Spermine modulation of the glutamate(NMDA) receptor is differentially responsive to conantokins in normal and Alzheimer's disease human cerebral cortex

The pharmacology of the N -methyl-d-aspartate (NMDA) receptor site was examined in pathologically affected and relatively spared regions of cerebral cortex tissue obtained at autopsy from Alzheimer's disease cases and matched controls. The affinity and density of the [H-3]MK-801 binding site were delineated along with the enhancement of [H-3]MK-801 binding by glutamate and spermine. Maximal enhancement induced by either ligand was regionally variable; glutamate-mediated maximal enhancement was higher in controls than in Alzheimer's cases in pathologically spared regions, whereas spermine-mediated maximal enhancement was higher in controls in areas susceptible to pathological damage. These and other data suggest that the subunit composition of NMDA receptors may be locally variable. Studies with modified conantokin-G (con-G) peptides showed that Ala(7)-con-G had higher affinity than Lys(7)-con-G, and also defined two distinct binding sites in controls. Nevertheless, the affinity for Lys(7)-con-G was higher overall in Alzheimer's brain than in control brain, whereas the reverse was true for Ala(7)-con-G. Over-excitation mediated by specific NMDA receptors might contribute to localized brain damage in Alzheimer's disease. Modified conantokins are useful for identifying the NMDA receptors involved, and may have potential as protective agents.

Deep and superficial fibers of the lumbar multifidus muscle are differentially active during voluntary arm movements

AB Study Design. A cross-sectional study was conducted. Objective. To determine the activity of the deep and superficial fibers of the lumbar multifidus during voluntary movement of the arm. Summary of Background Data. The multifidus contributes to stability of the lumbar spine. Because the deep and superficial parts of the multifidus are near the center of lumbar joint rotation, the superficial fibers are well suited to control spine orientation, and the deep fibers to control intervertebral movement. However, there currently are limited in vivo data to support this distinction. Methods. Electromyographic activity was recorded in both the deep and superficial multifidus, transversus abdominis, erector spinae, and deltoid using selective intramuscular electrodes and surface electrodes during single and repetitive arm movements. The latency of electromyographic onset in each muscle during single movements and the pattern of electromyographic activity during repetitive movements were compared between muscles. Results. With single arm movements, the onset of electromyography in the erector spinae and superficial multifidus relative to the deltoid was dependent on the direction of movement, but the onset in the deep multifidus and transversus abdominis was not. With repetitive arm movements, peaks in superficial multifidus and erector spinae electromyography occurred only during flexion for most subjects, whereas peaks in deep multifidus electromyography occurred during movement in both directions. Conclusions. The deep and superficial fibers of the multifidus are differentially active during single and repetitive movements of the arm. The data from this study support the hypothesis that the superficial multifidus contributes to the control of spine orientation, and that the deep multifidus has a role in controlling intersegmental motion. (C) 2002 Lippincott Williams & Wilkins, Inc.

Production of the baculovirus-expressed dengue virus glycoprotein NS1 can be improved dramatically with optimised regimes for fed-batch cultures and the addition of the insect moulting hormone, 20-Hydroxyecdysone

A perennial problem in recombinant protein expression is low yield of the product of interest. A strategy which has been shown to increase the production of baculovirus-expressed proteins is to utilise fed-batch cultures. One disadvantage of this approach is the time-consuming task of optimising the feeding strategy. Previously, a statistical optimisation routine was applied to develop a feeding strategy that increased the yield of beta-Galactosidase (beta-Gal) by 2.4-fold (Biotechnol. Bioeng, 59 (1998) 178). This involves the single addition of nutrient concentrates (amino acids, lipids. glucose and yeastolate ultrafiltrate) into Sf9 cell cultures grown in SF900II medium. In this study, it is demonstrated that this optimised fed-batch strategy developed for a high-yielding intracellular product beta-Gal could be applied successfully to a relatively low-yielding glycosylated and secreted product such as the dengue virus glycoprotein NS1. Optimised batch infections yielded 4 mug/ml of NS1 at a peak cell density of 4.2 x 10(6) cells/ml. In contrast. optimised fed-batch infections exhibited a 3-fold improvement in yield, with 12 mug ml of NS1 produced at a peak cell density of 11.3 x 10(6) cells/ml. No further improvements in yield were recorded when the feed volumes were doubled and the peak cell density was increased to 23 x 10(6) cells/ml, unless the cultures were stimulated by the addition of 4 mug/ml of 20-Hydroxyecdysone (an insect moulting hormone). In this case, the NS1 yield was increased to 20 mug/ml. which was nearly 5-fold higher than optimised batch cultures. (C) 2002 Elsevier Science B.V. All rights reserved.

