Species-conserved reconfigurations of brain network topology induced by ketamine.

Species-conserved (intermediate) phenotypes that can be quantified and compared across species offer important advantages for translational research and drug discovery. Here, we investigate the utility of network science methods to assess the pharmacological alterations of the large-scale architecture of brain networks in rats and humans. In a double-blind, placebo-controlled, cross-over study in humans and a placebo-controlled two-group study in rats, we demonstrate that the application of ketamine leads to a topological reconfiguration of large-scale brain networks towards less-integrated and more-segregated information processing in both the species. As these alterations are opposed to those commonly observed in patients suffering from depression, they might indicate systems-level correlates of the antidepressant effect of ketamine.

Evaluation of symptomatic drug effects in Alzheimer's disease: strategies for prediction of efficacy in humans.

In chronic diseases such as Alzheimer's disease (AD), the arsenal of biomarkers available to determine the effectiveness of symptomatic treatment is very limited. Interpretation of the results provided in literature is cumbersome and it becomes difficult to predict their standardization to a larger patient population. Indeed, cognitive assessment alone does not appear to have sufficient predictive value of drug efficacy in early clinical development of AD treatment. In recent years, research has contributed to the emergence of new tools to assess brain activity relying on innovative technologies of imaging and electrophysiology. However, the relevance of the use of these newer markers in treatment response assessment is waiting for validation. This review shows how the early clinical assessment of symptomatic drugs could benefit from the inclusion of suitable pharmacodynamic markers. This review also emphasizes the importance of re-evaluating a step-by-step strategy in drug development.

The low-frequency blood oxygenation level-dependent functional connectivity signature of the hippocampal-prefrontal network in the rat brain.

Interactions between the hippocampus and the prefrontal cortex (PFC) are of major interest in the neurobiology of psychiatric and neurodegenerative disorders and are central to many experimental rodent models. Non-invasive imaging techniques offer a translatable approach to probing this system if homologous features can be identified across species. The objective of the present study was to systematically characterize the rat brain connectivity signature derived from low-frequency resting blood oxygenation level-dependent (BOLD) oscillations associated with and within the hippocampal-prefrontal network, using an array of small seed locations within the relatively large anatomical structures comprising this system. A heterogeneous structure of functional connectivity, both between and within the hippocampal-prefrontal brain structures, was observed. In the hippocampal formation, the posterior (subiculum) region correlated more strongly than the anterior dorsal hippocampus with the PFC. A homologous relationship was found in the human hippocampus, with differential functional connectivity between hippocampal locations proximal to the fornix body relative to locations more distal being localized to the medial prefrontal regions in both species. The orbitofrontal cortex correlated more strongly with sensory cortices and a heterogeneous dependence of functional coupling on seed location was observed along the midline cingulate and retrosplenial cortices. These findings are all convergent with known anatomical connectivity, with stronger BOLD correlations corresponding to known monosynaptic connections. These functional connectivity relationships may provide a useful translatable probe of the hippocampal-prefrontal system for the further study of rodent models of disease and potential treatments, and inform electrode placement in electrophysiology to yield more precise descriptors of the circuits at risk in psychiatric disease.

The grey mouse lemur: a non-human primate model for ageing studies.

The use of non-human primate models is required to understand the ageing process and evaluate new therapies against age-associated pathologies. The present article summarizes all the contributions of the grey mouse lemur Microcebus murinus, a small nocturnal prosimian primate, to the understanding of the mechanisms of ageing. Results from studies of both healthy and pathological ageing research on the grey mouse lemur demonstrated that this animal is a unique model to study age-dependent changes in endocrine systems, biological rhythms, thermoregulation, sensorial, cerebral and cognitive functions.

Excessive insulin receptor serine phosphorylation in cultured fibroblasts and in skeletal muscle. A potential mechanism for insulin resistance in the polycystic ovary syndrome.

