Base-Sequence-Independent Efficient Redox Switching of Self-Assembled DNA Nanocages.

Stimuli responsivity has been extensively pursued in dynamic DNA nanotechnology, due to its incredible application potentials. Among diverse dynamic systems, redox-responsive DNA assembly holds great promise for broad applications, especially considering that redox processes widely exist in various physiological environments. However, only a few studies have been reported on redox-sensitive dynamic DNA assembly. Albeit ingenious, most of these studies are either dependent on the DNA sequence or involve chemical modification. Herein, a facile and universal mechanism to realize redox-responsive self-assembly of DNA nanocages (tetrahedron and cube) driven by the interconversion between cystamine and cysteamine toward dynamic DNA nanotechnology is reported.

The 55 kDa tissue transglutaminase cross-linking active isoform TG induces cell death.

Transamidations are calcium-dependent reactions catalyzed by transglutaminase enzymes. Tissue transglutaminase (TG2 or TGC) is a multifunctional protein with a controversial role in apoptosis. The cross-linking function of transglutaminase enzymes has been shown to play a role in cell death. Human breast cancer cell lines (MCF7 and T47D), which express low endogenous levels of transglutaminase, were transiently transfected with the cross-linking 55 kDa active TG isoform or its precursor the 80 kDa full-length TGC. The increased frequency of apoptosis correlated with the increase in transglutaminase expression and the highest rates of apoptosis were found in cells transfected with the potent TG isoform as compared to the full length TGC. The calcium ionophores A231827 and maitotoxin, which are known to induce transamidation, were found to promote apoptosis, whereas cystamine, an active transglutaminase inhibitor, blocked apoptosis due to the over-expression of the active TG isoform. This is the first time that TG has been used in cellular transfections and the results presented show that TG is a potent inducer of cell death. This finding may help to clarify the conflicting functions of TG in the induction of cell death. The TG-dependent irreversible cross-linking of intracellular proteins, a function previously assigned to TGC, represents an important biochemical event in the induction of the structural changes present in cells during apoptosis.

Mapping of O-GlcNAc sites of 20 S proteasome subunits and Hsp90 by a novel biotin-cystamine tag.

The post-translational modification of proteins with O-GlcNAc is involved in various cellular processes including signal transduction, transcription, translation, and nuclear transport. This transient protein modification enables cells or tissues to adapt to nutrient conditions or stress. O-Glycosylation of the 26 S proteasome ATPase subunit Rpt2 is known to influence the stability of proteins by reducing their proteasome-dependent degradation. In contrast, knowledge of the sites of O-GlcNAcylation on the subunits of the catalytic core of the 26 S proteasome, the 20 S proteasome, and the impact on proteasome activity is very limited. This is predominantly because O-GlcNAc modifications are often substoichiometric and because 20 S proteasomes represent a complex protein mixture of different subtypes. Therefore, identification of O-GlcNAcylation sites on proteasome subunits essentially requires effective enrichment strategies. Here we describe an adapted ß-elimination-based derivatization method of O-GlcNAc peptides using a novel biotin-cystamine tag. The specificity of the reaction was increased by differential isotopic labeling with either "light" biotin-cystamine or deuterated "heavy" biotin-cystamine. The enriched peptides were analyzed by LC-MALDI-TOF/TOF-MS and relatively quantified. The method was optimized using bovine α-crystallin and then applied to murine 20 S proteasomes isolated from spleen and brain and murine Hsp90 isolated from liver. Using this approach, we identified five novel and one known O-GlcNAc sites within the murine 20 S proteasome core complex that are located on five different subunits and in addition two novel O-GlcNAc sites on murine Hsp90ß, of which one corresponds to a previously described phosphorylation site.

Integrated electroosmotic perfusion of tissue with online microfluidic analysis to track the metabolism of cystamine, pantethine, and coenzyme A.

