Calcium activated proteolysis of fibrous proteins in central nervous system

A Ca²⁺ activated protease(s) capable of hydrolyzing several polypeptides at neutral pH including cytoskeletal proteins, actin, myosin, tubulin and neurofilament triplet was identified in calf brain cortex. The enzyme activity precipitates at 75 mM KCl, pH 6.5 – 7.0 and is inhibited by the sulfhydryl inhibitors, N-ethylmaleimide and para-chloromercuribenzoate and the protease inhibitors, antipain, pepstatin and leupeptin, leupeptin being the most effective.     

Interaction of phenolsulphonphthalein dyes with rabbit plasma and rabbit serum albumin.

Interaction of various substituted phenolsulphonphthalein dyes to rabbit plasma and rabbit serum albumin has been studied by ultrafiltration, equilibrium dialysis and spectrophotometry. The results obtained by ultrafiltration and equilibrium dialysis showed that the degree of binding of these dyes to protein increases in the following order: Phenol red less than bromophenol blue less than bromocresol green less than bromothymol blue. Analysis of binding results revealed that five molecules of bromothymol blue bound very strongly to a molecule of rabbit albumin, whereas only two and three molecules of bromophenol blue and bromocresol green strongly interact with the protein, respectively. It is suggested that strong binding of these substances to protein may be related to the hydrophobicity of these compounds. Finally, an attempt has been made to evaluate the possibility, whether the spectral changes occurred during interaction of dyes to albumin can be utilized for the determination of binding of these ligands to proteins.     

Low nitrogen stress regulates chlorophyll fluorescence in coordination with photosynthesis and Rubisco efficiency of rice

Nitrogen (N) is the basis of plant growth and development and, is considered as one of the priming agents to elevate a range of stresses. Plants use solar radiations through photosynthesis, which amasses the assimilatory components of crop yield to meet the global demand for food. Nitrogen is the main regulator in the allocation of photosynthetic apparatus which changes of the photosynthesis (P_n) and quantum yield (F_v/F_m) of the plant. In the present study, dynamics of the photosynthetic establishment, N-dependent relation with chlorophyll fluorescence attributes and Rubisco efficacy was evaluated in low-N tolerant (cv. CR Dhan 311) and low-N sensitive (cv. Rasi) rice cultivars under low-N and optimum-N conditions. There was a decrease in the stored leaf N under low-N condition, resulting in the decreased P_n and F_v/F_m efficiency of the plants through depletion in the activity and content of Rubisco. The P_n and F_v/F_m followed the parallel trend of leaf N content during low-N condition along with depletion of intercellular CO₂ concentration and overall conductance under low-N condition. Photosynthetic saturation curve cleared abrupt decrease of effective quantum yield in the low-N sensitive rice cultivar than the low-N tolerant rice. Also, the rapid light curve highlighted the unacclimated regulation of photochemical and non-photochemical quenching in the low-N condition. The low-N sensitive rice cultivar triumphed non-photochemical quenching, whereas the low-N tolerant rice cultivar rose gradually during the light curve. Our study suggested that the quantum yield is the key limitation for photosynthesis in low-N condition. Regulation of Rubisco, photochemical and non-photochemical quenching may help plants to grow under low-N level.

     

Optimization of an improved,efficient and rapid in vitro micropropagation protocol for Petunia hybrida Vilm. Cv. “Bravo”

An efficient protocol for in-vitro propagation of an important ornamental crop, Petunia hybrida Vilm. Cv. “Bravo” was developed. The explants that were used to carry out the experiment were Leaf segments, nodal segments and shoot tips. Nodal segments recorded highest per cent asepsis followed by shoot tips and leaf segments. Asepsis was found to be highest when the explants were sterilized with Fungicide (Carbendazim) 0.02% for the duration of 30 min followed by 0.1% HgCl₂ for duration of 10 min and then ethanol 70% for 10 s. Longer duration of the sterilant treatment showed more necrotic effects on the explants, thus mercuric chloride treatment when given for 5 min proved to be more effective in terms of survival of the explants. Maximum establishment per cent was recorded in Murashige and Skoog (MS) media fortified with BAP (1.5 mg L⁻¹) and IBA (0.5 mg L⁻¹) in shoot tips and nodal segments, i.e. 97.90 and 95.74% respectively. Callus was efficiently induced and developed when PGR amalgamation of BAP (0.1 mg L⁻¹) and 2,4-D (1.5mg L⁻¹) was used. Kinetin at the concentration of 2.0 mg L⁻¹ along with IBA at 0.5mg L⁻¹ recorded highest callus regeneration in both leaf and internodal segment derived callus. Maximum proliferation percent of shoots (97.90%), highest number of shoots (20.50 explant⁻¹) and maximum length of shoot (2.70 cm) was recorded in PGR combination of IBA and BAP both at 0.5 mg L⁻1 concentration level. Rhizogenesis was recorded to be highest in the MS media containing IBA 1.00 mg L⁻¹. Best hardening media which recorded maximum survival per cent 92.50% was noticed on the media formulation comprised of equal ratio of perlite and vermiculite mix, under poly house conditions.     

