Human endothelin converting enzyme-2 (ECE2): characterization of mRNA species and chromosomal localization.

Generation of the functionally pleiotropic members of the endothelin vasoactive peptide family is critically catalyzed by unique type II metalloproteases, termed endothelin converting enzymes (ECE). Isolation of human ECE-2 (EC 3.4.24.71) cDNAs revealed deduced open reading frames of 787 and 765 amino acids with approximately 60% identity with human ECE-1. Characterization of mRNA variants revealed mRNA structural diversity at the 5'-terminus. Two mRNA species exist containing distinct first and second exons. Furthermore, in one of these species, an in-frame deletion of the intracytoplasmic domain removed 29 amino acids. Because of the previously reported human genetic diseases ascribed to germline mutations of member genes of the endothelin family, ECE2 was localized in human chromosomes with fluorescence in situ hybridization and radiation hybrid mapping to 3q28-q29 and SHGC-20171/D3S1571, respectively.     

Assay of zofenopril and its active metabolite zofenoprilat by liquid chromatography coupled with tandem mass spectrometry

Zofenopril is a pro-drug designed to undergo metabolic hydrolysis yielding the active free sulfhydryl compound zofenoprilat, which is an angiotensin converting enzyme (ACE) inhibitor, endowed also with a marked cardioprotective activity. A simple, highly sensitive specific LC–MS–MS method was developed for the determination of zofenopril and zofenoprilat in human plasma. In order to prevent oxidative degradation of zofenoprilat and its internal standard, their free sulfhydryl groups were protected by treatment with N-ethylmaleimide (NEM), which produced the succinimide derivatives. The compounds and their corresponding fluorine derivatives, used as internal standards, were extracted from plasma with toluene. The reconstituted dried extracts were chromatographed and then monitored by a triple-stage-quadrupole instrument operating in the negative ion spray ionization mode. The method was validated over the concentration range of 1–300 ng/ml for zofenopril and 2–600 ng/ml for zofenoprilat. Inter- and intra-assay precision and accuracy of both zofenopril and zofenoprilat were better than 10%. The limit of quantitation was 1 ng/ml with zofenopril and 2 ng/ml with zofenoprilat. Extraction recovery proved to be on average 84.8% with zofenopril and 70.1% with zofenoprilat. Similar recoveries were shown by the above two internal standards. The method was applied to measure plasma concentrations of zofenopril and zofenoprilat in 18 healthy volunteers treated orally with zofenopril calcium salt at the dose of 60 mg.     

Identification of Tumor Antigens in the HLA Peptidome of Patient-derived Xenograft Tumors in Mouse

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Highlights

• •Sufficient tumor tissues are often unavailable large HLA peptidome discovery.
• •Using patient derived xenograft (PDX) tumors can overcome this limitation.
• •The large PDX HLA peptidomes expand significantly those of the original biopsies.
• •The HLA peptidomes of the PDX tumors included many tumor antigens.

     

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.     

Evaluation of a radioreceptor assay to assess exogenous estrogen activity in serum of patients with breast cancer.

A radioreceptor assay (RRA) for the determination of total estrogen activity, was set up and used to assess the possible presence of exogenous molecules with estrogen activity in serum; a comparison was made with the specific radioimmunoassay (RIA) for the endogenous estrogen 17-B estradiol (17-B-E2). The assay was first performed on sera from healthy people taking estrogens in the form of oral contraceptives or lotions for local application whose total estrogenic activity in the blood was assumed to be abnormal. The assay was then performed on serum from 98 patients with early breast cancer and 20 patients with metastasis, not undergoing hormone therapy. A higher estrogen activity was found in 2.5% of sera compared to the activity found using the RIA method which is specific for endogenous estrogen 17-B-E2, the RRA/17-B-E2 ratio being higher than 3. Increased estrogen activity was found in 10% serum samples from digoxin treated cardiopathic patients, with an RRA/17-B-E2 ratio ranging from 4.4 to 20. The RRA assay could prove useful for showing up exogenous estrogen activity from various sources (drugs, food) in sera of people in whom estrogen stimulation could be potentially dangerous (i.e. in patients with hormone-sensitive tumors). This exogenous activity could support a certain degree of neoplastic stimulation and, therefore, unfavourably condition the patients' therapeutic response.     

