required for stable association with dIRS. Coimmunoprecipitation experiments demonstrated insulin-dependent binding of dIRS to phosphatidylinositol 3-kinase and SHP2. However, we did not detect interactions with Grb2, SHC, or phospholipase C-gamma. Taken together with published genetic studies, these biochemical data support the hypothesis that dIRS functions directly downstream from the insulin receptor in Drosophila.

Inhibition of hepatitis C virus NS2/3 processing by NS4A peptides. Implications for control of viral processing.

The NS2/3 protease of hepatitis C virus is responsible for a single cleavage in the viral polyprotein between the nonstructural proteins NS2 and NS3. The minimal protein region necessary to catalyze this cleavage includes most of NS2 and the N-terminal one-third of NS3. Autocleavage reactions using NS2/3 protein translated in vitro are used here to investigate the inhibitory potential of peptides likely to affect the reaction. Peptides representing the cleaved sequence have no effect upon reaction rates, and the reaction rate is insensitive to dilution. Both results are consistent with prior suggestions that the NS2/3 cleavage is an intramolecular reaction. Surprisingly, peptides containing the 12-amino acid region of NS4A responsible for binding to NS3 inhibit the NS2/3 reaction with K(i) values as low as 3 microM. Unrelated peptide sequences of similar composition are not inhibitory, and neither are peptides containing incomplete segments of the NS4A region that binds to NS3. Inhibition of NS2/3 by NS4A peptides can be rationalized from the organizing effect of NS4A on the N terminus of NS3 (the NS2/3 cleavage point) as suggested by the known three-dimensional structure of the NS3 protease domain (Yan, Y., Li, Y., Munshi, S., Sardana, V., Cole, J. L., Sardana, M., Steinkuhler, C., Tomei, L., De Francesco, R., Kuo, L. C., and Chen, Z. (1998) Protein Sci. 7, 837-847). These findings may imply a sequential order to proteolytic maturation events in hepatitis C virus.

The effect of menopause and perimenopause on the course of epilepsy.

PURPOSE: The purpose of this study was to obtain preliminary information about the effect of menopause and perimenopause on the course of epilepsy, and to determine whether seizure type, use of hormone-replacement therapy (HRT), or a history of catamenial seizure pattern would influence this course. METHODS: We performed a questionnaire study of women with epilepsy currently in menopause and perimenopause, requesting information regarding the course of their epilepsy and treatment. Statistical analysis was performed by using Pearson chi2 with 95% confidence limits. RESULTS: Forty-two menopausal women (ages 41-86 years) responded. Twelve subjects reported no change in seizures at menopause, 17 reported a decrease in seizure frequency, and 13 reported an increase. Sixteen (38%) took synthetic HRT. Sixteen (38%) additional subjects (having some overlap with the HRT group) reported having a catamenial seizure pattern before menopause. HRT was significantly associated with an increase in seizures during perimenopause (p = 0.001). A history of catamenial seizure pattern was significantly associated with a decrease in seizures at menopause (p = 0.013). Thirty-nine perimenopausal women (ages 38-55 years) responded. Nine subjects reported no change in seizures at perimenopause, five reported a decrease in seizure frequency, and 25 reported an increase. Eight (15%) subjects took synthetic HRT, and 28 (72%) reported having a catamenial seizure pattern before menopause. HRT had no significant effect on seizures; however, a history of catamenial seizure pattern was significantly associated with an increase in seizures at perimenopause (p = 0.02). CONCLUSIONS: These pilot data suggest that synthetic HRT may be associated with an increase in seizure frequency in menopausal women with epilepsy. A catamenial seizure pattern may be associated with seizure decrease during menopause but with an increase during perimenopause.

Late-onset congenital adrenal hyperplasia: a treatable cause of anxiety.

BACKGROUND: Some intermediaries of cortisol synthesis, especially the sulfated ester of dehydroepiandrosterone (DHEAS), are picrotoxin-like antagonists of the gamma-aminobutyric acid A (GABA-A) receptor and exert potent anxiogenic effects. We report 5 men and 7 women with refractory anxiety disorders, who had late-onset congenital adrenal hyperplasia (CAH), and in whom interactions between neuroactive steroids and anomalous brain substrates may have participated in the pathophysiology and treatment of anxiety. METHODS: Twelve patients with refractory anxiety disorders as defined by DSM-IV had elevated DHEAS and specific enzyme deficiencies diagnostic of CAH. All were treated with adrenal suppressive therapy using ketoconazole or low (physiologic) dose glucocorticoids. Anxiety was rated by the Tension Scale of the Profile of Mood States (POMS Tension) questionnaire before and during hormonal treatment. RESULTS: Reduction of DHEAS was associated with lower anxiety scores in all twelve cases. POMS Tension scores decreased by 55%. Hormonal treatment, which failed to lower DHEAS, was ineffective. CONCLUSIONS: These findings suggest that late onset CAH can contribute to anxiety disorders and that adrenal suppressive therapy or inhibition of steroidogenesis with ketoconazole may be efficacious as adjuvant therapy.

