ELECTRON MICROSCOPE STUDIES OF SPERMATOZOA OF RHYNCHOSCIARA SP : I. Disruption of Microtubules by Various Treatments

The flagellar complex of the unusual motile spermatozoon of the fungus gnat, Rhynchosciara sp, does not conform to the usual "9 + 2" filament pattern but rather consists of over 350 pairs of filaments (doublet microtubules) distributed in a spiral array. Experiments were designed to disrupt and extract flagellar microtubular components from spermatozoa of the fungus gnat. Pepsin, chymotrypsin, potassium iodide, urea, and heat were used to extract specific portions of microtubule walls Such experiments provide information on the composition of the wall and the existence of wall sites selectively sensitive to various treatments Results obtained include: (a) doublet microtubules are comprised at least in part of protein, and all subunits are probably not identical; (b) a portion of the B subfiber is apparently more sensitive to disruption than other portions of the doublet microtubule; and (c) the ac cessory singlet microtubules may be chemically different from the doublet microtubules     

Structural Basis for the Association of MAP6 Protein with Microtubules and Its Regulation by Calmodulin

Microtubules are highly dynamic αβ-tubulin polymers. In vitro and in living cells, microtubules are most often cold- and nocodazole-sensitive. When present, the MAP6/STOP family of proteins protects microtubules from cold- and nocodazole-induced depolymerization but the molecular and structure determinants by which these proteins stabilize microtubules remain under debate. We show here that a short protein fragment from MAP6-N, which encompasses its Mn1 and Mn2 modules (MAP6(90–177)), recapitulates the function of the full-length MAP6-N protein toward microtubules, i.e. its ability to stabilize microtubules in vitro and in cultured cells in ice-cold conditions or in the presence of nocodazole. We further show for the first time, using biochemical assays and NMR spectroscopy, that these effects result from the binding of MAP6(90–177) to microtubules with a 1:1 MAP6(90–177):tubulin heterodimer stoichiometry. NMR data demonstrate that the binding of MAP6(90–177) to microtubules involve its two Mn modules but that a single one is also able to interact with microtubules in a closely similar manner. This suggests that the Mn modules represent each a full microtubule binding domain and that MAP6 proteins may stabilize microtubules by bridging tubulin heterodimers from adjacent protofilaments or within a protofilament. Finally, we demonstrate that Ca²⁺-calmodulin competes with microtubules for MAP6(90–177) binding and that the binding mode of MAP6(90–177) to microtubules and Ca²⁺-calmodulin involves a common stretch of amino acid residues on the MAP6(90–177) side. This result accounts for the regulation of microtubule stability in cold condition by Ca²⁺-calmodulin.     

The sequence of a region of bacteriophage phiX174 DNA coding for parts of genes A and B

The kinetics of microtubule assembly were investigated by monitoring changes in turbidity which result from the scattering of incident light by the polymer. These studies indicated that assembly occurred by a pathway involving a nucleation phase, followed by an elongation phase as evidenced by a lag in the polymerization kinetics, followed by a psuedo-first-order exponential increase in turbidity. Analytical ultracentrifugation of solutions polymerized to equilibrium showed that 6 S tubulin was the only species detectable in equilibrium with microtubules. Investigation of the elongation reaction in mixtures of 6 S tubulin and microtubule fragments demonstrated that: (1) the net rate of assembly was the sum of the rates of polymerization and depolymerization; (2) the rate of polymerization was proportional to the product of the microtubule number concentration and the 6 S tubulin concentration; and (3) the rate of depolymerization was proportional to the number concentration of microtubules. These results demonstrate that microtubule assembly occurs by a condensation polymerization mechanism consisting of distinct nucleation and elongation steps. Microtubules are initiated in a series of protein association reactions in a pathway that has not been fully elucidated. Elongation proceeds by the consecutive association of 6 S tubulin subunits onto the ends of existing microtubules. Similarly, depolymerization occurs by dissociation of 6 S subunits from the ends of microtubules. The rate constants measured for polymerization and depolymerization at 30 °C were 4 × 10⁶m^?1 s^?1 and 7 s^?1, respectively.     

