Protective effect of aminoguanidine against nephrotoxicity induced by cisplatin in normal rats

The effect of aminoguanidine (AG) on nephrotoxicity induced by cisplatin (CDDP) was investigated. A single dose of CDDP (7.5 mg/kg i.p.) induced nephrotoxicity, manifested biochemically by a significant elevation in serum urea, creatinine and a severe decrease in serum albumin. Moreover, marked increases in kidney weight, urine volume and urinary excretion of albumin were observed. Nephrotoxicity was further confirmed by a significant decrease in glutathione-S-transferase (GST, E.C. 2.5.1.18), glutathione peroxidase (GSH-Px, E.C. 1.11.1.9) and catalase (E.C. 1.11.1.6) and a significant increase in lipid peroxides measured as malondialdhyde (MDA) in kidney homogenates. Administration of AG (100 mg/kg per day p.o.) in drinking water 5 days before and 5 days after CDDP injection produced a significant protection against nephrotoxicity induced by CDDP. The amelioration of nephrotoxicity was evidenced by significant reductions in serum urea and creatinine concentrations. In addition, AG tended to normalize decreased levels of serum albumin. Urine volume, urinary excretions of albumin and GST and kidney weight were significantly decreased. Moreover, AG prevented the rise of MDA and the reduction of GST and GSH-Px activities in the kidney. These results suggest that AG has a protective effect on nephrotoxicity induced by CDDP and it may therefore improve the therapeutic index of CDDP.     

An intelligent biological information management system

MOTIVATION: As biomedical researchers are amassing a plethora of information in a variety of forms resulting from the advancements in biomedical research, there is a critical need for innovative information management and knowledge discovery tools to sift through these vast volumes of heterogeneous data and analysis tools. In this paper we present a general model for an information management system that is adaptable and scalable, followed by a detailed design and implementation of one component of the model. The prototype, called BioSifter, was applied to problems in the bioinformatics area. RESULTS: BioSifter was tested using 500 documents obtained from PubMed database on two biological problems related to genetic polymorphism and extracorporal shockwave lithotripsy. The results indicate that BioSifter is a powerful tool for biological researchers to automatically retrieve relevant text documents from biological literature based on their interest profile. The results also indicate that the first stage of information management process, i.e. data to information transformation, significantly reduces the size of the information space. The filtered data obtained through BioSifter is relevant as well as much smaller in dimension compared to all the retrieved data. This would in turn significantly reduce the complexity associated with the next level transformation, i.e. information to knowledge.     

Design,synthesis, and biological evaluation of a cephalosporin-monohydroguaiaretic acid prodrug activated by a monoclonal antibody-beta-lactamase conjugate

A novel cephalosporin derivative of monohydroguaiaretic acid (cephem-M(3)N, 7) was synthesized and found to possess anticancer activity against human leukemia (K562), breast carcinoma (MCF7), human lung cancer (A549), human colon cancer (Colo205) and pancreatic cancer cells (Capan2 and MiaPaCa2). A tumor targeting fusion protein (dsFv3-beta-lactamase) was also used in conjunction with cephem-based M(3)N 7 and its potency toward K562, MCF7, A549, Colo205, Capan2, and MiaPaCa2 was found to approach that of the free M(3)N (4). In the presence of dsFv3-beta-lactamase, tumor cells were found to be much more susceptible to conjugate 7 than normal human embryonic lung (HEL) cells and normal fibroblasts (Hef522). These notions provide a new approach to the use of nordihydroguaiaretic acid (NDGA) and its derivatives for antitumor therapy.     

Multiplex Pyrosequencing

We describe here the development of a new and simple single-tube multiplex Pyrosequencing assay. Genomic DNA or cDNA was employed to PCR amplify region(s) using biotinylated and normal primer(s). Subsequent to capture of PCR products on streptavidin-coated beads, single-stranded DNA separation and hybridization of multiple sequencing primers, Pyrosequencing was performed. The obtained pyrogram resulted in a unique pattern in which the intensity of the signal determined the number of incorporated nucleotide(s). Here, we demonstrate the use of this multiplex Pyrosequencing for single nucleotide polymorphisms genotyping and microbial typing.     

