Disruption of the anterior commissure in Olig2 deficient mice.

In the present study, we examined neural circuit formation in the forebrain of the Olig2 knockout (Olig2-KO) mouse model and found disruption of the anterior commissure at the late foetal stage. Axon bundles of the anterior commissure encountered the wall of the third ventricle and ceased axonal extension. L1-CAM immunohistochemistry showed that Olig2-KO mice lose decussation formation in the basal forebrain. DiI tracing revealed that the thin bundles of the anterior commissure axons crossed the midline but ceased further extension into the deep part of the contralateral side. Furthermore, some fractions of DiI-labelled axons were oriented dorsolaterally, which was not observed in the control mouse forebrain. The rostral part of the third ventricle was much wider in the Olig2-KO mice than in wild-type mice, which likely resulted in the delay of midline fusion and subsequent delay and malformation of the anterior commissure. We analysed gene expression alterations in the Olig2-KO mice using a public database and found multiple genes, which are related to axon guidance and epithelial-mesenchymal transition, showing subtle expression changes. These results suggest that Olig2 is essential for anterior commissure formation, likely by regulating multiple biological processes.

Cystathionine γ-Lyase Self-Inactivates by Polysulfidation during Cystine Metabolism.

Cystathionine γ-lyase (CSE) is an enzyme responsible for the biosynthesis of cysteine from cystathionine in the final step of the transsulfuration pathway. It also has ß-lyase activity toward cystine, generating cysteine persulfide (Cys-SSH). The chemical reactivity of Cys-SSH is thought to be involved in the catalytic activity of particular proteins via protein polysulfidation, the formation of -S-(S)n-H on their reactive cysteine residues. The Cys136/171 residues of CSE have been proposed to be redox-sensitive residues. Herein, we investigated whether CSE polysulfidation occurs at Cys136/171 during cystine metabolism. Transfection of wild-type CSE into COS-7 cells resulted in increased intracellular Cys-SSH production, which was significantly increased when Cys136Val or Cys136/171Val CSE mutants were transfected, instead of the wild-type enzyme. A biotin-polyethylene glycol-conjugated maleimide capture assay revealed that CSE polysulfidation occurs at Cys136 during cystine metabolism. In vitro incubation of CSE with CSE-enzymatically synthesized Cys-SSH resulted in the inhibition of Cys-SSH production. In contrast, the mutant CSEs (Cys136Val and Cys136/171Val) proved resistant to inhibition. The Cys-SSH-producing CSE activity of Cys136/171Val CSE was higher than that of the wild-type enzyme. Meanwhile, the cysteine-producing CSE activity of this mutant was equivalent to that of the wild-type enzyme. It is assumed that Cys-SSH-producing CSE activity could be auto-inactivated via the polysulfidation of the enzyme during cystine metabolism. Thus, the polysulfidation of CSE at the Cys136 residue may be an integral feature of cystine metabolism, which functions to down-regulate Cys-SSH synthesis by the enzyme.

Conserved and divergent functions of Pax6 underlie species-specific neurogenic patterns in the developing amniote brain.

The evolution of unique organ structures is associated with changes in conserved developmental programs. However, characterizing the functional conservation and variation of homologous transcription factors (TFs) that dictate species-specific cellular dynamics has remained elusive. Here, we dissect shared and divergent functions of Pax6 during amniote brain development. Comparative functional analyses revealed that the neurogenic function of Pax6 is highly conserved in the developing mouse and chick pallium, whereas stage-specific binary functions of Pax6 in neurogenesis are unique to mouse neuronal progenitors, consistent with Pax6-dependent temporal regulation of Notch signaling. Furthermore, we identified that Pax6-dependent enhancer activity of Dbx1 is extensively conserved between mammals and chick, although Dbx1 expression in the developing pallium is highly divergent in these species. Our results suggest that spatiotemporal changes in Pax6-dependent regulatory programs contributed to species-specific neurogenic patterns in mammalian and avian lineages, which underlie the morphological divergence of the amniote pallial architectures.

