Continued Tumor Reduction of Metastatic Pheochromocytoma/Paraganglioma Harboring Succinate Dehydrogenase Subunit B Mutations with Cyclical Chemotherapy

Patients harboring germline mutations in the succinate dehydrogenase complex subunit B (SDHB) gene present with pheochromocytomas and paragangliomas (PPGL) that are more likely malignant and clinically aggressive. The combination chemotherapy cyclophosphamide, vincristine, and dacarbazine (CVD) was retrospectively evaluated in patients with SDHB-associated metastatic PPGL.Query Twelve metastatic PPGL patients harboring SDHB mutations/polymorphisms with undetectable SDHB immunostaining were treated with CVD. CVD therapy consisted of 750 mg/m² cyclophosphamide with 1.4 mg/m² vincristine on day 1 and 600 mg/m² dacarbazine on days 1 and 2, every 21–28 days. Treatment outcome was determined by RECIST criteria as well as determination of response duration and progression-free and overall survivals. A median of 20.5 cycles (range 4–41) was administered. All patients had tumor reduction (12–100% by RECIST). Complete response was seen in two patients, while partial response was observed in 8. The median number of cycles to response was 5.5. Median duration of response was 478 days, with progression-free and overall survivals of 930 and 1190 days, respectively. Serial [¹⁸F]-fluorodeoxyglucose positron emission tomography and computed tomography imaging demonstrated continued incremental reduction in maximal standardized uptake values (SUV_max) values in 26/30 lesions. During treatment administration, the median SUV decreased from?> 25 to?< 6, indicating the efficacy of chemotherapy over a prolonged period of time. Prolonged therapy results in continued incremental tumor reduction, and is consistent with persistent drug sensitivity. CVD chemotherapy is recommended to be considered part of the initial management in patients with metastatic SDHB-related PPGL.     

“Protein aggregates” contain RNA and DNA,entrapped by misfolded proteins but largely rescued by slowing translational elongation

All neurodegenerative diseases feature aggregates, which usually contain disease‐specific diagnostic proteins; non‐protein constituents, however, have rarely been explored. Aggregates from SY5Y‐APP_Sw neuroblastoma, a cell model of familial Alzheimer''s disease, were crosslinked and sequences of linked peptides identified. We constructed a normalized “contactome” comprising 11 subnetworks, centered on 24 high‐connectivity hubs. Remarkably, all 24 are nucleic acid‐binding proteins. This led us to isolate and sequence RNA and DNA from Alzheimer''s and control aggregates. RNA fragments were mapped to the human genome by RNA‐seq and DNA by ChIP‐seq. Nearly all aggregate RNA sequences mapped to specific genes, whereas DNA fragments were predominantly intergenic. These nucleic acid mappings are all significantly nonrandom, making an artifactual origin extremely unlikely. RNA (mostly cytoplasmic) exceeded DNA (chiefly nuclear) by twofold to fivefold. RNA fragments recovered from AD tissue were ~1.5‐to 2.5‐fold more abundant than those recovered from control tissue, similar to the increase in protein. Aggregate abundances of specific RNA sequences were strikingly differential between cultured SY5Y‐APP_Sw glioblastoma cells expressing APOE3 vs. APOE4, consistent with APOE4 competition for E‐box/CLEAR motifs. We identified many G‐quadruplex and viral sequences within RNA and DNA of aggregates, suggesting that sequestration of viral genomes may have driven the evolution of disordered nucleic acid‐binding proteins. After RNA‐interference knockdown of the translational‐procession factor EEF2 to suppress translation in SY5Y‐APP_Sw cells, the RNA content of aggregates declined by >90%, while reducing protein content by only 30% and altering DNA content by ≤10%. This implies that cotranslational misfolding of nascent proteins may ensnare polysomes into aggregates, accounting for most of their RNA content.     

