Neurosecretory cells in the optic lobes of the brain and activity rhythms in Lycosa tarentula (Araneae: Lycosidae)

Several paired groups of neurosecretory cells (NS) were identified in the dorsal cortical neurons of the optic lobes of the brain of Lycosa tarentula (Araneae). Two large bottle-shaped cells (NS A1, A2) and a cluster of ca. 20 smaller cells (NS B) were found between the lamina and medulla of the anterior median eyes (AM). The forward oriented bundles of NS B axons run alongside large fibres linked to the synaptic zones of the indirect eyes. In front of the arcuate body, an islet of about 10 fusiform cells (NS C1) sends short axons close to the internal cortical border. Other large cells (NS C2, C3) are found from the medulla of the AM to the anterior border of the central body. Their long axons end deeply in the brain neuropil. NS B and C1 function synchronously. The secretory cycles of NS A1 and A2 seem to be in opposition. The activity of these three types of NS depends on the phase of the day. Anatomical relationships of NS A, B and C1 with visual afferent/efferent fibres via synaptic buttons indicate a role of these cells in the modulation of circadian rhythms of visual and locomotor activity. On the other hand, NS C2 and C3, the functioning of which is not synchronous, might be involved in the modulation or control of the elementary movements of L. tarentula when active or at rest.     

The structure and cytochemistry of the neurosecretory cells in Ocypoda platytarsis Milne Edwards (Crustacea: Brachyura).

Four types of NS cells ('A', 'B', 'C' and 'D') in the brain, thoracic ganglion and four types ('alpha', 'beta', 'gamma' and 'delta') in the eye-stalk of Ocypoda platytarsis have been encountered. The cells in the brain occur in groups. Five groups have been noticed in the brain. Of the five groups the ventro-median group is unpaired and homogenous. In the other four one is unpaired and three are paired, and all are heterogenous. In the eyestalk the NS cells are distributed in 6 groups. They are MEX1, MIX1, MTX1, MTX2, MTX3 and MTX4. Of these MEX1 and MTX4 are homogenous. A battery of histochemical tests applied to NS cells revealed that the NS material is rich in carbohydrate moiety, disulfide groups, lipids, phospholipids and RNA. It contains little sulfhydryl groups, protein bound NH2 groups and no tyrosine, tryptophan and arginine.     

Neurosecretory system of the ventral ganglia in the dragonfly,Orthetrum chrysis (selys) (Odonata: Libellulidae)

Summary The neurosecretory cells of the ventral ganglia in the adult dragonfly, Orthetrum chrysis, are classified into A, B, C1 and C2 cells. The neurosecretory material in the ventral ganglia is composed of PAS-positive material with 1-, 2-glycol groups and some proteins. The proteins rich in cystine or cysteine occur predominantly in the A cells, moderately in C cells and negligibly in B cells. Proteins containing arginine occur in A and B cells only, and those containing basic amino acids occur in C2 cells. The neurosecretory pathways and the neurohemal organs are also described.     

Morphological and histological studies on the neuroendocrine system of the sugarcane leaf hopper, Pyrilla perpusilla Wlk. (Fulgoridae: Hemoptera)

Neuroendocrine system of the sugarcane leaf hopper, Pyrilla perpusilla, has been studied by employing PAVB and AF techniques in situ and on sections. There are four groups of NS cells in the brain, a medial of 14--17 cells, lateral and an antero-ventral of 2 cells each and a latero-ventral of a single cell. The cells are of A and B types: tinctorially the A-cells are further subdivided into A1, A2 and A3 subtypes. Suboesophageal and thoracic ganglions have 2 groups of 2 and 3 cells, each of A and B types. Axons of the cells of cerebral groups converge to form a common pathway which emerge from the protocerebrum as NCC. NSM transports both inter and intracellularly. In CC stained colloids accumulate at the commissuris, the gland has two--A and B--types of intrinsic cells. CA is devoid of NSM. Though considerably small in size and have new NS cells, its NS pathways are easily demonstrated in situ. It is emphasized that size and number of neurosecretory axons is not a limitation of the in situ technique but the demonstration of the tract depends upon the physiological state of the animal at the time of fixation.     

