Purification, characterizations of a snake guard seeds lectin with antitumor activity against Ehrlich ascites carcinoma cells in vivo in mice

A lectin was purified (designated as TCSL) from the Snake guard seeds with molecular mass of 56±2 kDa containing two subunits (34±1 and 22±1 kDa.). TCSL exhibited high agglutination activity at the temperature range 30 to 70°C and did not lose its activity between pH 3.0 to 12.0. The lectin was stable in the presence of denaturants and agglutinated mouse, goat, cow, chicken and human erythrocytes. TCSL did not show antifungal activity whereas it agglutinated six pathogenic bacteria and showed less toxicity against brine shrimp nauplii with the LC50 of 261±29 μg/ml. TCSL showed 28% and 72% inhibition against Ehrlich ascites carcinoma (EAC) cells in vivo in mice when administered 1 mg/kg/day and 2 mg/kg/day (i.p.) respectively for five days. TCSL enhanced the number of macrophages remarkably in the normal mice. The lectin reduced the tumor burden to 62% of EAC cells and significantly increased the hemoglobin and RBC. Treating the EAC bearing mice with TCSL at 2 mg/Kg/day for ten days with a monitoring of 20 days decreased the total WBC towards the normal level and it increased the life span by 39%.     

A new species of the cicada genus Cicadatra Kolenati, 1857 (Hemiptera, Cicadidae) from Pakistan with a key to the known species of Pakistani Cicadatra

A new species of cicada, Cicadatra ziaraticasp. n., is described from Pakistan. Male genitalia, timbal and opercula are described and illustrated as important diagnostic characters. Biological notes are also provided. A key to the known Cicadatra of Pakistan is provided.     

Antibody Protection Reveals Extended Epitopes on the Human TSH Receptor

Stimulating, and some blocking, antibodies to the TSH receptor (TSHR) have conformation-dependent epitopes reported to involve primarily the leucine rich repeat region of the ectodomain (LRD). However, successful crystallization of TSHR residues 22-260 has omitted important extracellular non-LRD residues including the hinge region which connects the TSHR ectodomain to the transmembrane domain and which is involved in ligand induced signal transduction. The aim of the present study, therefore, was to determine if TSHR antibodies (TSHR-Abs) have non-LRD binding sites outside the LRD. To obtain this information we employed the method of epitope protection in which we first protected TSHR residues 1-412 with intact TSHR antibodies and then enzymatically digested the unprotected residues. Those peptides remaining were subsequently delineated by mass spectrometry. Fourteen out of 23 of the reported stimulating monoclonal TSHR-Ab crystal contact residues were protected by this technique which may reflect the higher binding energies of certain residues detected in this approach. Comparing the protected epitopes of two stimulating TSHR-Abs we found both similarities and differences but both antibodies also contacted the hinge region and the amino terminus of the TSHR following the signal peptide and encompassing cysteine box 1 which has previously been shown to be important for TSH binding and activation. A monoclonal blocking TSHR antibody revealed a similar pattern of binding regions but the residues that it contacted on the LRD were again distinct. These data demonstrated that conformationally dependent TSHR-Abs had epitopes not confined to the LRDs but also incorporated epitopes not revealed in the available crystal structure. Furthermore, the data also indicated that in addition to overlapping contact regions within the LRD, there are unique epitope patterns for each of the antibodies which may contribute to their functional heterogeneity.     

<Emphasis Type="Italic">Brevibacterium anseongense</Emphasis> sp. nov., isolated from soil of ginseng field

Gram-positive, aerobic, non-motile, pale-yellow, and rodshaped bacterium, designated as Gsoil 188^T, was isolated from the soil of a ginseng field in Pocheon, South Korea. A phylogenetic analysis based on 16S rRNA gene sequence comparison revealed that the strain formed a distinct lineage within the genus Brevibacterium and was most closely related to B. epidermidis NBRC 14811^T (98.4%), B. sediminis FXJ8.269^T (98.2%), B. avium NCFB 3055^T (98.1%), and B. oceani BBH7^T (98.1%), while it shared less than 98.1% identity with the other species of this genus. The DNA G + C content was 68.1 mol%. The predominant quinone was MK-8(H₂). The major fatty acids were anteiso-C_15:0 and anteiso-C_17:0. The cell wall peptidoglycan of strain Gsoil 188^T contained meso-diaminopimelic acid. The major polar lipids were phosphatidylglycerol, diphosphatidylglycerol, and an unidentified aminolipid. The physiological and biochemical characteristics, low DNA-DNA relatedness values, and taxonomic analysis allowed the differentiation of strain Gsoil 188^T from the other recognized species of the genus Brevibacterium. Therefore, strain Gsoil 188^T represents a novel species of the genus Brevibacterium, for which the name Brevibacterium anseongense sp. nov. is proposed, with the type strain Gsoil 188^T (= KACC 19439^T = LMG 30331^T).     

