Inflammatory expression profile in peripheral blood mononuclear cells from patients with Nasu-Hakola Disease.

Homozygous mutations in Triggering Receptor Expressed on Myeloid cells 2 gene (TREM2) are one of the major causes of Nasu Hakola Disease (NHD). We analysed Peripheral Blood Mononuclear Cells (PBMC) profile of 164 inflammatory factors in patients with NHD carrying the TREM2 Q33X mutation as compared with heterozygous and wild type individuals. Several molecules related to bone formation and angiogenesis were altered in NHD compared to non-carriers: Bone Morphogenetic Protein (BMP)-1 mRNA levels were significantly increased in PBMC (2.32 fold-increase; P = 0.01), as were Transforming Growth Factor Beta (TGFB)3 levels (1.51 fold-increase; P = 0.02). Conversely, CXCL5 and Pro Platelet Basic Protein (PPBP) were strongly downregulated (-28.26, -9.85 fold-decrease over non-carriers, respectively, P = 0.01), as well as Platelet Factor 4 Variant 1 (PF4V1; -41.44, P = 0.03). Among other inflammatory factors evaluated, Interleukin (IL)-15 and Tumor Necrosis Factor Superfamily Member (TNFSF)4 mRNA levels were decreased in NHD as compared with non-carriers (-2.25 and -3.87 fold-decrease, P = 0.01 and 0.001, respectively). In heterozygous individuals, no significant differences were observed, apart from IL-15 mRNA levels, that were decreased at the same extent as NHD (-2.05 fold-decrease over non-carriers, P = 0.002). We identified a signature in PBMC from patients with NHD consisting of strongly decreased mRNA levels of CXCL5, PPBP, PF4V1, mildly decreased IL-15 and TNFSF4 and mildly increased BMP-1 and TGFB3.

Inflammatory molecules in Frontotemporal Dementia: cerebrospinal fluid signature of progranulin mutation carriers.

Mutations in progranulin gene (GRN) are one of the major causes of autosomal dominant Frontotemporal Lobar Degeneration (FTLD). Progranulin displays anti-inflammatory properties and is likely a ligand of Tumor Necrosis Factor (TNF) receptor 2, expressed on microglia. A few cytokines and chemokines are altered in cerebrospinal fluid (CSF) from patients with sporadic FTLD, whereas no information is available in familial cases. We evaluated, through BioPlex, levels of 27 inflammatory molecules, including cytokines, chemokines, and related receptors, in CSF and matched serum, from FTLD patients carrying GRN mutations as compared with sporadic FTLD with no GRN mutations and controls. Mean±SD Monocyte Chemoattractant Protein-1 (MCP-1) levels were significantly increased in CSF from sporadic FTLD patients as compared with controls (334.27±151.5 versus 159.7±49pg/ml; P⩽0.05). In GRN mutation carriers versus controls, CSF levels of MCP-1 were unchanged, whereas Interferon-γ-inducible protein-10 (IP-10) levels were increased (809.17±240.0 versus 436.61±202.5pg/ml; P=0.012). In the same group, TNFα and Interleukin (IL)-15 levels were decreased (3.18±1.41 versus 35.68±30.5pg/ml; P=0.013 and 9.34±5.54 versus 19.15±10.03pg/ml; P=0.023, respectively). Conversely, Regulated upon Activation, Normal T-cell Expressed, and Secreted (RANTES) levels were decreased in patients, with or without mutations, as compared with controls (4.63±3.30 and 2.58±20 versus 87.57±70pg/ml, respectively; P<0.05). Moreover, IP-10, IL-15 and RANTES CSF levels were not influenced by age, whereas MCP-1 levels increased with age (ρ=0.48; P=0.007). In conclusion, inflammatory de-regulation was observed in both sporadic FTLD and GRN carriers compared to controls, with a specific inflammatory profile for the latter group.

Plasma microglial-derived extracellular vesicles are increased in frail patients with Mild Cognitive Impairment and exert a neurotoxic effect.