Characterization of a Chlamydomonas Insertional Mutant that Disrupts Flagellar Central Pair Microtubule-associated Structures

Two alleles at a new locus, central pair–associated complex 1 (CPC1), were selected in a screen for Chlamydomonas flagellar motility mutations. These mutations disrupt structures associated with central pair microtubules and reduce flagellar beat frequency, but do not prevent changes in flagellar activity associated with either photophobic responses or phototactic accumulation of live cells. Comparison of cpc1 and pf6 axonemes shows that cpc1 affects a row of projections along C1 microtubules distinct from those missing in pf6, and a row of thin fibers that form an arc between the two central pair microtubules. Electron microscopic images of the central pair in axonemes from radial spoke–defective strains reveal previously undescribed central pair structures, including projections extending laterally toward radial spoke heads, and a diagonal link between the C2 microtubule and the cpc1 projection. By SDS-PAGE, cpc1 axonemes show reductions of 350-, 265-, and 79-kD proteins. When extracted from wild-type axonemes, these three proteins cosediment on sucrose gradients with three other central pair proteins (135, 125, and 56 kD) in a 16S complex. Characterization of cpc1 provides new insights into the structure and biochemistry of the central pair apparatus, and into its function as a regulator of dynein-based motility.     

The Drosophila Gene abnormal spindle Encodes a Novel Microtubule-associated Protein That Associates with the Polar Regions of the Mitotic Spindle

abnormal spindle, a gene required for normal spindle structure and function in Drosophila melanogaster, lies immediately adjacent the gene tolloid at 96A/B. It encodes a 220-kD polypeptide with a predicted pI of 10.8. The recessive mutant allele asp¹ directs the synthesis of a COOH terminally truncated or internally deleted peptide of ~124 kD. Wild-type Asp protein copurifies with microtubules and is not released by salt concentrations known to dissociate most other microtubule-associated proteins. The bacterially expressed NH₂-terminal 512-amino acid peptide, which has a number of potential phosphorylation sites for p34^cdc2 and MAP kinases, strongly binds to microtubules. The central 579-amino acid segment of the molecule contains one short motif homologous to sequences in a number of actin bundling proteins and a second motif present at the calmodulin binding sites of several proteins. Immunofluorescence studies show that the wild-type Asp protein is localized to the polar regions of the spindle immediately surrounding the centrosome. These findings are discussed in relation to the known spindle abnormalities in asp mutants.     

The ras-related YPT1 gene product in yeast: A GTP-binding protein that might be involved in microtubule organization

The 23.5 kd protein product of the ras-related YPT1 gene of S. cerevisiae was found to be essential for cell growth. The loss of YPT1 function, studied in cells with the YPT1 gene on chromosome VI regulated by the galactose-inducible GAL10 promoter, led to arrested cells that were multibudded and exhibited a complete disorganization of microtubules and an apparent loss of nuclear integrity. The YPT protein binds GTP specifically. GTP binding of the protein is essential for its intracellular function. The Asn¹²¹→lle substitution, generated by site-directed mutagenesis, had a dominant lethal phenotype, the expression of the mutant protein led to binucleated cells and abnormal spindles. In contrast to the S. cerevisiae RAS1 and RAS2 gene products, the YPT protein seems to be involved, directly or indirectly, in microtubule organization and function.     

A carbamate herbicide causes microtubule and microfilament disruption and nuclear fragmentation in fibroblasts

Microtubule associated proteins (MAPs) are high molecular weight proteins that associate with microtubules during polymerization. This report describes a high molecular weight protein fraction with a molecular weight of approx. 290 000 from cultured mammalian fibroblasts that associates with polymerized rat brain tubulin. This protein(s), which is referred to as f-MAP, is enriched approx. 25-fold in a twice polymerized microtubules when compared with the original cell extract. Polymerization of rat brain extract in the presence of in vivo ³²P-labeled fibroblast extract reveals the presence of a ³²P-labeled protein in the polymerized pellet with the same electrophoretic mobility as f-MAP. The present study suggests that fibroblasts in culture contain a high molecular weight phosphoprotein with properties and a molecular weight very similar to the MAPs described in mammalian brain.     

