2C-B: a new psychoactive phenylethylamine recently discovered in Ecstasy tablets sold on the Swiss black market

Amyloid is a biophysically defined, biochemically non-uniform protein which is deposited in the tissue, forming a cross-beta configuration. In paraffin sections it is demonstrated using Congo red staining according to Puchtler et al. and a polarizing microscope with a tension-free clean optic system that shows a typical apple-green birefringence. The identification of amyloid has to be followed by immunostaining for AA, ATTR, A lambda, A kappa and A beta 2 microglobulin and possibly further types of amyloids for classification purposes. Considering the localization, expansion and type localized and generalized types probably can be differentiated. The latter should be correlated to a basic disease. The treatment of the basic disease is the only chance for reducing or eliminating the amyloid deposits. In this review, the modern morphological methods for demonstration and both immunohistological and clinical classification of amyloid and amyloidoses are presented. Rare types are mentioned in the tables.     

Direct effects of growth hormone (GH)-releasing hexapeptide (GHRP-6) and GH-releasing factor (GRF) on GH secretion from cultured porcine somatotropes

Growth hormone (GH)-releasing hexapeptide (GHRP-6) belongs to the expanding family of synthetic GH secretagogues (GHSs). Previous studies have shown that non-peptidyl GHRP-6 analogues stimulate GH release in vivo in pigs, and interact synergistically with GH-releasing factor (GRF), but its direct effects on porcine somatotropes have not been addressed hitherto. In the present study, we have evaluated the response of cultured porcine pituitary cells to GHRP-6, and its interaction with GRF and somatostatin (SRIF). Secretory response of somatotropes was assessed by using two distinct techniques. GH released by monolayer cell cultures was evaluated by enzyme immunoassay, whereas that secreted by individual somatotropes was measured by immunodensitometry using a cell blotting assay. Our results demonstrate that both GHRP-6 and GRF stimulated GH release from monolayer cultures at doses equal to or above 10(-9) M. Use of cell immunoblot assay demonstrated that, like GRF, the hexapeptide acts directly upon porcine somatotropes to exert its action. Moreover, regardless of the technique applied, combined administration of GHRP-6 (10(-6) or 10(-9) M) and GRF (10(-8) M) resulted in an additive, but not synergistic, stimulatory GH response. Finally, SRIF (10(-7) M) inhibited the stimulatory effect of GHRP-6 alone or in combination with GRF. These results indicate that GHRP-6 directly and effectively stimulates GH secretion from porcine somatotropes in vitro, and acts additively when coadministered with GRF. Therefore, the synergistic stimulatory effect of GHSs and GRF reported in vivo in this species might require additional factors that are lacking in the in vitro situation.     

Influence of carbon source on nitrate removal by nitrate-tolerant Klebsiella oxytoca CECT 4460 in batch and chemostat cultures

The nitrate-tolerant organism Klebsiella oxytoca CECT 4460 tolerates nitrate at concentrations up to 1 M and is used to treat wastewater with high nitrate loads in industrial wastewater treatment plants. We studied the influence of the C source (glycerol or sucrose or both) on the growth rate and the efficiency of nitrate removal under laboratory conditions. With sucrose as the sole C source the maximum specific growth rate was 0.3 h-1, whereas with glycerol it was 0.45 h-1. In batch cultures K. oxytoca cells grown on sucrose or glycerol were able to immediately use sucrose as a sole C source, suggesting that sucrose uptake and metabolism were constitutive. In contrast, glycerol uptake occurred preferentially in glycerol-grown cells. Independent of the preculture conditions, when sucrose and glycerol were added simultaneously to batch cultures, the sucrose was used first, and once the supply of sucrose was exhausted, the glycerol was consumed. Utilization of nitrate as an N source occurred without nitrite or ammonium accumulation when glycerol was used, but nitrite accumulated when sucrose was used. In chemostat cultures K. oxytoca CECT 4460 efficiently removed nitrate without accumulation of nitrate or ammonium when sucrose, glycerol, or mixtures of these two C sources were used. The growth yields and the efficiencies of C and N utilization were determined at different growth rates in chemostat cultures. Regardless of the C source, yield carbon (YC) ranged between 1.3 and 1.0 g (dry weight) per g of sucrose C or glycerol C consumed. Regardless of the specific growth rate and the C source, yield nitrogen (YN) ranged from 17.2 to 12.5 g (dry weight) per g of nitrate N consumed. In contrast to batch cultures, in continuous cultures glycerol and sucrose were utilized simultaneously, although the specific rate of sucrose consumption was higher than the specific rate of glycerol consumption. In continuous cultures double-nutrient-limited growth appeared with respect to the C/N ratio of the feed medium and the dilution rate, so that for a C/N ratio between 10 and 30 and a growth rate of 0.1 h-1 the process led to simultaneous and efficient removal of the C and N sources used. At a growth rate of 0.2 h-1 the zone of double limitation was between 8 and 11. This suggests that the regimen of double limitation is influenced by the C/N ratio and the growth rate. The results of these experiments were validated by pulse assays.     

The development of novel and selective p56lck tyrosine kinase inhibitors

Early T-cell receptor mediated signal transduction involves the activation of several tyrosine protein kinases. One of these tyrosine kinases, p56lck, is expressed primarily in T-cells and Natural Killer (NK) cells and has been shown to be critical for their proliferative and effector functions. Indandiones have been identified as a potent and selective chemical class that inhibits p56lck.     

