Proteome profiling of aging in mouse models: Differential expression of proteins involved in metabolism,transport, and stress response in kidney

Aging is a time‐dependent complex biological phenomenon observed in various organs and organelles of all living organisms. To understand the molecular mechanism of age‐associated functional loss in aging kidneys, we have analyzed the expression of proteins in the kidneys of young (19–22 wk) and old (24 months) C57/BL6 male mice using 2‐DE followed by LC‐MS/MS. We found that expression levels of 49 proteins were upregulated (p ≤ 0.05), while that of only ten proteins were downregulated (p ≤ 0.05) due to aging. The proteins identified belong to three broad functional categories: (i) metabolism (e.g., aldehyde dehydrogenase family, ATP synthase β‐subunit, malate dehydrogenase, NADH dehydrogenase (ubiquinone), hydroxy acid oxidase 2), (ii) transport (e.g., transferrin), and (iii) chaperone/stress response (e.g., Ig‐binding protein, low density lipoprotein receptor‐related protein associated protein 1, selenium‐binding proteins (SBPs)). Some proteins with unknown functions were also identified as being differentially expressed. ATP synthase β subunit, transferrin, fumarate hydratase, SBPs, and albumin are present in multiple forms, possibly arising due to proteolysis or PTMs. The above functional categories suggest specific mechanisms and pathways for age‐related kidney degeneration.     

Detoxification of Multiple Heavy Metals by a Half-Molecule ABC Transporter,HMT-1, and Coelomocytes of Caenorhabditis elegans

Background

Developing methods for protecting organisms in metal-polluted environments is contingent upon our understanding of cellular detoxification mechanisms. In this regard, half-molecule ATP-binding cassette (ABC) transporters of the HMT-1 subfamily are required for cadmium (Cd) detoxification. HMTs have conserved structural architecture that distinguishes them from other ABC transporters and allows the identification of homologs in genomes of different species including humans. We recently discovered that HMT-1 from the simple, unicellular organism, Schizosaccharomyces pombe, SpHMT1, acts independently of phytochelatin synthase (PCS) and detoxifies Cd, but not other heavy metals. Whether HMTs from multicellular organisms confer tolerance only to Cd or also to other heavy metals is not known.

Methodology/Principal Findings

Using molecular genetics approaches and functional in vivo assays we showed that HMT-1 from a multicellular organism, Caenorhabditis elegans, functions distinctly from its S. pombe counterpart in that in addition to Cd it confers tolerance to arsenic (As) and copper (Cu) while acting independently of pcs-1. Further investigation of hmt-1 and pcs-1 revealed that these genes are expressed in different cell types, supporting the notion that hmt-1 and pcs-1 operate in distinct detoxification pathways. Interestingly, pcs-1 and hmt-1 are co-expressed in highly endocytic C. elegans cells with unknown function, the coelomocytes. By analyzing heavy metal and oxidative stress sensitivities of the coelomocyte-deficient C. elegans strain we discovered that coelomocytes are essential mainly for detoxification of heavy metals, but not of oxidative stress, a by-product of heavy metal toxicity.

Conclusions/Significance

We established that HMT-1 from the multicellular organism confers tolerance to multiple heavy metals and is expressed in liver-like cells, the coelomocytes, as well as head neurons and intestinal cells, which are cell types that are affected by heavy metal poisoning in humans. We also showed that coelomocytes are involved in detoxification of heavy metals. Therefore, the HMT-1-dependent detoxification pathway and coelomocytes of C. elegans emerge as novel models for studies of heavy metal-promoted diseases.     