Elimination of Von Hippel-Lindau Function Perturbs Pancreas Endocrine Homeostasis in Mice

Mutations in the human homolog of the Vhlh gene [encoding the von-Hippel Lindau (VHL) protein] lead to tumor development. In mice, depletion of Vhlh in pancreatic ß-cells causes perturbed glucose homeostasis, but the role of this gene in other pancreatic cells is poorly understood. To investigate the function of VHL/HIF pathway in pancreatic cells, we inactivated Vhlh in the pancreatic epithelium as well as in the endocrine and exocrine lineages. Our results show that embryonic depletion of Vhlh within the pancreatic epithelium causes postnatal lethality due to severe hypoglycemia. The hypoglycemia is recapitulated in mice with endocrine-specific removal of Vhlh, while animals with loss of Vhlh predominantly in the exocrine compartment survive to adulthood with no overt defects in glucose metabolism. Mice with hypoglycemia display diminished insulin release in response to elevated glucose. Significantly, the glucagon response is impaired both in vivo (circulating glucagon levels) as well as in an in vitro secretion assay in isolated islets. Hypoxia also impairs glucagon secretion in a glucagon-expressing cell line in culture. Our results reveal a novel role for the hypoxia/HIF pathway in islet hormone secretion and maintenance of the fine balance that allows for the establishment of normoglycemia.     

Dormancy in peach (Prunus persica L.) flower buds

Morphological studies were carried out with peach flower buds collected monthly in 1989 and 1990, from two months before leaf fall (7 March) until two to three weeks before bloom (7/8 August). Chilled (2–4°C for 30 days) and unchilled buds were exposed to 20 to 25°C, 100% RH and continuous light. Gibberellin A₃ (3 ng or 30 ng) was applied to some of the non-chilled cuttings at three days intervals. Then, 12, 19, and 26 days after they were planted, the buds were sampled and processed for histological studies. Cultured flower buds (chilled or unchilled) had accelerated anther and gynoecium morphogenesis after 12 days under controlled conditions, compared to buds processed immediately after collection from the field. Chilling treatment augmented the bud culture effect, while Gibberellin A₃ applications to the excised buds retarded bud morphogenesis to a stage comparable to that of buds collected directly from the field. This, suggests that the comparatively high levels of Gibberellin A_1/3 we previously found in mid winter [15, 18] could be at least one of the factors that controls floral bud dormancy by retarding anther and gynoecium development.     

Characterization of the Actions of Toxins II-9.2.2 and II-10 from the Venom of the Scorpion Centruroides noxius on Transmitter Release from Mouse Brain Synaptosomes

Toxic peptides II-9.2.2 and II-10, purified from Centruroides noxius venom, bear highly homologous N-terminal amino acid sequences, and both toxins are lethal to mice. However, only toxin II-10 is active on the voltage-clamped squid axon, selectively decreasing the voltage-dependent Na+ current. Here, we have tested toxins II-9 and II-10 on synaptosomes from mouse brain: both toxins increased the release of gamma-[3H]aminobutyric acid ([3H]GABA). Their effect was completely blocked by tetrodotoxin or by the absence of external Na+. Also, both toxins increased Na+ permeability in isolated nerve terminals. Besides the observation that toxin II-9 is active on synaptosomes, the effect of toxin II-10 in this preparation is opposite to that observed in the squid axon. Thus, our results reflect functional differences between the populations of Na+ channels in mouse brain synaptosomes and in the squid axon. The release of GABA evoked by these toxins from synaptosomes required external Ca2+ and was blocked by Ca2+ channel blockers (verapamil and Co2+). This latter observation is in sharp contrast to the releasing action of veratrine, which evoked release even in the absence of external Ca2+. Furthermore, the action of both C. noxius toxins was potentiated by veratrine, a result suggesting they have different mechanisms of action. Among drugs that release neurotransmitters by increasing Na+ permeability, it is noteworthy that scorpion toxins are the only ones yet reported to have a strict requirement for external Ca2+.     

Elicitation of Necrosis in Vigna unguiculata Walp. by Homogeneous Aspergillus niger Endo-Polygalacturonase and by alpha-d-Galacturonate Oligomers

Endo-polygalacturonase (PG) was purified from a commercial preparation of Aspergillus niger pectinase by means of carboxymethylcellulose chromatography, preparative isoelectric focusing, and gel permeation through Sephadex G-50. The enzyme was electrophoretically homogeneous and consisted of a single polypeptide chain with a molecular weight of 33,500. The enzyme exhibited a specific activity significantly higher than those of purified polygalacturonases from phytopathogenic fungi. Galacturonate oligomers with a degree of polymerization higher than four appeared quickly as products of the enzymic hydrolysis of Napolygalacturonate. The oligomers were later degraded to di- and monogalacturonate. The homogeneous enzyme and growing mycelium of Aspergillus niger separately elicited a necrotic response in cowpea (Vigna unguiculata Walp.) pods. Heat-inactivated PG and PG inactivated with specific antibodies did not elicit necrosis, suggesting that the catalytic activity of the enzyme is necessary for its function as an elicitor. The PG-released oligosaccharides from Vigna cell wall and the galacturonides with a degree of polymerization greater than four separately elicited necrosis, whereas di- and monogalacturonate did not.