Extracellular vesicles (EVs) are mediators of cellular communication that can be released by almost all cell types in both physiological and pathological conditions and are present in most biological fluids. Such characteristics make them attractive in the research of biomarkers for age-related pathological conditions. Based on this, the aim of the present study was to examine the changes in EV concentration and size in the context of frailty, a geriatric syndrome associated with a progressive physical and cognitive decline. Specifically, total EVs and neural and microglial-derived EVs (NDVs and MDVs respectively) were investigated in plasma of frail and non-frail controls (CTRL), mild cognitive impairment (MCI) subjects, and in Alzheimer's disease (AD) patients. Results provided evidence that AD patients displayed diminished NDV concentration (3.61 × 109 ± 1.92 × 109 vs 7.16 × 109 ± 4.3 × 109 particles/ml) and showed high diagnostic performance. They are able to discriminate between AD and CTRL with an area under the curve of 0.80, a sensitivity of 78.95% and a specificity of 85.7%, considering the cut-off of 5.27 × 109 particles/ml. Importantly, we also found that MDV concentration was increased in frail MCI patients compared to CTRL (5.89 × 109 ± 3.98 × 109 vs 3.16 × 109 ± 3.04 × 109 particles/ml, P < 0.05) and showed high neurotoxic effect on neurons. MDV concentration discriminate frail MCI vs non-frail CTRL (AUC = 0.76) with a sensitivity of 80% and a specificity of 70%, considering the cut-off of 2.69 × 109 particles/ml. Altogether, these results demonstrated an alteration in NDV and MDV release during cognitive decline, providing important insight into the role of EVs in frailty status.

GABAergic neurons expressing p75 in rat substantia innominata and nucleus basalis.

In vitro findings suggested a role for the p75 neurotrophin receptor in the maturation of GABAergic neurons residing in the basal forebrain (BF), a brain area known to have p75 expression only on cholinergic neurons. We document here the presence of GABAergic neurons which express p75 in the BF in vivo. Colocalization of p75 with the cholinergic marker choline-acetyltransferase (ChAT) and/or the GABAergic marker glutamic acid decarboxylase-67 (GAD67) was investigated in the BF at birth, at two weeks, and in adulthood. A subset of GAD67(+) neurons was p75(+) (p75(+)/GAD67(+)) but ChAT(-) in the substantia innominata and nucleus basalis magnocellularis at birth, whereas all p75(+)/GAD67(+) neurons were also ChAT(+) from two weeks onward. These phenotypic features suggest that a subpopulation of GABAergic neurons could be sensitive to neurotrophins during brain maturation. To unravel this issue, we then pursued a functional analysis by assessing p75 expression profile, and its modulation by nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF) in primary BF cell cultures. NGF increased p75 expression exclusively in cholinergic neurons, whereas BDNF induced p75 expression only in a subset of GABAergic neurons (p75(+)/GAD67(+)/ChAT(-)) through a p75- and tyrosine-kinase-dependent mechanism. The latter findings point to a selective role of BDNF in the induction of p75 expression in BF GABAergic neurons. Altogether these results confirm the role of neurotrophins in the developing and mature circuitry of GABAergic neurons in the BF regions.

Progranulin gene variability increases the risk for primary progressive multiple sclerosis in males.

Progranulin (GRN) gene variability has been analyzed in a sample of 354 patients with multiple sclerosis (MS) compared with 343 controls. No significant differences were observed, but by stratifying according to MS subtypes, a significant increased frequency of the rs2879096 TT genotype was found in primary progressive MS (PPMS) patients versus controls (16.0 vs 3.5%, P=0.023, odds ratio (OR) 5.2, 95% confidence interval (CI) 1.2-21.4). In addition, in PPMS, an association with the C allele of rs4792938 was observed (55.3 vs 33.5%, P=0.011, OR 2.4, 95% CI 1.2-4.7). An independent population was studied as replication, failing to confirm results previously obtained. Stratifying according to gender, an association with rs4792938 C allele was found in male PPMS patients compared with controls (40.7 vs 26.9%, P=0.002, OR 1.87, 95% CI 1.2-2.8). An association with the rs2879096T allele was observed (29.2 in patients compared with 18.9% in controls, P=0.012, OR 1.77, 95% CI 1.1-2.8). Haplotype analysis showed that TC haplotype frequency is increased in PPMS male patients compared with male controls (25.7 vs 16.6%; P=0.02, OR 1.69, 95% CI 1.1-2.7), whereas the respective GC haplotype seems to exert a protective effect, as its frequency is decreased in patients compared with controls (55.8% vs 70.9%; P=0.001, OR 0.52, 95% CI 0.4-0.8). Therefore, GRN haplotypes likely influence the risk of developing PPMS in males.

In cow anterior pituitary, growth hormone and prolactin can be packed in separate granules of the same cell.

