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PubMed1998

Effects of nutrient deprivation and differentiation on the expression of growth-arrest genes (gas and gadd) in F9 embryonal carcinoma cells.

The growth-arrest genes (gas and gadd) are widely expressed during mammalian embryogenesis and may be useful as markers of nutritional stress in the embryo. F9 embryonal carcinoma cells have been used to characterize the effect of serum or amino acid deficiency on growth-arrest gene expression in a differentiating embryonic cell. The differentiation markers, homeobox B2 (HoxB2), collagen type IV and laminin B2, were not induced by growth arrest. Treatment with all-trans retinoic acid (RA) produced a dose-dependent increase in alkaline phosphatase activity, which was unchanged in lysine-deficient medium and reduced in low-serum medium. Low-serum medium also reduced HoxB2 expression. There was a transient 2-6-fold increase in mRNAs for C/EBP-beta, gadd153/CHOP-10 and gas5 genes 24 h after transfer to amino-acid-deficient media. The mRNAs for the gas2 and gas6 genes began to rise slowly by 5-10-fold after a delay of approx. 24 h. The transient increases did not occur in low-serum medium where there was a much smaller and slower increase. Differentiation caused 1-2-fold increases in gas2, gas3 and gas6 mRNA levels. The transient overexpression of gas5, gadd153/CHOP-10 and CCAAT-enhancer-binding protein-beta, and the later expression of gas6 mRNAs in response to amino acid deficiency, were not affected by differentiation. RA treatment increased the expression of gas3 and caused gas2 to be transiently overexpressed in amino-acid-deficient medium. Differentiation in serum-deficient medium did not significantly alter the levels of the growth-arrest gene mRNAs. These results show that in F9 cells the growth-arrest genes are expressed sequentially as a result of nutrient stress.

Amino Acids↗
PubMed1988

Properties of the insulin receptor ectodomain.

To study the properties of the extracellular insulin-binding domain of the human insulin receptor (hIR), we have expressed portions of the parent molecule in mammalian cells. Receptor cDNAs encoding the entire hIR ectodomain, the alpha subunit of the hIR alone, or a portion of the alpha subunit containing the cysteine-rich region were placed within an expression vector and in turn used to transfect CHO cells. Only cells expressing mRNA for the entire hIR ectodomain secreted hIR-related protein, suggesting that the truncated versions of this domain are unstable. The ectodomain molecules were extensively glycosylated, properly processed heterotetramers. Further, they bound insulin with an affinity similar to that of the intact hIR. In the electron microscope the secreted ectodomains appeared as discrete globular structures. After incubation with roughly equimolar quantities of insulin, the ectodomains associated to form loops or branched and folded linear macroarrays. However, these structures were not restricted to the specific ligand, insulin, since epidermal growth factor also produced the effect. Nevertheless, it seems that the receptor ectodomains can exist in two structural states. The conversion of the singular to the aggregated state may somehow be associated with transmembrane communication and activation of the biological response.

Animals↗
PubMed1998

A novel component involved in ubiquitination is required for development of Dictyostelium discoideum.

A novel component of the ubiquitination system, called NOSA, is essential for cellular differentiation in Dictyostelium discoideum. Disruption of nosA does not affect the growth rate but causes an arrest in development after the cells have aggregated. nosA contains seven exons and codes for a developmentally regulated 3.5-kb mRNA. The 125-kDa NOSA protein is present in the cytosol at constant levels during growth and development. The C-terminal region of NOSA has homology with ubiquitin fusion degradation protein-2 (UFD2) of Saccharomyces cerevisiae and putative homologs in Caenorhabditis elegans and humans. UFD2 is involved in the ubiquitin-mediated degradation of model substrates in which ubiquitin forms part of the translation product, but ufd2 mutants have no detected phenotype. In accord with the homology to UFD2, we found differences in the ubiquitination patterns between nosA mutants and their parental cell line. While general in vivo and in vitro ubiquitination is minimally affected, ubiquitination of individual proteins is altered throughout growth and development in nosA mutants. These findings suggest that events involving ubiquitination are critical for progression through the aggregate stage of the Dictyostelium life cycle.

Amino Acid Sequence↗
PubMed1999

Heat shock of HeLa cells inactivates a nuclear protein phosphatase specific for dephosphorylation of the C-terminal domain of RNA polymerase II.

