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.     

Detection of Plasma Protease Activity Using Microsphere-Cytometry Assays with E. coli Derived Substrates: VWF Proteolysis by ADAMTS13

Protease levels in human blood are often prognostic indicators of inflammatory, thrombotic or oncogenic disorders. The measurement of such enzyme activities in substrate-based assays is complicated due to the low prevalence of these enzymes and steric hindrance of the substrates by the more abundant blood proteins. To address these limitations, we developed a molecular construct that is suitable for microsphere-cytometer based assays in the milieu of human blood plasma. In this proof of principle study, we demonstrate the utility of this substrate to measure metalloprotease ADAMTS13 activity. The substrate, expressed in E. coli as a fusion protein, contains the partial A2-domain of von Willebrand factor (VWF amino acids 1594–1670) that is mutated to include a single primary amine at the N-terminus and free cysteines at the C-terminus. N-terminus fluorescence conjugation was possible using NHS (N-hydroxysuccinimide) chemistry. Maleimide-PEG(Polyethylene glycol)_n-biotin coupling at the C-terminus allowed biotinylation with variable PEG spacer lengths. Once bound to streptavidin-bearing microspheres, the substrate fluorescence signal decreased in proportion with ADAMTS13 concentration. Whereas recombinant ADAMTS13 activity could be quantified using substrates with all PEG repeat-lengths, only the construct with the longer 77 PEG-unit could quantify proteolysis in blood plasma. Using this longer substrate, plasma ADAMTS13 down to 5% of normal levels could be detected within 30 min. Such measurements could also be readily performed under conditions resembling hyperbilirubinemia. Enzyme catalytic activity was tuned by varying buffer calcium, with lower divalent ion concentrations enhancing cleavage. Overall, the study highlights the substrate design features important for the creation of efficient proteolysis assays in the setting of human plasma. In particular, it emphasizes the need to introduce PEG spacers in plasma-based experiments, a design attribute commonly ignored in immobilized peptide-substrate assays.     

Deliberately infecting healthy volunteers with malaria parasites: Perceptions and experiences of participants and other stakeholders in a Kenyan-based malaria infection study

Controlled human malaria infection (CHMI) studies involve the deliberate infection of healthy volunteers with malaria parasites under controlled conditions to study immune responses and/or test drug or vaccine efficacy. An empirical ethics study was embedded in a CHMI study at a Kenyan research programme to explore stakeholders’ perceptions and experiences of deliberate infection and moral implications of these. Data for this qualitative study were collected through focus group discussions, in-depth interviews and non-participant observation. Sixty-nine participants were involved, including CHMI study volunteers, community representatives and research staff. Data were managed using QSR Nvivo 10 and analysed using an inductive-deductive approach, guided by ethics literature. CHMI volunteers had reasonable understanding of the study procedures. Decisions to join were influenced by study incentives, trust in the research institution, their assessment of associated burdens and motivation to support malaria vaccine development. However, deliberate malaria infection was a highly unusual research strategy for volunteers, community representatives and some study staff. Volunteers’ experiences of physical, emotional and social burdens or harms were often greater than anticipated initially, and fluctuated over time, related to specific procedures and events. Although unlikely to deter volunteers' participation in similar studies in furture, we argue that the dissonance between level of understanding of the burdens involved and actual experiences are morally relevant in relation to community engagement, informed consent processes, and ongoing support for volunteers and research staff. We further argue that ethics oversight of CHMI studies should take account of these issues in deciding whether consent, engagement and the balance of benefits and harms are reasonable in a given context.     

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.     

Ontogeny of head and caudal fin shape of an apex marine predator: The tiger shark (Galeocerdo cuvier)

How morphology changes with size can have profound effects on the life history and ecology of an animal. For apex predators that can impact higher level ecosystem processes, such changes may have consequences for other species. Tiger sharks (Galeocerdo cuvier) are an apex predator in tropical seas, and, as adults, are highly migratory. However, little is known about ontogenetic changes in their body form, especially in relation to two aspects of shape that influence locomotion (caudal fin) and feeding (head shape). We captured digital images of the heads and caudal fins of live tiger sharks from Southern Florida and the Bahamas ranging in body size (hence age), and quantified shape of each using elliptical Fourier analysis. This revealed changes in the shape of the head and caudal fin of tiger sharks across ontogeny. Smaller juvenile tiger sharks show an asymmetrical tail with the dorsal (upper) lobe being substantially larger than the ventral (lower) lobe, and transition to more symmetrical tail in larger adults, although the upper lobe remains relatively larger in adults. The heads of juvenile tiger sharks are more conical, which transition to relatively broader heads over ontogeny. We interpret these changes as a result of two ecological transitions. First, adult tiger sharks can undertake extensive migrations and a more symmetrical tail could be more efficient for swimming longer distances, although we did not test this possibility. Second, adult tiger sharks expand their diet to consume larger and more diverse prey with age (turtles, mammals, and elasmobranchs), which requires substantially greater bite area and force to process. In contrast, juvenile tiger sharks consume smaller prey, such as fishes, crustaceans, and invertebrates. Our data reveal significant morphological shifts in an apex predator, which could have effects for other species that tiger sharks consume and interact with. J. Morphol. 277:556–564, 2016. © 2016 Wiley Periodicals, Inc.     

酸敏感离子通道与脑梗死

急性脑梗死约占全部脑卒中的70%,病死率和致残率高,且极易复发。但目前针对急性脑梗死在时间窗内溶栓、抗凝等治疗手段不能从根本上切实有效地修复受损脑组织,且伴有出血等风险。寻找脑梗死形成发展的原因并予以治疗迫在眉睫。酸中毒是引起缺血性脑损伤的重要机制。大量实验研究表明,酸中毒能加重神经元的缺血性损伤,且其梗死面积与酸中毒的程度直接相关。但缺血产生的酸中毒如何引起神经元损伤的确切机制尚不明确。最近研究发现酸中毒能激活一种在中枢及周围神经中广泛存在的膜通道,即酸敏感离子通道,它对Ca2+通透,能引起细胞内Ca2+超载,同时能激活胞内酶引起细胞内蛋白质、脂类及核酸的降解,加重缺血后脑损伤。本文就酸敏感离子通道1a与脑梗死做一综述。