Time-resolved long-lived infrared emission from bacteriorhodopsin during its photocycle

The infrared emission observed below 2000 cm(-1) upon exciting retinal in bacteriorhodopsin (bR) is found to have a rise time in the submicrosecond time regime and to relax with two exponential components on the submillisecond to millisecond time scale. These time scales, together with the assignment of this emission to hot vibrations from the all-trans retinal (in bR) and the 13-cis retinal (in the K intermediate), support the recent assignment of the J-intermediate as an electronically excited species (Atkinson et al., J. Phys. Chem. A. 104:4130-4139, 2000) rather than a vibrationally hot K intermediate. A discussion of these time scales of the observed infrared emission is given in terms of the competition between radiative and nonradiative relaxation processes of the vibrational states involved.     

Multiplexed genotyping with sequence-tagged molecular inversion probes

We report on the development of molecular inversion probe (MIP) genotyping, an efficient technology for large-scale single nucleotide polymorphism (SNP) analysis. This technique uses MIPs to produce inverted sequences, which undergo a unimolecular rearrangement and are then amplified by PCR using common primers and analyzed using universal sequence tag DNA microarrays, resulting in highly specific genotyping. With this technology, multiplex analysis of more than 1,000 probes in a single tube can be done using standard laboratory equipment. Genotypes are generated with a high call rate (95%) and high accuracy (>99%) as determined by independent sequencing.     

Effect of 50 Hz, 0.2 mT magnetic fields on RBC properties and heart functions of albino rats

In this work the effect of sinusoidal 50 Hz, 0.2 mT magnetic fields on the red blood cells (RBCs) and heart functions of Albino rats were investigated. Twenty-four male Albino rats were equally divided into four groups, A, B, C, and D. Animals from groups B were continuously exposed to the magnetic field for 15 days; and groups C and D, for 30 days. Group A was used as control. Animals from group D were kept after exposure to the magnetic field for a period of 45 days for delayed effect studies. The osmotic fragility and shape of RBCs' membrane and hemoglobin (Hb) structure tests were carried out for all groups. The dielectric relaxation of Hb molecules was measured in the frequency range of 0.1-10 MHz and the dielectric increment (Deltaepsilon), relaxation time (tau), molecular radius (r), and Cole-Cole parameter (alpha) were calculated for all groups. The ECG was measured for all animals before and after exposure to the magnetic field. The results indicated that exposure of the animals to 50 Hz, 0.2 mT magnetic fields resulted in the decrease of RBCs membrane elasticity and permeability and changes in the molecular structure of Hb. The ECG of the exposed animals was considerably altered. The data also indicated that there was no sign of repair in the newly generated RBCs structure and the ECG after removing the animals from the magnetic field, which indicates that the blood generating system was severely injured. The injuries in the heart of the animals were attributed to the loss of some physiological functions of the RBCs as a result of exposures of the rats to the magnetic field.     

Progesterone side-chain degradation by some species ofAspergillus flavus group

Seventy isolates belonging to 6 species and one variety of A. flavus group were shown to degrade the progesterone side-chain to yield delta 4-androstene-3,17-dione and testosterone. The isolates of five species (A. flavo-furcatis, A. flavus, A. oryzae, A. parasiticus and A. tamarii) possessed enzyme systems catalyzing the opening of ring D and formed testololactone as final steroid metabolite in addition to their ability to produce the above mentioned two products. 11 beta-Hydroxy-delta 4-androstene-3,17-dione was formed by only A. flavus and A. tamarii while 11 beta-hydroxytestosterone was produced by A. flavo-furcatis, A. parasiticus and A. subolivaceus. The chromatographic resolution of the mixture products obtained (when the selective isolate of each species reacted with 1 g of progesterone) revealed that 60-75% of progesterone was converted into delta 4-androstene-3,17-dione (8-30%), testosterone (7-33%), testololactone (14-37%) and other products (3-40%). The most bioconversion activity was exhibited by A. oryzae, followed by A. parasiticus. The highest values of delta 4-androstene-3,17-dione (30% of added progesterone) and testosterone (33%) were formed by A. flavus var. columnaris while those of testololactone (37%) were produced by A. oryzae. A systematic variation could be observed between the different tested species of A. flavus group with respect to the transformation reactions of progesterone. Comparative biotransformation results showed that essential differences exist between the tested species in this group; this biochemical differentiation may supplement the morphological and other physiological criteria used in the identification of the different species in the A. flavus group.     

