Thyroid status alters gill ionic metabolism and chloride cell morphology as evidenced by scanning electron microscopy in a teleost Anabas testudineus (Bloch): short and long term in vivo study

Gill is the main organ of osmotic regulation in teleosts and chloride cells are the sites of ion transport across gill epithelium. Thyroid hormones are implicated in the regulation of osmotic balance in teleosts also. Treatment with 6-propyl thiouracil (6-PTU) inhibited the membrane bound enzyme Na+K+ ATPase in the gill while triiodothyronine (T3) injection stimulated it in a short-term in vivo study in the teleost Anabas testudineus. Na+, K+ and Ca2+ ions were also decreased in the 6-PTU treated fish and the T3 treatment increased their concentrations in the gill lamellae. The gill morphology also changed according to the thyroid status in the long term study. 6-PTU treatment altered the typical serrated morphology of the gill lamellae, while the T3 treatment reversed it. T3 injection increased the density of pavement and chloride cells as evidenced by scanning electron microscopy. The results demonstrate that physiological status of the thyroid influences gill Na+ pump activity and chloride cell morphological changes. Further, the study suggests a regulatory role of T3 on gill ions (Na+, K+ and Ca2+), Na+K+ and Ca2+ ATPase activity and the different gill cell types in A. testudineus.     

Assembly of ovarian follicles in the caecilians Ichthyophis tricolor and Gegeneophis ramaswamii: light and transmission electron microscopic study

Though much is known about various aspects of reproductive biology of amphibia, there is little information on the cellular and mechanistic basis of assembly of ovarian follicles in this group. This is especially true of the caecilians. Therefore, taking advantage of the abundant distribution of caecilians in the Western Ghats of India, two species of caecilians, Ichthyophis tricolor and Gegeneophis ramaswamii, were subjected to light and transmission electron microscopic analysis to trace the sequential changes during the assembly of ovarian follicles. The paired ovaries of these caecilians are elongated sac-like structures each including numerous vitellogenic follicles. The follicles are connected by a connective tissue stroma. This stroma contains nests of oogonia, primary oocytes and pregranulosa cells as spatially separated nests. During assembly of follicles the oocytes increase in size and enter the meiotic prophase when the number of nucleoli in the nucleus increases. The mitochondrial cloud or Balbiani vitelline body, initially localized at one pole of the nucleus, disperses through out the cytoplasm subsequently. Synaptonemal complexes are prominent in the pachytene stage oocytes. The pregranulosa cells migrate through the connective tissue fibrils of the stroma and arrive at the vicinity of the meiotic prophase oocytes. On contacting the oocyte, the pregranulosa cells become cuboidal in shape, wrap the diplotene stage oocyte as a discontinuous layer and increase the content of cytoplasmic organelles and inclusions. The oocytes increase in size and are arrested in diplotene when the granulosa cells become flat and form a continuous layer. Soon a perivitelline space appears between the oolemma and granulosa cells, completing the process of assembly of follicles. Thus, the events in the establishment of follicles in the caecilian ovary are described.     

Cryptococcus laurentii Biofilms: Structure, Development and Antifungal Drug Resistance

A great number of fungal infections are related to biofilm formation on inert or biological surfaces, which are recalcitrant to most treatments and cause human mortality. Cryptococcus laurentii has been diagnosed as the aetiological pathogen able to cause human infections mainly in immunosuppressed patients and the spectrum of clinical manifestations ranges from skin lesions to fungaemia. The effect of temperature, pH and surface preconditioning on C. laurentii biofilm formation was determined by 2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide (XTT) reduction assay. Scanning electron microscopic (SEM) analysis of C. laurentii biofilms demonstrated surface topographies of profuse growth and dense colonization with extensive polymeric substances around the cells. In this study, we determined the activity of amphotericin B, itraconazole and fluconazole against C. laurentii free-living cells and biofilms. The activity of antifungals tested was greater against free-living cells, but sessile cells fell into the resistant range for these antifungal agents. Extracellular polymeric substances (EPS), comprising the matrix of C. laurentii biofilms, were isolated by ultrasonication. Fourier transform infrared spectroscopy (FT-IR) was performed with ethanol-precipitated and dried samples. Also, the multielement analysis of the EPS was performed by inductively coupled plasma optical emission spectroscopy (ICP-OES).     

RSC facilitates Rad59-dependent homologous recombination between sister chromatids by promoting cohesin loading at DNA double-strand breaks

Homologous recombination repairs DNA double-strand breaks by searching for, invading, and copying information from a homologous template, typically the homologous chromosome or sister chromatid. Tight wrapping of DNA around histone octamers, however, impedes access of repair proteins to DNA damage. To facilitate DNA repair, modifications of histones and energy-dependent remodeling of chromatin are required, but the precise mechanisms by which chromatin modification and remodeling enzymes contribute to homologous DNA repair are unknown. Here we have systematically assessed the role of budding yeast RSC (remodel structure of chromatin), an abundant, ATP-dependent chromatin-remodeling complex, in the cellular response to spontaneous and induced DNA damage. RSC physically interacts with the recombination protein Rad59 and functions in homologous recombination. Multiple recombination assays revealed that RSC is uniquely required for recombination between sister chromatids by virtue of its ability to recruit cohesin at DNA breaks and thereby promoting sister chromatid cohesion. This study provides molecular insights into how chromatin remodeling contributes to DNA repair and maintenance of chromatin fidelity in the face of DNA damage.     

