Mahjabeen Saleem

Glycosylated Alpha-1-acid glycoprotein 1 as a potential lung cancer serum biomarker

Presently existing screening approaches for lung cancer are not being proving sufficient and sensitive, so a study was conducted to identify disease related biomarker proteins for diagnostic applications. A total of 100 lung cancer patients (88 non-small cell lung cancer and 12 small cell lung cancer) and 50 healthy controls were included in this study. Serum samples of patients and healthy controls were subjected to a series of proteomic approaches and as a result of two dimensional gel electrophoresis, a ∼ 43 kDa protein was found to be differentially expressed compared to healthy controls. Quantitative profiling of two dimensional gels by Dymension software analysis displayed 3.58 fold increased expression of ∼ 43 kDa protein in squamous cell carcinoma and 2.92 fold in case of adenocarcinoma. Mass spectrometric analysis resulted in identification of 8 differentially expressed proteins, out of which human Alpha-1-acid glycoprotein 1 was targeted for further validations. This candidate protein exhibited N-linked glycosylation at five amino acid residues; 33, 56, 72, 93, and 103 with significant score of 0.66, 0.78, 0.78, 0.53 and 0.66, respectively. Sandwich ELISA quantified high serum levels of Alpha-1-acid glycoprotein 1 in squamous cell carcinoma (2.93 g/l ± 1.22) and adenocarcinoma (2.39 g/l ± 1.13) when compared with healthy controls (0.83 g/l ± 0.21). One-way ANOVA analysis predicted highly significant variation of Alpha-1-acid glycoprotein 1, among all the study types (F-value 65.37, p-value 0.000). This study may prove as a non-invasive, cost effective and sensitive scheme for diagnosis of lung cancer, by passing the expensive and painful screening procedures.

Production, Purification and Characterization of β-1,4-Endoglucanase from a Novel Bacterial Strain CTP-09 of a Bacillus sp.

Bacillus strain CTP-09 yielded maximum productivity (1120 IU/L.h) of extracellular endoglucanase (CMCase) on 0.5% cellobiose after 10 h fermentation at 55 degrees C. The purified enzyme is mono-meric in nature and exhibits stability up to 80 degrees C and over a pH range (6.0-9.0). Activation energy, enthalpy and entropy of catalysis, and inactivation indicated that this CMCase is highly thermos-table. Purified enzyme possessed high power of defibrillation of textile and was minutely inhibited by anionic detergent and oxidizing agent comparable with inhibition by commercial enzyme. This polypeptide could be exploited for mass production and application in local industries.

Biochemical characterization of fruit-specific pathogenesis-related antifungal protein from basrai banana

Pathogenesis-related/thaumatin like (PR-5/TL) antifungal protein from basrai banana was purified by using a simple protocol consisting of ammonium sulphate precipitation, affinity chromatography (Affi-gel blue gel), Q-Sepharose chromatography and gel filtration on Sephadex G-75. The purified protein with acidic character (pI 6.67) has molecular weight of 21.155 kDa, as determined by MALDI-TOF mass spectrometry. The purified protein shared N-terminal sequence homology with other TLPs. Crude banana extract inhibited the growth of Fusarium oxysporum, Aspergillus niger, Aspergillus fumigatus and Trichoderma viride with IC₅₀ values (determined by Probit analysis) 15 μM (slope=0.086, χ(2)=17.843, P=0.033), 17 μM (slope=0.183, χ(2)=61.533, P=0.011), 6.5 μM (slope=0.211, χ(2)=14.380, P=0.023) and 29.11 μM (slope=0.072, χ(2)=45.768, P=0.014). The purified antifungal protein repressed the growth of F. oxysporum, A. niger, A. fumigatus and T. viride with IC₅₀ values 9.7 μM (slope=0.056, χ(2)=11.538, P=0.021), 11.83 μM (slope=0.127, χ(2)=42.82, P=0.00), 4.61 μM (slope=0.150, χ(2)=10.199, P=0.017) and 21.43 μM (slope=0.053, χ(2)=33.693, P=0.00), respectively. The IC50 values of antifungal activity of crude banana extract were higher than the purified antifungal protein. It indicated that proteins in crude banana extract have antagonistic effect on the fungal growth. White bread is particularly vulnerable by fungal pathogens. Purified antifungal protein suppressed the growth of Aspergillus phoenicis and Aspergillus flavus on white bread suggesting that this protein can be used as a preservative in the bakery industry as well as in other relevant food processing industries.