We investigated the cellular mechanisms of the unique disorder of insulin action found in the polycystic ovary syndrome (PCOS). Approximately 50% of PCOS women (PCOS-Ser) had a significant increase in insulin-independent beta-subunit [32P]phosphate incorporation (3.7-fold, P < 0.05 vs other groups) in skin fibroblast insulin receptors that was present in serine residues while insulin-induced tyrosine phosphorylation was decreased (both P < 0.05 vs other groups). PCOS skeletal muscle insulin receptors had the same abnormal phosphorylation pattern. The remaining PCOS women (PCOS-n1) had basal and insulin-stimulated receptor autophosphorylation similar to control. Phosphorylation of the artificial substrate poly GLU4:TYR1 by the PCOS-Ser insulin receptors was significantly decreased (P < 0.05) compared to control and PCOS-n1 receptors. The factor responsible for excessive serine phosphorylation appeared to be extrinsic to the receptor since no insulin receptor gene mutations were identified, immunoprecipitation before autophosphorylation corrected the phosphorylation defect and control insulin receptors mixed with lectin eluates from affected PCOS fibroblasts displayed increased serine phosphorylation. Our findings suggest that increased insulin receptor serine phosphorylation decreases its protein tyrosine kinase activity and is one mechanism for the post-binding defect in insulin action characteristic of PCOS.

Regulation of growth hormone (GH), insulin-like growth factor (IGF)I, IGF binding proteins -1, -2, -3 and GH binding protein during progression of liver cirrhosis.

The aim of this study was to investigate the regulation of various proteins of the GHIGF axis during progression of liver failure and to search for potential prognostic markers of functional hepatic reserve. Serum levels of growth hormone (GH) and high affinity growth hormone binding protein (GHBP), insulin-like growth factor I (IGF-I) and IGF binding proteins (IGFBP) -1, -2 and -3 were determined in patients with liver cirrhosis. A continuous decline in the concentrations of IGF-I, IGFBP-3 and serum GH-binding activity (GHBP) was observed during progression of cirrhosis and the data correlated significantly with choline esterase, total serum protein and the Child score. In addition, GHBP showed a significant correlation with the enzymatic activity of glutamate dehydrogenase or transaminases and seems so to be influenced by the degree of liver cell damage. In contrast, IGFBP-1 and IGFBP-2 levels were significantly elevated in preterminal disease suggesting an upregulatory mechanism is still effective in this situation. Only when liver function had markedly deteriorated, the serum levels of these two parameters decreased again, possibly due to an impaired synthesis. The excellent correlation between the serum levels of IGF-I (r = -0.64, p < 0.001) or IGFBP-3 (r = -0.67, p < 0.001) and the Child score index suggests that they reflect the hepatic functions just as conventional indicators. For an appropriate interpretation of the liver function the measurement of the growth related peptides can be a valuable tool to estimate pathological alteration in the functional hepatic reserve or in the glucose homeostasis.

Absence of insulin receptor gene mutations in three insulin-resistant women with the polycystic ovary syndrome.

Women with polycystic ovary syndrome (PCOS) are markedly insulin-resistant, but the molecular mechanisms of these changes and their relationship to the hyperandrogenic state remain to be clarified. Mutations have recently been identified in the insulin receptor gene of patients with extreme forms of insulin resistance associated with hyperandrogenism (eg, type A insulin resistance), and these mutations account for the insulin resistance in such patients. We performed this study to determine whether mutations in the coding portion of the insulin receptor gene were responsible for insulin resistance in PCOS. Insulin binding studies using cultured skin fibroblasts of three obese (body mass index > 27 kg/m2) women with PCOS (ie, mild hyperandrogenemia and chronic anovulation of unknown etiology) and documented insulin resistance showed no apparent abnormalities in either the number or affinity of insulin binding sites. Direct sequencing of all 22 exons of the insulin receptor gene from two of the women with PCOS did not reveal any mutations. Furthermore, both alleles of the gene were expressed at equal levels. In a third insulin-resistant PCOS woman, there was no evidence for a mutation in the coding portion of the insulin receptor gene as determined by denaturing gradient gel electrophoresis (DGGE). We conclude that the insulin resistance in these PCOS women was caused by a defect extrinsic to the insulin receptor.

Did anxiety during the Gulf War cause premature delivery?

The Persian Gulf War presented Israel with a threat of chemical attacks on the home front. There is no doubt, according to many publications, that the missile attacks caused extensive long-term stress on the Israeli population. In this research, the connection between environmental stress and preterm delivery was studied. One thousand twenty-two deliveries during the war were compared to 1,027 deliveries in the previous year. The two groups showed no differences in the average gestational age, the rate of premature labor, and the cesarean section rate. The percentage of instrumental deliveries using forceps or vacuum was significantly higher (p < 0.05) in the war group.