We have developed an approach that integrates electroosmotic perfusion of tissue with a substrate-containing solution and online microfluidic analysis of products, in this case thiols. Using this approach we have tracked the metabolism of cystamine, pantethine and CoA in the extracellular space of organotypic hippocampal slice cultures (OHSCs). Currently, little is known about coenzyme A (CoA) biodegradation and even less is known about the regulation and kinetic characteristics for this sequential multienzyme reaction. We found that the steady state percentage yields of cysteamine from cystamine and pantethine during the transit through OHSCs were 91% ± 4% (SEM) and 0.01%-0.03%, respectively. The large difference in the yields of cysteamine can be used to explain the drugs' different toxicities and clinical effectiveness against cystinosis. The kinetic parameters of the enzyme reaction catalyzed by the ectoenzyme pantetheinase are KM,C/α = 4.4 ± 1.1 mM and Vmax,C = 29 ± 3 nM/s, where α is the percentage yield of pantethine to pantetheine through disulfide exchange. We estimate that the percentage yield of pantethine to pantetheine through disulfide exchange is approximately 0.5%. Based on the formation rate of cysteamine in the OHSCs, we obtained the overall apparent Michaelis constant and maximum reaction rate for sequential, extracellular CoA degradation in an in situ environment, which are K'M = 16 ± 4 µM, V'max = 7.1 ± 0.5 nM/s. Kinetic parameters obtained in situ, although difficult to measure, are better representations of the biochemical flux in the living organism than those from isolated enzymes in vitro.

SDC1-dependent TGM2 determines radiosensitivity in glioblastoma by coordinating EPG5-mediated fusion of autophagosomes with lysosomes.

Glioblastoma multiforme (GBM) is the most common brain malignancy insensitive to radiotherapy (RT). Although macroautophagy/autophagy was reported to be a fundamental factor prolonging the survival of tumors under radiotherapeutic stress, the autophagic biomarkers coordinated to radioresistance of GBM are still lacking in clinical practice. Here we established radioresistant GBM cells and identified their protein profiles using tandem mass tag (TMT) quantitative proteomic analysis. It was found that SDC1 and TGM2 proteins were overexpressed in radioresistant GBM cells and tissues and they contributed to the poor prognosis of RT. Knocking down SDC1 and TGM2 inhibited the fusion of autophagosomes with lysosomes and thus enhanced the radiosensitivity of GBM cells. After irradiation, TGM2 bound with SDC1 and transported it from the cell membrane to lysosomes, and then bound to LC3 through its two LC3-interacting regions (LIRs), coordinating the encounter between autophagosomes and lysosomes, which should be a prerequisite for lysosomal EPG5 to recognize LC3 and subsequently stabilize the STX17-SNAP29-VAMP8 QabcR SNARE complex assembly. Moreover, when combined with RT, cystamine dihydrochloride (a TGM2 inhibitor) extended the lifespan of GBM-bearing mice. Overall, our findings demonstrated the EPG5 tethering mode with SDC1 and TGM2 during the fusion of autophagosomes with lysosomes, providing new insights into the molecular mechanism and therapeutic target underlying radioresistant GBM.Abbreviations: BafA1: bafilomycin A1; CQ: chloroquine; Cys-D: cystamine dihydrochloride; EPG5: ectopic P-granules 5 autophagy tethering factor; GBM: glioblastoma multiforme; GFP: green fluorescent protein; LAMP2: lysosomal associated membrane protein 2; LIRs: LC3-interacting regions; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NC: negative control; RFP: red fluorescent protein; RT: radiotherapy; SDC1: syndecan 1; SNAP29: synaptosome associated protein 29; SQSTM1/p62: sequestosome 1; STX17: syntaxin 17; TGM2: transglutaminase 2; TMT: tandem mass tag; VAMP8: vesicle associated membrane protein 8; WT: wild type.

New mechanistic explanation for the localization of ulcers in the rat duodenum: role of iron and selective uptake of cysteamine.

Cysteamine, a coenzyme A metabolite, induces duodenal ulcers in rodents. Our recent studies showed that ulcer formation was aggravated by iron overload and diminished in iron deficiency. We hypothesized that cysteamine is selectively taken up in the duodenal mucosa, where iron absorption primarily occurs, and is transported by a carrier-mediated process. Here we report that cysteamine administration in rats leads to cysteamine accumulation in the proximal duodenum, where the highest concentration of iron in the gastrointestinal tract is found. In vitro, iron loading of intestinal epithelial cells (IEC-6) accelerated reactive oxygen species (ROS) production and increased [(14)C]cysteamine uptake. [(14)C]Cysteamine uptake by isolated gastrointestinal mucosal cells and by IEC-6 was pH-dependent and inhibited by unlabeled cysteamine. The uptake of [(14)C]cysteamine by IEC-6 was Na(+)-independent, saturable, inhibited by structural analogs, H(2)-histamine receptor antagonists, and organic cation transporter (OCT) inhibitors. OCT1 mRNA was markedly expressed in the rat duodenum and in IEC-6, and transfection of IEC-6 with OCT1 siRNA decreased OCT1 mRNA expression and inhibited [(14)C]cysteamine uptake. Cysteamine-induced duodenal ulcers were decreased in OCT1/2 knockout mice. These studies provide new insights into the mechanism of cysteamine absorption and demonstrate that intracellular iron plays a critical role in cysteamine uptake and in experimental duodenal ulcerogenesis.

