Design of cyclic and d‐amino acids containing peptidomimetics for inhibition of protein‐protein interactions of HER2‐HER3

HER2 receptors are surface proteins belonging to the epidermal growth factor family of receptors. Their numbers are elevated in breast, lung, and ovarian cancers. HER2‐positive cancers are aggressive, have higher mortality rate, and have a poor prognosis. We have designed peptidomimetics that bind to HER2 and block the HER2‐mediated dimerization of epidermal growth factor family of receptors. Among these, a symmetrical cyclic peptidomimetic (compound 18 ) exhibited antiproliferative activity in HER2‐overexpressing lung cancer cell lines with IC₅₀ values in the nanomolar concentration range. To improve the stability of the peptidomimetic, d ‐amino acids were introduced into the peptidomimetic, and several analogs of compound 18 were designed. Among the analogs of compound 18 , compound 32 , a cyclic, d ‐amino acid‐containing peptidomimetic, was found to have an IC₅₀ value in the nanomolar range in HER2‐overexpressing cancer cell lines. The antiproliferative activity of compound 32 was also measured by using a 3D cell culture model that mimics the in vivo conditions. The binding of compound 32 to the HER2 protein was studied by surface plasmon resonance. In vitro stability studies indicated that compound 32 was stable in serum for 48 hours and intact peptide was detectable in vivo for 12 hours. Results from our studies indicated that 1 of the d ‐amino acid analogs of 18 , compound 32 , binds to the HER2 extracellular domain, inhibiting the phosphorylation of kinase of HER2.     

Effect of viscosity and dielectric constant variation on fractional fluorescence quenching analysis of coumarin dye in binary solvent mixtures

Photo physical properties of fluorescent organic compounds give an immense improved knowledge on characteristics of excited state that is beneficial to devise innovate molecules and understand their performance in particular applications. Coumarin derivatives have been extensively investigated in this regard. This article narrates steady state fluorescence quenching measurements of a coumarin derivative namely 3‐hydroxy‐3‐[2‐oxo‐2‐(3‐oxo‐3H‐benzo[f]chromen‐2‐yl)‐ethyl]‐1,3‐dihydro‐indol‐2‐one (3HBCD) in a binary mixture of acetonitrile and 1,4‐dioxane. Aniline is used as quencher. Fluorescence intensity is large in acetonitrile and decreases as the percentage of 1,4‐dioxane in the solvent mixture increases. With modest quencher concentration a deviation towards the x axis is noticed in the Stern–Volmer (S–V) plots. This downward curvature is interpreted as due to the presence of 3HBCD in different conformers in the lowest energy level. Ground state intramolecular hydrogen bonding formation is observed due to the conformational changes in the solute. Figured estimations of various quenching parameters recommend that, while dynamic quenching prompts linearity in S–V plot at lower quencher concentration, increasing quenching efficiency with increasing medium viscosity suggests that reaction is not entirely controlled by material diffusion. Stern–Volmer constant increases with decreasing medium dielectric constant.     

Autophagy regulates cisplatin‐induced stemness and chemoresistance via the upregulation of CD44, ABCB1 and ADAM17 in oral squamous cell carcinoma

Objective

We inspected the relevance of CD44, ABCB1 and ADAM17 in OSCC stemness and deciphered the role of autophagy/mitophagy in regulating stemness and chemoresistance.

Material and methods

A retrospective analysis of CD44, ABCB1 and ADAM17 with respect to the various clinico‐pathological factors and their correlation was analysed in sixty OSCC samples. Furthermore, the stemness and chemoresistance were studied in resistant oral cancer cells using sphere formation assay, flow cytometry and florescence microscopy. The role of autophagy/mitophagy was investigated by transient transfection of siATG14, GFP‐LC3, tF‐LC3, mKeima‐Red‐Mito7 and Western blot analysis of autophagic and mitochondrial proteins.

Results

In OSCC, high CD44, ABCB1 and ADAM17 expressions were correlated with higher tumour grades and poor differentiation and show significant correlation in their co‐expression. In vitro and OSCC tissue double labelling confirmed that CD44⁺ cells co‐expresses ABCB1 and ADAM17. Further, cisplatin (CDDP)‐resistant FaDu cells displayed stem‐like features and higher CD44, ABCB1 and ADAM17 expression. Higher autophagic flux and mitophagy were observed in resistant FaDu cells as compared to parental cells, and inhibition of autophagy led to the decrease in stemness, restoration of mitochondrial proteins and reduced expression of CD44, ABCB1 and ADAM17.

Conclusion

The CD44⁺/ABCB1⁺/ADAM17⁺ expression in OSCC is associated with stemness and chemoresistance. Further, this study highlights the involvement of mitophagy in chemoresistance and autophagic regulation of stemness in OSCC.     

