Binding of biogenic and synthetic polyamines to β-lactoglobulin

The bindings of biogenic polyamines spermine (spm), spermidine (spmd) and synthetic polyamines 3,7,11,15-tetrazaheptadecane·4HCl (BE-333) and 3,7,11,15,19-pentazahenicosane·5HCl (BE-3333) with β-lactoglobulin (β-LG) were determined in aqueous solution. FTIR, UV-vis, CD and fluorescence spectroscopic methods as well as molecular modeling were used to determine the polyamine binding sites and the effect of polyamine complexation on protein stability and secondary structure. Structural analysis showed that polyamines bind β-LG via both hydrophilic and hydrophobic contacts. Stronger polyamine-protein complexes formed with synthetic polyamines than biogenic polyamines, with overall binding constants of K_spm-β-LG = 3.2(±0.6) × 10⁴ M⁻¹, K_spmd-β-LG = 1.8(±0.5) × 10⁴ M⁻¹, K_BE-333-β-LG = 5.8(±0.3) × 10⁴ M⁻¹ and K_{BE-3333-β-LG} = 6.2(±0.05) × 10⁴ M⁻¹. Molecular modeling showed the participation of several amino acids in the polyamine complexes with the following order of polyamine-protein binding affinity: BE-3333 > BE-333 > spermine > spermidine, which correlates with their positively charged amino group content. Alteration of protein conformation was observed with a reduction of β-sheet from 57% (free protein) to 55-51%, and a major increase of turn structure from 13% (free protein) to ∼21% in the polyamine-β-LG complexes, indicating a partial protein unfolding.     

Glucocorticoids Inhibit Basal and Hormone-Induced Serotonin Synthesis in Pancreatic Beta Cells

Diabetes is a major complication of chronic Glucocorticoids (GCs) treatment. GCs induce insulin resistance and also inhibit insulin secretion from pancreatic beta cells. Yet, a full understanding of this negative regulation remains to be deciphered. In the present study, we investigated whether GCs could inhibit serotonin synthesis in beta cell since this neurotransmitter has been shown to be involved in the regulation of insulin secretion. To this aim, serotonin synthesis was evaluated in vitro after treatment with GCs of either islets from CD1 mice or MIN6 cells, a beta-cell line. We also explored the effect of GCs on the stimulation of serotonin synthesis by several hormones such as prolactin and GLP 1. We finally studied this regulation in islet in two in vivo models: mice treated with GCs and with liraglutide, a GLP1 analog, and mice deleted for the glucocorticoid receptor in the pancreas. We showed in isolated islets and MIN6 cells that GCs decreased expression and activity of the two key enzymes of serotonin synthesis, Tryptophan Hydroxylase 1 (Tph1) and 2 (Tph2), leading to reduced serotonin contents. GCs also blocked the induction of serotonin synthesis by prolactin or by a previously unknown serotonin activator, the GLP-1 analog exendin-4. In vivo, activation of the Glucagon-like-Peptide-1 receptor with liraglutide during 4 weeks increased islet serotonin contents and GCs treatment prevented this increase. Finally, islets from mice deleted for the GR in the pancreas displayed an increased expression of Tph1 and Tph2 and a strong increased serotonin content per islet. In conclusion, our results demonstrate an original inhibition of serotonin synthesis by GCs, both in basal condition and after stimulation by prolactin or activators of the GLP-1 receptor. This regulation may contribute to the deleterious effects of GCs on beta cells.     

Treatment with captopril abrogates the altered expression of alpha1 macroglobulin and alpha1 antiproteinase in sera of spontaneously hypertensive rats

Background

Proteins that are associated with hypertension may be identified by comparing the 2-dimensional gel electrophoresis (2-DE) profiles of the sera of spontaneously hypertensive rats (SHR) with those generated from normotensive Spraque-Dawley rats (SDR).