Fine structure of the photogenous areas in the bioluminescent ophiuroidAmphipholis squamata (Echinodermata,Ophiuridea)

 Amphipholis squamata is a small bioluminescent ophiuroid whose arms are the only body part to produce light. The morphology of the arms was described paying particular attention to the spinal ganglia, viz the areas of most intense luminescence. Spinal ganglia consist of five different cell types (A–E) which were studied at different stages of the photogenous reaction. Type D cells have numerous irregularlyshaped vacuoles, widespread Golgi apparatus and well-developed rough endoplasmic reticulum (RER) that show obvious ultrastructural changes after luminescence. Type D cells appear, therefore, to be the best photocyte candidate. Type B and C cells were frequently observed in the nervous system outside spinal ganglia. Type A and E cells have not been described before. Type A cells are ciliated cells and type E cells extend long processes which are intimately associated with type D cells and epidermal ciliated cells. Both type A and type E cells could take part to the stimulation pathway that triggers luminescence. Accepted: 1 September 1996     

The nervous system of Microstomum lineare (Turbellaria,Macrostomida)

Summary The nervous system (NS) of Microstomum lineare (Turbellaria, Macrostomida) was studied by electron and light microscopy, combined with fluorescence histochemistry (Falck-Hillarp method for biogenic monoamines). The NS is primitively organized, with a bilobed brain, two lateral nerve cords lacking commissures, and peripheral nerve cells scattered along the nerve cords. The stomatogastric NS, with a pharyngeal nerve ring, is joined to the central NS by a pair of connective ganglia. A green fluorescence in all parts of the NS indicates catecholaminergic neurons as the dominant neuron type.Ultrastructurally, two types of neurons were identified on the basis of their vesicle content: 1. Aminergic (catecholaminergic) neurons containing densecore vesicles of varying electron-density and size, i.e., small dense-core vesicles (diameter 50–100 nm), vesicles with a highly electron-dense core (60–140 nm), and vesicles with an eccentric dense-core. 2. Presumed peptidergic neuro-secretory neurons containing large granular vesicles (diameter about 200 nm) in the stomatogastric NS and peripheral parts of the central NS. In light microscopy, paraldehyde-thionin stained neurons were observed in the same areas.     

Chinese Strains (Type 7) of JC Virus Are Afro-Asiatic in Origin But Are Phylogenetically Distinct from the Mongolian and Indian Strains (Type 2D) and the Korean and Japanese Strains (Type 2A)

We have further characterized the Asian genotypes (Types 2 and 7) and subtypes of JC virus (JCV). Urine samples from 224 individuals with Han and Mongolian populations were collected in five regions in eastern China: Kunming, Chengdu, Shenyang, Chifeng, and Manzhouli. Also, 99 urine samples were collected from coastal and hill groups in Kerala, southern India, and 23 urine samples from Seoul, Korea. PCR products of four typing fragments were sequenced, including two in the VP1 gene, as well as one each in the VT intergenic region and regulatory region. It was possible to clone and sequence a total of 42 JCV whole genomes (~5120 bp). Five genotypes of JCV (Types 7A, 7B, 7C, 2D, and 4) were found in China, four genotypes (Types 2D, 7C, 4, and 1B) in southern India, and three genotypes (Types 7B, 2A, and 1A) in Korea. Type 7A was most prevalent in South China (59–64%) and Type 7B was predominant in northeast China and Inner Mongolia (67–77%). Type 7C strains were spread throughout North and South China (3–14%), while Type 2D strains were found only in the two Mongolian groups (9–10%). In southern India, Type 2D was predominant in the coastal group (95%), and two major types, Type 7C (50%) and Type 2D (35%), were prevalent in the tribal hill groups. In Korea two major genotypes were found: Type 7B (50%) and Type 2A (43%). Phylogenetic reconstruction places the Chinese genotypes in the Afro-Asiatic supercluster, but distinct from the Mongolian and Indian strains (Type 2D), as well as the Korean and Japanese genotype (Type 2A) that predominates in the Americas.     