Down-regulation of retinoic acid receptor activity associated with decreased alpha and gamma isoforms expression in F9 embryonal carcinoma cells differentiated by retinoic acid.

F9 embryonal carcinoma cells differentiate in response to retinoic acid (RA). To investigate the regulation of RA receptors (RARs) expression during this process, cDNA probes specific for the major RAR isoforms were used. In contrast to the level of RAR beta 2 mRNA which was high in cells treated 5 days with RA and below detection in untreated cells, as previously described, the steady state levels of RAR alpha 1, alpha 2, gamma 1, and gamma 2 mRNAs were markedly decreased in the RA-differentiated cells as compared to untreated cells. The down-regulation of the RA-responsive system in differentiated cells was also evident in gel shift assays as a marked decrease in binding capacity to a retinoid acid response element (beta 2RARE), as well as in chloramphenicol acetyltransferase (CAT) assays as a sixfold decrease in RA-mediated transacting activity via this element. The down-regulation of RAR DNA-binding and transacting activity coincided with the burst in tissue plasminogen activator secretion and thus, occurred at the hinge between early and late differentiation. The down-regulation of RA responsiveness may constitute an important event in the transition between early and late differentiation stage in F9 cells.

Evaluation of a dual-color flow cytometry immunophenotyping panel in a multicenter quality assurance program.

A basic immunophenotyping panel that employed dual-color combinations of fluorescein isothiocyanate (FITC) and phycoerythrin (PE) conjugated monoclonal antibodies (mAb; FITC-CD45/PE-CD14, FITC-IgG1/PE-IgG2, FITC-CD3/PE-CD8, FITC-CD3/PE-CD4, FITC-CD3/PE-CD16 + PE-CD56, and PE-CD19) was utilized in a quality assurance program to determine whether the 4 laboratories participating in a multicenter AIDS study obtained similar lymphocyte subset percentage values for T cells, B cells, NK cells, and CD4+ and CD8+ T cells. Over a 1 1/2 year period, 78 shared peripheral blood specimens were prepared and analyzed in each laboratory. The CD45bright CD14- percentage for each specimen was used to correct that individual's lymphocyte subset values. Interlaboratory coefficients of variation (CV) for the human immunodeficiency virus type I (HIV) seronegative (n = 38) and HIV-seropositive (n = 40) specimens using this panel were < 3% for total T cells; < 5% for CD4+ T cells and CD8+ T cells; < or = 17% for B and NK cells; and < 8% for CD4T/CD8T ratios. The 6-tube basic immunophenotyping panel has several notable features: a) for clinical studies, it permits comprehensive evaluation of an individual's major lymphocyte subsets, i.e., T, B, NK, and CD4+ and CD8+ T cells; b) for interlaboratory proficiency testing programs, it allows the detection of differences among laboratories in measurements of several functionally distinct cell populations; and c) for within-sample quality assurance, it provides several quality control checks, including the lymphosum, i.e., the sum of an individual's corrected T+B+NK values, a sum that was generally 100 +/- 5% on the HIV-seronegative specimens analyzed in this study.

Measurement of insulin-like growth factor I (IGF-I) in normal adults, patients with liver cirrhosis and acromegaly: experience with a new competitive enzyme immunoassay.

A competitive enzyme immunoassay for the determination of human insulin-like growth factor I in microtiter plates was established. Using a polyclonal antiserum raised in rabbits against hIGF-I ovalbumin conjugate the assay system was able to detect IGF-I at a range of 12-800 pg/well with a sensitivity of 10 pg/well. It showed a low (< 0.5%) cross reactivity with hIGF-II. The serum concentrations of IGF-I found by EIA agreed well with those found in a conventional RIA (r = 0.965, p < 0.001). Effects of age and sex on IGF-I levels were studied in 260 normal adults. There was no evidence for sex differences but a steep decline of values from the third to the fourth and from the eight to the ninth decade, respectively. To asses the diagnostic capability of the IGF-I determination in liver cirrhosis, 71 sera of patients classified according to Child classes (A-C) were measured. Although significantly diminished concentrations were found in class B vs A and in class C vs B, the diagnostic sensitivity in cross-sectional examinations proved to be low (class A: 0.33, class B: 0.67). Only in the case of extensively destroyed liver parenchyma (Child C: 0.94) IGF-I was a good indicator of impaired hepatocellular capacity. In 29 patients with acromegaly serum IGF-I levels were investigated. All patients with active acromegaly showed increased IGF-I levels. In contrast, in inactive or weakly active acromegaly values were considerably lower.(ABSTRACT TRUNCATED AT 250 WORDS)

Stress and human reproduction.