MUC1 epithelial mucin (CD227) is expressed by activated dendritic cells

The MUC1 mucin (CD227) is a cell surface mucin originally thought to be restricted to epithelial tissues. We report that CD227 is expressed on human blood dendritic cells (DC) and monocyte-derived DC following in vitro activation. Freshly isolated murine splenic DC had very low levels of CD227; however, all DC expressed CD227 following in vitro culture. In the mouse spleen, CD227 was seen on clusters within the red pulp and surrounding the marginal zone in the white pulp. Additionally, we confirm CD227 expression by activated human T cells and show for the first time that the CD227 cytoplasmic domain is tyrosine-phosphorylated in activated T cells and DC and is associated with other phosphoproteins, indicating a role in signaling. The function of CD227 on DC and T cells requires further elucidation.

Use of heterologously-expressed cytochomre P450 and glutathione transferase enzymes in toxicity assays

Our groups have had a long-term interest in utilizing bacterial systems in the characterization of bioactivation and detoxication reactions catalyzed by cytochrome P450 (P450) and glutathione transferase (GST) enzymes. Bacterial systems remain the first choice for initial screens with new chemicals and have advantages, including high-throughput capability. Most human P450s of interest in toxicology have been readily expressed in Escherichia coli with only minor sequence modification. These enzymes can be readily purified and used in assays of activation of chemicals. Bicistronic systems have been developed in order to provide the auxiliary NADPH-P450 reductase. Alternative systems involve these enzymes expressed together within bacteria. In one approach, a lac selection system is used with E. coli and has been applied to the characterization of inhibitors of P450s 1A2 and 1131, as well as in basic studies involving random mutagenesis. Another approach utilizes induction of the SOS (umu) response in Salmonella typhimurium, and systems have now been developed with human P450s 1A1, 1A2, 1B1, 2C9, 2D6, 2E1, and 3A4, which have been used to report responses from heterocyclic amines. S. typhimurium his reporter systems have also been used with GSTs, first to demonstrate the role of rat GST 5-5 in the activation of dihalomethanes. These systems have been used to compare these GSTs with regard to activation of dihaloalkanes and potential toxicity. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

SOX8 is expressed during testis differentiation in mice and synergizes with SF1 to activate the Amh promoter in vitro

Sox8 is a member of the Sox family of developmental transcription factor genes and is closely related to Sox9, a key gene in the testis determination pathway in mammals. Like Sox9, Sox8 is expressed in the developing mouse testis around the time of sex determination, suggesting that it might play a role in regulating the expression of testis-specific genes. An early step in male sex differentiation is the expression of anti-Mullerian hormone (AMH) in Sertoli cells. Expression of the Amh gene during sex differentiation requires the interaction of several transcription factors, including SF1, SOX9, GATA4, WT1, and DAX1. Here we show that SOX8 may also be involved in regulating the expression of Amh. Expression of Sox8 begins just prior to that of Amh at 12 days post coitum (dpc) in mouse testes and continues beyond 16 dpc in Sertoli cells. In vitro assays showed that SOX8 binds specifically to SOX binding sites within the Amh minimal promoter and, like SOX9, acts synergistically with SF1 through direct protein-protein interaction to enhance Amh expression, albeit at lower levels compared with SOX9. SOX8 and SOX9 appear to have arisen from a common ancestral gene and may have retained some common functions during sexual development. Our data provide the first evidence that SOX8 may partially compensate for the reduced SOX9 activity in campomelic dysplasia and substitute for Sox9 where Sox9 is either not expressed or expressed too late to be involved in sex determination or regulation of Amh expression.