Ellagic acid improved diabetes mellitus-induced testicular damage and sperm abnormalities by activation of Nrf2

Diabetes mellitus induces testicular damage, increases sperm abnormalities, and impairs reproductive dysfunction due to induction of endocrine disturbance and testicular oxidative stress. This study evaluated the reproductive protective effect of ellagic acid (EA) against testicular damage and abnormalities in sperm parameters in Streptozotocin (STZ)-induced diabetic rats (T1DM) and examined some possible mechanisms of protection. Adult male rats were segregated into 5 groups (n = 12 rat/each) as control, control + EA (50 mg/kg/day), T1DM, T1DM + EA, and T1DM + EA + brusatol (an Nrf-2 inhibitor) (2 mg/twice/week). All treatments were conducted for 12 weeks, daily. EA preserved the structure of the seminiferous tubules, prevented the reduction in sperm count, motility, and viability, reduced sperm abnormalities, and downregulated testicular levels of cleaved caspase-3 and Bax in diabetic rats. In the control and diabetic rats, EA significantly increased the circulatory levels of testosterone, reduced serum levels of FSH and LH, and upregulated Bcl-2 and all steroidogenic genes (StAr, 3β-HSD1, and 11β-HSD1). Besides, it reduced levels of ROS and MDA but increased levels of GSH and MnSOD and the transactivation of Nrf2. All these biochemical alterations induced by EA were associated with increased activity and nuclear accumulation of Nrf2. However, all these effects afforded by EA were weakened in the presence of brusatol. In conclusion, EA could be an effective therapy to alleviated DM-induced reproductive toxicity and dysfunction in rats by a potent antioxidant potential mediated by the upregulation of Nrf2.     

Endophytic Fungal Flora from Roots and Fruits of an Indian Neem Plant <Emphasis Type="Italic">Azadirachta indica</Emphasis> A. Juss., and Impact of Culture Media on their Isolation

Azadirachta indica A. Juss. (neem), native to India, is well known worldwide for its insecticidal and ethanopharmacological properties. Although endophytic microbes are known from this plant as only leaves and stems were the subjects of past reports. Now, a variety of procedures and a number of different media were used to isolate the maximum number of endophytic fungi from unripe fruits and roots. A total of 272 isolates of 29 filamentous fungal taxa were isolated at rate of 68.0% from 400 samples of three different individual trees (at locations-Az1, Az2, Az3). Mycological agar (MCA) medium yielded the highest number of isolates (95, with a 14.50% isolation rate) with the greatest species richness. Mycelia Sterilia (1, 2, 3) accounted for 11.06%, Coelomycetes 7.25%, while Hyphomycetes showed the maximum number of representative isolates (81.69%). Mycelia-Sterilia (1, 2, 3), based on their 5.8S ITS 1, ITS2 and partial 18S and 28S rDNA sequences were identified as Fusarium solani (99%), Chaetomium globosum (93%) and Chaetomium globosum (93%) respectively. Humicola, Drechslera, Colletotrichum, and Scytalidium sp. were some of the peculiar fungal endophytes recovered from this plant.     

Distribution and biochemical properties of an M1-family aminopeptidase in Plasmodium falciparum indicate a role in vacuolar hemoglobin catabolism

Aminopeptidases catalyze N-terminal peptide bond hydrolysis and occupy many diverse roles across all domains of life. Here we present evidence that an M1-family aminopeptidase, PfA-M1, has been recruited to specialized roles in the human malaria parasite Plasmodium falciparum. PfA-M1 is abundant in two subcellular compartments in asexual intraerythrocytic parasites; that is, the food vacuole, where the catabolism of host hemoglobin takes place, and the nucleus. A unique N-terminal extension contributes to the observed dual targeting by providing a signal peptide and putative alternate translation initiation sites. PfA-M1 exists as two major isoforms, a nuclear 120-kDa species and a processed species consisting of a complex of 68- and 35-kDa fragments. PfA-M1 is both stable and active at the acidic pH of the food vacuole lumen. Determination of steady-state kinetic parameters for both aminoacyl-β-naphthylamide and unmodified dipeptide substrates over the pH range 5.0-8.5 reveals that k(cat) is relatively insensitive to pH, whereas K(m) increases at pH values below 6.5. At the pH of the food vacuole lumen (5.0-5.5), the catalytic efficiency of PfA-M1 remains high. Consistent with the kinetic data, the affinity of peptidic competitive inhibitors is diminished at acidic pH. Together, these results support a catalytic role for PfA-M1 in the food vacuole and indicate the importance of evaluating the potency of peptidic inhibitors at physiologically relevant pH values. They also suggest a second, distinct function for this enzyme in the parasite nucleus.     