DEB025 (Alisporivir) inhibits hepatitis C virus replication by preventing a cyclophilin A induced cis-trans isomerisation in domain II of NS5A

DEB025/Debio 025 (Alisporivir) is a cyclophilin (Cyp)-binding molecule with potent anti-hepatitis C virus (HCV) activity both in vitro and in vivo. It is currently being evaluated in phase II clinical trials. DEB025 binds to CypA, a peptidyl-prolyl cis-trans isomerase which is a crucial cofactor for HCV replication. Here we report that it was very difficult to select resistant replicons (genotype 1b) to DEB025, requiring an average of 20 weeks (four independent experiments), compared to the typically <2 weeks with protease or polymerase inhibitors. This indicates a high genetic barrier to resistance for DEB025. Mutation D320E in NS5A was the only mutation consistently selected in the replicon genome. This mutation alone conferred a low-level (3.9-fold) resistance. Replacing the NS5A gene (but not the NS5B gene) from the wild type (WT) genome with the corresponding sequence from the DEB025(res) replicon resulted in transfer of resistance. Cross-resistance with cyclosporine A (CsA) was observed, whereas NS3 protease and NS5B polymerase inhibitors retained WT-activity against DEB025(res) replicons. Unlike WT, DEB025(res) replicon replicated efficiently in CypA knock down cells. However, DEB025 disrupted the interaction between CypA and NS5A regardless of whether the NS5A protein was derived from WT or DEB025(res) replicon. NMR titration experiments with peptides derived from the WT or the DEB025(res) domain II of NS5A corroborated this observation in a quantitative manner. Interestingly, comparative NMR studies on two 20-mer NS5A peptides that contain D320 or E320 revealed a shift in population between the major and minor conformers. These data suggest that D320E conferred low-level resistance to DEB025 probably by reducing the need for CypA-dependent isomerisation of NS5A. Prolonged DEB025 treatment and multiple genotypic changes may be necessary to generate significant resistance to DEB025, underlying the high barrier to resistance.     

Natural suppressor (NS) activity from murine neonatal spleen is responsive to IFN-gamma

Natural suppressor (NS) cell activity is the ability of apparently unprimed "null" cells to nonspecifically suppress immune responses. Previously we have shown that NS cell activity from the spleens of mice undergoing chronic graft-vs-host disease (GVHD) is enhanced in vitro by activated T cell signals (e.g., Con A supernatant [CAS]). Here we asked if the naturally occurring suppressor activity found in the neonatal mouse spleen is caused by NS cells, and if so whether this NS activity is also responsive to T cell signals. Finally, we wanted to identify the material in the CAS to which the NS cells respond. Spleen cells from (BALB/c X B10.D2)F1 neonates contain potent, genetically unrestricted suppressor activity toward normal mitogen responses. The cells responsible for this suppression are nonadherent, Thy-, Ig- and are thus by definition NS cells. Neonatal spleen NS cells suppress the indicator Con A response of all mouse strains tested, but their behavior with regard to LPS responses is different. They significantly inhibit the indicator LPS response of allogeneic strains, but are less inhibitory of LPS-stimulated syngeneic (BALB/c X B10.D2)F1 and parental strains. However, the addition of CAS to these latter cultures enhances the NS inhibition of the LPS response to the level of suppression seen with a Con A response. Two lymphokines were able to replace the CAS. Recombinant interferon-gamma (rIFN-gamma) closely mimics the activity found with whole CAS, with low concentrations (1 U/well) being capable of enhancing the neonatal NS activity to near-maximal levels. Recombinant interleukin 2 (rIL 2) is also capable of stimulating the neonatal NS activity to near maximum. However, the rIL 2 must be added at much higher concentrations, taking greater than 50 U/well to get maximum activation of NS suppression. The addition of anti-IFN-gamma antiserum to these LPS suppression assays removes the ability of CAS to activate the neonatal NS cells. Anti-IFN-gamma antiserum also removes the ability of rIL 2 as well as rIFN-gamma to activate the NS cells. It thus appears that the rIL 2 is working by its ability to stimulate IFN-gamma production. Anti-IFN-gamma also removes the ability of the neonatal NS cells to suppress a Con A response. Therefore, it appears that neonatal splenic NS cells respond to, and are activated by, IFN-gamma to carry out their suppressive activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Neurosecretory cells in the frontal ganglion of the tobacco hornworm, Manduca sexta