Use of pH-Active Catechol-Bearing Polymeric Nanogels with Glutathione-Responsive Dissociation to Codeliver Bortezomib and Doxorubicin for the Synergistic Therapy of Cancer.

Synergistic therapy holds promising potential in cancer treatment. Here, the inclusion of catechol moieties, a disulfide cross-linked structure, and pendent carboxyl into the network of polymeric nanogels with glutathione (GSH)-responsive dissociation and pH-sensitive release is first disclosed for the codelivery of doxorubicin (DOX) and bortezomib (BTZ) in synergistic cancer therapy. The pendent carboxyl groups and catechol moieties are exploited to absorb DOX through electrostatic interaction and conjugate BTZ through boronate ester, respectively. Both electrostatic interactions and boronate ester are stable at neutral or alkaline pH, while they are instable in an acidic environment to further recover the activities of BTZ and DOX. The polymeric nanogels possess a superior stability to prevent the premature leakage of drugs in a physiological environment, while their structure is destroyed in response to a typical endogenous stimulus (GSH) to unload drugs. The dissociation of the drug-loaded nanogels accelerates the intracellular release of DOX and BTZ and further enhances the therapeutic efficacy. In vitro and in vivo investigations revealed that the dual-drug loaded polymeric nanogels exhibited a strong ability to suppress tumor growth. This study thus proposes a new perspective on the production of multifunctional polymeric nanogels through the introduction of different functional monomers.

Overcoming multidrug resistance by intracellular drug release and inhibiting p-glycoprotein efflux in breast cancer.

Doxorubicin (DOX) is limited to use in clinical practice because of poor targeting, serious side effects and multidrug resistance (MDR). Vitamin E and its derivatives are currently considered as hydrophobic material that can reverse tumor MDR by suppressing the action of p-glycoprotein (p-gp). Therefore, reduction-sensitive amphiphilic heparosan polysaccharide-cystamine-vitamin E succinate (KSV) copolymers were designed to reverse breast cancer MDR cells. The spherical micelles (DOX/KSV) micelles which had suitable particle size presented redox-sensitive release character. Simultaneously, DOX-loaded reduction insensitive heparosan-adipic dihydrazide-vitamin E succinate (KV) micellar system was designed as a control. DOX/KSV and DOX/KV micelles had the higher capability to overcome tumor MDR than that free DOX. However, DOX/KSV had the highest amount of cellular uptake which might be caused by the synergistic intracellular drug release and inhibition of p-gp expression. The mechanism experiments revealed that DOX/KSV could be fast disassembled to release DOX after internalization into tumor cells. Moreover, DOX/KSV produced more ROS than free DOX and DOX/KV resulting in enhanced anticancer effect. In vivo tumor-bearing mice study suggested that DOX/KSV micelles could efficiently enhance antitumor effect by overcoming tumor MDR and reduce toxicity of DOX. The DOX/KSV micelles could synergistically increase the therapeutic effect of chemotherapeutic drug on tumor MDR cells.

Cystamine metabolism and brain transport properties: clinical implications for neurodegenerative diseases.