Subtilase from Beauveria sp.: conformational and functional investigation of unusual stability

Retention of total activity of the subtilisin-like serine protease from Beauveria sp. MTCC 5184 (Bprot) in the vicinity of (1) 3 M GdnHCl for 12 h, (2) 50 % methanol and dimethyl sulfoxide each for 24 h, and (3) proteolytic enzymes (trypsin, chymotrypsin, and proteinase K) for 48 h led to expect the enzyme to be a kinetically stable protein. Also, the structure of the protein was stable at pH 2.0. Biophysical characterization and conformational transitions were monitored using steady-state and time-resolved fluorescence, FTIR, and CD spectroscopy. Single tryptophan in the protein exists as two conformers, in hydrophobic and polar environment. The secondary structure of Bprot was stable in 3 M GdnHCl as seen in far-UV CD spectra. The active fraction of Bprot obtained from size-exclusion chromatography in the presence of GdnHCl (1.0–3.0 M) eluted at reduced retention time. The peak area of inactive or denatured protein with the same retention time as that of native protein increased with increasing concentration of denaturant (1.0–4.0 M GdnHCl). However, the kinetics of GdnHCl-induced unfolding as studied from intrinsic fluorescence revealed k _unf of native protein to be 5.407 × 10^?5 s^?1 and a half-life of 3.56 h. The enzyme is thermodynamically stable in spite of being resistant to the denaturant, which could be due to the effect of GdnHCl imparting rigidity to the active fraction and simultaneously unfolding the partially unfolded protein that exists in equilibrium with the folded active protein. Thermal and pH denaturation of Bprot exhibited interesting structural transitions.     

Polymorphism of cytokine and innate immunity genes associated with bovine brucellosis in cattle

Genetic susceptibility to brucellosis is multifactorial, and it is known that impairment of the immune system could contribute to risk for getting brucellosis. The aim of the study was to find association of bovine brucellosis with 20 SNPs pertaining to bovine cytokine (IFNG, IFNGR1, IFNGR2, TNFA) and innate immunity (SLC11A1, TLR1, TLR4, and TLR9) genes using PCR-RFLP genotyping technique and it was observed that SLC11A1 (+1066 C/G), TLR1 (+1446 C/A), TLR1 (+1380 G/A), TLR4 (+10 C/T) and TLR4 (+399 C/T) loci were significantly (P ≤ 0.05) associated with bovine brucellosis. The odds ratios (OR) of CG and CC genotypes versus GG genotype were 0.31 (0.12–0.82; 95 % CI) and 0.18 (0.03–1.06; 95 % CI) at SLC11A1 (+1066 C/G) locus in cases of brucellosis affected cattle. For TLR1 (+1380 G/A) locus, the OR for AG and AA genotypes versus GG genotypes were 0.15 (0.05–0.44; 95 % CI) and 0.26 (0.04–1.47; 95 % CI) which indicated that proportion of GG homozygote was significantly higher in brucellosis affected animals as compared to control. At TLR1 (+1446 C/A) locus the OR of AC genotype versus CC genotype was 0.24 (0.08–0.68; 95 % CI) which revealed that relative proportion CC genotypes was significantly higher in case population. The TLR4 (+10 C/T) locus had three genotypes (TT, CT and CC) where OR of CT and CC genotypes versus TT genotype were near to zero. The OR of CT genotypes versus CC genotypes was 8.25 (0.94–71.92; 95 % CI) at TLR4 (+399 C/T) locus and indicated that CT genotype had higher odds of bovine brucellosis than control animals.     

