alpha-conotoxin EpI, a novel sulfated peptide from Conus episcopatus that selectively targets neuronal nicotinic acetylcholine receptors

We have isolated and characterized ol-conotoxin EpI, a novel sulfated peptide from the venom of the molluscivorous snail, Conus episcopatus, The peptide was classified as an cy-conotoxin based on sequence, disulfide connectivity, and pharmacological target. EpI has ho mology to sequences of previously described cu-conotoxins, particularly PnIA, PnIB, and ImI, However, EpI differs from previously reported conotoxins in that it has a sulfotyrosine residue, identified by amino acid analysis and mass spectrometry, Native EpI was shown to coelute with synthetic EpI, The peptide sequence is consistent with most, but not all, recognized criteria for predicting tyrosine sulfation sites in proteins and peptides, The activities of synthetic EpI and its unsulfated analogue [Tyr(15)]EpI were similar. Both peptides caused competitive inhibition of nicotine action on bovine adrenal chromaffin cells (neuronal nicotinic ACh receptors) but had no effect on the rat phrenic nerve-diaphragm (muscle nicotinic ACh receptors), Both EpI and [Tyr(15)]EpI partly inhibited acetylcholine-evoked currents in isolated parasympathetic neurons of rat intracardiac ganglia, These results indicate that EPI and [Tyr(15)]EpI selectively inhibit alpha 3 beta 2 and alpha 3 beta 4 nicotinic acetylcholine receptors.

A phase-segregated model for plant cell culture: The effect of cell volume fraction

Plant cells are characterized by low water content, so the fraction of cell volume (volume fraction) in a vessel is large compared with other cell systems, even if the cell concentrations are the same. Therefore, concentration of plant cells should preferably be expressed by the liquid volume basis rather than by the total vessel volume basis. In this paper, a new model is proposed to analyze behavior of a plant cell culture by dividing the cell suspension into the biotic- and abiotic-phases, Using this model, we analyzed the cell-growth and the alkaloid production by Catharanthus roseus, Large errors in the simulated results were observed if the phase-segregation was not considered.

Growth and lactic acid production in batch culture of Lactobacillus rhamnosus in a defined medium

To facilitate metabolic analysis, batch fermentations of Lactobacillus rhamnosus were carried out in a new defined medium. Biomass at 10.5 g/l and lactic acid at 67 g/l with a Y-P/S of 0.84 were achieved. The maximum specific growth rate and the average productivity were 0.49/h and 2.48 g/l.h, respectively. These are comparable to those of this organism and related organisms in complex media. Preliminary amino acid studies were also conducted, highlighting the importance of serine, asparagine, glutamine and cysteine. Kinetic analysis revealed that lactic acid production was predominantly growth-associated with growth associated and non-growth associated lactic acid constants of 0.389 mol/g-cell and 0.0025 mol/g-cell.h, respectively. Finally a kinetic model has been included to describe the fermentation of L. rhamnosus.

Novel guidance cues during neuronal pathfinding in the early scaffold of axon tracts in the rostral brain

A scaffold of axons consisting of a pair of longitudinal tracts and several commissures is established during early development of the vertebrate brain. We report here that NOC-2, a cell surface carbohydrate, is selectively expressed by a subpopulation of growing axons in this scaffold in Xenopus. NOC-2 is present on two glycoproteins, one of which is a novel glycoform of the neural cell adhesion molecule N-CAM. When the function of NOC-2 was perturbed using either soluble carbohydrates or anti-NOC-2 antibodies, axons expressing NOC-2 exhibited aberrant growth at specific points in their pathway. NOC-2 is the first-identified axon guidance molecule essential for development of the axon scaffold in the embryonic vertebrate brain.

p75 neurotrophin receptor-mediated neuronal death is promoted by Bcl-2 and prevented by Bcl-x(L)

The p75 neurotrophin receptor (p75NTR) has been shown to mediate neuronal death through an unknown pathway. We microinjected p75NTR expression plasmids into sensory neurons in the presence of growth factors and assessed the effect of the expressed proteins on cell survival. We show that, unlike other members of the TNFR family, p75NTR signals death through a unique caspase-dependent death pathway that does not involve the death domain and is differentially regulated by Bcl-2 family members: the anti-apoptotic molecule Bcl-2 both promoted, and was required for, p75NTR killing, whereas killing was inhibited by its homologue BcI-x(L). These results demonstrate that Bcl-2, through distinct molecular mechanisms, either promotes or inhibits neuronal death depending on the nature of the death stimulus.

