Complex regulation of membrane-type matrix metalloproteinase expression and matrix metalloproteinase-2 activation by concanavalin A in MDA-MB-231 human breast cancer cells

Matrix Metalloproteinase-2 (MMP-2) is secreted as a zymogen, the activation of which has been associated with metastatic progression in human breast cancer (HBC). Concanavalin A (Con A) has been found to induce activation of MMP-2 in invasive HBC cell lines. Con A effects on the expression of mRNA for membrane-type matrix metalloproteinase (MT-MMP), a newly described cell surface-associated MMP, showed a close temporal correlation with induction of MMP-2 activation. It is surprising that MT-MMP mRNA is constitutively present in the uninduced MDA-MB-231 cell, despite a lack of MMP-2 activation. We have used actinomycin D to demonstrate a partial requirement for de novo gene expression in the induction of MMP-2 activation by Con A in MDA-MB-231 HBC cells. Furthermore, this transcriptional response to Con A appeared to require the continued presence of Con A for its manifestation. The nontranscriptional component of the Con A induction manifests rapidly, is quite substantial, and persists strongly despite actinomycin D abrogation of both constitutive and Con A-induced MT-MMP. Cycloheximide analyses suggest that protein synthesis may be involved in this rapid transcription-independent response. These studies suggest that Con A induces MMP-2-activation in part by up-regulation of MT-MMP expression but has a more complicated mode of action, involving additional nontranscriptional effects, which apparently require protein synthesis.

Association of MMP-2 activation potential with metastatic progression in human breast cancer cell lines independent of MMP-2 production

Background: Expression of matrix metalloproteinase-2 (MMP-2), the 72-kd type IV collagenase/gelatinase, by cancer cells has been implicated in metastasis through cancer cell invasion of basement membranes mediated by degradation of collagen IV. However, the abundance of this latent proenzyme in normal tissues and fluids suggests that MMP-2 proenzyme utilization is limited by its physiological activation rather than expression alone. We previously reported activation of this proenzyme by normal and malignant fibroblastoid cells cultured on collagen I (vitrogen) gels. Purpose: Our purposes in this study were 1) to determine whether MMP-2 activation is restricted to the more invasive human breast cancer cell lines and 2) to localize the activating mechanism. Methods: Zymography was used to monitor MMP-2 activation through detection of latent MMP-2 (72 kd) and mature species of smaller molecular weight (59 or 62 kd). Human breast cancer cell lines cultured on plastic, vitrogen, and other matrices were thus screened for MMP- 2 activation. Collagen I-cultured cells were exposed to cycloheximide, a protein synthesis inhibitor, or to protease inhibitors to determine the nature of the MMP-2-activating mechanism. Triton X-114 (TX-114) detergent extracts from cells cultured on collagen I or plastic were incubated with latent MMP-2 and analyzed by zymography to localize the MMP-2 activator. Results: MMP-2 activation was only induced by collagen I culture in the more aggressive, highly invasive estrogen receptor-negative, vimentin-positive human breast cancer cell lines (Hs578T, MDA-MB-436, BT549, MDA-MB-231, MDA- MB-435, MCF-7(ADR)) and was independent of MMP-2 production. MMP-2 activation was detected in cells cultured on collagen I gels but not in those cultured on gelatin gels, Matrigel, or thin layers of collagen I or IV, gelatin, or fibronectin. Collagen-induced activation was specific for the enzyme species MMP-2, since MMP-9, the 92-kd type IV collagenase/gelatinase, was not activatable under similar conditions. MMP-2 activation was inhibited by cycloheximide and was sensitive to a metalloproteinase inhibitor but not to aspartyl, serine, or cysteinyl protease inhibitors. MMP-2 activation was detected in the hydrophobic, plasma membrane-enriched, TX-114 extracts from invasive collagen I-cultured cells. Conclusion: Collagen I-induced MMP-2 activation is restricted to highly invasive estrogen receptor-negative, vimentin-positive human breast cancer cell lines, is independent of MMP-2 production, and is associated with metastatic potential. Our findings are consistent with plasma membrane localization of the activator. Implications: The MMP-2 activation mechanism may represent a new target for diagnosis, prognosis, and treatment of human breast cancer.