Granzyme M: characterization with sites of post-translational modification and specific sites of interaction with substrates and inhibitors

Granzymes kill cells in a variety of ways. They induce mitochondrial dysfunction through caspase dependent and caspase-independent pathways and destroy DNA and the integrity of the nucleus. For gaining a better understanding of the molecular function of granzyme M and its NK cell specificity, structural characterization of this enzyme by molecular modeling as well as its detailed comparison with other granzymes is presented in this study. The study includes mode of action of granzyme M using cationic binding sites, substrate specificity, post-translational structural modification and its functional relationship and interaction of the enzyme with inhibitor in an attempt to explore how the activity of human granzyme M is controlled under physiological conditions. It is concluded from the present study that the post-translational modification, including Oglycosylation of serine, phosphorylation of serine and threonine and myristoylation of glycine, play an important role in the interaction of enzyme with the cell surface membrane and regulate protein trafficking and stability. Phosphorylated serine and threonine also plays a role in tumor elimination, viral clearance and tissue repair. In Gzm M there are cationic sites, cs1 and cs2 that may participate in binding of Gzm M to the cell surface, thereby promoting its uptake and eventual release into the cytoplasm. Gzm M shows apoptotic activity both by caspase dependent and independent pathways. Modeling of inhibitors bound to the granzyme active site shows that the dimer also contributes to substrate specificity in a unique manner by extending the active-site cleft.

Mutation in DsbA signal sequence hampers the SRP mechanism: A new strategy to combat virulence factor

Disulphide bond (Dsb) protein, characterized as an important virulence factor in gram negative bacteria. In this study, amino acid mutations in DsbA signal sequence (ss) and its effect on translocation of recombinant Ovine growth hormone (rOGH) was observed. Eight constructs were designed on the basis of increased hydrophobicity and showed that hydrophobicity and specificity of amino acid plays a crucial role in translocation of rOGH. Two DsbAss with the same hydropathy (1.539), one had alteration at -13 and second at -11 position; alanine (Ala) to isoleucine respectively were designed. The former DsbAss translocated rOGH from membrane to cytoplasmic fraction in E. coli as confirmed by SDS-PAGE, Western blot and molecular modelling analysis. MD simulations and binding free energy calculations evidenced that, altering Ala changed the orientation of signal peptide in the Ffh-M domain binding groove and hampered the process of translocation while change at position -11 pointed it outward. We hypothesize, amino acid and position of mutations in DsbAss can hinder the translocation process of signal recognition particle system, thus affecting the virulence of bacteria.

Molecular Characterization, Structural Modeling, and Evaluation of Antimicrobial Activity of Basrai Thaumatin-Like Protein against Fungal Infection

A thaumatin-like protein gene from Basrai banana was cloned and expressed in Escherichia coli. Amplified gene product was cloned into pTZ57R/T vector and subcloned into expression vector pET22b(+) and resulting pET22b-basrai TLP construct was introduced into E. coli BL21. Maximum protein expression was obtained at 0.7 mM IPTG concentration after 6 hours at 37°C. Western blot analysis showed the presence of approximately 20 kDa protein in induced cells. Basrai antifungal TLP was tried as pharmacological agent against fungal disease. Independently Basrai antifungal protein and amphotericin B exhibited their antifungal activity against A. fumigatus; however combined effect of both agents maximized activity against the pathogen. Docking studies were performed to evaluate the antimicrobial potential of TLP against A. fumigatus by probing binding pattern of antifungal protein with plasma membrane ergosterol of targeted fungal strain. Ice crystallization primarily damages frozen food items; however addition of antifreeze proteins limits the growth of ice crystal in frozen foods. The potential of Basrai TLP protein, as an antifreezing agent, in controlling the ice crystal formation in frozen yogurt was also studied. The scope of this study ranges from cost effective production of pharmaceutics to antifreezing and food preserving agent as well as other real life applications.