Development of potent antipseudomonal ß-lactams by means of polycarboxylation of aminopenicillins.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that presents a serious risk to immunosuppressed individuals and other extremely vulnerable patients such as those in intensive care units. The emergence of multidrug-resistant Pseudomonas strains has increased the need for new antipseudomonal agents. In this study, a series of amino group-modified aminopenicillin derivatives was synthesized that have different numbers of carboxyl groups and structurally resemble carboxypenicillin-ureidopenicillin hybrids, and their antipseudomonal activities were evaluated. Among the derivatives synthesized, diethylenetriaminepentaacetic acid (DTPA)-modified amoxicillin (DTPA-Amox) showed potent antipseudomonal activity, not only against the laboratory strain PAO1 but also against clinically isolated Pseudomonas strains that were resistant to piperacillin and carbenicillin. DTPA-Amox had no obvious cytotoxic effects on cultured mammalian cells. In addition, in an in vivo model of leukopenia, DTPA-Amox treatment produced a moderate but statistically significant improvement in the survival of mice with P. aeruginosa strain PAO1 infection. These data suggest that polycarboxylation by DTPA conjugation is an effective approach to enhance antipseudomonal activity of aminopenicillins.

[A case of scirrhous gastric cancer in which cancer cells were first identified in the ascites by immunostaining using the cell transfer technique].

This was a case of a woman in her 60s with the chief complaint of an abnormal stomach X-ray at the screening. Although suspected to be scirrhous gastric cancer, gastric biopsy revealed Group 1, and cytology in accumulated ascites and open surgery was initially Class II, but cancer cells in the ascites were confirmed for the first time by subsequent immunostaining using the cell transfer technique. Undifferentiated advanced gastric cancer, peritoneal dissemination, and lymphatic metastasis were pathologically observed. This case suggests the effectiveness of immunostaining when the results of ascites cytology are different from the clinical picture.

Involvement of protein disulfide isomerase in subtilase cytotoxin-induced cell death in HeLa cells.

Subtilase cytotoxin (SubAB) is a member of bacterial AB5 toxin produced by certain enterohemorrhagic E. coli strains which cleaves host chaperone BiP in endoplasmic reticulum (ER), leading to ER stress-mediated cytotoxicity. Previous study suggested that protein disulfide isomerase (PDI), an enzyme which catalyzes the formation and breakage of disulfide bonds in proteins, regulates AB5 toxin such as cholera toxin by unfolding of A subunit, leading to its translocation into cytosol to induce disease. Although SubAB targets ER and has similar A subunit to that of other AB5 toxins, it is unclear whether PDI can modulate the SubAB function. Here we determined the role of PDI on SubAB-induced BiP cleavage, ER stress response and cytotoxicity in HeLa cells. We found that PDI knockdown significantly suppressed SubAB-induced BiP cleavage and eIF2α phosphorylation. The accumulation of SubAB in ER was perturbed upon PDI knockdown. Finally, cell viability assay showed that PDI knockdown and PDI inhibitor canceled the SubAB-induced cytotoxicity. Present results suggested that SubAB, after cellular uptake, translocates into ER and interacts with BiP that might be modulated by PDI. Identification of pivotal role of host proteins on bacterial toxin to elicit its pathogenesis is necessary basis for development of potential chemotherapy and new diagnostic strategy for control of toxin-producing bacterial infections.

The evolution of basal progenitors in the developing non-mammalian brain.