Macromolecular and enzymatic abnormalities induced by a synthetic pyrethroid,Ripcord (Cypermethrin), in adult beetles of a stored grain pest,Tribolium castaneum (Herbst.) (Coleoptera: Tenebrionidae)

The effects of a synthetic pyrethroid insecticide, cypermethrin, administered as a formulation Ripcord 25EC (emulsified concentrate), to adult beetles of a stored grain pest, Tribolium castaneum, have been studied, with an objective to ascertain its toxicity on enzymes such as carbohydrases, phosphatases, dehydrogenases, aminotransferases, and concentration of various biochemical components such as monosaccharides, glycogen, cholesterol, nucleic acids, urea, total lipids, and total proteins. Almost all the enzymes and biochemical components were sensitive to sublethal doses of Ripcord 25 EC and these effects were found to be dependent on the duration of treatment. All carbohydrate metabolizing enzymes (amylase, invertase, lactase, maltase, lactate dehydrogenase) were elevated, except for trehalase, which was also elevated up to day 3 but returned to normal levels subsequently. Phosphatases (alkaline as well as acidic) were increased first and decreased thereafter, while isocitrate dehydrogenase decreased throughout the experimental period. Transaminases (aspartate aminotransferase and alanine aminotransferase) showed a decreasing trend. Of the other biochemical components tested, glucose content decreased during the first 3 days but increased subsequently. Fructose content showed an increase, while the glycogen content decreased throughout the study. Total lipid content was not disturbed up to day 3 but increased thereafter. Cholesterol content was depleted by day 7. Total proteins started decreasing from day 3 onwards, while soluble proteins were not affected. DNA, RNA, and urea contents exhibited elevated levels, while uric acid showed a decreasing trend. Sublethal doses of Ripcord, therefore, resulted in extensive enzyme induction, and utilization of carbohydrates, proteins, and lipids, in the given order, perhaps to produce extra energy to combat insecticidal stress. Arch. Insect Biochem. Physiol. 39:144–154, 1998. © 1998 Wiley-Liss, Inc.     

Allelopathic interference of alfalfa (<Emphasis Type="Italic">Medicago sativa</Emphasis> L.) genotypes to annual ryegrass (<Emphasis Type="Italic">Lolium rigidum</Emphasis>)

Alfalfa (Medicago sativa L.) genotypes at varying densities were investigated for allelopathic impact using annual ryegrass (Lolium rigidum) as the target species in a laboratory bioassay. Three densities (15, 30, and 50 seedlings/beaker) and 40 alfalfa genotypes were evaluated by the equal compartment agar method (ECAM). Alfalfa genotypes displayed a range of allelopathic interference in ryegrass seedlings, reducing root length from 5 to 65%. The growth of ryegrass decreased in response to increasing density of alfalfa seedlings. At the lowest density, Q75 and Titan9 were the least allelopathic genotypes. An overall inhibition index was calculated to rank each alfalfa genotype. Reduction in seed germination of annual ryegrass occurred in the presence of several alfalfa genotypes including Force 10, Haymaster7 and SARDI Five. A comprehensive metabolomic analysis using Quadruple Time of Flight (Q-TOF), was conducted to compare six alfalfa genotypes. Variation in chemical compounds was found between alfalfa root extracts and exudates and also between genotypes. Further individual compound assessments and quantitative study at greater chemical concentrations are needed to clarify the allelopathic activity. Considerable genetic variation exists among alfalfa genotypes for allelopathic activity creating the opportunity for its use in weed suppression through selection.     

Preparation and characterization of cockle shell aragonite nanocomposite porous 3D scaffolds for bone repair

The demands for applicable tissue-engineered scaffolds that can be used to repair load-bearing segmental bone defects (SBDs) is vital and in increasing demand. In this study, seven different combinations of 3 dimensional (3D) novel nanocomposite porous structured scaffolds were fabricated to rebuild SBDs using an extraordinary blend of cockle shells (CaCo₃) nanoparticles (CCN), gelatin, dextran and dextrin to structure an ideal bone scaffold with adequate degradation rate using the Freeze Drying Method (FDM) and labeled as 5211, 5400, 6211, 6300, 7101, 7200 and 8100. The micron sized cockle shells powder obtained (75 µm) was made into nanoparticles using mechano-chemical, top-down method of nanoparticles synthesis with the presence of the surfactant BS-12 (dodecyl dimethyl bataine). The phase purity and crystallographic structures, the chemical functionality and the thermal characterization of the scaffolds’ powder were recognized using X-Ray Diffractometer (XRD), Fourier transform infrared (FTIR) spectrophotometer and Differential Scanning Calorimetry (DSC) respectively. Characterizations of the scaffolds were assessed by Scanning Electron Microscopy (SEM), Degradation Manner, Water Absorption Test, Swelling Test, Mechanical Test and Porosity Test. Top-down method produced cockle shell nanoparticles having averagely range 37.8±3–55.2±9 nm in size, which were determined using Transmission Electron Microscope (TEM). A mainly aragonite form of calcium carbonate was identified in both XRD and FTIR for all scaffolds, while the melting (Tm) and transition (Tg) temperatures were identified using DSC with the range of Tm 62.4–75.5 °C and of Tg 230.6–232.5 °C. The newly prepared scaffolds were with the following characteristics: (i) good biocompatibility and biodegradability, (ii) appropriate surface chemistry and (iii) highly porous, with interconnected pore network. Engineering analyses showed that scaffold 5211 possessed 3D interconnected homogenous porous structure with a porosity of about 49%, pore sizes ranging from 8.97 to 337 µm, mechanical strength 20.3 MPa, Young's Modulus 271±63 MPa and enzymatic degradation rate 22.7 within 14 days.     