The frontal ganglion of the tobacco hornworm, Manduca sexta (L.), was found to contain two neurosecretory (NS) cells (max dia = 40–45 μm). The cytoplasmic inclusions of the NS cells were stained purple with paraldehyde fuchsin, and marked fluctuations in amounts of NS material in the perikarya were observed, depending upon the developmental status of the insect. The perikarya of NS cells in the frontal ganglia of starved larvae and diapause pupae contained large accumulations of NS material, whereas feeding larvae and developing pharate adults showed relatively low amounts of neurosecretion. Electron microscopy revealed large accumulations of NS granules (dia = 80–240 nm) in the frontal ganglia of diapause pupae, but only slight accumulations of granules were seen in the NS cells of developing larvae and pharate adults.It was concluded that axonal transport and release but not synthesis is shut down during starvation and diapause, leading to accumulation of NS material in the perikarya. It is also suggested that the failure of many investigators to differentiate NS cells in the frontal ganglion of various insects may have been due to the selection of very active stages when the amount of available NS material was too low to be visualized by conventional staining techniques.     

Neurosecretory cells in Dysdercus similis (Hemiptera)

The neurosecretory cells of Dysdercus similis have been described. "A", "B", "C" and "D" types of neurosecretory cells are present. The "A" type of cells of the pars-intercerebralis show cyclical secretion. When these cells show secretory activity during one to three days of emergence, they have scattered granules. The cells are seen packed with clumps of neurosecretory material when they are not secreting, and this is interpreted as a storage stage. The axons of these cells supply the corpora cardiaca and some neurosecretory material also reaches the corpus allatum. The release of this neurosecretory material can be correlated both with moulting in the young stages and later with reproduction in the adults.     

Cytochemistry of the neurosecretory cells in the crab Menippe rumphii (Fabricius, Crustacea: Brachyura)

In Menippe rumphii five types of neurosecretory cells are found in the cerebral, commissural and thoracic ganglia. Detailed cytochemical observations on the neurosecretory cells revealed that they have responded strongly to saliva resistant PAS staining. Among proteins those rich in disulfides and sulhydryl groups are observed. Greater amounts of cytoplasmic RNA are observed in the reproductive season. Considerable amounts of lipids and phospholipids are also observed in the AS cells. The cytochemical differences between the NS cells and the nonsecretory neurons are also discussed.     

Ultrastructural differentiation of Toxoplasma gondii schizonts (types B to E) and gamonts in the intestines of cats fed bradyzoites

The ultrastructural characterisitics of four types of Toxoplasma gondii schizonts (types B, C, D and E) and their merozoites, microgamonts and macrogamonts were compared in cats killed at days 1, 2, 4 and 6 after feeding tissues cysts from the brains of mice. Schizonts, merozoites and gamonts contained most of the ultrastructural features characteristic of the phylum Apicomplexa. All four types of schizonts developed within enterocytes or intraepithelial lymphocytes. Occasionally, type B and C schizonts developed within enterocytes that were displaced beneath the epithelium into the lamina propria. Type D and E schizonts and gamonts developed exclusively in the epithelium. Tachyzoites occurred exclusively within the lamina propria. Type B schizonts formed merozoites by endodyogeny, whereas types C to E developed by endopolygeny. The parasitophorous vacuoles surrounding type B and C schizonts consisted of a single membrane, whereas those surrounding types D and E schizonts were comprised of two to four electron-dense membranes. The parasitophorous vacuole of type B schizonts had an extensive tubulovesicular membrane network (TMN); the TMN was reduced or absent in type C schizonts and completely absent in types D and E schizonts and gamonts. Type B merozoites were ultrastructurally similar to tachyzoites, except that they were slightly larger. Type C merozoites exhibited a positive periodic acid-Schiff reaction by light microscopy and ultrastructurally contained amylopectin granules. Rhoptries were labyrinthine in type B merozoites but were electron-dense in types C-E. The development of microgamonts, macrogamont and oocysts is also described.     