The amplification of distinct neural stem/progenitor cell subtypes during embryogenesis is essential for the intricate brain structures present in various vertebrate species. For example, in both mammals and birds, proliferative neuronal progenitors transiently appear on the basal side of the ventricular zone of the telencephalon (basal progenitors), where they contribute to the enlargement of the neocortex and its homologous structures. In placental mammals, this proliferative cell population can be subdivided into several groups that include Tbr2(+) intermediate progenitors and basal radial glial cells (bRGs). Here, we report that basal progenitors in the developing avian pallium show unique morphological and molecular characteristics that resemble the characteristics of bRGs, a progenitor population that is abundant in gyrencephalic mammalian neocortex. Manipulation of LGN (Leu-Gly-Asn repeat-enriched protein) and Cdk4/cyclin D1, both essential regulators of neural progenitor dynamics, revealed that basal progenitors and Tbr2(+) cells are distinct cell lineages in the developing avian telencephalon. Furthermore, we identified a small population of subapical mitotic cells in the developing brains of a wide variety of amniotes and amphibians. Our results suggest that unique progenitor subtypes are amplified in mammalian and avian lineages by modifying common mechanisms of neural stem/progenitor regulation during amniote brain evolution.

Impact of Topoisomerase IIα, PTEN, ABCC1/MRP1, and KI67 on triple-negative breast cancer patients treated with neoadjuvant chemotherapy.

PURPOSE: Triple-negative breast cancer (TNBC) patients with residual disease following neoadjuvant chemotherapy (NAC) harbor higher risk of relapse, and eventual demise compared to those who achieve pathologic complete response. Therefore, in this study, we assessed a panel of molecules involved in key pathways of drug resistance and tumor progression before and after NAC in TNBC patients, in order to clarify the underlying mechanisms. METHODS: We studied 148 TNBC Japanese patients treated with anthracycline/taxane-based NAC. KI67, Topoisomerase IIα (TopoIIα), PTEN, p53, Bcl2, vimentin, ABCG2/BCRP1, ABCB1/MDR1, and ABCC1/MRP1 were immunolocalized in surgical pathology materials before and after NAC. RESULTS: The status of vimentin and increasing labeling index (LI) of TopoIIα and KI67 in biopsy specimens were significantly associated with those who responded to NAC treatment. The abundance of p53 (p = 0.003), ABCC1/MRP1 (p = 0.033), ABCB1/MDR1 (p = 0.022), and a loss of PTEN (p < 0.0001) in surgery specimens following treatment were associated with pathologic parameters. TopoIIα, PTEN, and ABCC1/MRP1 status predicted pathologic response. In addition, the status of PTEN, ABCC1/MRP1, ABCB1/MDR1, Bcl2, and vimentin in surgical specimens was also significantly associated with adverse clinicopathological factors in surgery specimens, suggesting that these alterations could be responsible for tumor relapse in TNBC patients. CONCLUSION: KI67, TopoIIα, PTEN, and ABCC1/MRP1 status could predict treatment response and/or eventual clinical outcomes. These results could also provide an insight into the mechanisms of drug resistance and relapse of TNBC patients receiving NAC.

Synthesis of Pegylated Manganese Protoporphyrin as a Catalase Mimic and Its Therapeutic Application to Acetaminophen-Induced Acute Liver Failure.

Metalloporphyrin derivatives have been investigated for their therapeutic potential for oxidative stress-related diseases because of their scavenging of reactive oxygen species (ROS). Here, we describe the synthesis, physicochemical properties, and ROS-scavenging activities of one such derivative-polyethylene glycol (PEG)-conjugated manganese protoporphyrin (PEG-MnPP). Carboxyl groups of the protoporphyrin ring at the C6 and C7 positions were first conjugated with ethylenediamine to introduce amino groups into the protoporphyrin structure. The amino groups were then reacted with succinimidyl PEG, with an average molecular weight of 2000, to obtain pegylated protoporphyrin (PEG-PP). Manganese was chelated to the protoporphyrin ring by incubating PEG-PP and manganese acetate in methanol. Dynamic light scattering and fluorescent spectrometry analyses revealed that PEG-MnPP self-assembled into nanoparticles in aqueous media with an apparent diameter of 70 nm. PEG-MnPP effectively eliminated hydrogen peroxide from cell culture media and protected cultured mammalian cells from toxic insults induced by hydrogen peroxide exposure or by 6-hydroxydopamine treatment. Intravenous administration of PEG-MnPP to mice significantly suppressed acute liver failure that had been induced by acetaminophen overdose. These data warrant additional investigation to study the therapeutic potential of PEG-MnPP as a water-soluble metalloporphyrin-based catalase mimic for oxidative stress-associated diseases.