The interaction between emotional stress and infertility has been investigated for many years. Many infertile couples show marked stress during infertility evaluation and treatment. Most of the investigations that were performed during the last two decades show that in the majority of cases stress is the result and not the cause of infertility. The biological interaction between stress and infertility is the result of the action of stress hormones at the brain level, especially on the hypothalamus-pituitary and on the female reproductive organs. Stress hormones such as catecholamines (adrenalin, nonadrenaline and dopamine) and the hypothalamic-pituitary-adrenal axis interact with hormones which are responsible for normal ovulatory cycles: i.e., gonadotropin releasing hormone (GnRH), prolactin, LH and FSH. Endogenous opiates and melatonin secretion are altered by stress and interfere with ovulation. Sympathetic innervation of the female reproductive system provides routes by which stress can influence fertility at the of the sex organs level. Infertility causes stress which is aggravated as time passes and the couple remains infertile. Among the causes of stress are the couple's isolation, life with unrealized potential and unborn child, disruption of day-to-day life during infertility evaluation and treatment, and the couple's feeling that they do not have control of their own lives. The IVF program is considered by many as the final step for the evaluation of the couples fertility potential, hence, couples participating in an IVF program are highly stressed, especially after a failed IVF cycle.

The native alpha 2 beta 2 tetramer is the only subunit structure of the insulin receptor in intact cells and purified receptor preparations.

The native subunit structure of the insulin receptor was reinvestigated by two-dimensional nonreducing/reducing gel electrophoresis. Human insulin receptor expressed in murine fibroblasts was found to be a single oligomer, the alpha 2 beta 2 heterotetramer. The structure was assessed using receptor metabolically labeled with [35S]methionine, and using receptor autophosphorylation at two levels of purification: the insulin affinity-purified receptor and the more commonly used wheat germ agglutinin-Sepharose-enriched fraction from whole membrane extracts. Lower molecular weight oligomers and free subunits were observed only upon heating the sample prior to electrophoresis. This artifact of sample handling was dependent upon three factors: (i) temperature, (ii) time of heating, and (iii) impurities typically present in partially purified receptor preparations. We conclude that the alpha 2 beta 2 tetramer is the only insulin receptor subunit structure native in intact cells and subsequently isolated from cell membranes.

Control of insulin receptor autophosphorylation by polypeptide substrates: inhibition and stimulation by interaction with the catalytic subunit.

Autophosphorylation of purified insulin receptor, in the absence of insulin, was stimulated by selected polypeptide substrates. In the presence of 1 microM insulin these peptides inhibited autophosphorylation. Stimulation was observed with reduced [S-(carboxamidomethyl)cysteinyl]lysozyme (RCAM-lysozyme) and three peptides generated by CNBr cleavage, V8 proteinase digestion and/or chemical modification. We also generated two peptide substrates from RCAM-lysozyme which did not stimulate receptor autophosphorylation and were very weak inhibitors. As a control peptide, the simple substrate angiotensin inhibited receptor autophosphorylation in the absence or presence of insulin. However, stimulatory peptide, but not insulin, significantly shifted the concentration dependence for inhibition by angiotensin. The stimulatory peptides also increased autophosphorylation of the cloned cytoplasmic domain of the kinase [R-BIRK; Villalba, M., Wente, S. R., Russell, D. S., Ahn, J., Reichelderfer, C. F., & Rosen, O. M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7848]. Therefore, stimulation occurs by interaction with the cytoplasmic process of the beta-subunit and not through interaction with the insulin binding alpha-subunit of the native receptor. Autophosphorylation was analyzed by mapping 32P-labeled tryptic phosphopeptides from the beta-subunit and from R-BIRK. Nearly identical phosphopeptide maps were found, comparing first, basal R-BIRK and basal native receptor, second, peptide- and insulin-stimulated native receptor, and third, peptide-stimulated R-BIRK and insulin-stimulated native receptor. Therefore, R-BIRK functions as a basal-state enzyme and can be stimulated in an insulin-like manner. On the basis of these observations, stimulation by insulin and by peptides yields similar functional results, but by apparently different mechanisms.