Activation of marginal zone B cells from lupus mice with type A(D) CpG-oligodeoxynucleotides

Several types of CpG-oligodeoxynucleotides (ODN) have been recently characterized. In mice, type A(D) CpG-ODNs primarily stimulate macrophages and dendritic cells, but fail to stimulate B cells. On the contrary, type B(K) CpG-ODNs are excellent B cell activators. Type C CpG-ODNs combine features of both types A(D) and B(K) CpG-ODNs. Despite cell type preferences, all CpG-ODNs require the presence of TLR9 for activation. In this study, we show that a subset of B cells from lupus mice responds to type A(D) CpG-ODN stimulation vigorously and directly with increased CD25 and CD86 expression and IL-10 secretion. Furthermore, these CpG-ODNs induce high surface IgM expression and promote 50- to 100-fold higher IgM and IgG3 secretion in lupus B cells than in controls. This response is similar to that seen with bacterial DNA stimulation of B cells. Type A(D)-responsive cells are enriched within lupus B cells with the marginal zone (MZ) phenotype. These cells are at least twice more numerous in lupus mice than in controls. The ability of lupus B cells to respond to type A(D) CpG-ODN stimulation is not due to differential TLR9 expression. Therefore, type A(D) CpG-ODNs may contribute to the lupus pathogenesis by inducing MZ-B cell activation, costimulatory molecule expression, and polyclonal Ig secretion. Through increased IL-10 secretion, MZ-B cells may also modify the activity of other cell types, particularly dendritic cells and macrophages.     

Graft-vs-host reactions (GVHR) across minor murine histocompatibility barriers. II. Development of natural suppressor cell activity

We explored the immunoincompetence of mice undergoing a chronic graft-vs-host reaction (GVHR) across minor histocompatibility barriers. BALB/c and B10.D2 mice are H-2d and mls b, and differ only with regard to minor histocompatibility antigens (MiHA). A large number of BALB/c mice were unirradiated or were irradiated with 300, 600, or 900 R. They then were injected with 5 X 10(7) spleen cells from either allogeneic B10.D2 or syngeneic BALB/c mice. The spleen cells from these recipient mice were assayed at various times post-irradiation/injection for their proliferative response to Con A and LPS, their ability to suppress the mitogen responses of normal spleen cells, and for the genetic specificity of this suppression. Spleen cells from BALB/c mice that had received 600 or 900 R (but not 0 or 300 R), and allogeneic B10.D2 lymphocytes, became very hyporesponsive to mitogens and became suppressive in vitro by days 7 to 10 post-irradiation/injection. These phenomena persisted for the entire 49 days of the experiment. After an initial period of splenomegaly, the spleens of these mice gradually became depleted of viable lymphocytes. Initial characterization of suppressor cells found in the spleens of GVH mice showed that they were not removed by treatment with anti-Thy-1.2 plus complement. GVH suppressors also were not adherent to plates coated with antiserum directed towards murine Ig. In addition, these cells did not adhere to plastic plates. Thus, we believe that the suppressor cells found in mice undergoing GVHD across MiHA are not mature T cells, B cells, or macrophages, but belong to a class of suppressor cells termed natural suppressor (NS). Genetic analysis of NS cell activity showed that as early as 10 days post-irradiation/injection, NS cells inhibited mitogen responses of all mouse strains tested, the exception being the relative difficulty in suppressing the LPS response of B10.D2 (syngeneic with donor cells). By day 42, this had developed into an almost complete inability to suppress a B10.D2 LPS response, although at this time NS cells were still capable of inhibiting all the other mitogen responses of all strains tested, including the Con A response of B10.D2 spleen cells. Moderate amounts of mitogen unresponsiveness and suppressor activity were seen in the syngeneic groups (BALB/c----BALB/c) but only if recipients received 600 or 900 R. This was a transient phenomenon that was maximal at day 14, and which we believe to be a similar but less severe degree of immunoincompetence when compared with that seen with allogeneic stimulation in the B10.D2----BALB/c GVH model.(ABSTRACT TRUNCATED AT 400 WORDS)     

Leucine incorporation in the ganglia of praying mantids during a learning process