Neuroprotection in a Novel Mouse Model of Multiple Sclerosis

Multiple sclerosis is an immune-mediated, demyelinating and neurodegenerative disease that currently lacks any neuroprotective treatments. Innovative neuroprotective trial designs are required to hasten the translational process of drug development. An ideal target to monitor the efficacy of strategies aimed at treating multiple sclerosis is the visual system, which is the most accessible part of the human central nervous system. A novel C57BL/6 mouse line was generated that expressed transgenes for a myelin oligodendrocyte glycoprotein-specific T cell receptor and a retinal ganglion cell restricted-Thy1 promoter-controlled cyan fluorescent protein. This model develops spontaneous or induced optic neuritis, in the absence of paralytic disease normally associated with most rodent autoimmune models of multiple sclerosis. Demyelination and neurodegeneration could be monitored longitudinally in the living animal using electrophysiology, visual sensitivity, confocal scanning laser ophthalmoscopy and optical coherence tomography all of which are relevant to human trials. This model offers many advantages, from a 3Rs, economic and scientific perspective, over classical experimental autoimmune encephalomyelitis models that are associated with substantial suffering of animals. Optic neuritis in this model led to inflammatory damage of axons in the optic nerve and subsequent loss of retinal ganglion cells in the retina. This was inhibited by the systemic administration of a sodium channel blocker (oxcarbazepine) or intraocular treatment with siRNA targeting caspase-2. These novel approaches have relevance to the future treatment of neurodegeneration of MS, which has so far evaded treatment.     

Antibacterial, antifungal and cytotoxic properties of some sulfonamide-derived chromones

A series of antibacterial and antifungal sulfonamide (sulfanilamide, sulfaguanidine, sulfamethaxozole, 4-aminoethylbenzene-sulfonamide and 4-amino-6-trifluoromethyl-benzene-1,3-disulfonamide) derived chromones, previously reported as inhibitors of carbonic anhydrase, have been screened for in-vitro antibacterial activity against four Gram-negative (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi and Shigella flexener) and two Gram-positive (Bacillus subtilis and Staphylococcus aureus) bacterial strains, and for in-vitro antifungal activity against Trichophyton longifusus, Candida albicans, Aspergillus flavus, Microsporum canis, Fusarium solani, Candida glaberata. All compounds (1)-(5) showed significant antibacterial activity against all four Gram-negative species and both Gram-positive species. However, three of them, (1), (4) and (5), were found to be comparatively much more active compared to (2) and (3). Of these, (5) was found to be the most active one. For antifungal activity, generally compounds (1) and (2) showed significant activity against more than three strains whereas (3)-(5) also showed significant activity against varied fungal strains. In the brine shrimp bioassay for in-vitro cytotoxic properties, only two compounds, (4) and (5) displayed potent cytotoxic activity, LD50 = 2.732 x 10(-4)M) and LD50 = 2.290 x 10(-4)M) respectively, against Artemia salina.     

Evaluation of physiological importance of metallothionein protein expressed by Tetrahymena cadmium metallothionein 1 (TMCd1) gene in Escherichia coli

TMCd1 is a cadmium inducible metallothionein (MT) gene. In the present study the TMCd1 gene of a ciliate protozoan has been expressed in E. coli and the function of the expressed TMCd1 protein as a metal-binding protein has been evaluated. The growth of E. coli cells expressing the GST fused TMCd1 proteins in the presence of cadmium metal clearly demonstrated the role of TMCd1 as a metal-binding protein. The metal accumulation experiments showed that the bacterial cells expressing the functional TMCd1 protein accumulated 19-fold more cadmium in contrast to control cells that lacked the TMCd1 protein expression. The results clearly demonstrate a physiological role of full length TMCd1 protein of a ciliate, expressed in E. coli, in cadmium metal sequestration and detoxification.