Cystamine has shown significant neuroprotective properties in preclinical studies of Parkinson's disease (PD) and Huntington's disease (HD). Cysteamine, its FDA-approved reduced form, is scheduled to be tested for clinical efficacy in HD patients. Here, we studied the key cystamine metabolites, namely cysteamine, hypotaurine and taurine, as well as cysteine, in order to identify which one is more distinctively responsible for the neuroprotective action of cystamine. After a single administration of cystamine (10, 50 or 200 mg/kg), naïve mice were perfused with phosphate-buffered saline (PBS) at 1, 3, 12, 24 or 48 h post-injection and brain and plasma samples were analyzed by two distinct HPLC methods. Although plasma levels remained under the detection threshold, significant increases in cysteamine brain levels were detected with the 50 and 200 mg/kg doses in mice perfused 1 and 3 h following cystamine injection. To further assess cysteamine as the candidate molecule for pre-clinical and clinical trials in PD, we evaluated its capacity to cross the blood brain barrier. Using an in situ cerebral perfusion technique, we determined that the brain transport coefficient (Clup) of cysteamine (259 µM) was 0.15 ± 0.02 µL/g/s and was increased up to 0.34 ± 0.07 µL/g/s when co-perfused in the presence of cysteine. Taken together, these results strongly suggest that cysteamine is the neuroactive metabolite of cystamine and may further support its therapeutic use in neurodegenerative diseases, particularly in HD and PD.

Relationship between three polymorphisms of methylenetetrahydrofolate reductase (MTHFR C677T, A1298C, and G1793A) gene and risk of prostate cancer: a case-control study.

BACKGROUND: We hypothesized that genetic polymorphisms in methylenetetrahydrofolate reductase (MTHFR) gene are associated with prostate cancer risk. METHODS: We genotyped three MTHFR polymorphisms (C677T, A1298C, and G1793A) and measured serum total homocysteine (tHcy), folate, and vitamin B12 levels in a case-control study of 174 cases and 348 normal healthy controls. The cancer-free controls were frequency matched to the cases by age (±2 years), educational level, occupational status, ethnicity, and smoking status. RESULTS: We found that the MTHFR 677TT and 1298CC genotypes were associated with an about 40% reduction in risk of prostate cancer (adjusted OR = 0.59, 95% CI = 0.41-0.94, and adjusted OR = 0.58, 95% CI = 0.32-0.91, respectively) compared to the 677CC, and 1298AA genotypes. The combined variant genotypes of 1298AC + 677CC were associated with a 30% reduction in risk of prostate cancer (OR = 0.70; 95% CI = 0.53-0.79). In contrast, the variant genotypes of 1793GA + 677CT were associated with slightly increased risk for prostate cancer (OR = 1.64; 95% CI = 0.86-2.15). Regarding prostate cancer aggressiveness, the 677TT genotype was associated with more than 50% decreased risk of high-grade prostate cancer (Gleason score >7) compared with the 677CC and 677CT genotypes (OR = 0.35, 95% CI = 0.24-0.64; P = 0.001). There was no significant difference in plasma levels of tHcy, folate, and vitamin B12 between the two groups with any genotypes. CONCLUSION: These data suggest that all three MTHFR polymorphisms may play a pivotal role in the developing prostate cancer. Larger studies in different ethnic populations and incorporating dietary folate intake are needed to replicate our findings.

Cystamine and cysteamine increase brain levels of BDNF in Huntington disease via HSJ1b and transglutaminase.

There is no treatment for the neurodegenerative disorder Huntington disease (HD). Cystamine is a candidate drug; however, the mechanisms by which it operates remain unclear. We show here that cystamine increases levels of the heat shock DnaJ-containing protein 1b (HSJ1b) that are low in HD patients. HSJ1b inhibits polyQ-huntingtin-induced death of striatal neurons and neuronal dysfunction in Caenorhabditis elegans. This neuroprotective effect involves stimulation of the secretory pathway through formation of clathrin-coated vesicles containing brain-derived neurotrophic factor (BDNF). Cystamine increases BDNF secretion from the Golgi region that is blocked by reducing HSJ1b levels or by overexpressing transglutaminase. We demonstrate that cysteamine, the FDA-approved reduced form of cystamine, is neuroprotective in HD mice by increasing BDNF levels in brain. Finally, cysteamine increases serum levels of BDNF in mouse and primate models of HD. Therefore, cysteamine is a potential treatment for HD, and serum BDNF levels can be used as a biomarker for drug efficacy.

The physiological role of the Gomori-positive glia: a new hypothesis.

On the basis of histological observations of the brains of intact animals and of those injected with a sulfur-containing material (cystamine) we propose that the main, if not unique, role of the Gomori-positive glia is to scavenge the brain for sulfur-containing material because such material when physiologically present (e.g. neurophysin) may give rise to free cysteine that is toxic to neurons, being a long-recognized neurotoxin.