Purification and characterisation of functional early pregnancy factor expressed in Sf9 insect cells and in Escherichia coli

Early pregnancy factor (EPF) is a secreted protein with growth regulatory and immunomodulatory properties. It is an extracellular form of the mitochondrial matrix protein chaperonin 10 (Cpn10), a molecular chaperone. An understanding of the mechanism of action of EPF and an exploration of therapeutic potential has been limited by availability of purified material. The present study was undertaken to develop a simple high-yielding procedure for preparation of material for structure/function studies, which could be scaled up for therapeutic application. Human EPF was expressed in Sf9 insect cells by baculovirus infection and in Escherichia coli using a heat inducible vector. A modified molecule with an additional N-terminal alanine was also expressed in E coli. The soluble protein was purified from cell lysates via anion exchange (negative-binding mode), cation exchange, and hydrophobic interaction chromatography, yielding similar to42 and 36 mg EPF from 300 ml bacterial and I L Sf9 cultures, respectively. The preparations were highly purified ( greater than or equal to99% purity on SDS-PAGE for the bacterial products and greater than or equal to97% for that of insect cells) and had the expected mass and heptameric structure under native conditions, as determined by mass spectrometry and gel permeation chromatography, respectively. All recombinant preparations exhibited activity in the EPF bioassay, the rosette inhibition test, with similar potency both to each other and to the native molecule. In two in vivo assays of immuno suppressive activity, the delayed-type hypersensitivity reaction and experimental autoimmune encephalomyelitis, the insect cell and modified bacterial products, both with N-terminal additions (acetylation or amino acid), exhibited similar levels of suppressive activity, but the bacterial product with no N-terminal modification had no effect in either assay. Studies by others have shown that N-terminal addition is not necessary for Cpn10 activity. By defining techniques for facile production of molecules with and without immunosuppressive properties, the present studies make it possible to explore mechanisms underlying the distinction between EPF and Cpn10 activity. (C) 2003 Elsevier Inc. All rights reserved.

Muscarinic and nicotinic ACh receptor activation differentially mobilize Ca2+ in rat intracardiac ganglion neurons

The origin of intracellular Ca2+ concentration ([Ca2+](i)) transients stimulated by nicotinic ( nAChR) and muscarinic ( mAChR) receptor activation was investigated in fura-2-loaded neonatal rat intracardiac neurons. ACh evoked [Ca2+](i) increases that were reduced to similar to 60% of control in the presence of either atropine ( 1 muM) or mecamylamine ( 3 muM) and to < 20% in the presence of both antagonists. Removal of external Ca2+ reduced ACh-induced responses to 58% of control, which was unchanged in the presence of mecamylamine but reduced to 5% of control by atropine. The nAChR-induced [Ca2+](i) response was reduced to 50% by 10 μM ryanodine, whereas the mAChR-induced response was unaffected by ryanodine, suggesting that Ca2+ release from ryanodine-sensitive Ca2+ stores may only contribute to the nAChR-induced [Ca2+](i) responses. Perforated-patch whole cell recording at - 60 mV shows that the rise in [Ca2+](i) is concomitant with slow outward currents on mAChR activation and with rapid inward currents after nAChR activation. In conclusion, different signaling pathways mediate the rise in [Ca2+](i) and membrane currents evoked by ACh binding to nicotinic and muscarinic receptors in rat intracardiac neurons.

Differential expression of CDC25 phosphatases splice variants in human breast cancer cells

Background: CDC25 phosphatases control cell cycle progression by activating cyclin dependent kinases. The three CDC25 isoforms encoding genes are submitted to alternative splicing events which generate at least two variants for CDC25A and five for both CDC25B and CDC25C. An over-expression of CDC25 was reported in several types of cancer, including breast cancer, and is often associated with a poor prognosis. Nevertheless, most of the previous studies did not address the expression of CDC25 splice variants. Here, we evaluated CDC25 spliced transcripts expression in anti-cancerous drug-sensitive and resistant breast cancer cell lines in order to identify potential breast cancer biomarkers. Methods: CDC25 splice variants mRNA levels were evaluated by semi-quantitative RT-PCR and by an original real-time RT-PCR assay. Results: CDC25 spliced transcripts are differentially expres-sed in the breast cancer cell lines studied. An up-regulation of CDC25A2 variant and an increase of the CDC25C5/C1 ratio are associated to the multidrug-resistance in VCREMS and DOXOR breast cancer cells, compared to their sensitive counterpart cell line MCF-7. Additionally, CDC25B2 tran-script is exclusively over-expressed in VCREMS resistant cells and could therefore be involved in the development of certain type of drug resistance. Conclusions: CDC25 splice variants could represent interesting potential breast cancer prognostic biomarkers.

