Synthesis and biological activity of linear and cyclic enkephalins modified at the Gly3-Phe4 amide bond

As part of our continuing effort to define structure-activity relationships for enkephalin and design enzymatically resistant analogs, we report the synthesis and biological activities of linear and cyclic enkephalin analogs modified at the Gly3-Phe4 amide bond. The partial retro-inverso enkephalin analog Tyr-D-Ala-gGly-(R,S)-mPhe-Leu-NH2 and its cyclic counterpart, Tyr-cyclo[D-A2 bu-gGly-(R,S)-mPhe-Leu-], were synthesized as diastereomeric mixtures using solution methodology. The racemic benzylmalonate allowed the linear analog to be synthesized by fragment coupling at the reversed bond. Cyclization of the second analog was carried out at high concentration, eliminating formation of polymer by the use of an insoluble base. All gem-diaminoalkyl residues were prepared by conversion of peptidyl amides with benzene iodonium bis(trifluoroacetate). Diastereomers of both compounds were separable by reverse phase HPLC but those of the linear compound racemized rapidly under conditions of testing and were therefore tested together. All analogs tested had activities ranging from 6 to 14% of the activity of Leu enkephalin, indicating that the Gly3-Phe4 amide bond is important, though not crucial, for receptor binding.     

The human ASM (adult skeletal muscle) gene: expression and chromosomal assignment to 11p15

A rat adult skeletal muscle probe (Asm15) originated from a rhabdomyosarcoma was used to isolate the human homologous sequence from a placenta cDNA library. Among several positive clones the longest EcoRI-EcoRI insert (ASM1) obtained was 1875 bp long with 72% homology with rat Asm15 cDNA sequence. Important variations of ASM1 RNA level were observed in different adult skeletal muscles. Expression of a 29kD ASM1 protein was demonstrated in human adult skeletal muscle lysates using an antiserum (PB1579) raised against the C terminal region of the rat Asm15 protein. The human ASM gene was assigned by somatic cell analysis with human (ASM1) and rat (Asm15) probes to chromosome 11, and by in situ hybridization with the human probe to 11p15, a chromosome region involved in human embryonal rhabdomyosarcomas. Except for the presence of a HindII restriction site, the results obtained for the restriction map and the sequence of ASM1 cDNA (data not shown) exhibited extensive homology with the human H19 DNA sequence which have been mapped with a mouse probe also in 11p15. This suggests that ASM/Asm and H19 may represent the same sequence (in this hypothesis the presence of the supplementary HindII site in our ASM1 probe is explained by polymorphic variability). However it was reported that human and mouse H19 mRNA did not encode for a protein but acted as an RNA molecule whereas in our present study ASM protein was detected in human adult skeletal muscle. This could be explained by important regulation of ASM protein expression during development and cell differentiation. However we cannot exclude for the different species studied (mouse, rat, and man) the hypothesis that H19 and ASM/Asm mRNA may represent two distinct messengers from the same gene or even from duplicated genes.     

Conscious Brain-to-Brain Communication in Humans Using Non-Invasive Technologies

Human sensory and motor systems provide the natural means for the exchange of information between individuals, and, hence, the basis for human civilization. The recent development of brain-computer interfaces (BCI) has provided an important element for the creation of brain-to-brain communication systems, and precise brain stimulation techniques are now available for the realization of non-invasive computer-brain interfaces (CBI). These technologies, BCI and CBI, can be combined to realize the vision of non-invasive, computer-mediated brain-to-brain (B2B) communication between subjects (hyperinteraction). Here we demonstrate the conscious transmission of information between human brains through the intact scalp and without intervention of motor or peripheral sensory systems. Pseudo-random binary streams encoding words were transmitted between the minds of emitter and receiver subjects separated by great distances, representing the realization of the first human brain-to-brain interface. In a series of experiments, we established internet-mediated B2B communication by combining a BCI based on voluntary motor imagery-controlled electroencephalographic (EEG) changes with a CBI inducing the conscious perception of phosphenes (light flashes) through neuronavigated, robotized transcranial magnetic stimulation (TMS), with special care taken to block sensory (tactile, visual or auditory) cues. Our results provide a critical proof-of-principle demonstration for the development of conscious B2B communication technologies. More fully developed, related implementations will open new research venues in cognitive, social and clinical neuroscience and the scientific study of consciousness. We envision that hyperinteraction technologies will eventually have a profound impact on the social structure of our civilization and raise important ethical issues.     

Production and characterization of human renin antibodies with region-oriented synthetic peptides

Polyclonal and monoclonal antibodies were raised against pure human renin, but nothing was known about the regions against which they were directed. Using a three-dimensional model of mouse submandibular renin, we selected seven peptide sequences as belonging to potential epitopes. The main criteria for their choice were the location of the peptide sequences near the catalytic region and on the surface of the renin molecule and their hydrophilicity. After transposition of the regions to the 340-amino acid sequence of human renin, the seven peptides (corresponding to amino acids 50-60, 63-71, 81-90, 118-126, 162-169, 247-255, and 287-295) were synthesized, coupled to bovine serum albumin, and injected into rabbits. Five of these peptides elicited antibodies, and 50-68% binding of the corresponding iodinated peptide was obtained with a 1:25 dilution of antiserum. The antisera titers ranged from 1:5,000 to 1:100,000 when tested by enzyme-linked immunosorbent assay. The same antisera bound 15-65% of labeled pure human renin at a final dilution of 1:2.5, the highest percentage being obtained with peptide 81-90 antiserum. At a 1:5 dilution, the five antisera inhibited renin activity by 23-68% in human plasma with a high renin activity (40 ng of angiotensin I/h/ml). At a final dilution of 1:50, peptide 81-90 antiserum was still capable of producing 25% inhibition. Purified IgG (0.6 mg) from this antiserum inhibited pure human renin activity by up to about 40%, as measured by its reaction with pure synthetic human tetradecapeptide substrate. Antigenic peptides that mimic a part of the human renin sequence, especially peptide 81-90 representing the "flap" covering the cleft between the two renin lobes, constitute promising tools for the development of a synthetic antirenin vaccine.     