Results

Five proteins of high abundance were found to be significantly altered when the 2-DE serum profiles of the SHR were compared to those that were similarly generated from the SDR. Analysis by mass spectrometry and database search identified the proteins as retinol binding protein 4, complement C3, albumin (19.9 kDa fragment), alpha1 macroglobulin and alpha1 antiproteinase, which are all known to be associated with hypertension. The altered expression of the two latter proteins was found to be abrogated when similar analysis was performed on sera of the SHR that were treated with captopril.

Conclusion

Our data suggests that serum alpha1 macroglobulin and alpha1 antiproteinase are potentially useful complementary biomolecular indicators for monitoring of hypertension.     

Genetic diversity analysis of Bt cotton genotypes in Pakistan using simple sequence repeat markers

The popularity of genetically modified insect resistant (Bt) cotton has promoted large scale monocultures, which is thought to worsen the problem of crop genetic homogeneity. Information on genetic diversity among Bt cotton varieties is lacking. We evaluated genetic divergence among 19 Bt cotton genotypes using simple sequence repeat (SSR) markers. Thirty-seven of 104 surveyed primers were found informative. Fifty-two primers selected on the basis of reported intra-hirsutum polymorphism in a cotton marker database showed a high degree of polymorphism, 56% compared to 13% for randomly selected primers. A total of 177 loci were amplified, with an average of 1.57 loci per primer, generating 38 markers. The amplicons ranged in size from 98 to 256 bp. The genetic similarities among the 19 genotypes ranged from 0.902 to 0.982, with an average of 0.947, revealing a lack of diversity. Similarities among genotypes from public sector organizations were higher than genotypes developed by private companies. Hybrids were found to be more distant compared to commercial cultivars and advanced breeding lines. Cluster analysis grouped the 19 Bt cotton genotypes into three major clusters and two independent entries. Cultivars IR-3701, Ali Akbar-802 and advanced breeding line VH-259 grouped in subcluster B2, with very narrow genetic distances despite dissimilar parentage. We found a very high level of similarity among Pakistani-bred Bt cotton varieties, which means that genetically diverse recurrent parents should be included to enhance genetic diversity. The intra-hirsutum polymorphic SSRs were found to be highly informative for molecular genetic diversity studies in these cotton varieties.     

Evaluation of genetic diversity of <Emphasis Type="Italic">Clinacanthus nutans</Emphasis> (Acanthaceaea) using RAPD,ISSR and RAMP markers

Three polymerase chain reaction (PCR) techniques were compared to analyse the genetic diversity of Clinacanthus nutans eight populations in the northern region of Peninsular Malaysia. The PCR techniques were random amplified polymorphic deoxyribonucleic acids (RAPD), inter-simple sequence repeats (ISSR) and random amplified microsatellite polymorphisms (RAMP). Leaf genomic DNA was PCR amplified using 17 RAPD, 8 ISSR and 136 RAMP primers . However, only 10 RAPD primers, 5 ISSR primers and 37 RAMP primers produced reproducible bands. The results were evaluated for polymorphic information content (PIC), marker index (MI) and resolving power (RP). The RAMP marker was the most useful marker compared to RAPD and ISSR markers because it showed the highest average value of PIC (0.25), MI (11.36) and RP (2.86). The genetic diversity showed a high percentage of polymorphism at the species level compared to the population level. Furthermore, analysis of molecular variance revealed that the genetic diversity was higher within populations, as compared to among populations of C. nutans. From the results, the RAMP technique was recommended for the analysis of genetic diversity of C. nutans.     