The ultrastructural localization of growth hormone and prolactin in cow anterior pituitary was studied by double immunocytochemical labeling using specific antibodies and protein A-gold particles of different sizes. The two hormones were found in specific somatotrophs and mammotrophs as well as in somatomammotropic cells which were multinucleated and predominantly arranged in clusters in the central area of the lobules. In these mixed cells the two hormones were packaged (a) in different granules of the same cell, (b) in the same granules where they were segregated in different portions of the granule content, or (c) in the same granules but evenly intermixed. The relative proportion of these three types of granules varied in somatomammotrophs of different animals. A single large Golgi complex was generally present in somatomammotrophs. Small, immature granules containing either growth hormone or prolactin or both hormones were found randomly distributed along Golgi stacks. This suggests that in these cells the two hormones are processed in the same Golgi cisternae and that mechanism(s) exist(s) to sort out the two hormones from each other.

Spatial changes in calcium signaling during the establishment of neuronal polarity and synaptogenesis.

Calcium imaging techniques were used to obtain a clear although indirect evidence about the distribution of functional glutamate receptors of NMDA and non-NMDA type in cultured hippocampal neurons during establishment of polarity and synaptogenesis. Glutamate receptors were expressed and were already functional as early as one day after plating. At this stage NMDA and non-NMDA receptors were distributed in all plasmalemmal areas. During the establishment of neuronal polarity, responses to either types of glutamate receptors became restricted to the soma and dendrites. Compartmentalization of glutamate receptors occurred at stages of development when synaptic vesicles were already fully segregated to the axon. Formation of synapses was accompanied by a further redistribution of receptors, which segregated to synapse-enriched portions of dendrites. Receptor compartmentalization and dendritic redistribution as well as accumulation of synaptic vesicles at synaptic sites occurred also in neurons cultured in the presence of either the sodium channel blocker tetrodotoxin or glutamate receptor antagonists. These results indicate that signals generated by neuronal electrical activity or receptor activation are not involved in the establishment of neuronal polarity and synaptogenesis.

Sorting of three secretory proteins to distinct secretory granules in acidophilic cells of cow anterior pituitary.

The distribution of three proteins discharged by regulated exocytosis--growth hormone (GH), prolactin (PRL), and secretogranin II (SgII)--was investigated by double immunolabeling of ultrathin frozen sections in the acidophilic cells of the bovine pituitary. In mammotrophs, heavy PRL labeling was observed over secretory granule matrices (including the immature matrices at the trans Golgi surface) and also over Golgi cisternae. In contrast, in somatotrophs heavy GH labeling was restricted to the granule matrices; vesicles and tubules at the trans Golgi region showed some and the Golgi cisternae only sparse labeling. All somatotrophs and mammotrophs were heavily positive for GH and PRL, respectively, and were found to contain small amounts of the other hormone as well, which, however, was almost completely absent from granules, and was more concentrated in the Golgi complex, admixed with the predominant hormone. Mixed somatomammotrophs (approximately 26% of the acidophilic cells) were heavily positive for both GH and PRL. Although admixed within Golgi cisternae, the two hormones were stored separately within distinct granule types. A third type of granule was found to contain SgII. Spillage of small amounts of each of the three secretory proteins into granules containing predominantly another protein was common, but true intermixing (i.e., coexistence within single granules of comparable amounts of two proteins) was very rare. It is concluded that in the regulated pathway of acidophilic pituitary, cell mechanisms exist that cause sorting of the three secretory proteins investigated. Such mechanisms operate beyond the Golgi cisternae, possibly at the sites where condensation of secretion products into granule matrices takes place.

Purification and characterization of two plasma membrane domains from ejaculated bull spermatozoa.