l-Phenylalanyl-l-Glutamate-Stimulated, Chloride-Dependent Glutamate Binding Represents Glutamate Sequestration Mediated by an Exchange System

Stimulation of glutamate binding by the dipeptide L-phenylalanyl-L-glutamate (Phe-Glu) was inhibited by the peptidase inhibitor bestatin, suggesting that the stimulation was caused by glutamate liberated from the dipeptide and not by the dipeptide itself. It further suggests that this form of glutamate binding should be reinterpreted as glutamate sequestration and that stimulation of binding both by dipeptides and after preincubation with high concentrations of glutamate is likely to be due to counterflow accumulation. Several other criteria indicate that most of glutamate binding stimulated by chloride represents glutamate sequestration: Binding is reduced when the osmolarity of the incubation medium is increased, when membranes incubated with [3H]glutamate are lysed before filtration, and when membranes are made permeable by transient exposure to saponin. Moreover, dissociation of bound glutamate after a 100-fold dilution of the incubation medium is accelerated about 50 times by the addition of glutamate to the dilution medium. This result would be anomalous if glutamate were bound to a receptor site; it suggests instead that glutamate is transported in and out of membrane vesicles by a transport system that preferentially mediates exchange between internal and external glutamate. Glutamate binding contains a component of glutamate sequestration even when measured in the absence of chloride. Sequestration is adequately abolished only after treating membranes with detergents; even extensive lysis, sonication, and freezing/thawing may be insufficient.     

Cytotoxicity of a novel lipid-like bacterial product

A highly cytotoxic, lipid-like compound was isolated from a Serratia marcescens strain currently under identification. We have named the compound DCX for its direct cytotoxic activity on various cell types in culture. DCX was purified by preparative thin layer chromatography from chloroform: methanol = 4:1 extracts of whole bacteria, and is chromatographically homogeneous. The effect of DCX on cells is dose, time, and temperature dependent. DCX is particularly toxic to the mastocytoma cell line P815 (TD50 = 75 pg/ml). Three other malignant or transformed murine cell lines were sensitive to the cytotoxic action of DCX. The effect of DCX was also tested on normal cells (human gingival fibroblasts), which showed greater resistance to DCX than the other cells tested.     

Crystal structure of cytochrome c peroxidase compound I

We have compared the 2.5-A crystal structure of yeast cytochrome c peroxidase (CCP) with that of its semistable two-equivalent oxidized intermediate, compound I, by difference Fourier and least-squares refinement methods. Both structures were observed at -15 degrees C. The difference Fourier map reveals that formation of compound I causes only small positional adjustments of a few tenths of an angstrom. The map's most pronounced feature is a pair of positive and negative peaks bracketing the heme iron position. Least-squares refinement shows that the iron atom moves about 0.2 A toward the distal side of the heme. No significant difference density is evident near the side chains of Trp-51 or Met-172, each of which has been proposed to be the site of the electron paramagnetic resonance (EPR) active radical in compound I. However, the second most prominent feature of difference density is a negative peak near the side chain of Thr-180, which, according to the results of least-squares refinement, moves by 0.15 A in the direction of Met-230. These observations, together with the results of mutagenesis experiments [Fishel, L. A., Villafranca, J. E., Mauro, J. M., & Kraut, J. (1987) Biochemistry 26, 351-360; Goodin, D. B., Mauk, A. G., & Smith, M. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 1295-1299] in which Trp-51 and Met-172 have been replaced without loss of the EPR radical signal in compound I, lead us to consider the possibility that the radical site lies within a cluster composed of the side chains of Met-230, Met-231, and Trp-191.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mapping of the gene for anti-müllerian hormone to the short arm of human chromosome 19

The gene coding for human anti-Müllerian hormone (AMH) was localized to subbands p13.2----p13.3 on chromosome 19, using in situ hybridization and Southern blot analysis of a panel of man-mouse and man-hamster somatic cell hybrids.     

Detection of ionizing radiations with the SOS Chromotest, a bacterial short-term test for genotoxic agents

The effects of 3 types of ionizing radiation, gamma-rays, neutrons and accelerated alpha-particles, were examined using the SOS Chromotest, a bacterial colorimetric assay for genotoxic agents based on the measurement of the SOS response in Escherichia coli. The SOS Chromotest appeared to be a sensitive and simple assay to detect quantitatively these radiations as well as their biological effects. The range of adsorbed doses for which induction was observed was similar for the 3 types of radiation, the minimum inducing doses being in the order of 2.5-5 Gy. We discuss the possible use of these observations to study the molecular action of radiations and to compare their genotoxic effects with those of chemicals.