Microtubule reassembly in vitro of Strongylocentrotus purpuratus sperm tail outer doublet tubulin

Strongylocentrotus purpuratus outer doublet microtubules were prepared by extraction of sperm tail axonemes with 0.6 m-KCl. Sonication of the outer doublet microtubules in 5 mm-2-(N-morpholino)ethanesulphonic acid, 1 mm-ethyleneglycol-bis-(β-aminoethyl ether) N,N′-tetraacetic acid, 1 inm-MgSO₄ (pH 6.7) solubilized up to 35% of the outer doublet protein, depending on the power input, in a manner which was non-selective for either subfiber. Tubulin comprised 75 to 85% of the total solubilized protein in a 200,000 g supernatant obtained from the sonicated suspension. Colchicine-binding assays demonstrated that the tubulin was largely in a native form (K_A = 10⁶, liters mole^?; 0.74 mole of colchicine bound per mole of tubulin at infinite concentration of colchicine).Microtubule self-assembly from the 200,000 g supernatants in the absence of added seeds or glycerol was quantitated by light-scattering at 350 nm. The critical protein concentration for assembly was 0.55 mg ml^?1 at 37 °C and the reaction occurred optimally in the presence of 2 mm-GTP and 150 mm-KCl. The solubilized outer doublet tubulin formed singlet microtubules upon reassembly under our in vitro conditions. The authenticity of the microtubules was verified by both negative stain and thin-section electron microscopy. Polymerization was prevented by colchicine and podophyllotoxin, and depolymerization occurred rapidly on cooling the microtubules to 0 °C.The susceptibility of the reassembled microtubules to low temperature suggested that they could be “recycled” by the warm assembly-cold disassembly procedure developed for vertebrate brain (Borisy et al., 1974). Twice recycled outer doublet tubulin was devoid of high molecular weight microtubule-associated proteins, as judged by gel electrophoresis in the presence of sodium dodecyl sulfate. However, trace amounts (less than 5%) of intermediate molecular weight material was visible on heavily overloaded gels. The function of this material is uncertain, but it is not chemically equivalent to the tau factor of vertebrate brain (Weingarten et al., 1975), since it cannot be separated from the tubulin by phosphocellulose adsorption. In addition, phosphocellulose-treated tubulin reassembled to the same extent as untreated tubulin, suggesting that the reassembly of outer doublet tubulin does not require the protein equivalents of brain microtubule-associated proteins or tau factor. If accessory proteins are required for the reassembly of outer doublet tubulin, they are not removed by phosphocellulose under the conditions employed, and they must comprise less than 5% of the total protein.     

Evaluating fermentation effects on cell growth and crude extract metabolic activity for improved yeast cell-free protein synthesis

Saccharomyces cerevisiae is a promising source organism for the development of a practical, eukaryotic crude extract based cell-free protein synthesis (CFPS) system. Crude extract CFPS systems represent a snapshot of the active metabolism in vivo, in response to the growth environment at the time of harvest. Therefore, fermentation plays a central role in determining metabolic activity in vitro. Here, we developed a fermentation protocol using chemically defined media to maximize extract performance for S. cerevisiae-based CFPS. Using this new protocol, we obtained a 4-fold increase in protein synthesis yields with extracts derived from wild-type S288c as compared to a previously developed protocol that uses complex growth media. The final luciferase yield in our new method was 8.86 ± 0.28 μg mL⁻¹ in a 4 h batch reaction. For each of the extracts processed under different fermentation conditions, synthesized protein, precursor monomers (amino acids), and energy substrates (nucleotides) were evaluated to analyze the effect of the changes in the growth environment on cell-free metabolism. This study underscores the critical role fermentation plays in preparing crude extract for CFPS reactions and offers a simple strategy to regulate desired metabolic activity for cell-free synthetic biology applications based on crude cell extracts.     