Plasma membranes were detached from ejaculated bull spermatozoa by a brief sonication in a moderately hypotonic medium, and the released plasma membranes were partially purified by differential centrifugation. The resulting fraction was enriched 8- and 15-fold in alkaline phosphatase and 5' nucleotidase activities, respectively, compared with the starting sonicated spermatozoa. This total plasma membrane fraction was separated into two distinct fractions by equilibrium density centrifugation on a continuous linear sucrose gradient. Two peaks of light scattering material were formed at densities of 1.117 and 1.148 g/ml. The denser peak contained most of the protein of the plasma membrane fraction, whereas nearly all the concanavalin A binding activity was found in the lighter peak. The two bands had distinctly different polypeptide compositions when analyzed by SDS PAGE. Polyclonal antibodies were raised in rabbits against a major integral membrane glycoprotein of each fraction (Mr of 92,000 in the light peak and 98,000 in the dense peak). The two antigens were detected on the surface of intact spermatozoa by indirect immunofluorescence microscopy. The 92-kD protein (present in the lighter band) was detected only on the plasma membrane of the acrosomal and anterior postacrosomal regions of the head. The 98-kD antigen, present in the heavier band, was localized to the surface of the postacrosomal region of the head, to the principal piece of the tail, and to the connecting piece between the head and tail. The exclusive localization of the 92-kD polypeptide to the surface of the anterior portion of the head was confirmed by immunoelectron microscopy. These data show that the two fractions isolated on the sucrose gradient originate from different regions of the sperm cell plasma membrane.

The major tyrosine-sulfated protein of the bovine anterior pituitary is a secretory protein present in gonadotrophs, thyrotrophs, mammotrophs, and corticotrophs.

The anterior pituitary is a complex secretory tissue known to contain several sulfated macromolecules. In the present study, we identified the major tyrosine-sulfated protein of the bovine anterior pituitary and investigated its cellular and subcellular localization. This protein consisted of two tyrosine-sulfated polypeptides of molecular weight 86,000 and 84,000 that were highly homologous to each other. In agreement with previous biochemical studies, the tyrosine-sulfated protein of Mr 86,000/84,000 was found to be secretory, as it was observed in the matrix of secretory granules by immunoelectron microscopy. Immunofluorescence studies indicated that the tyrosine-sulfated, secretory protein of Mr 86,000/84,000, referred to as TSP 86/84, was present in all endocrine cells except for some somatotrophic cells. Higher levels of immunoreactivity for TSP 86/84 were observed in gonadotrophic and thyrotrophic than in mammotrophic and corticotrophic cells. This appeared to result from the occurrence of TSP 86/84 in all secretory granules of the former cells and in only some secretory granules of the latter cells. We discuss the possibility that TSP 86/84 may have a role in the packaging of several distinct peptides hormones into secretory granules. One, though not the only, possible function of tyrosine sulfation may concern the sorting of this protein in the Golgi complex.

Ca2+ waves in PC12 neurites: a bidirectional, receptor-oriented form of Ca2+ signaling.

Spatial and temporal aspects of Ca2+ signaling were investigated in PC12 cells differentiated with nerve growth factor, the well known nerve cell model. Activation of receptors coupled to polyphosphoinositide hydrolysis gave rise in a high proportion of the cells to Ca2+ waves propagating non decrementally and at constant speed (2-4 microns/s at 18 degrees C and approximately 10-fold faster at 37 degrees C) along the neurites. These waves relied entirely on the release of Ca2+ from intracellular stores since they could be generated even when the cells were incubated in Ca(2+)-free medium. In contrast, when the cells were depolarized with high K+ in Ca(2+)-containing medium, increases of cytosolic Ca2+ occurred in the neurites but failed to evolve into waves. Depending on the receptor agonist employed (bradykinin and carbachol versus ATP) the orientation of the waves could be opposite, from the neurite tip to the cell body or vice versa, suggesting different and specific distribution of the responsible surface receptors. Cytosolic Ca2+ imaging results, together with studies of inositol 1,4,5-trisphosphate generation in intact cells and inositol 1,4,5-trisphosphate-induced Ca2+ release from microsomes, revealed the sustaining process of the waves to be discharge of Ca2+ from the inositol 1,4,5-trisphosphate- (and not the ryanodine-) sensitive stores distributed along the neurites. The activation of the cognate receptor appears to result from the coordinate action of the second messenger and Ca2+. Because of their properties and orientation, the waves could participate in the control of not only conventional cell activities, but also excitability and differential processing of inputs, and thus of electrochemical computation in nerve cells.

[Ca2+]i imaging in PC12 cells: multiple response patterns to receptor activation reveal new aspects of transmembrane signaling.

Fura-2 imaging microscopy was used to study [Ca2+]i in nerve growth factor-differentiated PC12 cells exposed to agonists (bradykinin, carbamylcholine, and ATP) binding to receptors coupled to polyphosphoinositide hydrolysis. With all the treatments employed, the response to an individual agonist was often incomplete, i.e., composed of either release from intracellular stores or influx only. In individual cells the responses were closely similar when only one and the same agonist was employed, and markedly heterogeneous, with considerable variation of the release/influx ratio, when different agonists were delivered in sequence. In a recently isolated PC12 cell clone, heterogeneity of the receptor-induced [Ca2+]i responses was markedly lower than in the overall population, although the release/influx ratio was still variable. We conclude that the large response heterogeneity observed in the overall PC12 cell population is due (a) to the coexistence of multiple clones; and (b) to the variable activation of intracellular transduction mechanisms.