The significance of lipid accumulation in breast carcinoma cells through perilipin 2 and its clinicopathological significance.

Both systemic and intratumoral lipid metabolism have been recently reported to play pivotal roles in both tumor development and progression in various human malignancies including breast cancer. However, its details have remained largely unknown in breast cancer patients. Therefore, in this study, we focused on perilipin 2, which is involved in constituting the intracellular lipid composition. Perilipin 2 was first immunolocalized in 105 cases of breast cancer. The status of perilipin 2 immunoreactivity was significantly positively associated with histological grade, Ki-67 labeling index and HER2 status and negatively with estrogen receptor status of these patients. Subsequent in vitro study also revealed that its mRNA expression in triple negative breast carcinoma cells was higher than cells of other subtypes. We then examined the correlation between perilipin 2 immunoreactivity and intracellular lipid droplet evaluated by Oil-red O stating in 13 cases of breast carcinoma tissues. A significantly positive correlation was detected between the status of perilipin 2 and Oil-red O staining. These findings above did indicate that perilipin 2 could represent the status of intracellular lipid droplets in surgical pathology specimens of breast cancer and perilipin 2 was also associated with its more aggressive biological phenotypes.

NG2 expression in NG2 glia is regulated by binding of SoxE and bHLH transcription factors to a Cspg4 intronic enhancer.

NG2 is a type 1 integral membrane glycoprotein encoded by the Cspg4 gene. It is expressed on glial progenitor cells known as NG2 glial cells or oligodendrocyte precursor cells that exist widely throughout the developing and mature central nervous system and vascular mural cells but not on mature oligodendrocytes, astrocytes, microglia, neurons, or neural stem cells. Hence NG2 is widely used as a marker for NG2 glia in the rodent and human. The regulatory elements of the mouse Cspg4 gene and its flanking sequences have been used successfully to target reporter and Cre recombinase to NG2 glia in transgenic mice when used in a large 200 kb bacterial artificial chromosome cassette containing the 38 kb Cspg4 gene in the center. Despite the tightly regulated cell type- and stage-specific expression of NG2 in the brain and spinal cord, the mechanisms that regulate its transcription have remained unknown. Here, we describe a 1.45 kb intronic enhancer of the mouse Cspg4 gene that directed transcription of EGFP reporter to NG2 glia but not to pericytes in vitro and in transgenic mice. The 1.45 kb enhancer contained binding sites for SoxE and basic helix-loop-helix transcription factors, and its enhancer activity was augmented cooperatively by these factors, whose respective binding elements were found in close proximity to each other. Mutations in these binding elements abrogated the enhancer activity when tested in the postnatal mouse brain.

The Future Vocation of Neural Stem Cells: Lineage Commitment in Brain Development and Evolution.

Understanding the fate commitment of neural stem cells is critical to identify the regulatory mechanisms in developing brains. Genetic lineage-tracing has provided a powerful strategy to unveil the heterogeneous nature of stem cells and their descendants. However, recent studies have reported controversial data regarding the heterogeneity of neural stem cells in the developing mouse neocortex, which prevents a decisive conclusion on this issue. Here, we review the progress that has been made using lineage-tracing analyses of the developing neocortex and discuss stem cell heterogeneity from the viewpoint of comparative and evolutionary biology.

Origin of Oligodendrocytes in the Vertebrate Optic Nerve: A Review.