Mal3, the Fission Yeast Homologue of the Human APC-interacting Protein EB-1 Is Required for Microtubule Integrity and the Maintenance of Cell Form

Through a screen designed to isolate novel fission yeast genes required for chromosome segregation, we have identified mal3⁺. The mal3-1 mutation decreased the transmission fidelity of a nonessential minichromosome and altered sensitivity to microtubule-destabilizing drugs. Sequence analysis revealed that the 35-kD Mal3 is a member of an evolutionary conserved protein family. Its human counterpart EB-1 was identified in an interaction screen with the tumour suppressor protein APC. EB-1 was able to substitute for the complete loss of the mal3⁺ gene product suggesting that the two proteins might have similar functions. Cells containing a mal3 null allele were viable but showed a variety of phenotypes, including impaired control of cell shape. A fusion protein of Mal3 with the Aequorea victoria green fluorescent protein led to in vivo visualization of both cytoplasmic and mitotic microtubule structures indicating association of Mal3 with microtubules. The absence of Mal3 protein led to abnormally short, often faint cytoplasmic microtubules as seen by indirect antitubulin immunofluorescence. While loss of the mal3⁺ gene product had no gross effect on mitotic spindle morphology, overexpression of mal3⁺ compromised spindle formation and function and led to severe growth inhibition and abnormal cell morphology. We propose that Mal3 plays a role in regulating the integrity of microtubules possibly by influencing their stability.     

α-Bungarotoxin binding in the central nervous system of Limulus polyphemus

The binding of ¹²⁵I-labeled α-bungarotoxin in the central nervous system of the horseshoe crab, Limulus polyphemus, was investigated. Comparative binding studies in various tissues of L. polyphemus demonstrated a selective association of the toxin with nervous tissues. The greatest enrichment of toxin binding in subcellular fractions of brain tissue was observed in a fraction enriched in mitochondria and acetylcholinesterase-containing membranes. Autoradiographic studies revealed the localization of α-bungarotoxin binding to the longitudinal connectives and neuropile regions of the abdominal ganglia. Three toxin binding components with approximate sedimentation coefficients of 9 S, 15.4 S and 17.4 S were present in solubilized extracts of brain tissue. ¹²⁵I-labeled α-bungarotoxin binding to these components was inhibited 72%, 47%, 9% and 0% by 10 μM concentrations of (+)-tubocurarine, nicotine, scopolamine and pilocarpine, respectively. The apparent formation of the 15.4 S and 17.4 S proteins from the 9 S protein was obtained. The 15.4 S and 17.4 S components are suggested as aggregates of the 9 S protein. This 9 S protein is proposed as an acetylcholine receptor from the central nervous system of L. polyphemus.     

Kinesin-8 from Fission Yeast: A Heterodimeric, Plus-End–directed Motor that Can Couple Microtubule Depolymerization to Cargo Movement

Fission yeast expresses two kinesin-8s, previously identified and characterized as products of the klp5⁺ and klp6⁺ genes. These polypeptides colocalize throughout the vegetative cell cycle as they bind cytoplasmic microtubules during interphase, spindle microtubules, and/or kinetochores during early mitosis, and the interpolar spindle as it elongates in anaphase B. Here, we describe in vitro properties of these motor proteins and some truncated versions expressed in either bacteria or Sf9 cells. The motor-plus-neck domain of Klp6p formed soluble dimers that cross-linked microtubules and showed both microtubule-activated ATPase and plus-end–directed motor activities. Full-length Klp5p and Klp6p, coexpressed in Sf9 cells, formed soluble heterodimers with the same activities. The latter recombinant protein could also couple microbeads to the ends of shortening microtubules and use energy from tubulin depolymerization to pull a load in the minus end direction. These results, together with the spindle localizations of these proteins in vivo and their requirement for cell viability in the absence of the Dam1/DASH kinetochore complex, support the hypothesis that fission yeast kinesin-8 contributes both to chromosome congression to the metaphase plate and to the coupling of spindle microtubules to kinetochores during anaphase A.     

Mechanism of cytotoxicity of methylmercury

Mechanism of methylmercury cytotoxicity was investigated with special reference to its preferential action on microtubules and protein biosynthesis in cultured cells. The tubulin synthesis analyzed by autoradiography of two-dimensional electropherogram using³⁵S-methionine was inhibited by 50–70% in mouse glioma cells exposed to 5×10^?6 M methylmercury for 3 h, which almost completely depolymerized microtubules. Total protein synthesis monitored by incorporation of labeled methionine into acid insoluble fraction was decreased slightly but significantly and the protein bands other than tubulin on gradient urea-PAGE gel appeared to remain unchanged under the experimental condition used. These results suggest that the inhibition of protein synthesis observed on exposure to methylmercury can be ascribed, at least partly, to a possible autoregulatory depression in tubulin synthesis owing to the increase in the pool of tubulin subunits resulted from microtubule depolymerization by methylmercury.     