Regulation of turnover and number of acetylcholine receptors at neuromuscular junctions.

The number and metabolic stability of acetylcholine receptors (AChRs) at neuromuscular junctions of rat tibialis anterior (TA) and soleus (SOL) muscles were examined after denervation, paralysis by continuous application of tetrodotoxin to the nerve, or denervation and direct stimulation of the muscle through implanted electrodes. After 18 days of denervation AChR half-life declined from about 10 days to 2.3 days (TA) or 3.6 days (SOL) and after 18 days of nerve conduction block to 3.1 days (TA). In contrast, the total number of AChRs per endplate was unaffected by these treatments. Denervation for 33 days had no further effect on AChR half-life but reduced the total number of AChRs to about 54% (SOL) or 38% (TA) of normal. Direct stimulation of the 33-day denervated SOL from day 18 restored normal AChR stability and counteracted muscle atrophy but had no effect on the decline in AChR number. The results indicate that motoneurons control the stability of junctional AChRs through evoked muscle activity and the number of junctional AChRs through trophic factors.

Efficacy of cancer chemotherapy in relation to synchronization of cortisol rhythm, immune status and psychospiritual profile in metastatic non-small cell lung cancer.

BACKGROUND: The prognosis of cancer and the efficacy of the various anticancer therapies depend not only on tumor characteristics, but also on the endocrine and immune status of patients. Moreover, studies have shown that the clinical course of the neoplastic disease is also influenced by the psychospiritual status of patients. It is thus probable that the influence of psychospirituality on tumor growth may be mediated by the immunoneuroendocrine system, as demonstrated by the recent advances in psychoneuroendocrinological research. However, at present there are only few data on the possible link between the psychospiritual status and immunoendocrine functions of cancer patients. This study was carried out to investigate the relationships existing among the psychospiritual profile, cortisol rhythm and lymphocyte number before and after chemotherapy, and the efficacy of chemotherapy itself in advanced cancer patients. PATIENTS AND METHODS: The study included 30 consecutive metastatic non-small cell lung cancer patients under chemotherapeutic treatment with cisplatin plus gemcitabine. The psychobiological investigations consisted of lymphocyte count, cortisol circadian rhythm, psychological profile using Rorschach test, and spiritual score, as assessed by a specific clinical test for spirituality. The control group consisted of 100 healthy volunteers. The patients who achieved a tumor regression, showed a significantly higher pre-treatment lymphocyte count and significantly lower alteration of the cortisol rhythm with respect to those who had no benefit from chemotherapy. Moreover, the lymphocyte mean number increased during chemotherapy in responder patients, whereas it progressively diminished in those who had disease progression. Lymphocytopenia and alterations of the cortisol rhythm prior to chemotherapy were associated with a loss of the psychosexual identity according the Rorschach test. Moreover, the mean spiritual score was lower in patients than in controls, although the difference was not significant. Finally, a low spiritual score prior to therapy was associated with a higher frequency of lymphocytopenia and cortisol rhythm alteration, as well as with a lower efficacy of chemotherapy itself. CONCLUSION: This preliminary study would suggest that the psychospiritual status of cancer patients may influence the efficacy of chemotherapy through the immunoneuroendocrine system.

Immune and endocrine mechanisms of advanced cancer-related hypercortisolemia.