Interaction of insulin receptor substrate 3 with insulin receptor, insulin receptor-related receptor, insulin-like growth factor-1 receptor, and downstream signaling proteins.

Insulin receptor substrates (IRS) mediate biological actions of insulin, growth factors, and cytokines. All four mammalian IRS proteins contain pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains at their N termini. However, the molecules diverge in their C-terminal sequences. IRS3 is considerably shorter than IRS1, IRS2, and IRS4, and is predicted to interact with a distinct group of downstream signaling molecules. In the present study, we investigated interactions of IRS3 with various signaling molecules. The PTB domain of mIRS3 is necessary and sufficient for binding to the juxtamembrane NPXpY motif of the insulin receptor in the yeast two-hybrid system. This interaction is stronger if the PH domain or the C-terminal phosphorylation domain is retained in the construct. As determined in a modified yeast two-hybrid system, mIRS3 bound strongly to the p85 subunit of phosphatidylinositol 3-kinase. Although high affinity interaction required the presence of at least two of the four YXXM motifs in mIRS3, there was not a requirement for specific YXXM motifs. mIRS3 also bound to SHP2, Grb2, Nck, and Shc, but less strongly than to p85. Studies in COS-7 cells demonstrated that deletion of either the PH or the PTB domain abolished insulin-stimulated phosphorylation of mIRS3. Insulin stimulation promoted the association of mIRS3 with p85, SHP2, Nck, and Shc. Despite weak association between mIRS3 and Grb2, this interaction was not increased by insulin, and may not be mediated by the SH2 domain of Grb2. Thus, in contrast to other IRS proteins, mIRS3 appears to have greater specificity for activation of the phosphatidylinositol 3-kinase pathway rather than the Grb2/Ras pathway.

Action of insulin receptor substrate-3 (IRS-3) and IRS-4 to stimulate translocation of GLUT4 in rat adipose cells.

The insulin receptor initiates insulin action by phosphorylating multiple intracellular substrates. Previously, we have demonstrated that insulin receptor substrates (IRS)-1 and -2 can mediate insulin's action to promote translocation of GLUT4 glucose transporters to the cell surface in rat adipose cells. Although IRS-1, -2, and -4 are similar in overall structure, IRS-3 is approximately 50% shorter and differs with respect to sites of tyrosine phosphorylation. Nevertheless, as demonstrated in this study, both IRS-3 and IRS-4 can also stimulate translocation of GLUT4. Rat adipose cells were cotransfected with expression vectors for hemagglutinin (HA) epitope-tagged GLUT4 (GLUT4-HA) and human IRS-1, murine IRS-3, or human IRS-4. Overexpression of IRS-1 led to a 2-fold increase in cell surface GLUT4-HA in cells incubated in the absence of insulin; overexpression of either IRS-3 or IRS-4 elicited a larger increase in cell surface GLUT4-HA. Indeed, the effect of IRS-3 in the absence of insulin was approximately 40% greater than the effect of a maximally stimulating concentration of insulin in cells not overexpressing IRS proteins. Because phosphatidylinositol (PI) 3-kinase is essential for insulin-stimulated translocation of GLUT4, we also studied a mutant IRS-3 molecule (IRS-3-F4) in which Phe was substituted for Tyr in all four YXXM motifs (the phosphorylation sites predicted to bind to and activate PI 3-kinase). Interestingly, overexpression of IRS-3-F4 did not promote translocation of GLUT4-HA, but actually inhibited the ability of insulin to stimulate translocation of GLUT4-HA to the cell surface. Our data suggest that IRS-3 and IRS-4 are capable of mediating PI 3-kinase-dependent metabolic actions of insulin in adipose cells, and that IRS proteins play a physiological role in mediating translocation of GLUT4.

Hormones and cognition: current concepts and issues in neuropsychology.

This article provides an extensive and comprehensive review of the effects of hormones on cognition. Studies detailing specific neurocognitive functions affected by variation in hormone levels across the life span are presented. Dysregulation of hormone levels is considered from models of both normal and diseased functioning. Patterns of cognitive dysfunction are described for a range of syndromes involving the neuroendocrine system, and evidence of specific neurophysiological mechanisms that can account for these findings is outlined. This review includes discussion of treatment outcomes and the permanency of endocrine-related cognitive dysfunction. The authors present a set of guidelines for clinical neuropsychologists to use for assessment of patients with neuroendocrine system dysfunction. Clinical and methodological issues in research and treatment settings are discussed.

Testosterone versus testosterone and testolactone in treating reproductive and sexual dysfunction in men with epilepsy and hypogonadism.

Antiepileptic drug-induced reductions in serum levels of biologically active testosterone and elevations in serum estradiol (E2) may contribute to sexual dysfunction among men with epilepsy. Treatment using a combination of testosterone and the aromatase inhibitor testolactone may have significantly better effects on sexual function and also seizure frequency than testosterone alone.

Apolipoprotein E epsilon4 associated with chronic traumatic brain injury in boxing.