Efficiency of RAPD,ISSR and ITS markers in detecting genetic variability among Salacia species sampled from the Western Ghats of Karnataka

Diversity and phylogenetic relationship between four closely related Salacia species, i.e., Salacia chinensis, Salacia macrosperma, Salacia fruticosa and Salacia oblonga, collected from the Western Ghats of Karnataka, India, was assessed. Ten each of RAPD and ISSR primers generated a total of 76 and 68 loci, generating polymorphisms of 92.21 and 89.71%, respectively. Maximum likelihood analysis of the ITS sequences revealed three clades. Dendrogram analyses of RAPD and ISSR revealed two and four clusters, respectively. Overall polymorphism revealed by RAPD was 41.45?±?10%, ISSR was 33.58?±?6.52%, and ITS was 25.50?±?17.25%. Molecular variance revealed significant variance within and among the Salacia species. Tajima’s D neutrality test and Fu’s Fs were negative for all four species, implying presences of rare alleles and population expansion. Comparative study of RAPD, ISSR and ITS for Salacia species has given an insight into the efficiency of each technique in detecting diversity within and among the population sampled in the Western Ghats of Karnataka.

     

Elucidation of the anticancer potential and tubulin isotype-specific interactions of β-sitosterol

Beta-sitosterol (β-SITO), a phytosterol present in many edible vegetables, has been reported to possess antineoplastic properties and cancer treatment potential. We have shown previously that it binds at a unique site (the ‘SITO-site’) compared to the colchicine binding site at the interface of α- and β-tubulin. In this study, we investigated the anticancer efficacy of β-SITO against invasive breast carcinoma using MCF-7 cells. Since ‘isotypes’ of β-tubulin show tissue-specific expression and many are associated with cancer drug resistance, using computer-assisted docking and atomistic molecular dynamic simulations, we also examined its binding interactions to all known isotypes of β-tubulin in αβ-tubulin dimer. β-SITO inhibited MCF-7 cell viability by up to 50%, compared to vehicle-treated control cells. Indicating its antimetastatic potential, the phytosterol strongly inhibited cell migration. Immunofluorescence imaging of β-SITO-treated MCF-7 cells exhibited disruption of the microtubules and chromosome organization. Far-UV circular dichroism spectra indicated loss of helical stability in tubulin when bound to β-SITO. Docking and MD simulation studies, combined with MM-PBSA and MM-GBSA calculations revealed that β-SITO preferentially binds with specific β-tubulin isotypes (β_II and β_III) in the αβ-tubulin dimer. Both these β-tubulin isotypes have been implicated in drug resistance against tubulin-targeted chemotherapeutics. Our data show the tubulin-targeted anticancer potential of β-SITO, and its potential clinical utility against β_II and β_III isotype-overexpressing neoplasms.     

Photorespiratory nitrogen cycle – A critical evaluation

The photorespiratory nitrogen cycle proposed by Keys et al. (Nature 275: 741–743, 1978) involved formation of glycine by transamination of glyoxylate in the peroxisomes utilizing glutamate. Subsequently, glycine is oxidized to ammonia, serine and CO₂ in the mitochondria. The ammonia is reassimilated via the GS/GOGAT pathway generating glutamate. In this article, experimental evidence which suggests the occurrence of alternative mechanisms of glycolate and serine synthesis as well as of CO₂ and ammonia evolution is discussed. The problem of utilization of NADH coupled to ATP synthesis during photosynthesis is still unresolved, which complicates the glycine oxidation reaction in light. Further, factors are presented that determine the availability of amino donors in the peroxisomes and of amino acids viz., glycine, serine and glutamate for the operation of the photorespiratory N cycle. Recent evidence regarding the role of formate arising out of the reaction of glyoxylate with H₂O₂ in the regulation of photosynthetic electron flow in the Hill reaction, as well as of photorespiratory substrates functioning as carbon sources for the citric acid cycle in the light or for export to the growing tissues, suggests that the role of photo-respiration in plant metabolism needs to be reexamined.     