BACKGROUND: Cancer progression depend on the immune and endocrine status of the patients. In particular, it has been observed that abnormally high levels of cortisol and/or an altered circadian secretion are associated with a poor prognosis in advanced cancer patients. The present study was performed to establish whether cancer-induced hypercortisolemia depends on an activation of the hypothalamic-pituitary axis or on a direct adrenal stimulation by inflammatory cytokines, such as IL-6, which have been proven to induce cortisol secretion. PATIENTS AND METHODS: The study included 50 metastatic solid tumor patients, who were evaluated before the onset of chemotherapy. Venous blood samples were collected in the morning to measure IL-10, IL-6, ACTH and cortisol serum levels. Moreover, to analyze its circadian secretion, cortisol levels were also evaluated on venous blood samples collected at 4.00 p.m. RESULTS: Abnormally high morning levels of cortisol were observed in 19/50 (38%) patients. Moreover, a lack of a normal circadian rhythm of cortisol was seen in 8/50 (16%) patients. None of the patients showed high levels of ACTH. Abnormally high concentrations of IL-6 and IL-10 were present in 21/50 (42%) and in 14/50 (28%) patients, respectively. Mean serum levels of both IL-6 and IL-10 were significantly higher in patients with hypercortisolemia than in those with normal cortisol values (p<0.005 and p<0.001, respectively). According to previous clinical studies, these results confirm that the advanced neoplastic disease may be associated with enhanced cortisol levels and alterations of its circadian secretion. The lack of enhanced ACTH secretion excludes the possibility that the abnormal cortisol production is due to the activation of the hypothalamic-pituitary axis. On the contrary, the evidence of significantly higher concentrations of IL-6 in hypercortisolemic patients would suggest that cancer-related enhanced cortisol production may depend on a direct adrenal stimulation by IL-6 itself The well-demonstrated stimulatory role of cortisol on IL-10 production would explain the enhanced IL-10 secretion in hypercortisolemic patients. CONCLUSION: Cancer-related hypercortisolemia would seem to depend on alterations of the feedback mechanisms between endocrine and cytokine secretions, occurring in the neoplastic disease.

A new chemical etching process to improve endosseous implant osseointegration: in vitro evaluation on human osteoblast-like cells.

The development of novel mechanical and chemical surface modification treatments to improve the osteointegration properties of osseointegrated dental implants is nowadays a topic of great applicative interest. The aim of the present study was to analyse the role of surface topography and chemistry of four different surface treatments on titanium by an in vitro human osteosarcoma immortalised cell line model (MG63). The surface treatments considered were (a) machined titanium, (b) chemical etched on machined titanium, (c) sandblasted titanium and (d) chemical etching on sandblasted titanium. Chemical and physical surface properties were investigated by Scanning Electron Microscopy, Thin Film-X ray Diffraction and by Laser Profilometry. The in vitro biological response was characterised using the MG63 cell line by elution cytotoxicity tests, cell morphology, adhesion, proliferation activity, alkaline phosphatase activity and total DNA content in order to show a relationship between osteoblast response and surface features. Chemical and physical characterisation showed that the considered treatments differently modify the surface morphology in the micro and sub-micrometric scale. Although some differences in alkaline phosphatase activity were observed in the biological characterisation, depending on the specific material's surface finishing, the results showed that cells were well responsive on all the tested materials and grew and differentiated with similar proliferation rate.

Mechanisms controlling sensitivity to platinum complexes: role of p53 and DNA mismatch repair.

Although cisplatin is effective in the treatment of different types of tumors, resistance to treatment is a major limitation. In an attempt of overcoming resistance mechanisms, a large effort has been made to generate compounds with a different geometry. At present, the most clinically relevant compounds include mononuclear (i.e. oxaliplatin) as well as multinuclear platinum complexes (i.e. BBR 3464). The mechanisms of cellular response to platinum complexes have not been completely elucidated. Among the main pathways affecting cell sensitivity of these drugs a role for p53 has been proposed at least for cisplatin and BBR 3464. Our results indicate that, also in the case of oxaliplatin, cytotoxicity is modulated by this pathway. Indeed, the effect of oxaliplatin could be reduced in tumor cells expressing mutant p53. The DNA mismatch repair system also appears to be critical in regulating cellular sensitivity to cisplatin because the loss of DNA mismatch repair results in low level of resistance to cisplatin, but not to oxaliplatin. Thus, platinum compounds are endowed with differential capability to activate pathways of p53-dependent or independent apoptosis, and differential recognition by specific cellular systems is likely to be the critical determinant of the cell fate (death/survival) after drug exposure. Further molecular studies are required to better define the precise contribution of such pathways to the cellular responses of the clinically relevant platinum complexes. A complete understanding of the molecular basis of sensitivity to platinum drugs is expected to provide useful insights for the optimization of tumor treatment.

Ultrastructural aspects of acetylcholine receptor turnover at the normal end-plate and in autoimmune myasthenia gravis.