Purification and Biochemical Characterization of a Pathogenesis Related Endochitinase Arachis hypogaea

Plants protect themselves by producing phytoalexins and pathogenesis related (PR) proteins in response to different physical and biological stresses as well as against pathological attack. In the present study, chitinase, a PR protein, from peanut seeds was purified to homogeneity by ammonium sulfate precipitation, gel filtration chromatography on Sephadex G-75 column and ion exchange chromatography on Q-Sepharose column. Chitinase was identified as monomeric protein with molecular mass of 30 kDa by SDS-PAGE. Chitinase activity was also evaluated on chitin agar plates showing distinct zones. The optimum pH and temperature of purified chitinase activity were found to be 5.2 and 35°C, respectively. The enzyme was stable within broad pH range of 3.6 to 7.6 and up to 55°C temperature. Amongst the various substrates, acid swollen chitin was found to be the best substrate for chitinase when used at the concentration of 1% exhibiting its high specificity in catalyzing glycosidic bonds between N-acetylglucosamine residues. Chitinase activity of the purified enzyme was inhibited by Hg and Ag at 1 mM concentration and enhanced by Ca, Mg. The increase in the presence of 2-mercaptomethanol demonstrates the existence of sulfhydryl groups in enzyme active site. The enzyme exhibited a strong inhibitory action towards a variety of fungal species including Fusarium oxysporum, Trichoderma reesei, Aspergillus flavus and A. niger. Purified chitinase showed fungicidal properties against A. flavus on white bread and therefore, could be applied in bakery products to inhibit the formation and growth of fungal colonies.

Mass spectrometric identification, characterization and validation of the haptoglobin β-chain protein as a lung cancer serum biomarker

Lung cancer is the major contributor to overall cancer-related mortality. Biomarkers are important in early detection and prognosis, in addition to developing treatment regimes, which may improve the patient survival rates. Biomarkers may also assist in investigating the in depth metabolic pathways and in establishing a set of therapeutic agents leading to early detection of the disease. The present study was designed to identify and confirm a lung cancer protein biomarker and to correlate the differential expression of the protein to a particular histological disease type. A total of 100 lung cancer patients and 50 healthy controls were included in the present study and were categorized into the two main histological types of lung cancer; non‑small cell lung cancer (NSCLC; n=88) and small cell lung cancer (SCLC; n=12). NSCLC was further subclassified into three histological types; adenocarcinoma (n=34), squamous cell carcinoma (n=48) and large cell carcinoma (n=6). The patient and control serum samples underwent sodium dodecyl sulphate polyacrylamide gel electrophoresis characterization followed by two‑dimensional gel electrophoresis. Following mass spectrometry, human haptoglobin was identified with a mass of ~42‑46 kDa and an isoelectric point (pI) of ~5.5‑6.2. The experimental mass of the protein was found to be 45.8 kDa with a pI of 6.13. The matrix‑assisted laser desorption/ionization time‑of‑flight/time‑of‑flight data exhibited spectral peaks of 1146.134, 1724.191, 1345.339 and 2210.319 m/z and Mascot search analysis identified these peaks as haptoglobin (accession no. P00738; Mascot score 87; sequence coverage 23%). This protein was significantly overexpressed in squamous cell carcinoma and adenocarcinoma, as compared with the control. The present study described differentially expressed human haptoglobin as a lung cancer serum protein biomarker, which may serve as a diagnostic and therapeutic target and set a standard criteria for the evaluation of histological types of lung cancer compared with other disease types.

Binding properties of beetal recombinant caprine growth hormone to Bovidae liver microsomal membranes

The aim of the study was to illustrate the radio-receptor assay of beetal recombinant caprine growth hormone (rcGH). Tracer ( 125 I-rcGH) was prepared by iodinating beetal rcGH with iodine-125 and its biological activity was analyzed by rabbit anti-rcGH antibodies. Liver microsomal membranes of the Bovidae species (caprine, ovine and bovine) were prepared to study the binding properties of beetal rcGH. The tracer binding was dependent on receptor protein concentration, tracer counts, temperature, time of incubation and assay pH. The maximum percentage specific binding of the tracer to the microsomal membrane was 45% in 32 h at 4°C. In cross-reactive study, human GH competed and displaced the 125 I-rcGH by binding effectively to the cGH receptor. Scatchard analysis of the rcGH binding suggested a single class of binding site to the caprine microsomal membrane with an affinity of 337.1 ± 82.94 × 10 9 M -1 and capacity of 57.61 ± 4.23 fmol mg -1 while rcGH binding affinity and capacity to the ovine and bovine receptor protein showed 365.4 ± 66.82 × 10 9 M -1 , 57.69 ± 3.23 (fmol mg -1 ) and 392.6 ± 56.25 × 10 9 M -1 , 56.8 ± 2.56 (fmol mg -1 ) respectively. This study provides data for beetal rcGH interaction with microsomal membrane that shall be beneficial to study hormone receptor interactions of other Bovidae species. Keywords: Beetal recombinant caprine growth hormone, Bovidae, iodine-125, microsomal membrane, receptor protein African Journal of Biotechnology , Vol 13(30) 3081-3091