Leucine incorporation into four ganglia (‘brain’. B; prothoracic ganglion, P; mesothoracic ganglion, M; and metathoracic ganglion, T) was studied in mantids (Stagmatoptera biocellata) trained not to attack a black star figure in movement. There were two experimental groups, i.e. LM and WH experimental groups, and one control group. The LM and WH groups differed in the experimental conditions of training in such a way that both had similar motor activity and sensorial stimulation but only one of them evinced learning (Maldonado and Tablante, 1975).After training, incorporation of |¹⁴C| leucine into P and B was greater in experimental than in control animals. This result is not necessarily related to learning because no difference was found between LM and WH experimental groups. The metabolic gradient of the four ganglia of the experimental animals was P→B→M→T, whereas that found for the control group was M→T→P→B. The authors interpret these findings as supporting a hypothesis previously set forth that correlates the metabolic gradient in the four ganglia with differences regarding control of motor activity and/or sensorial input.Experiments involving double labelling and electrophoresis indicated that the P and B changes after training involved all the proteins, and were not restricted to one or a few protein species.     

Neuroendocrine control of reproduction in the male penaeid prawn,Parapenaeopsis hardwickii (Miers) (Crustacea,Decapoda, Penaeidae)

Effects of bilateral eyestalks ablation and injections of eyestalks, (Es) brain (Br) and thoracic ganglia (ThG) extract separately on the androgenic gland and testis development of Es-ablated (Experiment 1) and normal (Experiment 2) P. hardwickii, were investigated. The androgenic gland activity increased significantly in prawns having Es-ablated (Group II), and eyestalkless (Groups VI & VII) and normal (Groups D & E) prawns received Br and ThG extracts, separately, as compared to their respective controls (Groups I & A). Injection of unboiled Es extract in eyestalkless (Group V) and normal (Group C) prawns arrested the androgenic gland activity. Simultaneously, there was a significant (p<0.05) increase in the diameter of testicular follicles, testis weight, testis index and number of mature spermatocytes per follicle in the prawns of groups II, VI & VII of experiment 1 and of groups D & E of experiment 2 when compared to their respective controls. Injection of Es extract into the eyestalkless (Group V) and normal (Group C) prawns ceased testicular development. A significant (<0.05) decrease in testicular protein and midgut gland glycogen and lipid was found in groups II, VI & VII of experiment 1 and in groups D & E of experiment 2, but the glycogen and lipid content of the testis increased. These results indicate that the hormones released from the neuroendocrine centres regulate androgenic gland activity and spermatogenesis.     

Chromosome banding in Cacopsylla mali (Schmidberger) and Cacopsylla sorbi (Linnaeus) (Homoptera, Psyllidae) with polymorphic sex chromosomes

C-banding and Ag staining were applied to Cacopsylla sorbi and C. mali. The aim was to discover some additional cytological markers to follow the pathways of the sex determination system transformation in the evolution of the species. Of three karyotype patterns so far described in the literature for these species--a type A (XO), B (neoXY) and C (neoX1X2Y), only the last two types (B and C) were found. All 6 studied Cacopsylla sorbi from Finland had a karyotype of type B (2n = 20 + XY), while C. mali had both types. Type B (2n = 22 + XY) was observed in 31 males, whereas type C (2n = 20 + neoX1X2Y) in the remaining four. The karyotype of C. sorbi was found to be characterized by a very small amount of C-positive material, localized in a telomere of the Y chromosome. The karyotype of C. mali was also characterized by a very small amount of C-banded material. Both the sex chromosomes and the autosomes displayed a marked polymorphism of C-positive bands within different individuals and even the same individual. In both species the nucleolus was located in the telomere of a middle sized autosomal till diplotene inclusive. The C-banding and Ag staining in the studied Cacopsylla species did not provide any additional cytological markers for an understanding of the pathways of sex determination system transformation in the evolution of the species.     

17beta-estradiol regulation of human growth hormone (hGH), insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding protein-3 (IGFBP-3) axis in hypoestrogenic, hypergonadotropic women