Immunohistochemical localization of adrenocorticotropin and melanocyte stimulating hormone in pars intermedia of rat hypophysis

Summary The sites of production of adrenocorticotropin (ACTH) and melanocyte stimulating hormone (MSH) are studied by the immunoglobulin-peroxidase bridge technique, using antisera prepared against synthetic porcine 1–24 and 17–39 ACTH, and bovine MSH on the rat adenohypophysis. Presence of ACTH all over the pars intermedia (PI) is indicated by staining with antisera _p 1–24 and _p 17-3-9 ACTH. There are darkly stained ACTH cells in the PI and pars tuberalis (PT), similar to those in the pars distalis (PD). With higher dilutions of the ACTH antiserum, staining intensity disappears or reduces markedly in majority of the PI cells, whereas, the ACTH cells in the PI, PD and PT do not vary much in their staining intensity. Therefore, it is concluded that majority of the PI glandular cells (light glandular and dark cells) contain less corticotropin than the ACTH cells. From these observations, it seems to me that the major amount of corticotropin is supplied by the ACTH cells of the PD, PI and PT, and less by the light glandular and dark cells of the PI. The antiserum is ineffective after absorption, so the staining reaction appears to be specific for _p 1–24 and _b 17–39 ACTH.Presence of MSH all over the PI is indicated by staining with antisera to bovine MSH. Majority of the PI cells are highly stained even with higher dilution of the antiserum. The unstained cells in the PI seem to be ACTH cells and/or marginal cuboidal cells. The antiserum was ineffective after absorption, so the staining reaction appears to be specific for _b MSH.Control over the PD corticotropin through the median eminence portal circulation and the PI and PT control through nervous system is also discussed.This study was supported by MRC of Canada Grant nos. MA-3759, and MA-5160.The author gratefully wishes to thank Drs. P. Desaulles and W. Rittel (CIBA, Basle, Switzerland) for the synthetic _p 1–24 ACTH and _b MSH, Dr. R. F. Phifer for _p 17–39 ACTH, and Dr. S. S. Spicer for providing samples of rabbit anti-porcine 17–39 ACTH and anti-human ACTH sera, Drs. George Sétáló and Paul Nakane for their valuable advice. He also acknowledges the help of Mr. Shankar Nayak to prepare the antisera and the skilful technical assistance of Miss. Elise Poiré.     

Development of doubled haploids from an elite <Emphasis Type="Italic">indica</Emphasis> rice hybrid (BS6444G) using anther culture

A total of 200 doubled haploids (DHs) were generated from an elite rice hybrid, ‘BS6444G’ for which an androgenic method was developed by manipulating the physical and chemical factors. The spike pretreated at 10?°C for 7–8 days was effective for callusing and green plant regeneration. The maximum callus frequency was achieved when the anthers cultured in N6 medium supplemented with 2.0 mg L^?1 2,4-diclorophenoxyacetic acid, 0.5 mg L^?1 6-benzylaminopurine and 3% maltose. Calli induced in N6 media also showed significant green shoot regeneration in MS medium supplemented with 0.5 mg L^?1 1-napthalene acetic acid, 0.5 mg L^?1 kinetin, 1.5 mg L^?1 benzylaminopurine and 3% sucrose producing 210 green plants. Assessment of the ploidy status showed 95.71% fertile diploids and 4.2% polyploids; no haploids were observed. A total of 38 sequence-tagged microsatellite (STMS) markers proved able to discriminate a heterozygote from all the 200 DHs. The DHs grown in the field showed significant variation for their agronomic traits. Comparison of traits with control indicates homogeneity within each DH line and significant variance of traits between DH lines. Nine DH lines produce higher grain yield than the hybrid parent which suggests the possibility of exploiting hybrid vigor in indica rice through the development of DH lines of high yielding hybrids.     

Pterostilbene ameliorates intracerebroventricular streptozotocin induced memory decline in rats

There is strong evidence that mitochondrial dysfunction mediated oxidative stress results in aging and energy metabolism deficits thus playing a prime role in pathogenesis of Alzheimer’s disease, neuronal death and cognitive dysfunction. Evidences accrued in empirical studies suggest the antioxidant, anticancer and anti-inflammatory activities of the phytochemical pterostilbene (PTS). PTS also exhibits favourable pharmacokinetic attributes compared to other stilbenes. Hence, in the present study, we explored the neuroprotective role of PTS in ameliorating the intracerebroventricular administered streptozotocin (STZ) induced memory decline in rats. PTS at doses of 10, 30 and 50 mg/kg, was administered orally to STZ administered Sprague–Dawley (SD) rats. The learning and memory tests, Morris water maze test and novel object recognition test were performed which revealed improved cognition on PTS treatment. Further, there was an overall improvement in brain antioxidant parameters like elevated catalase and superoxide dismutase activities, GSH levels, lowered levels of nitrites, lipid peroxides and carbonylated proteins. There was improved cholinergic transmission as evident by decreased acetylcholinesterase activities. The action of ATPases (Na⁺ K⁺, Ca²⁺ and Mg²⁺) indicating the maintenance of cell membrane potential was also augmented. mRNA expression of battery of genes involved in cellular mitochondrial biogenesis and inflammation showed variations which extrapolate to hike in mitochondrial biogenesis and abated inflammation. The histological findings corroborated the effective role of PTS in countering STZ induced structural aberrations in brain.