Engineering a cleavable disulfide bond into a natural product siderophore using precursor-directed biosynthesis.

An analogue of the bacterial siderophore desferrioxamine B (DFOB) containing a disulfide motif in the backbone was produced from Streptomyces pilosus cultures supplemented with cystamine. Cystamine competed against native 1,5-diaminopentane during assembly. DFOB-(SS)1[001] and its complexes with Fe(iii) or Ga(iii) were cleaved upon incubation with dithiothreitol. Compounds such as DFOB-(SS)1[001] and its thiol-containing cleavage products could expand antibiotic strategies and Au-S-based nanotechnologies.

Vanin-1-/- mice exhibit a glutathione-mediated tissue resistance to oxidative stress.

Vanin-1 is an epithelial ectoenzyme with pantetheinase activity and generating the amino-thiol cysteamine through the metabolism of pantothenic acid (vitamin B(5)). Here we show that Vanin-1(-/-) mice, which lack cysteamine in tissues, exhibit resistance to oxidative injury induced by whole-body gamma-irradiation or paraquat. This protection is correlated with reduced apoptosis and inflammation and is reversed by treating mutant animals with cystamine. The better tolerance of the Vanin-1(-/-) mice is associated with an enhanced gamma-glutamylcysteine synthetase activity in liver, probably due to the absence of cysteamine and leading to elevated stores of glutathione (GSH), the most potent cellular antioxidant. Consequently, Vanin-1(-/-) mice maintain a more reducing environment in tissue after exposure to irradiation. In normal mice, we found a stress-induced biphasic expression of Vanin-1 regulated via antioxidant response elements in its promoter region. This process should finely tune the redox environment and thus change an early inflammatory process into a late tissue repair process. We propose Vanin-1 as a key molecule to regulate the GSH-dependent response to oxidative injury in tissue at the epithelial level. Therefore, Vanin/pantetheinase inhibitors could be useful for treatment of damage due to irradiation and pro-oxidant inducers.

A Pharmacogenetic Discovery: Cystamine Protects Against Haloperidol-Induced Toxicity and Ischemic Brain Injury.

Haloperidol is an effective antipsychotic agent, but it causes Parkinsonian-like extrapyramidal symptoms in the majority of treated subjects. To address this treatment-limiting toxicity, we analyzed a murine genetic model of haloperidol-induced toxicity (HIT). Analysis of a panel of consomic strains indicated that a genetic factor on chromosome 10 had a significant effect on susceptibility to HIT. We analyzed a whole-genome SNP database to identify allelic variants that were uniquely present on chromosome 10 in the strain that was previously shown to exhibit the highest level of susceptibility to HIT. This analysis implicated allelic variation within pantetheinase genes (Vnn1 and Vnn3), which we propose impaired the biosynthesis of cysteamine, could affect susceptibility to HIT. We demonstrate that administration of cystamine, which is rapidly metabolized to cysteamine, could completely prevent HIT in the murine model. Many of the haloperidol-induced gene expression changes in the striatum of the susceptible strain were reversed by cystamine coadministration. Since cystamine administration has previously been shown to have other neuroprotective actions, we investigated whether cystamine administration could have a broader neuroprotective effect. Cystamine administration caused a 23% reduction in infarct volume after experimentally induced cerebral ischemia. Characterization of this novel pharmacogenetic factor for HIT has identified a new approach for preventing the treatment-limiting toxicity of an antipsychotic agent, which could also be used to reduce the extent of brain damage after stroke.

Introduction of sulfhydryl groups into proteins at carboxyl sites.

A two-step procedure for introduction of sulfhydryl groups at protein carboxyl groups is described. The resultant proteins contain 2-aminoethanethiol residues bound by amide linkages to the protein carboxyl groups. First an amide bond is formed between a carboxyl group of the protein and one of the amino groups of cystamine. Then the disulfide bond is reduced with dithiothreitol, yielding the amide of 2-aminoethanethiol. This procedure was used to incorporate sulfhydryl groups into carbonic anhydrase and adrenocorticotropic hormone. The effect of carbodiimide concentration and pH of the coupling reaction on stoichiometry of sulfhydryl group incorporation was examined. The method was used to prepare bovine carbonic anhydrase containing up to nine sulfhydryl groups per molecule with no loss of enzymatic activity and biologically active adrenocorticotropic hormone containing one sulfhydryl group per molecule.

