Suppression of glymphatic fluid transport in a mouse model of Alzheimer's disease.

Glymphatic transport, defined as cerebrospinal fluid (CSF) peri-arterial inflow into brain, and interstitial fluid (ISF) clearance, is reduced in the aging brain. However, it is unclear whether glymphatic transport affects the distribution of soluble Aß in Alzheimer's disease (AD). In wild type mice, we show that Aß40 (fluorescently labeled Aß40 or unlabeled Aß40), was distributed from CSF to brain, via the peri-arterial space, and associated with neurons. In contrast, Aß42 was mostly restricted to the peri-arterial space due mainly to its greater propensity to oligomerize when compared to Aß40. Interestingly, pretreatment with Aß40 in the CSF, but not Aß42, reduced CSF transport into brain. In APP/PS1 mice, a model of AD, with and without extensive amyloid-ß deposits, glymphatic transport was reduced, due to the accumulation of toxic Aß species, such as soluble oligomers. CSF-derived Aß40 co-localizes with existing endogenous vascular and parenchymal amyloid-ß plaques, and thus, may contribute to the progression of both cerebral amyloid angiopathy and parenchymal Aß accumulation. Importantly, glymphatic failure preceded significant amyloid-ß deposits, and thus, may be an early biomarker of AD. By extension, restoring glymphatic inflow and ISF clearance are potential therapeutic targets to slow the onset and progression of AD.

Bioactive compound loaded stable silver nanoparticle synthesis from microwave irradiated aqueous extracellular leaf extracts of Naringi crenulata and its wound healing activity in experimental rat model.

An efficient and eco-friendly protocol for the synthesis of bioactive silver nanoparticles was developed using Naringi crenulata leaf extracts via microwave irradiation method. Silver nanoparticles were synthesized by treating N. crenulata leaf extracts with 1mM of aqueous silver nitrate solution. An effective bioactive compound such as alkaloids, phenols, saponins and quinines present in the N. crenulata reduces the Ag(+) into Ag(0). The synthesized silver nanoparticles were monitored by UV-vis spectrophotometer and further characterized by X-ray diffraction (XRD), Fourier Transform Infra Red (FTIR), Energy-dispersive X-ray spectroscopy (EDX) and field emission scanning electron microscopy (FESEM). UV-vis spectroscopy showed maximum absorbance at 390nm due to surface plasmon resonance of AgNPs. From FESEM results, an average crystal size of the synthesized nanoparticle was 72-98nm. FT-IR results showed sharp absorption peaks and they were assigned to phosphine, alkyl halides and sulfonate groups. Silver nanoparticles synthesized were generally found to be spherical and cubic shape. Topical application of ointment prepared from silver nanoparticles of N. crenulata were formulated and evaluated in vivo using the excision wound healing model on Wistar albino rats. The measurement of the wound areas was performed on 3rd, 6th, 9th, 12th and 15th days and the percentage of wound closures was calculated accordingly. By the 15th day, the ointment base containing 5% (w/w) of silver nanoparticles showed 100% wound healing activity compared with that of the reference as well as control bases. The results strongly suggested that the batch C ointment containing silver nanaoparticles synthesized from the leaf extracts of N. crenulata was found to be very effective in wound repair and encourages harnessing the potentials of the plant biomolecules loaded silver nanoparticle in the treatment of tropical diseases including wound healing.

Seasonal variations in physico-chemical characteristics of pond and ground water of Tiruchirapalli, India.

The aim of this study was to assess the open pond and groundwater quality of Tiruchirapalli city of Tamil Nadu, India. The groundwater quality viz., pH, electrical conductivity, total hardness, calcium ion, magnesium ion, chloride, carbonate, bicarbonate, inorganic nitrate, nitrite, phosphate, ammonia and reactive silicate were analysed with respect to various seasons and recorded in the range of 7.1 to 8.1, 97.67 to 533.67 mhos cm(-1), 7.07 to 186 mg l(-1), 4.67 and 112.0 mg l(-1), 2.40 to 92.80 mg l(-1), 15.23 to 661.73 mg l(-1), 60 to 480 mg l(-1), 22.7 to 544.9 mg l(-1), 15.33 to 68.00 mg l(-1), 0.001 to 0.480 mg l(-1), 0.01 to 0.42 mg l(-1), 0.02 to 0.75 mg l(-1) and 1.1 to 2.96 mg l(-1) respectively. The present findings concluded that the quality of ground waters can be considered suitable for human consumption. But the pond water available in and around Tiruchirappalli city was not fit for human usage, agricultural or industrial purposes.