Ameliorative Effect of Chrysin on Adenine-Induced Chronic Kidney Disease in Rats

Chrysin (5, 7- dihydroxyflavone) is a flavonoid with several pharmacological properties that include antioxidant, anti-inflammatory and antiapoptotic activities. in this work, we investigated some effects of three graded oral doses of chrysin (10, 50 and 250 mg/kg) on kidney structure and function in rats with experimental chronic renal disease (CKD) induced by adenine (0.25% w/w in feed for 35 days), which is known to involve inflammation and oxidative stress. Using several indices in plasma, urine and kidney homogenates, adenine was found to impair kidney function as it lowered creatinine clearance and increased plasma concentrations of creatinine, urea, neutrophil gelatinase-associated lipocalin and N-Acetyl-beta-D-glucosaminidase activity. Furthermore, it raised plasma concentrations of the uremic toxin indoxyl sulfate, some inflammatory cytokines and urinary albumin concentration. Renal morphology was severely damaged and histopathological markers of inflammation and fibrosis were especially increased. In renal homogenates, antioxidant indices, including superoxide dismutase and catalase activities, total antioxidant capacity and reduced glutathione were all adversely affected. Most of these adenine – induced actions were moderately and dose -dependently mitigated by chrysin, especially at the highest dose. Chrysin did not cause any overt adverse effect on the treated rats. The results suggest that different doses of chrysin produce variable salutary effects against adenine-induced CKD in rats, and that, pending further pharmacological and toxicological studies, its usability as a possible ameliorative agent in human CKD should be considered.     

Functional Proteomics Identifies Acinus L as a Direct Insulin- and Amino Acid-Dependent Mammalian Target of Rapamycin Complex 1 (mTORC1) Substrate