OBJECTIVE: Ovarian hormonal function may be as important contributing factor to hGH-IGF-I-IGFBP-3 axis as age. AIM: To examine plasma hGH, IGF-1 and IGFBP-3 levels in women with premature ovarian failure compared to healthy normal controls and postmenopausal ones. PATIENTS: Group A-15 women with premature ovarian failure (POF) (mean: age 38.9+/-5.2 years, FSH 101.4+/-29.0 IU/l; 17beta-estradiol 22.5+/-14.6 ng/l). Group B consisted of 15 menopausal women (mean: age 54.7+/-2.7 years; FSH 81.9+/-32.1 IU/l; 17beta-estradiol 17.1+/- 8.0 ng/l). Group C - controls - 15 normally menstruating women (mean: age 37.1+/-9.0 years; FSH 6.2+/-1.0 IU/l; 17beta-estradiol 144.8+/-117.1 ng/l). METHODS: Body mass and BMI were measured. Basic fasting plasma hGH, IGF-I, IGFBP-3, insulin, testosterone and LH as well as prolactin (PRL), FSH and estradiol were assessed by RIA kits. Statistical analysis. Shapiro-Wilk test, Mann-Whitney u-test, Spearman rang correlation coefficient, stepwise multiple regression. RESULTS: Mean serum IGF-I level was the lowest (p<0.005) in group B (172.0+/-54.6 microg/l) and the highest in group C (273.6+/-109.0 microg/l). The mean plasma IGF-I level in group A was similar (NS) (208.3+/-66.5 microg/l) to that found in group B and lower (p<0.02) compared with that in group C. The lowest (p<0.005) serum IGFBP-3 level was found in group B (3.1+/-0.7 microg/l) compared to group C (4.4+/-0.3 microg/l). The mean plasma IGFBP-3 level (3.1+/-1.0 microg/l) in group A was lower than in group C (p<0.005) but identical as in group B. No statistically significant differences between groups were observed in mean hGH levels. Women in group A and C were younger (p<0.001) than those in group B. The lowest mean estradiol level was found in groups A and B. The highest was in group C (p<0.001). Mean plasma LH and FSH levels were higher (p<0.001) in groups A and B vs group C. In group C there were links between IGF-I and age (r=-0.60; p=0.014) The IGF-I/age relation disappeared in the groups A and B (rA=-0.26; rB=0.10; NS). The same regards IGFBP-3/ age link (rA=-0.44, NS; rB=0,31;NS). Estradiol level was related to hGH levels in group C (r=-0.54; p<0.05). In none of groups hGH/IGF-1 as well as IGFBP-3/hGH relations were found. Prolactin accounted for 69% of the variance in IGF-I level in the group B (p=0.003) and for 24% in group A (NS). Testosterone accounted for 88% (p=0.004) of the variance in IGF-I level in group B and IGFBP-3 was responsible for 86% (p=0.038) of the variance in IGF-I level in group C. Again IGFBP-3 was responsible for 47% (p=0.023) in group A and for 49% (p=0.04) in group B of the hGH variance. CONCLUSIONS: 17b-estradiol may be as important contributor to insulin-like growth factor-I (IGF-I) plasma level as age in hypoestrogenic, hypogonadotropic women.     

Hepatitis C virus (HCV) NS5A binds RNA-dependent RNA polymerase (RdRP) NS5B and modulates RNA-dependent RNA polymerase activity.

Hepatitis C virus (HCV) NS5B is RNA-dependent RNA polymerase (RdRP), the essential catalytic enzyme for HCV replication. Recently, NS5A has been reported to be important for the establishment of HCV replication in vitro by the adaptive mutations, although its role in viral replication remains uncertain. Here we report that purified bacterial recombinant NS5A and NS5B directly interact with each other in vitro, detected by glutathione S-transferase (GST) pull-down assay. Furthermore, complex formation of these proteins transiently coexpressed in mammalian cells was detected by coprecipitation. Using terminally and internally truncated NS5A, two discontinuous regions of NS5A (amino acids 105-162 and 277-334) outside of the adaptive mutations were identified to be independently essential for the binding both in vivo and in vitro (Yamashita, T., Kaneko, S., Shirota, Y., Qin, W., Nomura, T., Kobayashi, K., and Mkyrakami, S. (1998) J. Biol. Chem. 273, 15479-15486). We previously examined the effect of His-NS5A on RdRP activity of the soluble recombinant NS5Bt in vitro (see Yamashita et al. above). Wild NS5A weakly stimulated at first (when less than 0.1 molar ratio to NS5B) and then inhibited the NS5Bt RdRP activity in a dose-dependent manner. The internal deletion mutants defective in NS5B binding exhibited no inhibitory effect, indicating that the NS5B binding is necessary for the inhibition. Taken together, our results support the idea that NS5A modulates HCV replication as a component of replication complex.