Post-translational disulfide modifications in cell signaling--role of inter-protein, intra-protein, S-glutathionyl, and S-cysteaminyl disulfide modifications in signal transmission.

Cell signaling entails a host of post-translational modifications of effector-proteins. These modifications control signal transmission by regulating the activity, localization or half-life of the effector-protein. Prominent oxidative modifications induced by cell-signaling reactive oxygen species (ROS) are cysteinyl modifications such as S-nitrosylation, sulfenic acid and disulfide formation. Disulfides protect protein sulfhydryls against oxidative destruction and simultaneously influence cell signaling by engaging redox-regulatory sulfhydryls in effector-proteins. The types of disulfides implicated in signaling span (1) protein S-glutathionylation, e.g. as a novel mode of Ras activation through S-glutathionylation at Cys-118 in response to a hydrogen-peroxide burst, (2) intra-protein disulfides, e.g. in the regulation of the stability of the protein phosphatase Cdc25C by hydrogen-peroxide, (3) inter-protein disulfides, e.g. in the hydrogen peroxide-mediated inactivation of receptor protein-tyrosine phosphatase alpha (RPTPalpha) by dimerization and (4) protein S-cysteaminylation by cystamine. Cystamine is a byproduct of pantetheinase-catalyzed pantothenic acid recycling from pantetheine for biosynthesis of Coenzyme A (CoA), a ubiquitous and metabolically indispensable cofactor. Cystamine inactivates protein kinase C-epsilon (PKCepsilon), gamma-glutamylcysteine synthetase and tissue transglutaminase by S-cysteaminylation-triggered mechanisms. The importance of protein S-cysteaminylation in signal transmission in vivo is evident from the ability of cystamine administration to rescue the intestinal inflammatory-response deficit of pantetheinase knockout mice. These mice lack the predominant epithelial pantetheinase isoform and have sharply reduced levels of cystamine/cysteamine in epithelial tissues. In addition, intraperitoneal administration of cystamine significantly delays neurodegenerative pathogenesis in a Huntington's disease mouse model. Thus, cystamine may serve as a prototype for the development of novel therapeutics that target effector-proteins regulated by S-cysteaminylation.

The uptake and metabolism of cystamine and taurine by isolated perfused rat and rabbit lungs.

Cystamine has been reported to be taken up and metabolized to taurine by the rat lung slices. The objectives of the present study were to compare the uptake and metabolism of cystamine and taurine in isolated perfused lungs of rats and rabbits and examine the action of glutathione (GSH) on these processes. The uptake and metabolism of [14C]cystamine and [14C]taurine were studied at 20 microM concentrations each in isolated, ventilated, perfused rat and rabbit lungs. In some experiments, 1 microM GSH was included in the perfusate prior to the addition of cystamine. The perfusate and lung homogenate samples were analyzed for cystamine and its metabolites. [14C]cystamine uptake with and without GSH was 13 and 14% in rat lungs and 37 and 32% in rabbit lungs. [14C]taurine uptake was 10% in rat and 37% in rabbit lungs. The levels of radiolabeled cystamine and its metabolites were (nmol/g lung): 20.0 +/- 10.0 and 11.5 +/- 7.0 cystamine, 4.7 +/- 0.5 and 3.2 +/- 0.5 hypotaurine and 56.0 +/- 16.0 and 49.4 +/- 6.0 taurine, for rat and rabbit lungs, respectively, when perfused without GSH; and 18.0 +/- 1.0 and 2.5 +/- 0.5 cystamine, 6.6 +/- 0.5 and 18 +/- 10 hypotaurine and 60.0 +/- 12.0 and 33.6 +/- 9.0 taurine, when perfused with GSH, for rats and rabbit lungs, respectively. Taurine did not undergo any further metabolism in either of the lungs. These studies show that cystamine is taken up and metabolized to taurine via hypotaurine by both rat and rabbit lungs in a manner similar to that seen in rat lung slices. However, rat lungs have much greater capacity to metabolize cystamine to taurine than rabbit. Inclusion of GSH did not significantly alter the ability of lungs to sequester cystamine from the perfusate but the metabolism of hypotaurine to taurine was markedly decreased in rabbit lungs. Taurine was not metabolized any further. It is concluded that rat and rabbit lungs take up cystamine from the systemic circulation, metabolize it via hypotaurine to taurine, and effuse most of the latter in to the circulation.