Conformational characterization of human eukaryotic initiation factor 2α: A single tryptophan protein

The α-subunit of the human eukaryotic initiation factor 2 (heIF2α), a GTP binding protein, plays a major role in the initiation of protein synthesis. During various cytoplasmic stresses, eIF2α gets phosphorylated by eIF2α-specific kinases resulting in inhibition of protein synthesis. The cloned and over expressed heIF2α, a protein with a single tryptophan (trp) residue was examined for its conformational characteristics using steady-state and time-resolved tryptophan fluorescence, circular dichroism (CD) and hydrophobic dye binding. The steady-state fluorescence spectrum, fluorescence lifetimes (τ₁ = 1.13 ns and τ₂ = 4.74 ns) and solute quenching studies revealed the presence of trp conformers in hydrophobic and differential polar environment at any given time. Estimation of the α-helix and β-sheet content showed: (i) more compact structure at pH 2.0, (ii) distorted α-helix and rearranged β-sheet in presence of 4 M guanidine hydrochloride and (iii) retention of more than 50% ordered structure at 95 °C. Hydrophobic dye binding to the protein with loosened tertiary structure was observed at pH 2.0 indicating the existence of a molten globule-like structure. These observations indicate the inherent structural stability of the protein under various denaturing conditions.     

Insulin regulates ionic metabolism in a fresh water teleost, anabas testudineus (bloch)

Short term effects of insulin on total brain and branchial Na+K+ ATPase, Ca2+ ATPase and Na+, K+ and Ca2+ ions were investigated in A. testudineus. The increase in brain Ca2+ ATPase after alloxan treatment may account for an increased amount of intracellular calcium required for biochemical events taking place inside the cells. Branchial Na+K+ATPase was significantly stimulated while Ca2+ ATPase significantly inhibited after alloxan treatment. This suggests that alloxan exerts its inhibitory effect on the ATP-driven Ca2+ transport via; its action on the Ca2+ pump protein rather than the membrane permeability to Ca2+. The increased activity of brain Na+K+ ATPase at 3 and 24 hr by insulin to alloxan pretreated fish may account for the stimulated co-transport of glucose and its utilization for energy requirements and the excitatory action on neurons in the brain. The elevated brain Ca2+ ATPase may be due to the role of calcium as a second messenger in hormone action. At 24 hr, the activity of branchial Na+K+ ATPase and Ca2+ ATPase in alloxan pretreated specimens was significantly stimulated by insulin. This may be due to increased synthesis of these enzyme units. Administration of insulin (lU/fish) in normal fish significantly inhibited the activity of brain and branchial Na+K+ ATPase while brain Ca2+ ATPase showed a stimulatory effect at 3 and 24 hr compared to control. Inhibition of total branchial Ca2+ ATPase activity by insulin may be due to increased Ca2+ concentration. Higher plasma glucose level in alloxan treated groups confirms the diabetic effect of alloxan. Insulin reverses this effect. The possible mechanism by which insulin controls Na+K+ ATPase activity appears to be tissue specific. The results seem to be the first report on the effect of insulin on ATPase activity in a teleost. These data are consistent with the hypothesis that insulin performs a role in hydro mineral regulation in freshwater teleosts.     

A dual protease approach for expression and affinity purification of recombinant proteins

We describe a new method for affinity purification of recombinant proteins using a dual protease protocol. Escherichia coli maltose binding protein (MBP) is employed as an N-terminal tag to increase the yield and solubility of its fusion partners. The MBP moiety is then removed by rhinovirus 3C protease, prior to purification, to yield an N-terminally His₆-tagged protein. Proteins that are only temporarily rendered soluble by fusing them to MBP are readily identified at this stage because they will precipitate after the MBP tag is removed by 3C protease. The remaining soluble His₆-tagged protein, if any, is subsequently purified by immobilized metal affinity chromatography (IMAC). Finally, the N-terminal His₆ tag is removed by His₆-tagged tobacco etch virus (TEV) protease to yield the native recombinant protein, and the His₆-tagged contaminants are removed by adsorption during a second round of IMAC, leaving only the untagged recombinant protein in the column effluent. The generic strategy described here saves time and effort by removing insoluble aggregates at an early stage in the process while also reducing the tendency of MBP to “stick” to its fusion partners during affinity purification.     

Ultrastructural observations of previtellogenic ovarian follicles of the caecilians Ichthyophis tricolor and Gegeneophis ramaswamii

The ultrastructural organization of the previtellogenic follicles of the caecilians Ichthyophis tricolor and Gegeneophis ramaswamii, of the Western Ghats of India, were observed. Both species follow a similar seasonal reproductive pattern. The ovaries contain primordial follicles throughout the year with previtellogenic, vitellogenic, or postvitellogenic follicles, depending upon the reproductive status. The just-recruited primordial follicle includes an oocyte surrounded by a single layer of follicle and thecal cells. The differentiation of the theca into externa and interna layers, the follicle cells into dark and light variants, and the appearance of primordial yolk platelets and mitochondrial clouds in the ooplasm mark the transition of the primordial follicle into a previtellogenic follicle. During further development of the previtellogenic follicle the following changes occur: i) the theca loses distinction as externa and interna; ii) all the follicle cells become the dark variant and increase in the complexity of ultrastructural organization; iii) the nucleus of the oocyte transforms into the germinal vesicle and there is amplification of the nucleoli; iv) the primordial yolk platelets of the cortical cytoplasm of the oocyte increase in abundance; v) the mitochondrial clouds fragment and the mitochondria move away from the clouds, leaving behind the cementing matrix, which contains membrane-bound vesicles of various sizes, either empty or filled with a lipid material; vi) the perivitelline space appears first as troughs at the junctional points between the follicle cells and oocyte, which subsequently spread all around to become continuous; vii) macrovilli and microvilli develop from the follicle cells and oocyte, respectively; and viii) the precursor material of the vitelline envelop arrives at the perivitelline space. The sequential changes in the previtellogenic follicles of two species of caecilians are described.