One of the unsolved problems in the research field of oligodendrocyte (OL) development has been the site(s) of origin of optic nerve OLs and its precursor cells (OPCs). It is generally accepted that OLs in the optic nerve are derived from the brain, and thus optic nerve OLs are immigrant cells. We previously demonstrated the brain origin of optic nerve OPCs in chick embryos. However, the site of optic nerve OPC origin has not been examined experimentally in developing rodents for the past two decades. We have recently reported that optic nerve OPCs in mice arise in the preoptic area by E12.5 and gradually migrate caudally and enter the optic nerve. These OPCs give rise to myelinating OLs in the optic nerve in the postnatal or adult stages. Surprisingly, there are species differences with respect to the origin of optic nerve OPCs between chicks and mice. Here, we summarize the site of OPC origin in the optic nerve based on our own previous and recent results, and discuss possible mechanisms underlying these species differences.

Gold Coating of Silver Nanoplates for Enhanced Dispersion Stability and Efficient Antimicrobial Activity against Intracellular Bacteria.

Silver nanoparticles have antibacterial activity. However, the nanoparticles are unstable and easily form aggregates, which decreases their antibacterial activity. To improve the dispersion stability of silver nanoparticles in aqueous media and to increase their effectiveness as antibacterial agents, we coated triangular plate-like silver nanoparticles (silver nanoplates, Ag NPLs) with one or two layers of gold atoms (Ag@Au1L NPLs and Ag@Au2L NPLs, respectively). These gold coatings improved the dispersion stability in aqueous media with high salt concentrations. Ag@Au1L NPLs showed stronger antibacterial activity on pathogenic bacteria than Ag NPLs and Ag@Au2L NPLs. Furthermore, the Ag@Au1L NPLs decreased the number of bacteria in RAW 264.7 cells. The Ag@Au1L NPLs displayed no cytotoxicity towards RAW 264.7 cells and could be used as antibacterial agents for intracellular bacterial infections.

Exploring Protein⁻Protein Interaction in the Study of Hormone-Dependent Cancers.

Estrogen receptors promote target gene transcription when they form a dimer, in which two identical (homodimer) or different (heterodimer) proteins are bound to each other. In hormone-dependent cancers, hormone receptor dimerization plays pivotal roles, not only in the pathogenesis or development of the tumors, but also in the development of therapeutic resistance. Protein⁻protein interactions (PPIs), including dimerization and complex formation, have been also well-known to be required for proteins to exert their functions. The methods which could detect PPIs are genetic engineering (i.e., resonance energy transfer) and/or antibody technology (i.e., co-immunoprecipitation) using cultured cells. In addition, visualization of the target proteins in tissues can be performed using antigen⁻antibody reactions, as in immunohistochemistry. Furthermore, development of microscopic techniques (i.e., electron microscopy and confocal laser microscopy) has made it possible to visualize intracellular and/or intranuclear organelles. We have recently reported the visualization of estrogen receptor dimers in breast cancer tissues by using the in situ proximity ligation assay (PLA). PLA was developed along the lines of antibody technology development, and this assay has made it possible to visualize PPIs in archival tissue specimens. Localization of PPI in organelles has also become possible using super-resolution microscopes exceeding the resolution limit of conventional microscopes. Therefore, in this review, we summarize the methodologies used for studying PPIs in both cells and tissues, and review the recently reported studies on PPIs of hormones.

Sulfatide species with various fatty acid chains in oligodendrocytes at different developmental stages determined by imaging mass spectrometry.