Collagen biosynthesis in cultured rat testicular Sertoli and peritubular myoid cells

The incorporation of 3H-proline into protein was regarded as a measure of total protein synthesis and the incorporation into hydroxyproline as indicative of collagen synthesis. Relative collagen synthesis (expressed as percent of total protein synthesized) by Sertoli and peritubular myoid cells cultured from 20-22 day old rat testis was estimated. In both secreted and cellular pools, relative collagen synthesis by Sertoli cells was significantly greater than by peritubular myoid cells. Coculture of Sertoli and myoid cells resulted in a significant increase in relative collagen synthesis when compared to monocultures of each cell type. Addition of serum to peritubular myoid cells resulted in a stronger stimulation of relative collagen production. Sertoli cell extracellular matrix inhibited relative collagen synthesis by peritubular myoid cells in the presence or absence of serum. Radioactivity into hydroxyproline as corrected per cellular DNA also showed similar results. Immunolocalization studies confirmed that both cell types synthesize type I and type IV collagens. These results indicate that stimulation of collagen synthesis observed in Sertoli-myoid cell cocultures is due to humoral interactions, rather than extracellular matrix, and Sertoli cell extracellular matrix regulates serum-induced increase in collagen synthesis by peritubular myoid cells.

When supply does not meet demand-ER stress and plant programmed cell death

The endoplasmic reticulum (ER) is the central organelle in the eukaryotic secretory pathway. The ER functions in protein synthesis and maturation and is crucial for proper maintenance of cellular homeostasis and adaptation to adverse environments. Acting as a cellular sentinel, the ER is exquisitely sensitive to changing environments principally via the ER quality control machinery. When perturbed, ER-stress triggers a tightly regulated and highly conserved, signal transduction pathway known as the unfolded protein response (UPR) that prevents the dangerous accumulation of unfolded/misfolded proteins. In situations where excessive UPR activity surpasses threshold levels, cells deteriorate and eventually trigger programmed cell death (PCD) as a way for the organism to cope with dysfunctional or toxic signals. The programmed cell death that results from excessive ER stress in mammalian systems contributes to several important diseases including hypoxia, neurodegeneration, and diabetes. Importantly, hallmark features and markers of cell death that are associated with ER stress in mammals are also found in plants. In particular, there is a common, conserved set of chaperones that modulate ER cell death signaling. Here we review the elements of plant cell death responses to ER stress and note that an increasing number of plant-pathogen interactions are being identified in which the host ER is targeted by plant pathogens to establish compatibility.

With great structure comes great functionality: Understanding and emulating spider silk

The overarching aim of biomimetic approaches to materials synthesis is to mimic simultaneously the structure and function of a natural material, in such a way that these functional properties can be systematically tailored and optimized. In the case of synthetic spider silk fibers, to date functionalities have largely focused on mechanical properties. A rapidly expanding body of literature documents this work, building on the emerging knowledge of structure–function relationships in native spider silks, and the spinning processes used to create them. Here, we describe some of the benchmark achievements reported until now, with a focus on the last five years. Progress in protein synthesis, notably the expression on full-size spidroins, has driven substantial improvements in synthetic spider silk performance. Spinning technology, however, lags behind and is a major limiting factor in biomimetic production. We also discuss applications for synthetic silk that primarily capitalize on its nonmechanical attributes, and that exploit the remarkable range of structures that can be formed from a synthetic silk feedstock.

Impact of rRNA methylations on ribosome recycling and fidelity of initiation in Escherichia coli

Ribosomal RNA (rRNA) contains a number of modified nucleosides in functionally important regions including the intersubunit bridge regions. As the activity of ribosome recycling factor (RRF) in separating the large and the small subunits of the ribosome involves disruption of intersubunit bridges, we investigated the impact of rRNA methylations on ribosome recycling. We show that deficiency of rRNA methylations, especially at positions 1518 and 1519 of 16S rRNA near the interface with the 50S subunit and in the vicinity of the IF3 binding site, adversely affects the efficiency of RRF-mediated ribosome recycling. In addition, we show that a compromise in the RRF activity affords increased initiation with a mutant tRNA(fMet) wherein the three consecutive G-C base pairs ((29)GGG(31):39CCC41), a highly conserved feature of the initiator tRNAs, were mutated to those found in the elongator tRNA(Met) ((29)UCA(31):(39)psi GA(41)). This observation has allowed us to uncover a new role of RRF as a factor that contributes to fidelity of initiator tRNA selection on the ribosome. We discuss these and earlier findings to propose that RRF plays a crucial role during all the steps of protein synthesis.