Reversible phosphorylation of the C-terminal domain (CTD) of the largest RNA polymerase II (RNAP II) subunit plays a key role in gene expression. Stresses such as heat shock result in marked changes in CTD phosphorylation as well as in major alterations in gene expression. CTD kinases and CTD phosphatase(s) contribute in mediating differential CTD phosphory-lation. We now report that heat shock of HeLa cells at temperatures as mild as 41 degreesC results in a decrease in CTD phosphatase activity in cell extracts. The obser-vation that this CTD phosphatase interacts with the RAP74 subunit of the general transcription factor TFIIF suggests that it corresponds to the previously charac-terized major CTD phosphatase. This conclusion is also supported by the finding that the distribution of the 150 kDa subunit of CTD phosphatase in cells is altered by heat shock. Although CTD phosphatase is found predominantly in low salt extracts in unstressed cells, immunofluorescence microscopy indicates that its intracellular localization is nuclear. The decrease in CTD phosphatase activity correlates with a decrease in amount of 150 kDa phosphatase subunit in the extracts. During heat shock, CTD phosphatase switches to an insoluble form which remains aggregated to the nuclear matrix fraction. In contrast, heat shock did not result in a redistribution of RAP74, indicating that not all nuclear proteins aggregate under these conditions. Accordingly, the heat-inactivation of both the CTD phosphatase and the TFIIH-associated CTD kinase might contribute to the selective synthesis of heat-shock mRNAs.

Cell Line↗
PubMed2005

CDK5 regulates cell-cell and cell-matrix adhesion in human keratinocytes.

BACKGROUND: CDK5 is a member of proline-directed serine/threonine kinases. Although its cDNA was originally cloned as a homologue to those for the other members of the cyclin-dependent kinase (CDK) family, CDK5 has been shown to function differently from other CDKs. CDK5 is activated by non-cyclin partners, p35 and p39, and important during brain development by influencing adhesion, migration and differentiation of neurones. OBJECTIVES: We sought to investigate the expression and functions of CDK5 in human keratinocytes. METHODS: Expression of CDK5/p35, interaction of CDK5/p35 with adhesion molecules, and its roles in cell-cell and cell-matrix adhesion were studied by reverse transcriptase-polymerase chain reaction, immunoblotting and aggregation/adhesion assays in primary cultured normal human keratinocytes from infant foreskins and a human keratinocyte HaCaT cell line. Localization of CDK5 and p35 in normal human epidermis and psoriatic epidermis was studied by immunohistochemistry. RESULTS: Both CDK5 and p35 were expressed in primary cultured keratinocytes, HaCaT cells and normal human epidermis. Roscovitine, an inhibitor of CDK5, enhanced Ca2+-dependent (cadherin-dependent) aggregation of HaCaT cells whereas it inhibited adhesion of HaCaT cells to fibronectin associated with reduced active states of beta1 integrin. Interestingly, psoriatic skin showed reduced CDK5 and p35 expression in the lower half of the epidermis, which might be associated with decreased amount of activated beta1 integrin in the epidermis of psoriatic skin. CONCLUSIONS: CDK5/p35 may be involved in cell-cell and cell-matrix adhesion in human keratinocytes by differently regulating cadherins and integrins. Furthermore, reduced expression of CDK5/p35 in the epidermis might be involved in the pathogenesis of psoriasis.

Calcium↗
PubMed1986

Oligonucleotide binding to the coat protein disk of tobacco mosaic virus. Possible steps in the mechanism of assembly.

Binding of the oligoribonucleotides AAG, AAGAAG and AAGAAGUUG to the disk aggregate of tobacco mosaic virus coat protein has been studied in solution under conditions favourable for virus assembly. The two longer oligomers bind strongly with Kd around 1 microM, approach complete saturation of binding sites and cause the formation of long, nicked helical rods resembling the virus. It is suggested that the binding of these oligomers, with sequences chosen from the assembly origin of the viral RNA, simulates the tobacco mosaic virus assembly process. No binding could be detected for AAG, indicating that chain length is a crucial determinant in the interaction. The binding of AAGAAG to coat protein crystals is very much weaker than that observed in solution, and the crystals crack at high oligomer concentrations. The corresponding oligodeoxyribonucleotide, d(AAGAAG), shows no binding to the protein in solution; the interaction is extremely specific for RNA.

Base Sequence↗
PubMed1982

Transcriptional control of gene expression during development of Dictyostelium discoideum.