Signaling of neuronal cell death by the p75NTR neurotrophin receptor

The neurotrophin receptor (p75NTR) is best known for mediating tropic support by participating in the formation of high-affinity nerve growth factor (NGF) receptor complexes with trkA, however, p75NTR more recently has been shown to act as a bona fide death-signaling receptor, which can signal independently of trkA. This article discusses the evidence for an active role of p75NTR in neuronal cell death and the mechanisms controlling this process, including roles for Bcl-2 family members, the c-jun stress kinase JNK, the transcription factor nuclear factor kappa B (NF kappa B), and caspases.

Altered adhesion, proliferation and death in neural cultures from adults with schizophrenia

The causes of schizophrenia are unknown, but there is evidence linking subtle deviations in neural development with schizophrenia. Embryonic brain development cannot be studied in an adult with schizophrenia, but neurogenesis and early events in neuronal differentiation can be investigated throughout adult life in the human olfactory epithelium. Our past research has demonstrated that neuronal cultures can be derived from biopsy of the human adult olfactory epithelium. In the present study, we examined mechanisms related to neurogenesis and neuronal differentiation in adults with schizophrenia versus well controls. Forty biopsies were collected under local anaesthesia from ten individuals with DSM III-R schizophrenia and ten age- and sex-matched well controls. All patients, except one, were receiving antipsychotic medication at the time of the biopsy, Immunostaining for neuronal markers indicated that neurogenesis occurred in the biopsies from both patients and controls since all contained cells expressing tubulin and/or olfactory marker protein. The major findings of this study are: 1. biopsies from patients with schizophrenia showed a significantly reduced ability to attach to the culture slide: 29.9% of patient biopsies attached compared to 73.5% of control biopsies; 2. biopsies from patients with schizophrenia had a significantly greater proportion of cells undergoing mitosis: 0.69% in the patients compared to 0.29% in the controls; and 3. dopamine (10 mu M) significantly increased the proportion of apoptotic cells in the control cultures but significantly decreased the proportion in patients' cultures. (C) 1999 Elsevier Science B.V. All rights reserved.

Single amino acid substitutions in alpha-conotoxin PnIA shift selectivity for subtypes of the mammalian neuronal nicotinic acetylcholine receptor

The alpha-conotoxins, a class of nicotinic acetylcholine receptor (nAChR) antagonists, are emerging as important probes of the role played by different nAChR subtypes in cell function and communication, In this study, the native alpha-conotoxins PnIA and PnIB were found to cause concentration-dependent inhibition of the ACh-induced current in all rat parasympathetic neurons examined, with IC50 values of 14 and 33 nM, and a maximal reduction in current amplitude of 87% and 71%, respectively. The modified alpha-conotoxin [N11S]PnIA reduced the ACh-induced current with an IC50 value of 375 nM and a maximally effective concentration caused 91% block, [A10L]PnIA was the most potent inhibitor, reducing the ACh-induced current in similar to 80% of neurons, with an IC50 value of 1.4 nM and 46% maximal block of the total current, The residual current was not inhibited further by alpha-bungarotoxin, but was further reduced by the cu-conotoxins PnIA or PnIB, and by mecamylamine. H-1 NMR studies indicate that PnIA, PnIB, and the analogues, [A10L]PnIA and [N11S]PnIA, have identical backbone structures. We propose that positions 10 and II of PnIA and PnIB influence potency and determine selectivity among alpha 7 and other nAChR subtypes, including alpha 3 beta 2 and alpha 3 beta 4, Four distinct components of the nicotinic ACh-induced current in mammalian parasympathetic neurons have been dissected with these conopeptides.

Leu(10) of alpha-conotoxin PnIB confers potency for neuronal nicotinic responses in bovine chromaffin cells

Two alpha-conotoxins PnIA and PnIB (previously reported as being mollusc specific) which differ in only two amino acid residues (AN versus LS at residues 10 and 11, respectively), show markedly different inhibition of the neuronal nicotinic acetylcholine receptor response in bovine chromaffin cells, a mammalian preparation. Whereas alpha-conotoxin PnIB completely inhibits the nicotine-evoked catecholamine release at 10 mu M, with IC50 = 0.7 mu M, alpha-conotoxin PnIA is some 30-40 times less potent. Two peptide analogues, [A10L]PnIA and [N11S]PnIA were synthesized to investigate the extent to which each residue contributes to activity. [A10L]PnIA (IC50 = 2.0 mu M) completely inhibits catecholamine release at 10 mu M whereas [N11S]PnIA shows Little inhibition. In contrast, none of the peptides inhibit muscle-type nicotinic responses in the rat hemi-diaphragm preparation. We conclude that the enhanced potency of alpha-conotoxin PnIB over alpha-conotoxin PnIA in the neuronal-type nicotinic response is principally determined by the larger, more hydrophobic leucine residue at position 10 in alpha-conotoxin PnIB. (C) 2000 Elsevier Science B.V. All rights reserved.