Chemical Modification Studies of Gelonin - Involvement of Arginine Residues in Biological-Activity

Gelonin inhibits protein synthesis by inactivating the eukaryotic 60 S ribosomal subunit by an unknown mechanism. The protein was purified in high yield by a new method using Cibacron blue F3GA-Sepharose. Chemical modification studies reveal that arginine residues are essential for biological activity.

Co-induction of sucrose transport and invertase activities in a thermophilic fungus,Thermomyces lanuginosus

The incorporation of sucrose into the thermophilic fungus,Thermomyces lanuginosus, occurred only in mycelia previously exposed to sucrose or raffinose. Sucrose uptake and invertase were inducible. Both activities appeared in sucrose-induced mycelia at about the same time. Both activities declined almost simultaneously following the exhaustion of sucrose in the medium. The sucrose-induced uptake system was specific for \beta -fructofuranosides as revealed by competition with various sugars. The induction of sucrose uptake system was blocked by cycloheximide, showing that it was dependent on new protein synthesis. Transport of sucrose did not seem to be dependent on ATP. Rather, uptake of this sugar seemed to be driven by a proton gradient across the plasma membrane. The uptake system showed Michaelis-Menten kinetics.

Parameter estimation and sensitivity analysis of fat deposition models in beef steers using acslXtreme.

The Davis Growth Model (a dynamic steer growth model encompassing 4 fat deposition models) is currently being used by the phenotypic prediction program of the Cooperative Research Centre (CRC) for Beef Genetic Technologies to predict P8 fat (mm) in beef cattle to assist beef producers meet market specifications. The concepts of cellular hyperplasia and hypertrophy are integral components of the Davis Growth Model. The net synthesis of total body fat (kg) is calculated from the net energy available after accounting tor energy needs for maintenance and protein synthesis. Total body fat (kg) is then partitioned into 4 fat depots (intermuscular, intramuscular, subcutaneous, and visceral). This paper reports on the parameter estimation and sensitivity analysis of the DNA (deoxyribonucleic acid) logistic growth equations and the fat deposition first-order differential equations in the Davis Growth Model using acslXtreme (Hunstville, AL, USA, Xcellon). The DNA and fat deposition parameter coefficients were found to be important determinants of model function; the DNA parameter coefficients with days on feed >100 days and the fat deposition parameter coefficients for all days on feed. The generalized NL2SOL optimization algorithm had the fastest processing time and the minimum number of objective function evaluations when estimating the 4 fat deposition parameter coefficients with 2 observed values (initial and final fat). The subcutaneous fat parameter coefficient did indicate a metabolic difference for frame sizes. The results look promising and the prototype Davis Growth Model has the potential to assist the beef industry meet market specifications.

Heat exposure and hypothyroid conditions decrease hydrogen peroxide generation in liver mitochondria

Exposure of rats to heat (39 +/- 1 degree C) decreased H2O2 generation in mitochondria of the liver, but not of the kidney or the heart. The effect was obtained with three substrates, succinate, glycerol 1-phosphate and choline, with a decrease to 50% in the first 2-3 days of exposure, and a further decrease on longer exposure. The dehydrogenase activity with only glycerol 1-phosphate decreased, which is indicative of the hypothyroid condition, whereas choline dehydrogenase activity remained unchanged and that of succinate dehydrogenase decreased on long exposure. The serum concentration of thyroxine decreased in heat-exposed rats. Thyroxine treatment of rats increased H2O2 generation. Hypothyroid conditions obtained by treatment with propylthiouracil or thyroidectomy caused a decrease in H2O2 generation and changes in dehydrogenase activities similar to those with heat exposure. Treatment of heat-exposed or thyroidectomized rats with thyroxine stimulated H2O2 generation by a mechanism apparently involving fresh protein synthesis. The results indicate that H2O2 generation in mitochondria of heat-exposed animals is determined by thyroid status.