Mechanical regulation of osteoclastic genes in human osteoblasts

Bone adaptation to mechanical load is accompanied by changes in gene expression of bone-forming cells. Less is known about mechanical effects on factors controlling bone resorption by osteoclasts. Therefore, we studied the influence of mechanical loading on several key genes modulating osteoclastogenesis. Human osteoblasts were subjected to various cell stretching protocols. Quantitative RT-PCR was used to evaluate gene expression. Cell stretching resulted in a significant up-regulation of receptor activator of nuclear factor-κB ligand (RANKL) immediate after intermittent loading (3 × 3 h, 3 × 6 h, magnitude 1%). Continuous loading, however, had no effect on RANKL expression. The expression of osteoprotegerin (OPG), macrophage-colony stimulating factor (M-CSF), and osteoclast inhibitory lectin (OCIL) was not significantly altered. The data suggested that mechanical loading could influence osteoclasts recruitment by modulating RANKL expression in human osteoblasts and that the effects might be strictly dependent on the quality of loading.     

Detection of bacterial blight resistance genes in basmati rice landraces

Aromatic basmati rice is vulnerable to bacterial blight disease. Genes conferring resistance to bacterial blight have been identified in coarse rice; however, their incorporation into basmati varieties compromises the prized basmati aroma. We identified bacterial blight resistance genes Xa4, xa5, Xa7, and xa13 in 52 basmati landraces and five basmati cultivars using PCR markers. The Xa7 gene was found to be the most prevalent among the cultivars and landraces. The cultivars Basmati-385 and Basmati-2000 also contained the Xa4 gene; however, xa5 and xa13 were confined to landraces only. Ten landraces were found to have multiple resistance genes. Landraces Basmati-106, Basmati-189 and Basmati-208 contained Xa4 and Xa7 genes. Whereas, landraces Basmati-122, Basmati-427, Basmati-433 were observed to have xa5 and Xa7 genes. Landraces Basmati-48, Basmati-51A, Basmati-334, and Basmati-370A possessed Xa7 and xa13 genes. The use of landraces containing recessive genes xa5 and xa13 as donor parents in hybridization with cultivars Basmati-385 and Basmati-2000, which contain the genes Xa4 and Xa7, will expedite efforts to develop bacterial blight-resistant basmati rice cultivars through marker assisted selection, based on a pyramiding approach, without compromising aroma and grain quality.     

Christia vespertilionis extract induced antiproliferation and apoptosis in breast cancer (MCF7) cells

Molecular Biology Reports - C. vespertiliomis extracts were evaluated for antiproliferative and apoptosis effect on breast cancer (MCF7) cells. The leaves extracts were analysed for its...     

Mechanism of Interaction of Al3+ with the Proteins Composition of Photosystem II

The inhibitory effect of Al3+on photosystem II (PSII) electron transport was investigated using several biophysical and biochemical techniques such as oxygen evolution, chlorophyll fluorescence induction and emission, SDS-polyacrylamide and native green gel electrophoresis, and FTIR spectroscopy. In order to understand the mechanism of its inhibitory action, we have analyzed the interaction of this toxic cation with proteins subunits of PSII submembrane fractions isolated from spinach. Our results show that Al ³⁺, especially above 3 mM, strongly inhibits oxygen evolution and affects the advancement of the S states of the Mn₄O₅Ca cluster. This inhibition was due to the release of the extrinsic polypeptides and the disorganization of the Mn4O5Ca cluster associated with the oxygen evolving complex (OEC) of PSII. This fact was accompanied by a significant decline of maximum quantum yield of PSII (F_v/F_m) together with a strong damping of the chlorophyll a fluorescence induction. The energy transfer from light harvesting antenna to reaction centers of PSII was impaired following the alteration of the light harvesting complex of photosystem II (LHCII). The latter result was revealed by the drop of chlorophyll fluorescence emission spectra at low temperature (77 K), increase of F₀ and confirmed by the native green gel electrophoresis. FTIR measurements indicated that the interaction of Al ³⁺ with the intrinsic and extrinsic polypeptides of PSII induces major alterations of the protein secondary structure leading to conformational changes. This was reflected by a major reduction of α-helix with an increase of β-sheet and random coil structures in Al ³⁺-PSII complexes. These structural changes are closely related with the functional alteration of PSII activity revealed by the inhibition of the electron transport chain of PSII.