Cysteamine-induced decrease of somatostatin in rat brain synaptosomes in vitro.

The mechanism of somatostatin depletion induced by cysteamine [2-mercaptoethylamine (CySH)] was studied in isolated nerve endings (synaptosomes) from rat brain in vitro. A dose-dependent reduction of somatostatin-like immunoreactivity (SLI) was observed which reached its maximal extent (41%) at a concentration of 300 microM CySH after 1-5 min. There was no release of somatostatin into the incubation medium. CySH at concentrations of up to 10 mM did not interfere in the RIA. Among a variety of compounds, structurally related to CySH 4-aminothiophenol, 2-aminothiophenol and N,N-dimethylaminothiol exhibited the highest efficacy in decreasing somatostatin (60%, 50%, 30%, respectively, at 10 mM and 10 min). The disulfide form of CySH cystamine and dimercaprol resulted in about 15% reduction after 10-min incubation, whereas taurine, alanine, cysteine, and mercaptoethanol were inactive. A saturable, sodium-dependent uptake process was found for the disulfide form of [35S]CySH cystamine [Michaelis-Menten constant (Km) = 18.6 microM, maximum velocity (Vmax) = 2.3 nmol/mg protein X 3 min) which was inhibited by cysteine (87% at 1 mM). [35S]CySH, at concentrations of 20 microM or less, was not stable in buffer solution. It underwent considerable nonenzymatic conversion into its dimeric form (60% at 37 C and 3 min), however it exhibited the same kinetic data for its uptake. Size exclusion HPLC of purified hypothalamic synaptosomes revealed a major SLI peak coeluting with synthetic somatostatin-14 and two minor peaks representing somatostatin-28 and a 13,000 mol wt protein. The three molecular forms of somatostatin were reduced to varied extent by CySH (somatostatin-14 by about 70%, somatostatin-28 by 15%, and the high mol wt form by 30%). Our experiments suggest that high affinity uptake of CySH may precede its action in decreasing somatostatin levels. Increased release or inhibition of synthesis of somatostatin have been excluded as possible mechanisms. It is suggested that SLI is equally affected in nerve endings and in perikarya.

Benextramine-neuropeptide Y receptor interactions: contribution of the benzylic moieties to [3H]neuropeptide Y displacement activity.

Analogs of N,N'-bis[6-[(2-methoxybenzyl)amino]hex-1-yl]cystamine (benextramine, BXT, 2) were synthesized using solution-phase peptide synthesis methodology and analyzed for activity in displacing specifically bound 1 nM N-[propionyl-3H]neuropeptide Y([3H]NPY) from benextramine-sensitive neuropeptide Y (NPY) binding sites in rat brain. Our new synthetic approach to these analogs began with the acylation of cystamine with the N-hydroxysuccinimide ester of tert-butyloxycarbonyl (t-Boc) protected 6-aminohexanoic acid, followed by deprotection of the t-Boc groups with 4 N HCl in dioxane. Acylation of this symmetric diamine with N-hydroxysuccinimide esters of appropriately substituted benzoic acids, followed by reduction of the resultant tetraamides with diborane in refluxing THF, afforded the target compounds. The BXT analog lacking the benzylic group (i.e., compound 11) had no [3H]NPY displacement activity at concentrations up to 1.4 x 10(-3) M. The 9-fold range in activities observed for the ortho, meta, and para regioisomers of the methoxy, chloro, and hydroxy benextramine analogs at benextramine-sensitive NPY rat brain binding sites does not differ from the range of potencies observed at alpha-adrenoceptors. However, the order of potencies at [3H]NPY sites differs from the order of potencies at alpha-adrenoceptors, with the m-methoxyphenyl (9a), m-hydroxyphenyl (10b), and 2-naphthyl (9f) analogs being the most active at [3H]NPY binding sites. The present results demonstrate the importance of the benzylic moiety for BXT's NPY antagonist activity, and suggest that the BXT binding site on the NPY receptor is significantly distinct from that on the alpha-adrenoceptor.