OSL and thermally assisted OSL response in dental enamel for its possible application in retrospective dosimetry

Dental enamel was studied for its thermoluminescence (TL) and optically stimulated luminescence (OSL) defects. The TL studies showed a wide glow curve with multiple peaks. The thermally assisted OSL (TA-OSL) studies showed that the integrated TA-OSL and thus OSL signal increases with readout temperature between 100 and 250 °C, due to the temperature dependence of OSL. The thermally assisted energy E _A associated with this increase is found to be 0.21 ± 0.015 eV. On the other hand, the signal intensity decreases with temperature between 260 and 450 °C. This decrease could be due to depletion of OSL active traps or possible thermal quenching. The increase of the OSL signal at increased temperature can be used to enhance the sensitivity of dental enamel for ex vivo measurements in retrospective dosimetry. The emission and excitation spectra of its luminescence centers were studied by photoluminescence and were found to be at 412 and 324 nm, respectively. It was found to possess multiple OSL active traps having closely lying photoionization cross sections characterized by continuous wave OSL and nonlinear OSL methods. The investigated dental enamel samples showed a linear OSL dose response up to 500 Gy. The dose threshold was found to be 100 mGy using a highly sensitive compact OSL reader with blue LED (470 nm) stimulation.     

A Cell-Based High-Throughput Screen for Novel Chemical Inducers of Fetal Hemoglobin for Treatment of Hemoglobinopathies

Decades of research have established that the most effective treatment for sickle cell disease (SCD) is increased fetal hemoglobin (HbF). Identification of a drug specific for inducing γ-globin expression in pediatric and adult patients, with minimal off-target effects, continues to be an elusive goal. One hurdle has been an assay amenable to a high-throughput screen (HTS) of chemicals that displays a robust γ-globin off-on switch to identify potential lead compounds. Assay systems developed in our labs to understand the mechanisms underlying the γ- to β-globin gene expression switch during development has allowed us to generate a cell-based assay that was adapted for a HTS of 121,035 compounds. Using chemical inducer of dimerization (CID)-dependent bone marrow cells (BMCs) derived from human γ-globin promoter-firefly luciferase β-globin promoter-Renilla luciferase β-globin yeast artificial chromosome (γ-luc β-luc β-YAC) transgenic mice, we were able to identify 232 lead chemical compounds that induced γ-globin 2-fold or higher, with minimal or no β-globin induction, minimal cytotoxicity and that did not directly influence the luciferase enzyme. Secondary assays in CID-dependent wild-type β-YAC BMCs and human primary erythroid progenitor cells confirmed the induction profiles of seven of the 232 hits that were cherry-picked for further analysis.     

Novel KRAS Gene Mutations in Sporadic Colorectal Cancer

Introduction

In this article, we report 7 novel KRAS gene mutations discovered while retrospectively studying the prevalence and pattern of KRAS mutations in cancerous tissue obtained from 56 Saudi sporadic colorectal cancer patients from the Eastern Province.

Methods

Genomic DNA was extracted from formalin-fixed, paraffin-embedded cancerous and noncancerous colorectal tissues. Successful and specific PCR products were then bi-directionally sequenced to detect exon 4 mutations while Mutector II Detection Kits were used for identifying mutations in codons 12, 13 and 61. The functional impact of the novel mutations was assessed using bioinformatics tools and molecular modeling.

Results

KRAS gene mutations were detected in the cancer tissue of 24 cases (42.85%). Of these, 11 had exon 4 mutations (19.64%). They harbored 8 different mutations all of which except two altered the KRAS protein amino acid sequence and all except one were novel as revealed by COSMIC database. The detected novel mutations were found to be somatic. One mutation is predicted to be benign. The remaining mutations are predicted to cause substantial changes in the protein structure. Of these, the Q150X nonsense mutation is the second truncating mutation to be reported in colorectal cancer in the literature.

Conclusions

Our discovery of novel exon 4 KRAS mutations that are, so far, unique to Saudi colorectal cancer patients may be attributed to environmental factors and/or racial/ethnic variations due to genetic differences. Alternatively, it may be related to paucity of clinical studies on mutations other than those in codons 12, 13, 61 and 146. Further KRAS testing on a large number of patients of various ethnicities, particularly beyond the most common hotspot alleles in exons 2 and 3 is needed to assess the prevalence and explore the exact prognostic and predictive significance of the discovered novel mutations as well as their possible role in colorectal carcinogenesis.