Ultrastructural Changes of the Mitochondrion During the Life Cycle of Trypanosoma brucei

The mitochondrion is crucial for ATP generation by oxidative phosphorylation, among other processes. Cristae are invaginations of the mitochondrial inner membrane that house nearly all the macromolecular complexes that perform oxidative phosphorylation. The unicellular parasite Trypanosoma brucei undergoes during its life cycle extensive remodeling of its single mitochondrion, which reflects major changes in its energy metabolism. While the bloodstream form (BSF) generates ATP exclusively by substrate-level phosphorylation and has a morphologically highly reduced mitochondrion, the insect-dwelling procyclic form (PCF) performs oxidative phosphorylation and has an expanded and reticulated organelle. Here, we have performed high-resolution 3D reconstruction of BSF and PCF mitochondria, with a particular focus on their cristae. By measuring the volumes and surface areas of these structures in complete or nearly complete cells, we have found that mitochondrial cristae are more prominent in BSF than previously thought and their biogenesis seems to be maintained during the cell cycle. Furthermore, PCF cristae exhibit a surprising range of volumes in situ, implying that each crista is acting as an independent bioenergetic unit. Cristae appear to be particularly enriched in the region of the organelle between the nucleus and kinetoplast, the mitochondrial genome, suggesting this part has distinctive properties.     

Mn3O4 nanoparticles: Synthesis,characterization and their antimicrobial and anticancer activity against A549 and MCF-7 cell lines

Due to their inexpensive and eco-friendly nature, and existence of manganese in various oxidation states and their natural abundance have attained significant attention for the formation of Mn₃O₄ nanoparticles (Mn₃O₄ NPs). Herein, we report the preparation of Mn₃O₄ nanoparticles using manganese nitrate as a precursor material by utilization of a precipitation technique. The as-prepared Mn₃O₄ nanoparticles (Mn₃O₄ NPs) were characterized by using X-ray powder diffraction (XRD), UV–Visible spectroscopy (UV–Vis), High-Resolution Transmission electron microscopy (HRTEM), Field emission scanning electron microscopy (FESEM), Thermal gravimetric analysis (TGA) and Fourier-transform infrared spectroscopy (FT-IR). The antimicrobial properties of the as-synthesized Mn₃O₄ nanoparticles were investigated against numerous bacterial and fungal strains including S. aureus, E. coli, B. subtilis, P. aeruginosa, A. flavus and C. albicans. The Mn₃O₄ NPs inhibited the growth of S. aureus with a minimum inhibitory concentration (MIC) of 40 μg/ml and C. albicans with a MIC of 15 μg/ml. Furthermore, the Mn₃O₄ NPs anti-cancer activity was examined using MTT essay against A549 lung and MCF-7 breast cancer cell lines. The Mn₃O₄ NPs revealed significant activity against the examined cancer cell lines A549 and MCF-7. The IC₅₀ values of Mn₃O₄ NPs with A549 cell line was found at concentration of 98 µg/mL and MCF-7 cell line was found at concentration of 25 µg/mL.     

Antibacterial Schiff Bases of Oxalyl-hydrazine/diamide Incorporating Pyrrolyl and Salicylyl Moieties and of Their Zinc(II) Complexes

Schiff bases derived from oxaldiamide/oxalylhydrazine and pyrrol-2-carbaldehyde, or salicylaldehyde respectively, as well as their Zn(II) complexes have been prepared and tested as antibacterial agents. These Schiff bases function as tetradentate ligands, forming octahedral Zn(II) complexes. The ketonic form for the diamide derived Schiff base and the enolic form of the hydrazide derived Schiff base were the preferred tautomers for coordination of the metal ions. The title compounds and their Zn(II) derivatives were evaluated for antibacterial activity against several bacterial strains which easily develop resistance to classical antibiotics, such as Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. Some of them showed promising biological activity in inhibiting the growth of such organisms.     