CONTEXT: Given the similarities between Alzheimer disease and dementia pugilistica, we evaluated the relationship between apolipoprotein E (APOE) genotype and chronic traumatic brain injury (CTBI) in boxers to determine whether there is a genetic susceptibility to the effects of head trauma. OBJECTIVE: To assess the relationship between CTBI and APOE genotype in boxers. DESIGN AND SETTING: Clinical characterization of 24 volunteer and 6 referred boxers in an outpatient setting. PARTICIPANTS: Thirty professional boxers aged 23 to 76 years underwent neurologic and behavioral assessment in conjunction with APOE genotyping. MAIN OUTCOME MEASURES: Apolipoprotein E genotype was examined in relationship to measures of CTBI. A 10-point clinical rating scale (0-9), the Chronic Brain Injury (CBI) scale, was devised to assess the severity of traumatic encephalopathy associated with boxing. Boxers with abnormal CTBI scores were further classified on the basis of whether their impairments were possibly or probably related to boxing. Scores were analyzed in relation to boxing exposure (number of bouts) and APOE genotype. RESULTS: Among the 30 boxers, 11 were found to be normal (CBI score=0), 12 showed mild deficits (CBI score=1-2), 4 were moderately impaired (CBI score=3-4), and 3 showed signs of severe impairment (CBI score > 4). High-exposure boxers (ie, those with > or = 12 professional bouts) had significantly higher CBI scores (mean [SD], 2.6 [1.9]) than low-exposure boxers (mean [SD], 0.3 [0.7]) (P<.001), indicating that neurologic impairment as measured by the CBI scale seems related to boxing exposure. The APOE genotype frequencies of the study population were approximately the same as those found in the general population. Boxers with low exposure had mean CBI scores of 0.33, irrespective of APOE genotype. However, high-exposure boxers with an APOE epsilon4 allele had significantly greater CBI scores (mean [SD], 3.9 [2.3]) than high-exposure boxers without APOE epsilon4 (mean [SD], 1.8 [1.2]) (P=.04). All boxers with severe impairment possessed at least 1 APOE epsilon4 allele. The tendency for greater CTBI among those with both high exposure and an epsilon4 allele was statistically significant at the P<.001 level. CONCLUSIONS: These preliminary findings suggest that possession of an APOE epsilon4 allele may be associated with increased severity of chronic neurologic deficits in high-exposure boxers.

Home health services in New Hampshire.

While home health services have traditionally been an underused component of the health care system, current trends suggest the desirability of expanding these services. These trends include an increase in the number of elderly who need the benefits of home care, the recognition that long-term chronic illnesses require appropriate management at home, and concern that patients have access to care at the level most appropriate to their illnesses. In New Hampshire, 41 certified home health agencies offer services. Little systematic research has been conducted on the kinds of services they provide and the patients seen by their staffs. Patient encounter data were collected from a sample of eight agencies for a 4-week period. Staff of the agencies used the patient contact record developed by the National Functional Task Analysis Cooperative Study to collect data. The data reflected differences among the agencies in the size of the populations they serve, organizational characteristics, reasons for patients' visits, expected sources of the revenue that supported them, and the diagnosis of the patients they cared for. The agencies served areas with populations ranging from 1,000 to 40,000. The staffs ranged from 1 to 14 full-time persons. Two were public agencies; the others had voluntary sponsorship. When data on reasons for visits were averaged for the eight agencies, it was shown that 72% of the visits were made for disease control activities such as care for a chronic or acute condition or for treatment or a laboratory test. Disease prevention activities such as a checkup for adults, children, prenatal or postnatal care, or health education accounted for only 24% of the visits. This result may indicate that, in areas short of physician manpower, the community health nurse is taking on increasing responsibility for medical care as well as health and education. Reimbursement for the visits came from Medicare, 25%; Medicaid-welfare, 14%; the patients, 18%; and health insurance, 3%. For 35% of the visits there was no charge; they were underwritten by community resources.

Financial impact of physician assistants on medical practice.

A study of revenues generated and expenses incurred by 12 physician assistants (MEDEX) who had held salaried positions for at least one year was conducted to determine their financial impact on primary-care practices. Daily charge logs were used to make annual estimates of MEDEX-generated revenues. One method of estimating annual revenues produced a mean of $28,190 per year, and a second method yielded a figure of $30,210 per year. Financial statements were used in two different ways to estimate annual expenses related to the employment of the MEDEX. One procedure indicated average costs of employment were $15,900, and the other $20,100. Ten of the 12 practices in the study experienced substantial gains of estimated revenue over expenses ascribed to the activities of the MEDEX.

Impact of physician's assistants on patient visits in ambulatory care practices.

The addition of a physician's assistant to an ambulatory care practice increases the practice's productivity. Practices using physician's assistants (medexes) had a 12% increase in the number of patient visits during the first year of training and 1 3/4 years later had an average increase of 37%. The medex by himself provided care to 28% of the patients and, in company with the physician, to another 10%. No consistent changes across practices were noted in patient waiting times or time physicians spend with patients.