During development of the cellular slime mold Dictyostelium discoideum, approximately 2,000 to 3,000 regulated mRNAs are induced when amoebae enter multicellular aggregates. We used in vitro transcription in isolated nuclei to follow the synthesis of individual mRNA precursors during development; these were quantitated by hybridization to cloned cDNAs or genomic DNAs. Those RNAs that are present at all stages of development--the common RNAs--were transcribed by nuclei from cells at all stages of development. By contrast, those RNAs that are present only after cells begin to aggregate--here called aggregation stage RNAs--were transcribed only by nuclei from cells at the aggregation and postaggregation stages of development. The temporal pattern of in vitro transcription correlated well with the time course of accumulation of different aggregation stage mRNAs. Continued expression of aggregation stage genes normally depends upon cell-to-cell contact or cyclic AMP (cAMP); when cells are disaggregated, the regulated mRNAs are rapidly and specifically degraded. When cAMP is subsequently added to the disaggregated cells, most of the mRNAs reaccumulate. We show here that disaggregation reduced 2- to 10-fold the relative transcription of several aggregation stage RNAs, whereas addition of cAMP to disaggregated cells reinduced the level of regulated gene transcription to values approximating those found in normal postaggregation cells. These results indicate that a representative set of Dictyostelium aggregation stage genes are under transcriptional control; both the transcription and the stability of these mRNAs require either continued cell-to-cell interactions or cAMP.

Cell Nucleus↗
PubMed1992

An inhibitor of collagen-stimulated platelet activation from the salivary glands of the Haementeria officinalis leech. II. Cloning of the cDNA and expression.

Salivary glands of the leech Haementeria officinalis contain a protein, leech antiplatelet protein (LAPP), that specifically blocks collagen-mediated platelet aggregation (Connolly, T. M., Jacobs, J. W., and Condra, C. (1992) J. Biol. Chem. 267, 6893-6898). Degenerate oligonucleotides whose sequences were derived from two short peptides from V8 digests of the native LAPP were used as primers to generate a polymerase chain reaction (PCR) product which contains the cDNA region coding for the sequence between these two peptides. Using this PCR product as a hybridization probe, phage containing cDNA clones were isolated containing the entire deduced amino acid sequence for LAPP. Computer analysis of the amino acid sequence predicts a peptidase cleavage site between a 21-residue pre-peptide and a mature protein of 126 amino acids. A DNA insert to express the predicted mature LAPP protein was generated by PCR amplification using phage-derived cDNA clones as a substrate. This insert encoded a fusion protein with the leader sequence of the yeast alpha mating factor and the mature LAPP cDNA. These PCR products were cloned into the yeast expression vector pKH4 alpha 2. A KEX 2 Lys-Arg endopeptidase cleavage site was placed NH2-terminal to the predicted mature protein. This vector transfected into the yeast Saccharomyces cerevisiae directs expression of a secreted mature protein at levels up to 200 mg of LAPP/liter of culture medium. The recombinant protein was comparable to native LAPP in its electrophoretic mobility, its reactivity with anti-LAPP antisera, and its biological activity including inhibition of collagen-stimulated platelet aggregation and the adhesion of platelets to collagen. Availability of significant quantities of recombinant LAPP opens the way to further biochemical structure/function studies and to studies on the effects of an inhibitor of collagen-stimulated platelet aggregation in vivo.

Amino Acid Sequence↗
PubMed2002

Expression of osteopontin in rat kidneys: induction during ethylene glycol induced calcium oxalate nephrolithiasis.

PURPOSE: Osteopontin is a well-known component of stone matrix and a strong inhibitor of the nucleation, growth and aggregation of calcium oxalate crystals in vitro. To understand its involvement in vivo in calcium oxalate nephrolithiasis we investigated the renal expression and urinary excretion of osteopontin in normal rats, and rats with hyperoxaluria and calcium oxalate crystal deposits in the kidneys. MATERIALS AND METHODS: Calcium oxalate nephrolithiasis was induced by administering ethylene glycol. Immunohistochemistry and in situ hybridization were done to localize osteopontin and osteopontin messenger RNA in the kidneys, while sensitive reverse transcriptase quantitative competitive template polymerase chain reaction was performed to detect and quantify osteopontin messenger RNA expression. Urinary excretion was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis, and then quantified by densitometry of the Western blots. RESULTS: Osteopontin expression in the kidneys was significantly increased after hyperoxaluria and it increased further after the deposition of calcium oxalate crystals in the kidneys. Urinary excretion of osteopontin increased concomitantly. The results reveal differences in renal responses after exposure to oxalate and calcium oxalate crystals. In normal kidneys osteopontin expression was limited to a small number of cells of the thin limbs of the loop of Henle and papillary surface epithelium. During hyperoxaluria osteopontin expression in the kidneys was increased but still mostly limited to cells of the thin limb and papillary surface epithelium. However, after calcium oxalate crystal deposition osteopontin expression was observed throughout the kidneys, including segments of the proximal tubules. CONCLUSIONS: In response to exposure to oxalate and calcium oxalate crystals renal epithelial cells increase the production of osteopontin, which may have a significant role in calcium oxalate nephrolithiasis.