The influence of burden of care and perceived stigma on expressed emotions of relatives of stable persons with schizophrenia in Nigerian semi-urban/urban settings

RESUMO: Schizophrenia’s burden defines experience of family members and is associated with high level of distress. Courtesy stigma, a distress concept, worsens caregivers’ burden of care and impacts on schizophrenia. Expressed emotion (EE), another family variable, impacts on schizophrenia. However, relationship between EE, burden of care and stigma has been little explored in western literature but not in sub-Saharan Africa particularly Nigeria. This study explored the impact of burden of care and courtesy stigma on EE among caregivers of persons with schizophrenia in urban and semi-urban settings in Nigeria. Fifty caregivers each from semi-urban and urban areas completed a socio-demographic schedule, family questionnaire, burden interview schedule and perceived devaluation and discrimination scale. The caregivers had a mean age of 42 (± 15.6) years. Majority were females (57%), married (49%), from Yoruba ethnic group (68%), monogamous family (73%) and Christians (82%). A higher proportion of the whole sample (53%) had tertiary education. Three out of ten were sole caregivers. Seventy three (73%) lived with the person they cared for. The average number of hours spent per week by a caregiver with a person with schizophrenia was 35 hours. The urban sample had significantly higher proportion of carers with high global expressed emotion (72.7%) than the semi-urban sample (27.3%). The odds of a caregiver in an urban setting exhibiting high expressed emotion are 4.202 times higher than the odds of caregiver in a semi-urban setting. Additionally, there was significance difference between the urban and semi-urban caregivers in discrimination dimension. High levels of subjective and objective burden were associated with high levels of critical comments. In conclusion, this study is the first demonstration of urban-semi-urban difference in expressed emotion in an African country and its findings provide further support to hypothesized relationship between components of EE and burden of care.

Protease-activated receptor (PAR)-1 and PAR-2 protect rat astrocytes from apoptotic cell death via differentially regulating JNK isoform-specific release of the chemokine GRO/CINC-1 : two novel protective pathways in brain

Protease-activated receptor; c-Jun N-terminal kinase (JNK); thrombin; neuroprotection; siRNA

Social chromosome variants differentially affect queen determination and the survival of workers in the fire ant Solenopsis invicta.

Intraspecific variation in social organization is common, yet the underlying causes are rarely known. An exception is the fire ant Solenopsis invicta in which the existence of two distinct forms of social colony organization is under the control of the two variants of a pair of social chromosomes, SB and Sb. Colonies containing exclusively SB/SB workers accept only one single queen and she must be SB/SB. By contrast, when colonies contain more than 10% of SB/Sb workers, they accept several queens but only SB/Sb queens. The variants of the social chromosome are associated with several additional important phenotypic differences, including the size, fecundity and dispersal strategies of queens, aggressiveness of workers, and sperm count in males. However, little is known about whether social chromosome variants affect fitness in other life stages. Here, we perform experiments to determine whether differential selection occurs during development and in adult workers. We find evidence that the Sb variant of the social chromosome increases the likelihood of female brood to develop into queens and that adult SB/Sb workers, the workers that cull SB/SB queens, are overrepresented in comparison to SB/SB workers. This demonstrates that supergenes such as the social chromosome can have complex effects on phenotypes at various stages of development.

Liprin (beta)1 is highly expressed in lymphatic vasculature and is important for lymphatic vessel integrity.

The lymphatic vasculature is important for the regulation of tissue fluid homeostasis, immune response, and lipid absorption, and the development of in vitro models should allow for a better understanding of the mechanisms regulating lymphatic vascular growth, repair, and function. Here we report isolation and characterization of lymphatic endothelial cells from human intestine and show that intestinal lymphatic endothelial cells have a related but distinct gene expression profile from human dermal lymphatic endothelial cells. Furthermore, we identify liprin beta1, a member of the family of LAR transmembrane tyrosine phosphatase-interacting proteins, as highly expressed in intestinal lymphatic endothelial cells in vitro and lymphatic vasculature in vivo, and show that it plays an important role in the maintenance of lymphatic vessel integrity in Xenopus tadpoles.