Role of neurotrophin receptor p75NTR in mediating neuronal cell death following injury

1. The neurotrophin receptor p75NTR has been shown to mediate neuronal cell death after nerve injury. 2. Down-regulation of p75NTR by antisense oligonucleotides is able to inhibit both sensory and motor neuron death and this treatment is more effective than treatment with growth factors. 3. p75NTR induces cell death by a unique death signalling pathway involving transcription factors (nuclear factor kappa B and c-jun), Bcl-2 family members and caspases.

Chopper, a new death domain of the p75 neurotrophin receptor that mediates rapid neuronal cell death

The cytoplasmic juxtamembrane region of the p75 neurotrophin receptor (p75(NTR)) has been found to be necessary and sufficient to initiate neural cell death. The region was named Chopper to distinguish it from CD95-like death domains. A 29-amino acid peptide corresponding to the Chopper region induced caspase- and calpain-mediated death in a variety of neural and nonneural cell types and was not inhibited by signaling through Trk (unlike killing by full-length p75(NTR)). Chopper triggered cell death only when bound to the plasma membrane by a lipid anchor, whereas non-anchored Chopper acted in a dominant-negative manner, blocking p75(NTR)-mediated death both in vitro and in vivo. Removal of the ectodomain of p75(NTR) increased the potency of Chopper activity, suggesting that it regulates the association of Chopper with downstream signaling proteins.

Structured modeling of recombinant protein production in batch and fed-batch culture of baculovirus-infected insect cells

The infection of insect cells with baculovirus was described in a mathematical model as a part of the structured dynamic model describing whole animal cell metabolism. The model presented here is capable of simulating cell population dynamics, the concentrations of extracellular and intracellular viral components, and the heterologous product titers. The model describes the whole processes of viral infection and the effect of the infection on the host cell metabolism. Dynamic simulation of the model in batch and fed-batch mode gave good agreement between model predictions and experimental data. Optimum conditions for insect cell culture and viral infection in batch and fed-batch culture were studied using the model.

Novel omega-conotoxins from Conus catus discriminate among neuronal calcium channel subtypes

omega -Conotoxins selective for N-type calcium channels are useful in the management of severe pain. In an attempt to expand the therapeutic potential of this class, four new omega -conotoxins (CVIA-D) have been discovered in the venom of the piscivorous cone snail, Conus catus, using assay-guided fractionation and gene cloning. Compared with other omega -conotoxins, CVID has a novel loop 4 sequence and the highest selectivity for N-type over P/Q-type calcium channels in radioligand binding assays. CVIA-D also inhibited contractions of electrically stimulated rat vas deferens. In electrophysiological studies, omega -conotoxins CVID and MVIIA had similar potencies to inhibit current through central (alpha (1B-d)) and peripheral (alpha (1B-b)) splice variants of the rat N-type calcium channels when coexpressed with rat beta (3) in Xenopus oocytes, However, the potency of CVID and MVIIA increased when alpha (1B-d) and alpha (1B-b) were expressed in the absence of rat beta (3), an effect most pronounced for CVID at alpha (1B-d) (up to 540-fold) and least pronounced for MVIIA at alpha (1B-d) (3-fold). The novel selectivity of CVID may have therapeutic implications. H-1 NMR studies reveal that CMD possesses a combination of unique structural features, including two hydrogen bonds that stabilize loop 2 and place loop 2 proximal to loop 4, creating a globular surface that is rigid and well defined.

Neuronal sodium-channel alpha 1-subunit mutations in generalized epilepsy with febrile seizures plus

Generalized epilepsy with febrile seizures plus (GEFS+) is a familial epilepsy syndrome characterized by the presence of febrile and afebrile seizures. The first gene, GEFS1, was mapped to chromosome 19q and was identified as the sodium-channel beta1-subunit, SCN1B. A second locus on chromosome 2q, GEFS2, was recently identified as the sodium-channel alpha1-subunit, SCN1A. Single-stranded conformation analysis (SSCA) of SCN1A was performed in 53 unrelated index cases to estimate the frequency of mutations in patients with GEFS+. No mutations were found in 17 isolated cases of GEFS+. Three novel SCN1A mutations-D188V, V1353L, and I1656M-were found in 36 familial cases; of the remaining 33 families, 3 had mutations in SCN1B. On the basis of SSCA, the combined frequency of SCN1A and SCN1B mutations in familial cases of GEFS+ was found to be 17%.