AP2alpha alters the transcriptional activity and stability of p53.

AP2alpha and p53 form nuclear complexes that establish a functional partnership, which regulates the expression of certain genes involved in cell growth and metastasis. The growth effects of AP2alpha are mediated through p21WAF1/CIP1 and the ability for AP2alpha to coactivate p21 requires p53. Herein, we have localized the AP2-binding region of p53 to amino acids 305-375. Analysis of 26 distinct p53 alleles established a correlation between AP2alpha binding and transcriptional coactivation. The L350P point mutation was the only nonbinding allele that retained normal transcriptional activity by reporter assay. Although both wild-type and L350P alleles facilitated binding of AP2alpha to the p21 promoter, the L350P allele was significantly reduced in its ability to induce the endogenous p21 gene, demonstrating a striking difference in activity comparing reporter assays with activation of endogenous p53 target genes. Interestingly, expression of AP2 in the absence of radiation repressed p53-mediated induction of p21 and this effect was explained by a reduction in p53 stability induced by AP2alpha overexpression. We conclude that AP2alpha has competing effects on p53 activity through coactivation and decreased stability. These findings may provide a mechanism to account for the discrepancies reported for the association between AP2 and p21 expression in tumor tissue.

alpha-Adrenoceptor antagonists in the treatment of benign prostate hyperplasia.

Benign prostate hyperplasia (BPH) is a common malady affecting elderly men throughout the world, and it is the most common cause of voiding dysfunctions in man. The disease becomes prevalent, as the mean age of the population increases. In BPH the glandular cells and myofibroblasts of periurethral and transition zones proliferate excessively. This excessive growth of tissue mass physically compresses the urethra, leading to urinary obstruction and lower urinary tract symptoms (LUTS). Another major factor that contributes to LUTS in BPH is the increase in smooth muscle tone, a dynamic component. Contraction of the prostate gland is mainly under the control of the autonomic system and is mediated through alpha-adrenoceptors. In addition to alpha1-adrenoceptors, there are many other components that influence growth and contraction of the prostate gland, resulting in the development of BPH and LUTS. The prostate gland is contracted on stimulating 5-HT, endothelin, tachykinin, and muscarinic cholinergic receptors. Growth of the prostate gland is under the control of androgens, endothelin growth factor, vasoactive intestinal peptide, nerve growth factor, and growth hormone. The combination of excessive growth and the tone produced by different components results in LUTS and BPH. alpha1-Adrenoceptor antagonists were successfully used in the treatment of BPH to relieve the LUTS. The high selectivity of alpha1A-adrenoceptor antagonists reflects the uroselectivity. The uroselective compounds like terazosin, doxazosin, tamsulosin, and alfuzosin are in clinical use. Other new potent uroselective compounds, which are analogs of nigulidipine, 5-methyl-urapidil, or naftopidil, are in clinical trials. In addition to alpha-antagonists, antiandrogens (5alpha-reductase inhibitors) and polyherbal formulations are used to treat BPH. The success of BPH treatment lies in the development of new chemical entities which are efficacious as well as more uroselective and hence have least side effects.

Gbeta gamma isoforms selectively rescue plasma membrane localization and palmitoylation of mutant Galphas and Galphaq.

Mutation of Galpha(q) or Galpha(s) N-terminal contact sites for Gbetagamma resulted in alpha subunits that failed to localize at the plasma membrane or undergo palmitoylation when expressed in HEK293 cells. We now show that overexpression of specific betagamma subunits can recover plasma membrane localization and palmitoylation of the betagamma-binding-deficient mutants of alpha(s) or alpha(q). Thus, the betagamma-binding-defective alpha is completely dependent on co-expression of exogenous betagamma for proper membrane localization. In this report, we examined the ability of beta(1-5) in combination with gamma(2) or gamma(3) to promote proper localization and palmitoylation of mutant alpha(s) or alpha(q). Immunofluorescence localization, cellular fractionation, and palmitate labeling revealed distinct subtype-specific differences in betagamma interactions with alpha subunits. These studies demonstrate that 1) alpha and betagamma reciprocally promote the plasma membrane targeting of the other subunit; 2) beta(5), when co-expressed with gamma(2) or gamma(3), fails to localize to the plasma membrane or promote plasma membrane localization of mutant alpha(s) or alpha(q); 3) beta(3) is deficient in promoting plasma membrane localization of mutant alpha(s) and alpha(q), whereas beta(4) is deficient in promoting plasma membrane localization of mutant alpha(q); 4) both palmitoylation and interactions with betagamma are required for plasma membrane localization of alpha.