Animals↗
PubMed2004

Mechanistic aspects of Escherichia coli photodynamic inactivation by cationic tetra-meso(N-methylpyridyl)porphine.

The mechanistic aspects of Escherichia coli photodynamic inactivation (PDI) have been studied in bacteria expressing the reporter protein GFP, following transfection with wild type pGFP plasmid and treatment with the hydrophilic cationic sensitizer tetra-meso(N-methyl-4-pyridyl)porphine tetratosylate (TMPyP). Cell survival and morphology during PDI were correlated with plasmid-GFP degradation in comparison to DNA and RNA strand-breaks, while photobleaching of the GFP chromophore was used to monitor protein photodamage. Singlet oxygen generated upon TMPyP photoactivation interacted with target nucleic acid polymers in a drug-and light-dose dependent manner. The hierarchy and cascade of the photodamage was in the order: genomic-DNA > total RNA > plasmid-DNA, as revealed by specific extraction and agarose electrophoresis. The notable resistance of the plasmid DNA in comparison to genomic DNA has implications for PDI of antibiotic-resistant bacteria. Re-growth of the treated cells in fresh medium showed structural features of an SOS response. Under these conditions, DNA repair machinery was initiated by typical alignment of DNA-protein co-aggregates accompanied by lateral assembly of ribosomes, apart from damaged DNA-arrays, as depicted by electron microscopy. GFP-TMPyP interactions were demonstrated by double green and red fluorescence on electrophoresis plates analyzed by spectral imaging. Photobleaching measurements revealed specific GFP photodamage directly related to PDI of the E. coli. The kinetics of both the GFP photobleaching and the K(+) efflux, representing photodamage to cytosolic proteins and membrane damage, respectively, were found to be similar. The survival curves were correlated to chromosomal degradation and ultrastructural damage. We conclude that TMPyP-dependent PDI of E. coli is primarily dependent on genomic DNA photodamage rather than on protein or membrane malfunctions.

Escherichia coli↗
PubMed1968

Effects of the hepatotoxic agents retrorsine and aflatoxin B1 on hepatic protein synthesis in the rat.

1. Aflatoxin and the pyrrolizidine alkaloid retrorsine inhibited the incorporation of labelled amino acids into rat liver and plasma proteins in vivo. Inhibition was greater and detected earlier with retrorsine (1hr.) than with aflatoxin (3hr.). 2. Both toxins affected the liver ribosomal aggregates, causing increases in the proportion of monomers plus dimers. The effect of retrorsine was greater than that of aflatoxin. 3. Incorporation of labelled amino acids into proteins of cell-free preparations of liver from rats treated with aflatoxin was lower than in control preparations. The main site of inhibition appeared to be the ribosomes. 4. Both toxins inhibited the incorporation of orotate into liver nuclear RNA 1hr. after administration.

Aflatoxins↗
PubMed1979

Identification and characterization of a 19-S complex containing a 27 000-Mr protein in Artemia salina.

The cytoplasm of the cryptobiotic Artemia salina gastrula contains a large quantity of a unique 19-S complex. This particle is a specific aggregated form of a 27 000-Mr protein, having a molecular weight of 10(6) and an apparent buoyant density of 1.25 -- 1.26 g/cm3 in sucrose and 1.31 g/cm3 in CsCl. The relative quantity of this 19-S complex decreases significantly with respect to 80-S monoribosomes during the postgastrula development. Biochemical and immunological studies indicate that the 27 000-Mr protein is one of the RNA-binding proteins [Ovchinnikov et al., FEBS Lett. 88, 21 -- 26 (1978)] but is absent in the protein components associated with poly(A)-containing ribonucleoprotein complexes. The possibility is also suggested that the 27 000-Mr protein and Artemia elongation factor eEF-Ts might be related to each other on the basis of amino acid composition and immunological cross-reactivity.

Alkylation↗
PubMed1999

Expression of genes for proinflammatory cytokines in alveolar macrophages during propofol and isoflurane anesthesia.