Characterization of protoporphyrin as the physiological regulator of delta-aminolevulinate dehydratase in Neurospora crassa

In Neurospora crassa, the activity of δ-aminolevulinate dehydratase, the second and rate-limiting enzyme of the heme-biosynthetic pathway, is low in normal cells compared to the activity detected in plants, animals and bacteria. The activity is almost undetectable when Neurospora crassa is grown under iron-deficient conditions. The enzyme activity increases strikingly on addition of iron to iron-deficient cultures. This increase can be blocked by the addition of protoporphyrin, the penultimate product of the heme-biosynthetic pathway, to the cultures. The question whether iron directly acts at the genetic level or acts merely by removing protoporphyrin, converting the latter into heme prosthetic groups of hemoproteins, has been investigated by studying the effect of inhibition of heme synthesis on the induction of δ-aminolevulinate dehydratase. It has been found that treatments with levulinic acid or cyanide which inhibit the formation of the porphyrin moiety, induce δ-aminolevulinate dehydratase, whereas treatments which inhibit at a step after protoporphyrin formation (iron-deficiency and cobalt treatment) repress the enzyme. The endogenous levels of protoporphyrin are strictly controlled: a decrease below the optimum level causing induction and an increase above the optimum level leading to repression of δ-aminolevulinate dehydratase. Levulinic acid and cyanide can induce the enzyme in iron-deficient cultures in the absence of added iron, indicating that the metal iron acts only by converting protoporphyrin to heme fixed in hemoproteins in Neurospora crassa. Therefore it is suggested that protoporphyrin is the physiological regulator of δ-aminolevulinate dehydratase in Neurospora crassa.

Molecular genetic studies on nemaline myopathy and related disorders

Nemaline myopathy (NM) is a rare muscle disorder characterised by muscle weakness and nemaline bodies in striated muscle tissue. Nemaline bodies are derived from sarcomeric Z discs and may be detected by light microscopy. The disease can be divided into six subclasses varying from very severe, in some cases lethal forms to milder forms. NM is usually the consequence of a gene mutation and the mode of inheritance varies between NM subclasses and different families. Mutations in six genes are known to cause NM; nebulin (NEB), alpha-actin, alpha-tropomyosin (TPM3), troponin T1, beta-tropomyosin (TPM2) and cofilin 2, of which nebulin and -actin are the most common. One of the main interests of my research is NEB. Nebulin is a giant muscle protein (600-900 kDa) expressed mainly in the thin filaments of striated muscle. Mutations in NEB are the main cause of autosomal recessive NM. The gene consists of 183 exons. Thus being gigantic, NEB is very challenging to investigate. NEB was screened for mutations using denaturing High Performance Liquid Chromatography (dHPLC) and sequencing. DNA samples from 44 families were included in this study, and we found and published 45 different mutations in them. To date, we have identified 115 mutations in NEB in a total of 96 families. In addition, we determined the occurrence in a world-wide sample cohort of a 2.5 kb deletion containing NEB exon 55 identified in the Ashkenazi Jewish population. In order to find the seventh putative NM gene a genome-wide linkage study was performed in a series of Turkish families. In two of these families, we identified a homozygous mutation disrupting the termination signal of the TPM3 gene, a previously known NM-causing gene. This mutation is likely a founder mutation in the Turkish population. In addition, we described a novel recessively inherited distal myopathy, named distal nebulin myopathy, caused by two different homozygous missense mutations in NEB in six Finnish patients. Both mutations, when combined in compound heterozygous form with a more disruptive mutation, are known to cause NM. This study consisted of molecular genetic mutation analyses, light and electron microscopic studies of muscle biopsies, muscle imaging and clinical examination of patients. In these patients the distribution of muscle weakness was different from NM. Nemaline bodies were not detectable with routine light microscopy, and they were inconspicuous or absent even using electron microscopy. No genetic cause was known to underlie cap myopathy, a congenital myopathy characterised by cap-like structures in the muscle fibres, until we identified a deletion of one codon of the TPM2 gene, in a 30-year-old cap myopathy patient. This mutation does not change the reading frame of the gene, but a deletion of one amino acid does affect the conformation of the protein produced. In summary, this thesis describes a novel distal myopathy caused by mutations in the nebulin gene, several novel nebulin mutations associated with nemaline myopathy, the first molecular genetic cause of cap myopathy, i.e. a mutation in the beta-tropomyosin gene, and a founder mutation in the alpha-tropomyosin gene underlying autosomal recessive nemaline myopathy in the Turkish population.

Induction of carnitine acetyltransferase by clofibrate in rat liver

Administration of the anti-hypercholesterolaemic drug clofibrate to the rat increases the activity of carnitine acetyltransferase (acetyl-CoA-carnitine -acetyltransferase, EC 2.3.1.7) in liver and kidney. The drug-mediated increase in enzyme activity in hepatic mitochondria shows a time lag during which the activity increases in the microsomal and peroxisomal fractions. The enzyme induced in the particulate fractions is identical with one normally present in mitochondria. The increase in enzyme activity is prevented by inhibitors of RNA and general protein synthesis. Mitochondrial protein-synthetic machinery does not appear to be involved in the process. Immunoprecipitation shows increased concentration of the enzyme protein in hepatic mitochondria isolated from drug-treated animals. In these animals, the rate of synthesis of the enzyme is increased 7-fold.