Overexpression of 14-3-3ζ Promotes Tau Phosphorylation at Ser262 and Accelerates Proteosomal Degradation of Synaptophysin in Rat Primary Hippocampal Neurons

β-amyloid peptide accumulation, tau hyperphosphorylation, and synapse loss are characteristic neuropathological symptoms of Alzheimer’s disease (AD). Tau hyperphosphorylation is suggested to inhibit the association of tau with microtubules, making microtubules unstable and causing neurodegeneration. The mechanism of tau phosphorylation in AD brain, therefore, is of considerable significance. Although PHF-tau is phosphorylated at over 40 Ser/Thr sites, Ser²⁶² phosphorylation was shown to mediate β-amyloid neurotoxicity and formation of toxic tau lesions in the brain. In vitro, PKA is one of the kinases that phosphorylates tau at Ser²⁶², but the mechanism by which it phosphorylates tau in AD brain is not very clear. 14-3-3ζ is associated with neurofibrillary tangles and is upregulated in AD brain. In this study, we show that 14-3-3ζ promotes tau phosphorylation at Ser²⁶² by PKA in differentiating neurons. When overexpressed in rat hippocampal primary neurons, 14-3-3ζ causes an increase in Ser²⁶² phosphorylation, a decrease in the amount of microtubule-bound tau, a reduction in the amount of polymerized microtubules, as well as microtubule instability. More importantly, the level of pre-synaptic protein synaptophysin was significantly reduced. Downregulation of synaptophysin in 14-3-3ζ overexpressing neurons was mitigated by inhibiting the proteosome, indicating that 14-3-3ζ promotes proteosomal degradation of synaptophysin. When 14-3-3ζ overexpressing neurons were treated with the microtubule stabilizing drug taxol, tau Ser²⁶² phosphorylation decreased and synaptophysin level was restored. Our data demonstrate that overexpression of 14-3-3ζ accelerates proteosomal turnover of synaptophysin by promoting the destabilization of microtubules. Synaptophysin is involved in synapse formation and neurotransmitter release. Our results suggest that 14-3-3ζ may cause synaptic pathology by reducing synaptophysin levels in the brains of patients suffering from AD.     

Free and centrosome-attached microtubules: Quantitative analysis and modeling of two-component system

In cultivated in vitro interphase animal cells, microtubules form a network whose density is highest in the central cell area, in the region of centrosome, and decreases towards the cell periphery. Since identification of individual microtubules in the central cell area is significantly difficult and more often is impossible, there are several approaches to studying microtubules in the internal cell cytoplasm. These approaches are based on a decrease of microtubule density—both real, due to their partial depolymerization (by the action of cold temperatures or cytostatics), or apparent, due to a decrease of cell thickness (by photobleaching of preexisting microtubules and analysis of newly formed ones). In the present work, we propose a method based on the determination of optical density which allows evaluation of the state of the cytoplasmic microtubule system as a whole. The method consists of a comparison of the dependences describing changes of the microtubule optical density from the cell center to the periphery in controls and in experiments. Analysis of living cells by the proposed method has shown that the character of curves describing the decrease of optical density from the cell center to its periphery is different for various cell types; the dependence can be described both as an exponential regression (the CHO cell line) and as a linear regression (the NIH-3T3 and REF cell lines). Our previous studies have allowed the suggestion that the character of the dependence is determined by the ratio of free and centrosome-attached microtubules and by the position of their ends in the cell cytoplasm. To test this hypothesis, we considered model systems with all microtubules assumed to be in a straight orientation and divergent radially from the centrosome, but with different arrangements of plus-and minus-ends. In the model system, in which all the microtubule minus-ends are attached to the centrosome while the plus-ends are at different distances from it, the microtubule density is described by the exponential (f(x) = ae ^?bx ). Introduction of free microtubules into the system leads to a change of the character of this dependence, and the system in which the concentration of free microtubules with minus ends located at different distances from the cytoplasm is 5 times higher than that of the centrosome-attached microtubules is described by the linear regression equation (f(x) = k ^* x + b), which corresponds to the experimentally obtained dependences for 3T3 and REF cells. Thus, we believe that even in cells with a radial microtubule system, free microtubules may constitute the majority.     