Conformational states of the insulin receptor.

Insulin binding to the alpha-subunit of the purified insulin receptor changed the interaction between beta-subunits. This conformational change was demonstrated after labeling the receptor's beta-subunit by autophosphorylation in the absence of insulin, and then crosslinking the subunits to each other with bis (sulfosuccinimidyl) suberate. The convalent oligomers were resolved by reduction and denaturing gel electrophoresis. Insulin increased the rate of crosslinking, especially the formation of beta-beta dimers. These results support a conformational change following insulin binding, and may reflect the insulin-induced activation of autophosphorylation.

On the specificity of the aminophenazone breath test.

In 15 patients suffering from ulcer of the stomach or the duodenum, but without liver disease, the demethylation capacity was studied by the use of 14C-aminophenazone before and under the treatment with the H2-blocker cimetidine in daily doses of 1.0 g. As a measure of demethylation the expired 14CO2 (DPM/mmol CO2/70 kg body weight) was used before and 1 h after intake. Under cimetidine the mean 14CO2-value decreased from 724.7 DPM (SD 127.7 DPM) to 404.1 DPM (SD 153.1 DPM). In 3 patients the cimetidine medication was continued for additional 6 to 8 weeks with a dosage of 0.4 per day. In these subjects a mean of 533.2 DPM was measured. The enzyme activities (ALAT, ASAT, AP, AAP, gamma-GT, ChE) and the albumin and bilirubin concentration in serum measured concomitantly to the ABT did not differ from control values. Thus, the ABT is a useful test to assess changes of the demethylation capacity that can be caused by drugs or xenobiotics without affecting biochemical parameters used for diagnosis of liver diseases. The ABT might be a useful and simple technique to discover environmental disorders due to occupational conditions.

[Value of the 14C-aminophenazone inhalation test: inhibition of the demethylation function of the liver by cimetidine]. / Zur Wertigkeit des 14C-Aminophenazon-Atemtests: Hemmung der Demethylierungsfunktion der Leber durch Cimetidin.

On 15 patients with healthy liver and with a peptic ulcer the demethylation capacity of the liver was measured with the 14C-aminophenazone breathing test before the treatment and during the treatment with the H2-antagonist cimetidine (daily dose 1.0 g). As measure of the demethylation capacity the breathed out 14CO2 (DPM/mmol CO2/70 kg body weight) before and 1 hour after intake was chosen. Under influence of cimetidine the mean 14CO2-value of 724.7 DPM (S.D. 127.7) decreased to 404.1 DPM (S. D. 153.1). The enzyme activities (ALAT, ASAT, AP, AAP, GGT, CHE) and the concentrations of serum bilirubin and serum albumin remained unchanged and did not correlate mutually and with the 14CO2-data. The results confirm the independence of the aminophenazone test as technique for the recognition of disturbance of the MFO-system.

A simple method for routine determination of the metabolic liver capacity: the aminopyrine breath test.

In 16 healthy volunteers and in 39 patients with liver diseases (fatty liver, chronic persistent and chronic active hepatitis, hepatic cirrhosis) a simplified aminopyrine breath test (ABT) was carried out using a "tracer" dose of 3 mg (111 kBq) 14C-aminopyrine. The exhaled 14CO2 measured 1 h after intake amounted to values between 771 and 1337 DPM/mmol CO2/70 kg body weight in healthy controls. The amount of exhaled 14CO2 decreased in the order: fatty liver greater than chronic, active hepatitis greater than active, compensated cirrhosis greater than active, decompensated cirrhosis. Between the values of ABT and various conventional laboratory liver tests (alanine-aminotransferase, alanine-aminopeptidase, aspartate-aminotransferase, gamma-glutamyltransferase, total serum bilirubin) significant correlations were found (r = 0.6019 to 0.7765, n = 55; p less than 0.001). The proposed modification of the breath test is of advantage in that it requires a very low dose of aminopyrine and is easily practicable.