HSO3-3-galactosylceramide (Sulfatide) species comprise the major glycosphingolipid components of oligodendrocytes and myelin and play functional roles in the regulation of oligodendrocyte maturation and myelin formation. Although various sulfatide species contain different fatty acids, it is unclear how these sulfatide species affect oligodendrogenesis and myelination. The O4 monoclonal antibody reaction with sulfatide has been widely used as a useful marker for oligodendrocytes and myelin. However, sulfatide synthesis during the pro-oligodendroblast stage, where differentiation into the oligodendrocyte lineage has already occurred, has not been examined. Notably, this stage comprises O4-positive cells. In this study, we identified a sulfatide species from the pro-oligodendroblast-to-myelination stage by imaging mass spectrometry. The results demonstrated that short-chain sulfatides with 16 carbon non-hydroxylated fatty acids (C16) and 18 carbon non-hydroxylated fatty acids (C18) or 18 carbon hydroxylated fatty acids (C18-OH) existed in restricted regions of the early embryonic spinal cord, where pro-oligodendroblasts initially appear, and co-localized with Olig2-positive pro-oligodendroblasts. C18 and C18-OH sulfatides also existed in isolated pro-oligodendroblasts. C22-OH sulfatide became predominant later in oligodendrocyte development and the longer C24 sulfatide was predominant in the adult brain. Additionally, the presence of each sulfatide species in a different area of the adult brain was demonstrated by imaging mass spectrometry at an increased lateral resolution. These findings indicated that O4 recognized sulfatides with short-chain fatty acids in pro-oligodendroblasts. Moreover, the fatty acid chain of the sulfatide became longer as the oligodendrocyte matured. Therefore, individual sulfatide species may have unique roles in oligodendrocyte maturation and myelination. Read the Editorial Highlight for this article on page 356.

Development of the prethalamus is crucial for thalamocortical projection formation and is regulated by Olig2.

Thalamocortical axons (TCAs) pass through the prethalamus in the first step of their neural circuit formation. Although it has been supposed that the prethalamus is an intermediate target for thalamocortical projection formation, much less is known about the molecular mechanisms of this targeting. Here, we demonstrated the functional implications of the prethalamus in the formation of this neural circuit. We show that Olig2 transcription factor, which is expressed in the ventricular zone (VZ) of prosomere 3, regulates prethalamus formation, and loss of Olig2 results in reduced prethalamus size in early development, which is accompanied by expansion of the thalamic eminence (TE). Extension of TCAs is disorganized in the Olig2-KO dorsal thalamus, and initial elongation of TCAs is retarded in the Olig2-KO forebrain. Microarray analysis demonstrated upregulation of several axon guidance molecules, including Epha3 and Epha5, in the Olig2-KO basal forebrain. In situ hybridization showed that the prethalamus in the wild type excluded the expression of Epha3 and Epha5, whereas loss of Olig2 resulted in reduction of this Ephas-negative area and the corresponding expansion of the Ephas-positive TE. Dissociated cultures of thalamic progenitor cells demonstrated that substrate-bound EphA3 suppresses neurite extension from dorsal thalamic neurons. These results indicate that Olig2 is involved in correct formation of the prethalamus, which leads to exclusion of the EphA3-expressing region and is crucial for proper TCA formation. Our observation is the first report showing the molecular mechanisms underlying how the prethalamus acts on initial thalamocortical projection formation.

The Dorsoventral Boundary of the Germinal Zone is a Specialized Niche for the Generation of Cortical Oligodendrocytes during a Restricted Temporal Window.

Oligodendrocyte precursor cells (OPCs) appear in the late embryonic brain, mature to become oligodendrocytes (OLs), and form myelin in the postnatal brain. Recently, it has been proposed that early-born OPCs derived from the ventral forebrain are eradicated postnatally and that late-born OLs predominate in the cortex of the adult mouse brain. However, intrinsic and extrinsic factors that specify the ability of self-renewing multipotent neural stem cells in the embryonic brain to generate cortical OL-lineage cells remain largely unknown. Using an inducible Cre/loxP system to permanently label Nestin- and Olig2-lineage cells, we identified that cortical OL-lineage cells start differentiating from neural stem cells within a restricted temporal window just prior to E16.5 through P10. We then showed, by means of electroporation of a Cre expression plasmid into the VZ/SVZ of E15.5 reporter mouse brains, that neural precursor cells in the dorsal VZ/SVZ are inhibited by Wnt signaling from contributing to cortical OLs in the adult brain. In contrast, neural precursor cells present in the dorsoventral boundary VZ/SVZ produce a significant amount of OLs in the adult cortex. Our results suggest that neural stem cells at this boundary are uniquely specialized to produce myelin-forming OLs in the cortex.