Effects of peptide YY and its analogues on chloride ion secretion in fed and fasted rat jejunum

The aim of our study was to determine whether a meal modifies the antisecretory response induced by PYY and the structural requirements to elicit antisecretory effects of analogue PYY(22–36) for potential antidiarrhea therapy. The variations in short-circuit current (Isc) due to the modification of ionic transport across the rat intestine were assessed in vitro, using Ussing chambers. In fasted rats, PYY induced a dose- and time-dependent reduction in Isc, with a sensitivity threshold at 5 × 10⁻¹¹ M (ΔIsc −2 ± 0.5 μA/cm²). The reduction was maximal at 10⁻⁷ M (Isc −23 ± 2 μA/cm²), and the concentration producing half-maximal inhibition was 10⁻⁹ M. At 10⁻⁷ M, reduction of Isc by PYY reached 90% of response to 5 × 10⁻⁵ M bumetanide. The PYY effect was partly reversed by 10⁻⁵ M forskolin (Isc +13.43 ± 2.91 μA/h·cm², p < 0.05) or 10⁻³ M dibutyryl adenosine 3′,5′ cyclic monophosphate (Isc +12 ± 1.69 μA/cm², p < 0.05). Naloxone and tetrodotoxin did not alter the effect of PYY. In addition, PYY and its analogue P915 reduced net chloride ion secretion to 2.85 and 2.29 μEq/cm² (p < 0.05), respectively. The antisecretory effect of PYY was accompanied by dose- and time-dependent desensitization when jejunum was prestimulated by a lower dose of peptide. The antisecretory potencies exhibited by PYY analogues required both a C-terminal fragment (22–36) and an aromatic amino acid residue (Trp or Phe) at position 27. At 10⁻⁷ M the biological activity of PYY was lower in fed than fasted rats (p < 0.001). Our results confirm the antisecretory effect of PYY, but show that the fed period is accompanied by desensitization, similar to the transient desensitization observed in the fasted period with cumulative doses. This suggests that PYY may act as a physiological mediator that reduces intestinal secretion.     

Using a 3-O-sulfated heparin octasaccharide to inhibit the entry of herpes simplex virus type 1

Heparan sulfate (HS) is a highly sulfated polysaccharide and is present in large quantities on the cell surface and in the extracellular matrix. Herpes simplex virus type 1 (HSV-1) utilizes a specialized cell surface HS, known as 3-O-sulfated HS, as an entry receptor to establish infection. Here, we exploit an approach to inhibiting HSV-1 infection by using a 3-O-sulfated octasaccharide, mimicking the active domain of the entry receptor. The 3-O-sulfated octasaccharide was synthesized by incubating a heparin octasaccharide (3-OH octasaccharide) with HS 3-O-sulfotransferase isoform 3. The resultant 3-O-sulfated octasaccharide has a structure of Delta UA2S-GlcNS6S-IdoUA2S-GlcNS6S-IdoUA2S-GlcNS3S6S-IdoUA2S-GlcNS6S (where Delta UA is 4-deoxy-alpha-L-threo-hex-4-enopyranosyluronic acid, GlcN is D-glucosamine, and IdoUA is L-iduronic acid). Results from cell-based assays revealed that the 3-O-sulfated octasaccharide has stronger activity in blocking HSV-1 infection than that of the 3-OH octasaccharide, suggesting that the inhibition of HSV-1 infection requires a unique sulfation moiety. Our results suggest the feasibility of inhibiting HSV-1 infection by blocking viral entry with a specific oligosaccharide.     