A Role for Katanin-mediated Axonemal Severing during Chlamydomonas Deflagellation

Deflagellation of Chlamydomonas reinhardtii, and other flagellated and ciliated cells, is a highly specific process that involves signal-induced severing of the outer doublet microtubules at a precise site in the transition region between the axoneme and basal body. Although the machinery of deflagellation is activated by Ca²⁺, the mechanism of microtubule severing is unknown. Severing of singlet microtubules has been observed in vitro to be catalyzed by katanin, a heterodimeric adenosine triphosphatase that can remove tubulin subunits from the walls of stable microtubules. We found that purified katanin induced an ATP-dependent severing of the Chlamydomonas axoneme. Using Western blot analysis and indirect immunofluorescence, we demonstrate that Chlamydomonas expresses a protein that is recognized by an anti-human katanin antibody and that this protein is localized, at least in part, to the basal body complex. Using an in vitro severing assay, we show that the protein(s) responsible for Ca²⁺-activated outer doublet severing purify with the flagellar-basal body complex. Furthermore, deflagellation of purified flagellar-basal body complexes is significantly blocked by the anti-katanin antibody. Taken together, these data suggest that a katanin-like mechanism may mediate the severing of the outer doublet microtubules during Chlamydomonas deflagellation.     

Administration of the hot-water extract of Spirulina platensis enhanced the immune response of white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus

White shrimp Litopenaeus vannamei which had been injected with the hot-water extract of Spirulina platensis at 6, 10, and 20 μg g^?1, or immersed in aerated seawater containing extract at 200, 400, and 600 mg L^?1 were challenged with Vibrio alginolyticus at 1.5 × 10⁶ or 1.4 × 10⁶ colony-forming units (cfu) shrimp^?1, and then placed in seawater. Survival rates of shrimp that received the extract of S. platensis at 6–20 μg g^?1, and those of shrimp immersed in seawater containing the extract at 400 and 600 mg L^?1 were significantly higher than those of control shrimp after 24–96 and 48–96 h, respectively. In a separate experiment, the hyaline cell (HC) count, granular cell (GC, including semi-granular cell) count, total haemocyte count (THC), phenoloxidase (PO) activity, respiratory burst (RB), superoxide dismutase (SOD) activity, glutathione peroxidase (GPx) activity, and lysozyme activity were measured when shrimp were injected with the extract at 6, 10, and 20 μg g^?1, and immersed in seawater containing the extract at 200, 400, and 600 mg L^?1. These parameters directly increased with the concentration, and significantly increased when shrimp were immersed in the seawater containing the extract at 0.5–4 h. L. vannamei that received all doses of the extract via injection or via immersion all had increased phagocytic activity and clearance efficiency to V. alginolyticus at 12–72 h and 3–4 h, respectively. It was concluded that L. vannamei that received the hot-water extract of S. platensis had enhanced innate immunity and increased resistance against V. alginolyticus infection.     

AUR1, a novel gene conferring aureobasidin resistance onSaccharomyces cerevisiae: a study of defective morphologies in Aur1p-depleted cells