Influence of harvesting strategy on nutrient supply and production of dairy cows consuming diets based on grass and red clover silage

The main objective of this thesis was to elucidate the effects of regrowth grass silage and red clover silage on nutrient supply and milk production of dairy cows as compared with primary growth grass silages. In the first experiment (publication I), two primary growth and four regrowth grass silages were harvested at two stages of growth. These six silages were fed to 24 lactating dairy cows with two levels of concentrate allowance. Silage intake and energy corrected milk yield (ECM) responses, and the range in these response variables between the diets, were smaller when regrowth silages rather than primary growth silages were fed. Milk production of dairy cows reflected the intake of metabolizable energy (ME), and no differences in the ME utilization were found between the diets based on silages harvested from primary growth and regrowth. The ECM response to increased concentrate allowance was, on average, greater when regrowth rather than primary growth silages were fed. In the second experiment (publication II), two silages from primary growth and two from regrowth used in I were fed to rumen cannulated lactating dairy cows. Cows consumed less feed dry matter (DM), energy and protein, and produced less milk, when fed diets based on regrowth silages rather than primary growth silages. Lower milk production responses of regrowth grass silage diets were mainly due to the lower silage DM intake, and could not be accounted for by differences in energy or protein utilization. Regrowth grass silage intake was not limited due to neutral detergent fibre (NDF) digestion or rumen fill or passage kinetics. However, lower intake may be at least partly attributable to plant diseases such as leaf spot infections, dead deteriorating material or abundance of weeds, which are all higher in regrowth compared with primary growth, and increase with advancing regrowth. In the third experiment (publications III and IV), red clover silages and grass silages harvested at two stages of growth, and a mixed diet of red clover and grass silages, were fed to five rumen cannulated lactating dairy cows. In spite of the lower average ME intake for red clover diets, the ECM production remained unchanged suggesting more efficient utilisation of ME for red clover diets compared with grass diets. Intake of N, and omasal canal flows of total non-ammonia N (NAN), microbial and non-microbial NAN were higher for red clover than for grass silage diets, but were not affected by forage maturity. Delaying the harvest tended to decrease DM intake of grass silage and increase that of red clover silage. The digestion rate of potentially digestible NDF was faster for red clover diets than for grass silage diets. Delaying the harvest decreased the digestion rate for grass but increased it for red clover silage diets. The low intake of early-cut red clover silage could not be explained by silage digestibility, fermentation quality, or rumen fill but was most likely related to the nutritionally suboptimal diet composition because inclusion of moderate quality grass silage in mixed diet increased silage DM intake. Despite the higher total amino acid supply of cows fed red clover versus grass silage diets, further milk production responses on red clover diets were possibly compromised by an inadequate supply of methionine as evidenced by lower methionine concentration in the amino acid profile of omasal digesta and plasma. Increasing the maturity of ensiled red clover does not seem to affect silage DM intake as consistently as that of grasses. The efficiency of N utilization for milk protein synthesis was lower for red clover diets than for grass diets. It was negatively related to diet crude protein concentration similarly to grass silage diets.

Immunologic studies on nucleic acids and their components. I. An analysis of the specificity of anti-deoxyadenylate antibodies by a membrane-binding technique

Anti-deoxyadenylate antibodies were produced in rabbits by injecting a conjugate of deoxyadenosine 5′-phosphate with bovine serum albumin. The antisera, as analyzed by double diffusion in agar and the quantitative precipitin reaction, showed hapten-specific antibodies. The specific interaction between [3H]deoxyadenylate and antiserum was studied by a sensitive nitrocellulose membrane-binding assay. The specificity of the antibodies was analyzed by measuring the effectiveness of other nucleotides or derivatives to inhibit the hapten-antibody binding. The requirements for recognition by the antibody sites were studied by using a series of naturally occurring nucleic acid components as well as some synthetic derivatives as inhibitors. The antibodies were found to show a high degree of specificity for the whole nucleotide, the base, sugar and phosphate playing almost equally important roles. There was cross reactivity with other mononucleotides, although of a low order. The antibodies were able to react with DNA and tRNA.