Connecting species’ geographical distributions to environmental variables: range maps versus observed points of occurrence

Connecting the geographical occurrence of a species with underlying environmental variables is fundamental for many analyses of life history evolution and for modeling species distributions for both basic and practical ends. However, raw distributional information comes principally in two forms: points of occurrence (specific geographical coordinates where a species has been observed), and expert-prepared range maps. Each form has potential short-comings: range maps tend to overestimate the true occurrence of a species, whereas occurrence points (because of their frequent non-random spatial distribution) tend to underestimate it. Whereas previous comparisons of the two forms have focused on how they may differ when estimating species richness, less attention has been paid to the extent to which the two forms actually differ in their representation of a species’ environmental associations. We assess such differences using the globally distributed avian order Galliformes (294 species). For each species we overlaid range maps obtained from IUCN and point-of-occurrence data obtained from GBIF on global maps of four climate variables and elevation. Over all species, the median difference in distribution centroids was 234 km, and median values of all five environmental variables were highly correlated, although there were a few species outliers for each variable. We also acquired species’ elevational distribution mid-points (mid-point between minimum and maximum elevational extent) from the literature; median elevations from point occurrences and ranges were consistently lower (median −420 m) than mid-points. We concluded that in most cases occurrence points were likely to produce better estimates of underlying environmental variables than range maps, although differences were often slight. We also concluded that elevational range mid-points were biased high, and that elevation distributions based on either points or range maps provided better estimates.     

Quinonoid and other constituents of Aristea ecklonii

Plumbagin, 3,3′-biplumbagin, 8,8′-biplumbagin, neoisoshinanolone and sitosterol were isolated from the leaves and rhizomes of Aristea ecklonii. The rhizomes also contained α-spinasterol. This is the first report of plumbagin, previously found in several dicotyledonous families, in a monocotyledon.     

Deciphering the mode of action,structural and biochemical analysis of heparinase II/III (<Emphasis Type="Italic">Ps</Emphasis>PL12a) a new member of family 12 polysaccharide lyase from <Emphasis Type="Italic">Pseudopedobacter saltans</Emphasis>

Heparinases are widely used for production of clinically and therapeutically important bioactive oligosaccharides and in analyzing the polydisperse, heterogeneous, and complex structures of heparin/heparan sulfate. In the present study, the gene (1911 bp) encoding heparinase II/III of family 12 polysaccharide lyase (PsPL12a) from Pseudopedobacter saltans was cloned, expressed, and biochemically and functionally characterized. The purified enzyme PsPL12a of molecular size approximately 76 kDa exhibited maximum activity in the temperature range 45–50 °C and at pH 6.0. PsPL12a gave maximum activity at 1% (w/v) heparin under optimum conditions. The kinetic parameters, K _m and V_max, for PsPL12a were 4.6?±?0.5 mg/ml and 70?±?2 U/mg, respectively. Ten millimolars of each Mg²⁺ and Mn²⁺ ions enhanced PsPL12a activity by 80%, whereas Ni²⁺ inhibited by 75% and Co²⁺ by 10%, and EDTA completely inactivated the enzyme. Protein melting curve of PsPL12a gave a single peak at 55 °C and 10 mM Mg²⁺ ions and shifted the peak to 60 °C. The secondary structure analysis of PsPL12a by CD showed 65.12% α-helix, 11.84% β-strand, and 23.04% random coil. The degradation products of heparin by PsPL12a analyzed by ESI-MS spectra displayed peaks corresponding to heparin di-, tetra-, penta-, and hexa-saccharides revealing the endolytic mode of enzyme action. Heparinase II/III (PsPL12a) from P. saltans can be used for production of low molecular weight heparin oligosaccharides for their utilization as anticoagulants. This is the first report on heparinase cloned from P. saltans.     

Specificity of HCPTP variants toward EphA2 tyrosines by quantitative selected reaction monitoring