Interaction with Gbetagamma is required for membrane targeting and palmitoylation of Galpha(s) and Galpha(q).

Peripheral membrane proteins utilize a variety of mechanisms to attach tightly, and often reversibly, to cellular membranes. The covalent lipid modifications, myristoylation and palmitoylation, are critical for plasma membrane localization of heterotrimeric G protein alpha subunits. For alpha(s) and alpha(q), two subunits that are palmitoylated but not myristoylated, we examined the importance of interacting with the G protein betagamma dimer for their proper plasma membrane localization and palmitoylation. Conserved alpha subunit N-terminal amino acids predicted to mediate binding to betagamma were mutated to create a series of betagamma binding region mutants expressed in HEK293 cells. These alpha(s) and alpha(q) mutants were found in soluble rather than particulate fractions, and they no longer localized to plasma membranes as demonstrated by immunofluorescence microscopy. The mutations also inhibited incorporation of radiolabeled palmitate into the proteins and abrogated their signaling ability. Additional alpha(q) mutants, which contain these mutations but are modified by both myristate and palmitate, retained their localization to plasma membranes and ability to undergo palmitoylation. These findings identify binding to betagamma as a critical membrane attachment signal for alpha(s) and alpha(q) and as a prerequisite for their palmitoylation, while myristoylation can restore membrane localization and palmitoylation of betagamma binding-deficient alpha(q) subunits.

Binding characteristics of Ustilago maydis topoisomerase I to DNA containing secondary structures.

The binding affinity of purified native Ustilago maydis topoisomerase I enzyme for radiolabeled DNA substrates with various secondary structures was determined by gel shift and equilibrium binding analysis. Topoisomerase I exhibited cooperativity in binding to DNA regardless of the substrate structure. Further analysis demonstrated that cruciform DNA has two populations of binding sites for topoisomerase I while the other substrates (single-stranded DNA, DNA molecules containing six or one mismatched base pairs, hairpin, and fully homologous duplex DNA) have a single population of binding sites. The affinity of topoisomerase I for cruciform was found to be an order of magnitude higher affinity than for any of the other substrates. The high affinity of topoisomerase I for cruciform and specificity of topoisomerase I-cruciform structure interaction were confirmed by competition experiments. These studies demonstrate the high affinity of topoisomerase I for cruciform structure.

The topoisomerase I gene from Ustilago maydis: sequence, disruption and mutant phenotype.

The Ustilago maydis genomic TOP1 gene encoding DNA topoisomerase I was cloned by amplifying a gene fragment using the polymerase chain reaction, and using this fragment to search a genomic DNA library by hybridization. The predicted peptide sequence exhibited 30-40% identity to other eukaryotic TOP1 genes, yet differed in several features. First, an unusually long acidic region was identified near the amino terminus (28/29 residues are acidic), which resembles other nucleolar peptide motifs. Second, an atypical carboxy-terminal 'tail', absent in other TOP1 genes, followed the active site tyrosine residue. A top1 gene disruption mutant was constructed by replacing the genomic TOP1 gene, with a top1::HygR null allele. This mutant lost the abundant topoisomerase I activity evident in wild-type U.maydis, and displayed a subtle coloration phenotype evident during cell senescence.

DNA relaxation mediated by Ustilago maydis type I topoisomerase; modulation by chromatin associated proteins.

Ustilago maydis topoisomerase I relaxes superhelical DNA in the absence of any co-factors. The reaction reaches a defined end-point proportional to the amount of enzyme added and an analysis of the reaction by Hill plot transformation indicates that at least two molecules of topoisomerase must interact with the DNA to catalyze relaxation. The addition of purified Ustilago histone H1 reduces the stoichiometric amount of topoisomerase I required by 50%. H1 histone may function to enhance DNA relaxation through a cooperative mechanism. The purified HMG-like protein from Ustilago also enhances DNA relaxation mediated by the topoisomerase. Whereas H1 stimulates topo I-mediated DNA relaxation through a processive mode, the HMG-like protein enhances through a distributive mechanism. Taken together, these results demonstrate that the interaction of chromosomal proteins with topoisomerase can influence DNA topology, and mechanisms are proposed to explain this enhancement.