Synthesis and Characterization of 8-Nitroguanosine 3',5'-Cyclic Monophosphorothioate Rp-Isomer as a Potent Inhibitor of Protein Kinase G1α.

Guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinases (PKG) are kinases regulating diverse physiological functions including vascular smooth muscle relaxation, neuronal synaptic plasticity, and platelet activities. Certain PKG inhibitors, such as Rp-diastereomers of derivatives of guanosine 3',5'-cyclic monophosphorothioate (Rp-cGMPS), have been designed and used to study PKG-regulated cell signaling. 8-Nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP) is an endogenous cGMP derivative formed as a result of excess production of reactive oxygen species and nitric oxide. 8-Nitro-cGMP causes persistent activation of PKG1α through covalent attachment of cGMP moieties to cysteine residues of the enzyme (i.e., the process called protein S-guanylation). In this study, we synthesized a nitrated analogue of Rp-cGMPS, 8-nitroguanosine 3',5'-cyclic monophosphorothioate Rp-isomer (Rp-8-nitro-cGMPS), and investigated its effects on PKG1α activity. We synthesized Rp-8-nitro-cGMPS by reacting Rp-8-bromoguanosine 3',5'-cyclic monophosphorothioate (Rp-8-bromo-cGMPS) with sodium nitrite. Rp-8-Nitro-cGMPS reacted with the thiol compounds cysteine and glutathione to form Rp-8-thioalkoxy-cGMPS adducts to a similar extent as did 8-nitro-cGMP. As an important finding, a protein S-guanylation-like modification was clearly observed, by using Western blotting, in the reaction between recombinant PKG1α and Rp-8-nitro-cGMPS. Rp-8-Nitro-cGMPS inhibited PKG1α activity with an inhibitory constant of 22 µM in a competitive manner. An organ bath assay with mouse aorta demonstrated that Rp-8-nitro-cGMPS inhibited vascular relaxation induced by acetylcholine or 8-bromo-cGMP more than Rp-8-bromo-cGMPS did. These findings suggest that Rp-8-nitro-cGMPS inhibits PKG through induction of an S-guanylation-like modification by attaching the Rp-cGMPS moiety to the enzyme. Additional study is warranted to explore the potential application of Rp-8-nitro-cGMPS to biochemical and therapeutic research involving PKG1α activation.

Reactive cysteine persulfides and S-polythiolation regulate oxidative stress and redox signaling.

Using methodology developed herein, it is found that reactive persulfides and polysulfides are formed endogenously from both small molecule species and proteins in high amounts in mammalian cells and tissues. These reactive sulfur species were biosynthesized by two major sulfurtransferases: cystathionine ß-synthase and cystathionine γ-lyase. Quantitation of these species indicates that high concentrations of glutathione persulfide (perhydropersulfide >100 µM) and other cysteine persulfide and polysulfide derivatives in peptides/proteins were endogenously produced and maintained in the plasma, cells, and tissues of mammals (rodent and human). It is expected that persulfides are especially nucleophilic and reducing. This view was found to be the case, because they quickly react with H2O2 and a recently described biologically generated electrophile 8-nitroguanosine 3',5'-cyclic monophosphate. These results indicate that persulfides are potentially important signaling/effector species, and because H2S can be generated from persulfide degradation, much of the reported biological activity associated with H2S may actually be that of persulfides. That is, H2S may act primarily as a marker for the biologically active of persulfide species.