EphA2 receptor tyrosine kinase and the human cytoplasmic protein tyrosine phosphatase (HCPTP) are overexpressed in a number of epithelial cancers. Overexpressed EphA2 in these cancers shows a significant decrease in phosphotyrosine content which results in suppression of receptor signaling and endocytosis and an increase in metastatic potential. The decreased phosphotyrosine content of EphA2 has been associated with decreased contact with its ligand, ephrin A1 and dephosphorylation by HCPTP. Potential specificity of the two HCPTP variants for tyrosines on EphA2 has not been investigated. We have used a mass spectrometry assay to measure relative rates of dephosphorylation for the two HCPTP variants at phosphotyrosine sites associated with control of the EphA2 kinase activity or interaction with downstream targets. Our results suggest that although both variants dephosphorylate the EphA2 receptor, the rate and specificity of dephosphorylation for specific tyrosines are different for HCPTP-A and HCPTP-B. The SAM domain tyrosine Y960 which has been implicated in downstream PI3K signaling is dephosphorylated exclusively by HCPTP-B. The activation loop tyrosine (Y772) which directly controls kinase activity is dephosphorylated about six times faster by HCPTP-A. In contrast, the juxtamembrane tyrosines (Y575, Y588 and Y594) which are implicated in both control of kinase activity and downstream signaling are dephosphorylated by both variants with similar rates. This difference in preference for dephosphorylation sites on EphA2 not only illuminates the different roles of the two variants of the phosphatase in EphA2 signaling, but also explains why both HCPTP variants are highly conserved in most mammals.     

N-α-biotinylated-neuropeptide Y analogs: Syntheses, cardiovascular properties, and application to cardiac NPY receptor visualization

Two monobiotinylated analogs of neuropeptide Y (NPY) were synthesized by coupling the N-hydroxysuccinimidyl esters of biotin and (6-biotinylamido)-hexanoic acid, respectively, to the free alpha-NH2 group of the side chain protected NPY peptide resin. Crude peptides obtained by HF cleavage were purified by RPLC and their integrities were confirmed by amino acid and mass spectral analysis. As with NPY, both biotinylated analogs inhibited 125I-NPY binding and adenylate cyclase activity of rat cardiac ventricular membranes in a dose-dependent manner. N-alpha-[(6-biotinylamido)-hexanoyl]-NPY exhibited potencies comparable to that of NPY whereas N-alpha-biotinyl-NPY was slightly less potent. In the in vivo experiments, however, both the biotinylated analogs exhibited responses comparable to NPY in increasing arterial blood pressure and decreasing heart rate in anesthetized rats. The responses of the biotinyl analogs were longer lasting than those of NPY. Histochemical studies revealed that N-alpha-[(6-biotinylamido)-hexanoyl]-NPY could label the NPY receptors in rat cardiac ventricular tissues. This labeling was specific since intact NPY inhibited the staining. These studies show that biotinyl-NPY analogs exhibit biological potencies comparable to intact NPY and can therefore be used to further probe the NPY-receptor interaction.     

Interaction of NPY compounds with the rat glucocorticoid-induced receptor (GIR) reveals similarity to the NPY-Y2 receptor

The rat glucocorticoid-induced receptor (rGIR) is an orphan G protein-coupled receptor awaiting pharmacological characterization. Among known receptors, rGIR exhibits highest sequence similarity to the neuropeptide Y (NPY)-Y(2) receptor (38-40%). The pharmacological profile of rGIR was investigated using (125)I-PYY(3-36), a Y(2)-preferring radioligand and several NPY analogs. rGIR displayed a similar displacement profile as reported for the Y(2) receptor, in that the Y(2)-selective C terminus fragments of NPY and PYY (NPY(3-36) and PYY(3-36)) showed high affinity binding and activation of rGIR (low nanomolar range). The rank order potency for displacement was NPY(3-36)>PYY(3-36)=NPY>NPY(13-36)>Ac, Leu NPY(24-36)>[D-Trp(32)]-NPY>Leu(31), Pro(34)-NPY=hPP. NPY and Y(2)-selective agonists NPY(3-36) and PYY(3-36) led to significant activation of (35)S-GTPgammaS binding to rGIR transfected cells. BIIE0246, a specific Y(2) antagonist, displaced (125)I-PYY(3-36) binding to rGIR with high affinity (95nM). Activation of (35)S-GTPgammaS binding by Y(2)-selective agonist in rGIR transfected cells was also completely abolished by BIIE0246. Our data report, for the first time, an interaction of NPY ligands with rGIR expressed in vitro, and indicate similarities between GIR and the NPY-Y(2) receptor.