The serine/threonine kinase mammalian target of rapamycin (mTOR) governs growth, metabolism, and aging in response to insulin and amino acids (aa), and is often activated in metabolic disorders and cancer. Much is known about the regulatory signaling network that encompasses mTOR, but surprisingly few direct mTOR substrates have been established to date. To tackle this gap in our knowledge, we took advantage of a combined quantitative phosphoproteomic and interactomic strategy. We analyzed the insulin- and aa-responsive phosphoproteome upon inhibition of the mTOR complex 1 (mTORC1) component raptor, and investigated in parallel the interactome of endogenous mTOR. By overlaying these two datasets, we identified acinus L as a potential novel mTORC1 target. We confirmed acinus L as a direct mTORC1 substrate by co-immunoprecipitation and MS-enhanced kinase assays. Our study delineates a triple proteomics strategy of combined phosphoproteomics, interactomics, and MS-enhanced kinase assays for the de novo-identification of mTOR network components, and provides a rich source of potential novel mTOR interactors and targets for future investigation.The serine/threonine kinase mammalian target of rapamycin (mTOR)¹ is conserved in all eukaryotes from yeast to mammals (1). mTOR is a central controller of cellular growth, whole body metabolism, and aging, and is frequently deregulated in metabolic diseases and cancer (2). Consequently, mTOR as well as its upstream and downstream cues are prime candidates for targeted drug development to alleviate the causes and symptoms of age-related diseases (3, 4). The identification of novel mTOR regulators and effectors thus remains a major goal in biomedical research. A vast body of literature describes a complex signaling network around mTOR. However, our current comparatively detailed knowledge of mTOR''s upstream cues contrasts with a rather limited set of known direct mTOR substrates.mTOR exists in two structurally and functionally distinct multiprotein complexes, termed mTORC1 and mTORC2. Both complexes contain mTOR kinase as well as the proteins mLST8 (mammalian lethal with SEC thirteen 8) (5–7), and deptor (DEP domain-containing mTOR-interacting protein) (8). mTORC1 contains the specific scaffold protein raptor (regulatory-associated protein of mTOR) (9, 10), whereas mTORC2 contains the specific binding partners rictor (rapamycin-insensitive companion of mTOR) (5–7), mSIN1 (TORC2 subunit MAPKAP1) (11–13), and PRR5/L (proline rich protein 5/-like) (14–16). The small macrolide rapamycin acutely inhibits mTORC1, but can also have long-term effects on mTORC2 (17, 18). More recently, ATP-analogs (19) that block both mTOR complexes, such as Torin 1 (20), have been developed. As rapamycin has already been available for several decades, our knowledge of signaling events associated with mTORC1 as well as its metabolic inputs and outputs is much broader as compared with mTORC2. mTORC1 responds to growth factors (insulin), nutrients (amino acids, aa) and energy (ATP). In response, mTORC1 activates anabolic processes (protein, lipid, nucleotide synthesis) and blocks catabolic processes (autophagy) to ultimately allow cellular growth (21). The insulin signal is transduced to mTORC1 via the insulin receptor (IR), and the insulin receptor substrate (IRS), which associates with class I phosphoinositide 3-kinases (PI3Ks). Subsequent phosphatidylinositol 3,4,5 trisphosphate (PIP3) binding leads to relocalization of the AGC kinases phosphoinositide-dependent protein kinase 1 (PDK1) and Akt (also termed protein kinase B, PKB) to the plasma membrane, where PDK1 phosphorylates Akt at T308 (22, 23). In response, Akt phosphorylates and inhibits the heterocomplex formed by the tuberous sclerosis complex proteins 1 and 2 (TSC1-TSC2) (24, 25). TSC1-TSC2 is the inhibitory, GTPase-activating protein for the mTORC1-inducing GTPase Ras homolog enriched in brain (rheb) (26–30), which activates mTORC1 at the lysosome. mTORC1 localization depends on the presence of aa, which in a rag GTPase-dependent manner induce mTORC1 relocalization to lysosomes (31, 32). Low energy levels are sensed by the AMP-dependent kinase (AMPK), which in turn phosphorylates the TSC1-TSC2 complex (33) and raptor (34), thereby inhibiting mTORC1.mTORC1 phosphorylates its well-described downstream substrate S6-kinase (S6K) at T389, the proline-rich Akt substrate of 40 kDa (PRAS40) at S183, and the translational repressor 4E-binding protein (4E-BP) at T37/46 (35–41). Unphosphorylated 4E-BP binds and inhibits the translation initiation factor 4G (eIF4G), which within the eIF4F complex mediates the scanning process of the ribosome to reach the start codon. Phosphorylation by mTORC1 inhibits 4E-BP''s interaction with eIF4E, thus allowing for assembly of eIF4F, and translation initiation (42, 43). More recently, also the IR-activating growth factor receptor-bound protein 10 (Grb10) (44, 45), the autophagy-initiating Unc-51-like kinase ULK1 (46), and the trifunctional enzymatic complex CAD composed of carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase (47, 48), which is required for nucleotide synthesis, have been described as direct mTORC1 substrates.mTORC2 activation is mostly described to be mediated by insulin, and this is mediated by a PI3K variant that is distinct from the PI3K upstream of mTORC1 (49, 50). Furthermore, mTORC2 responds to aa (5, 51). In response, mTORC2 phosphorylates the AGC kinases Akt at S473 (52–55), and serum and glucocorticoid kinase SGK (56) and protein kinase C alpha (PKCalpha) (7) within their hydrophobic motifs (57, 58), to control cellular motility (5–7), hepatic glycolysis, and lipogenesis (59). In addition, mTOR autophosphorylation at S2481 has been established as an mTORC2 readout in several cell lines including HeLa cells (49).Given the multiplicity of effects via which mTOR controls cellular and organismal growth and metabolism, it is surprising that only relatively few direct mTOR substrates have been established to date. Proteomic studies are widely used to identify novel interactors and substrates of protein kinases. Two studies have recently shed light on the interaction of rapamycin and ATP-analog mTOR inhibitors with TSC2 inhibition in mammalian cells (44, 45), and one study has analyzed the effects of raptor and rictor knockouts in non-stimulated cells (48).In this work, we report a functional proteomics approach to study mTORC1 substrates. We used an inducible raptor knockdown to inhibit mTORC1 in HeLa cells, and analyzed the effect in combination with insulin and aa induction by quantitative phosphoproteomics using stable isotope labeling by amino acids in cell culture (SILAC) (60). In parallel, we purified endogenous mTOR complexes and studied the interactome of mTOR by SILAC-MS. Through comparative data evaluation, we identified acinus L as a potential novel aa/insulin-sensitive mTOR substrate. We further validated acinus L by co-immunoprecipitation and MS-enhanced kinase assays as a new direct mTORC1 substrate.