Acetylcholine receptor (AChR) deficiency at the myasthenic end-plate has been attributed to complement-mediated lysis of the junctional folds and to increased fractional degradation rate of AChR cross-linked by myasthenic immunoglobulin. This paper addresses the manner in which AChR is internalized and degraded at the normal end-plate and provides morphologic evidence for accelerated AChR degradation at the end-plate of rats with experimental autoimmune myasthenia gravis (EAMG). We sequentially traced the fate of end-plate AChR labeled in vivo with intramuscularly-injected peroxidase-alpha-bungarotoxin (PBGT) in control rats and rats with chronic EAMG. At both control and EAMG end-plates, AChR is internalized by endocytosis. The endocytosed vesicles containing AChR are transferred into the lysosomal compartment which extends from the junctional folds into the junctional sarcoplasm. Regardless of whether the initial intensity of the reaction for AChR at the EAMG end-plate appeared normal or reduced. AChR disappeared more rapidly from the EAMG than from the control end-plates. Despite the accelerated fractional turnover rate of end-plate AChR in EAMG, the postsynaptic membrane surface which could be labeled with PBGT for AChR remained unchanged over a 120-hour period. These data suggest that end-plate AChR is at a steady state in chronic EAMG.

Deprivation of growth hormone-releasing hormone early in the rat's neonatal life permanently affects somatotropic function.

This work investigated in rats whether passive immunization against the endogenous GHRF in the early postnatal period led to permanent alterations of somatotropic function, similar to those observed in several human growth disorders, e.g. constitutional growth delay (CGD). On postnatal days 1, 2, 4, 6, 8, and 10, rats were given an anti-GHRF-serum (GHRH-Ab, 100 microliters/rat, sc) and were tested 1, 30, and 60 days after this treatment for basal and GHRH-stimulated GH secretion both in vivo and in vitro. GHRH-Ab reduced both basal and GHRF-stimulated GH secretion at all intervals and induced marked and chronic impairment of growth rate. The following differences were observed in the GHRH-Ab treated rats compared to normal rabbit serum-treated controls: 1) GH biosynthesis (incorporation of L-[3H]leucine into the electrophoretic band of GH): reduction of about 70%, 1 day but not 30 days after treatment; 2) Pituitary weight: significant reduction in absolute weight (30-40%) at all posttreatment intervals, and relative weight, 1 and 30 days after treatment. 3) Pituitary GH concentration: significant reduction in GH content (about 40%) but not concentration, at all posttreatment intervals; 4) Percentage of somatotrophs (immunocytochemistry): about 40% reduction 1 day, but not 30 and 60 days after treatment; 5) Hypothalamic somatostatin messenger RNA (mRNA) levels in situ hybridization): selective reduction (40%) in the periventricular nucleus 1 day but not 30 days after treatment; 6) Hypothalamic somatostatin cell number (immunocytochemistry): no significant changes in any hypothalamic area at any interval; 7) Pituitary somatostatin binding (in situ autoradiography): significant reduction, 1 day and 30 days after treatment; 8) Somatostatin inhibition of GH release "in vitro": somatostatin effect on GH release was reduced 30 days after treatment. These and previous data indicate that: 1) Transient deprivation of GHRF in the immediate postnatal period of the rat leads to permanent impairment of growth rate and somatotropic function; 2) GHRF deficiency itself or through reduction of GH secretion impairs somatostatin functions temporarily in the hypothalamus and permanently in the pituitary; 3) This rat model may mimic some forms of growth disorders in humans and holds promise as useful tools for investigating the underlying pathophysiological mechanisms.

The Sso7d DNA-binding protein from Sulfolobus solfataricus has ribonuclease activity.

Sso7d is a small, basic, abundant protein from the thermoacidophilic archaeon Sulfolobus solfataricus. Previous research has shown that Sso7d can bind double-stranded DNA without sequence specificity by placing its triple-stranded beta-sheet across the minor groove. We previously found RNase activity both in preparations of Sso7d purified from its natural source and in recombinant, purified protein expressed in Escherichia coli. This paper provides conclusive evidence that supports the assignment of RNase activity to Sso7d, shown by the total absence of activity in the single-point mutants E35L and K12L, despite the preservation of their overall structure under the assay conditions. In keeping with our observation that the residues putatively involved in RNase activity and those playing a role in DNA binding are located on different surfaces of the molecule, the activity was not impaired in the presence of DNA. If a small synthetic RNA was used as a substrate, Sso7d attacked both predicted double- and single-stranded RNA stretches, with no evident preference for specific sequences or individual bases. Apparently, the more readily attacked bonds were those intrinsically more unstable.