The copines, a novel class of C2 domain-containing, calcium-dependent, phospholipid-binding proteins conserved from Paramecium to humans

In an attempt to identify proteins that might underlie membrane trafficking processes in ciliates, calcium-dependent, phospholipid-binding proteins were isolated from extracts of Paramecium tetraurelia. The major protein obtained, named copine, had a mass of 55 kDa, bound phosphatidylserine but not phosphatidylcholine at micromolar levels of calcium but not magnesium, and promoted lipid vesicle aggregation. The sequence of a 920-base pair partial cDNA revealed that copine is a novel protein that contains a C2 domain likely to be responsible for its membrane active properties. Paramecium was found to have two closely related copine genes, CPN1 and CPN2. Current sequence data bases indicate the presence of multiple copine homologs in green plants, nematodes, and humans. The full-length sequences reveal that copines consist of two C2 domains at the N terminus followed by a domain similar to the A domain that mediates interactions between integrins and extracellular ligands. A human homolog, copine I, was expressed in bacteria as a fusion protein with glutathione S-transferase. This recombinant protein exhibited calcium-dependent phospholipid binding properties similar to those of Paramecium copine. An antiserum raised against a fragment of human copine I was used to identify chromobindin 17, a secretory vesicle-binding protein, as a copine. This association with secretory vesicles, as well the general ability of copines to bind phospholipid bilayers in a calcium-dependent manner, suggests that these proteins may function in membrane trafficking.     

Functional MRI of the lumbar spine in erect position in a superconducting open-configuration MR system: preliminary results

High-level penicillin resistance in pneumococci is due to alterations in penicillin-binding proteins (PBPs) 2X, 2B, and 1A. We have sequenced the penicillin-binding domain of PBP 1A from penicillin-resistant South African pneumococcal isolates and have identified amino acid substitutions which are common to all the resistant isolates analyzed. Site-directed mutagenesis was then used to determine whether particular amino acid substitutions at specific positions in PBP 1A mediate penicillin resistance. PCR was used to isolate PBP 2X, 2B, and 1A genes from clinical isolate 8303 (penicillin MIC, 4 micrograms/ml). These wild-type PBP genes were cloned into pGEM-3Zf and were used as the transforming DNA. Susceptible strain R6 (MIC, 0.015 microgram/ml) was first transformed with PBP 2X and 2B DNA, resulting in PBP 2X/2B-R6 transformants for which MICs were 0.25 microgram/ml. When further transformed with PBP 1A DNA, 2X/2B/1A-R6 transformants for which MICs were 1.5 micrograms/ml were obtained. Site-directed mutagenesis of the PBP 1A gene from isolate 8303 was then used to reverse particular amino acid substitutions, followed by transformation of PBP 2X/2B-R6 transformants with the mutagenized PBP 1A DNA. For PBP 2X/2B/1A-R6 transformants, the introduction of the reversal of Thr-371 by Ser or Ala in PBP 1A decreased the MIC from 1.5 to 0.5 micrograms/ml, whereas the reversal of four consecutive amino acid substitutions (Thr-574 by Asn, Ser-575 by Thr, Gln-576 by Gly, and Phe-577 by Tyr) decreased the MIC from 1.5 to 0.375 micrograms/ml. These data reveal that amino acid residue 371 and residues 574 to 577 of PBP 1A are important positions in PBP 1A with respect to the interaction with penicillin and the development of resistance.     

Phosphatidylinositol-dependent membrane fusion induced by a putative fusogenic sequence of Ebola virus

The membrane-interacting abilities of three sequences representing the putative fusogenic subdomain of the Ebola virus transmembrane protein have been investigated. In the presence of calcium, the sequence EBO(GE) (GAAIGLAWIPYFGPAAE) efficiently fused unilamellar vesicles composed of phosphatidylcholine, phosphatidylethanolamine, cholesterol, and phosphatidylinositol (molar ratio, 2:1:1:0.5), a mixture that roughly resembles the lipid composition of the hepatocyte plasma membrane. Analysis of the lipid dependence of the process demonstrated that the fusion activity of EBO(GE) was promoted by phosphatidylinositol but not by other acidic phospholipids. In comparison, EBO(EA) (EGAAIGLAWIPYFGPAA) and EBO(EE) (EGAAIGLAWIPYFGPAAE) sequences, which are similar to EBO(GE) except that they bear the negatively charged glutamate residue at the N terminus and at both the N and C termini, respectively, induced fusion to a lesser extent. As revealed by binding experiments, the glutamate residue at the N terminus severely impaired peptide-vesicle interaction. In addition, the fusion-competent EBO(GE) sequence did not associate significantly with vesicles lacking phosphatidylinositol. Tryptophan fluorescence quenching by vesicles containing brominated phospholipids indicated that the EBO(GE) peptide penetrated to the acyl chain level only when the membranes contained phosphatidylinositol. We conclude that binding and further penetration of the Ebola virus putative fusion peptide into membranes might be governed by the nature of the N-terminal residue and by the presence of phosphatidylinositol in the target membrane. Moreover, since insertion of such a peptide leads to membrane destabilization and fusion, the present data would be compatible with the involvement of this sequence in Ebola virus fusion.     

The relation between electric auditory brain stem and cognitive responses and speech perception in cochlear implant users

Electrically evoked brainstem responses (EABR) and event-related cortical potentials were recorded in seven postlingually deaf adults who were experienced users of a Nucleus multichannel cochlear implant. The patients were divided into two subgroups: good performers and moderate performers. Poor EABR were found in two of the moderate performers. The latencies and amplitudes of the cortical N1 P2 complex in the good performers were within the same range as those of subjects with normal hearing, but were deviant in the group of moderate performers. This may indicate disturbed cochleotopical organization of the auditory cortex in the latter group. P300 measurements in the good performers showed normal latencies, whereas in the moderate performers they were prolonged. The results suggest that the outcomes of electrophysiological measurements to assess the integrity of a patient's auditory neural system on a brainstem and a cortical level, are related to the patient's performance with the cochlear implant.