Europium Nanoparticle-Based High Performing Immunoassay for the Screening of Treponemal Antibodies

Treponema pallidum subspecies pallidum (Tp) is the causative agent of syphilis which mainly spreads through sexual contact, blood transfusion and perinatal route. In order to curtail the spread of the infection and to clinically manage the disease, timely, accurate and reliable diagnosis is very important. We have developed an immunoassay for the detection of treponemal antibodies in human serum or plasma samples. In vivo biotinylated and non-biotinylated versions of the recombinant antigen were designed by the fusion of three Tp-specific antigens namely Tp15, Tp17 and Tp47. These fusion antigens were expressed in E. coli and purified using single-step metal affinity chromatography. Biotinylated fusion antigen immobilized on streptavidin coated plate was used to capture the treponemal antibodies and the non-biotinylated antigen coated on europium nanoparticles was used as tracer. Assays with two different incubation times of 10 min and 1 h were developed, and following the incubation the europium fluorescence was measured using time-resolved fluorometry. The developed time-resolved fluorometric (TRF) immunoassays were evaluated with in-house and commercial serum/plasma sample panels. For well-established treponemal antibodies positive or negative samples, the sensitivity of TRF immunoassay with 10 min incubation time was 97.4%, and of TRF immunoassay with 1 h incubation time was 98.7%, and the specificities of both the TRF immunoassays were 99.2%. For the samples with discordant results with the reference assays, both the TRF immunoassays showed better specificity than the Enzygnost syphilis enzyme immunoassay as a screening test. The two different incubation times did not have any significant effect on the signal to cutoff (S/Co) ratios obtained with the two immunoassays (p = 0.06). Our results indicate that the developed immunoassay with a short incubation time of 10 min has the potential to be used in clinical laboratories and in blood-bank settings as a screening test for treponemal antibodies.     

An Alteration in ELMOD3, an Arl2 GTPase-Activating Protein,Is Associated with Hearing Impairment in Humans

Exome sequencing coupled with homozygosity mapping was used to identify a transition mutation (c.794T>C; p.Leu265Ser) in ELMOD3 at the DFNB88 locus that is associated with nonsyndromic deafness in a large Pakistani family, PKDF468. The affected individuals of this family exhibited pre-lingual, severe-to-profound degrees of mixed hearing loss. ELMOD3 belongs to the engulfment and cell motility (ELMO) family, which consists of six paralogs in mammals. Several members of the ELMO family have been shown to regulate a subset of GTPases within the Ras superfamily. However, ELMOD3 is a largely uncharacterized protein that has no previously known biochemical activities. We found that in rodents, within the sensory epithelia of the inner ear, ELMOD3 appears most pronounced in the stereocilia of cochlear hair cells. Fluorescently tagged ELMOD3 co-localized with the actin cytoskeleton in MDCK cells and actin-based microvilli of LLC-PK1-CL4 epithelial cells. The p.Leu265Ser mutation in the ELMO domain impaired each of these activities. Super-resolution imaging revealed instances of close association of ELMOD3 with actin at the plasma membrane of MDCK cells. Furthermore, recombinant human GST-ELMOD3 exhibited GTPase activating protein (GAP) activity against the Arl2 GTPase, which was completely abolished by the p.Leu265Ser mutation. Collectively, our data provide the first insights into the expression and biochemical properties of ELMOD3 and highlight its functional links to sound perception and actin cytoskeleton.     

<i>Aspergillus tubingensis</i> Causes Leaf Spot of Cotton (<i>Gossypium hirsutum</i> L.) in Pakistan

Cotton (Gossypium hirsutum L.) is a key fiber crop of great commercial importance. Numerous phytopathogens decimate crop production by causing various diseases. During July-August 2018, leaf spot symptoms were recurrently observed on cotton leaves in Rahim Yar Khan, Pakistan and adjacent areas. Infected leaf samples were collected and plated on potato dextrose agar (PDA) media. Causal agent of cotton leaf spot was isolated, characterized and identified as Aspergillus tubingensis based on morphological and microscopic observations. Conclusive identification of pathogen was done on the comparative molecular analysis of CaM and β-tubulin gene sequences. BLAST analysis of both sequenced genes showed 99% similarity with A. tubingensis. Koch’s postulates were followed to confirm the pathogenicity of the isolated fungus. Healthy plants were inoculated with fungus and similar disease symptoms were observed. Fungus was re-isolated and identified to be identical to the inoculated fungus. To our knowledge, this is the first report describing the involvement of A. tubingensis in causing leaf spot disease of cotton in Pakistan and around the world.