Aureobasidin A (AbA), a cyclic depsipeptide produced byAureobasidium pullulans R106, is highly toxic to fungi includingSaccharomyces cerevisiae. We isolated several dominant mutants ofS. cerevisiae which are resistant to more than 25 µg/ml of AbA. From a genomic library of one suchAUR1 mutant, theAUR1 ^R (foraureobasidinresistant) mutant gene was isolated as a gene that confers resistance to AbA on wild-type cells. Its nucleotide sequence showed that the predicted polypeptide is a hydrophobic protein composed of 401 amino acids, which contains several possible transmembrane domains and at least one predicted N-linked glycosylation site. Comparison of the mutant gene with the wild-typeaur1 ⁺ gene revealed that the substitution of Phe at position 158 by Tyr is responsible for acquisition of AbA resistance. We suggest that the gene product of the wild-typeaur1 ⁺ is a target for AbA on the basis of following results. Firstly, cells that overexpress the wild-typeaur1 ⁺ gene become resistant to AbA, just as cells with anAUR1 ^R mutation do. Secondly, disruption of theaur1 ⁺ gene demonstrated that it is essential for growth. Thirdly, in the cells with a disruptedaur1 locus, pleiotropic morphological changes including disappearance of microtubules, degradation of tubulin and abnormal deposition of chitin were observed. Some of these abnormalities are also observed when wild-type cells are treated with AbA. The abnormality in microtubules suggests that the Aur1 protein is involved in microtubule organization and stabilization.     

Hepatoprotective Effects of Solanum nigrum Linn Fruits Against Cadmium Chloride Toxicity in Albino Rats

The present work is aimed to investigate the toxicity of 1/20 LD₅₀ of cadmium chloride (CdCl₂) on male albino rats by oral ingestion and to determine the hepatoprotective effect of Solanum nigrum Linn (SN) dried fruits and their ethanolic extract against CdCl₂ toxicity using biochemical parameters. Rats were divided into six groups; the first group is control, second group is CdCl₂-intoxicated rats, third group is fed with a semi-modified diet with S. nigrum fruits, fourth group rats ingested with dried extract, and intoxicated rats (groups 5 and 6) were treated with fruits and ethanolic extract of S. nigrum, respectively. The results showed that rats exposed to CdCl₂ induced remarkable decrease in body weight gain, feed efficiency, and Hb, Hct, RBC, and WBC count and MCHC, but increase in MCV and MCH values. In the case of plasma enzymes, there were significant stimulations observed in ALT and AST, acid phosphatase, alkaline phosphatase, and LDH activities of CdCl₂-intoxicated rats (group 2) compared to control (group 1). Plasma protein profile showed decreases in total soluble protein and albumin; also globulin content was decreased by CdCl₂ ingestion. Under the same condition, plasma total bilirubin and glucose levels were increased in group 2. In addition, lipid peroxidation and antioxidative system (GSH, catalase, and SOD) of liver were harmed by CdCl₂ ingestion. Whereas, normal rats treated with SN showed insignificant changes in groups 3 and 4 as compared to control (group 1). The treatment with dried fruits and their ethanolic extract in CdCl₂-intoxicated rats (groups 5 and 6) ameliorated and improved these harmful effects in all above parameters either for blood or liver. The results of this study suggest the protective effect of S. nigrum against liver injury happened by CdCl₂ which may be attributed to its hepatoprotective activity and thereby.     

Loss of S6K1 But Not S6K2 in the Tumor Microenvironment Suppresses Tumor Growth by Attenuating Tumor Angiogenesis

Two isoforms of the 70-kDa ribosomal protein S6 kinase, S6K1 and S6K2, have been identified and are considered key downstream effectors of the mTOR signaling pathway, which is involved in tumor growth and progression. However, their biological roles in the tumor microenvironment are poorly understood. In this study, utilizing tumor xenograft models in S6k1^−/− and S6k2^−/− mice, we show that loss of S6K1 but not S6K2 in the tumor stroma suppresses tumor growth, accompanied by attenuated tumor angiogenesis. We found that while S6K1 depletion had no effect on the proangiogenic phenotype of endothelial cells, the growth and angiogenesis of tumor xenografts were significantly reduced in wild-type mice upon reconstitution with S6K1-deficient bone marrow cells. Furthermore, upon S6K1 loss, induction of both mRNA and protein levels of Hif-1α and those of the downstream target, Vegf, was compromised in bone marrow–derived macrophages stimulated with lactate. These findings indicate that S6K1 but not S6K2 contributes to establishing a microenvironment that favors tumor growth through mediating angiogenesis, and suggest that attenuated tumor angiogenesis upon loss of S6K1 in the tumor stroma is, at least in part, attributable to impaired upregulation of Vegf in tumor-associated macrophages.