UNLABELLED: Anesthesia and surgery induce macrophage aggregation and neutrophil influx, responses that characterize an inflammatory reaction in the distal airway. We thus evaluated the time-dependent expression of genes for proinflammatory cytokines during propofol and isoflurane anesthesia. We studied patients anesthetized with propofol (n = 20) or isoflurane (n = 20). Alveolar macrophages were harvested by bronchoalveolar lavage immediately, 2, 4, and 6 h after induction of anesthesia, and at the end of surgery. RNA was extracted from harvested cells and cDNA was synthesized by reverse transcription. Expression of interleukin-1beta (IL-1beta), IL-6, IL-8, interferon gamma, and tumor necrosis factor-alpha was measured by semiquantitative polymerase chain reaction using beta-actin as an internal standard. We observed two 10-fold increases in gene expression of all proinflammatory cytokines except IL-6. The increases in IL-8 and interferon gamma were 1.5-3 times greater during isoflurane than propofol anesthesia. Expression of the genes for IL-1beta and tumor necrosis factor-alpha was similar with each anesthetic. Our data thus indicate that the pulmonary inflammatory response accompanying anesthesia and surgery is accompanied by the expression of proinflammatory cytokines, and that this expression was in some cases greater during isoflurane than propofol anesthesia. IMPLICATIONS: Gene expression of proinflammatory cytokines in alveolar macrophages increased significantly over time. The increases were greater during isoflurane than propofol anesthesia, suggesting that inflammatory responses at transcriptional levels in alveolar macrophages are modulated by the type and duration of anesthesia.

Anesthesia↗
PubMed2001

Polysaccharide--polynucleotide complexes. 2. Complementary polynucleotide mimic behavior of the natural polysaccharide schizophyllan in the macromolecular complex with single-stranded RNA and DNA.

Schizophyllan is an extracellular polysaccharide consisting of a beta-1,3-D-glucan main chain and exists as a triple helix in water and as a single chain in dimethyl sulfoxide (DMSO). When the single chain of schizophyllan (s-SPG) was mixed with poly(C), poly(A), poly(dA), or poly(dT), they form a macromolecular complex. On the other hand, poly(G), poly(U), poly(I), poly(dG), and poly(dC) do not. This nucleotide specificity evidences that the hydrogen bonds are essential to form the complex, because the former nucleotides have an unoccupied hydrogen-bonding site and the latter ones use the hydrogen-bonding sites in the intramolecular aggregation (i.e., such as the G quartet for poly(G) and poly(dG) and the U hairpin for poly(U)). The hypochromic effect and the increment in the circular dichroism (CD) intensity are observed in accordance with the complex formation. These facts indicate that the base stacking is enhanced in the complex. The solvent-composition (DMSO/water) dependence demonstrates that the hydrophobic interaction is important to form the complex as well as the hydrogen-bonding interaction. With increasing temperature the complex dissociates cooperatively and the melting curve enables the thermodynamic parameters to be evaluated (delta H = -60 to 70 kcal mol-1 and delta S = -150 to 200 cal mol-1 K-1). These values are comparable with those for double helix DNA. Namely, the complex can be characterized by enhancement of the base stacking, cooperative dissociation, the similar thermodynamic parameters to DNA, and combination of the hydrogen-bonding and hydrophobic interactions to form the higher-order structure. These facts surprisingly coincide with characters of the double helix of DNA. In other words, the s-SPG molecule behaves as if it were a complementary polynucleotide chain for the corresponding polynucleotide. Furthermore, stoichiometric study suggested that the complex structure is a triple helix consisting of two s-SPG and one poly(C) or poly(A) chains.

Carbohydrate Conformation↗
PubMed1998

The expression of PDGF-B chain mRNA in lung tissue from rats repeatedly infected with mycoplasma pneumoniae.

In order to investigate the role played by platelet derived growth factor-BB (PDGF-BB) in the pathogenesis of pulmonary interstitial fibrosis in rats repeatedly infected with mycoplasma pneumoniae (MP), a rat MP infection model was developed by infecting rats with MP for 9 times during a period of 24 weeks with a technique of ultrasonic nebulizing inhalation. Then in situ hybridization was performed with PDGF-B chain cDNA probe and the results were quantitatively analyzed to measure the changes in PDGF-B chain mRNA expression in the lung tissue. The results showed that: (1) MP polymerase chain reaction (PCR) tests showed positive results in the bronchoalveolar lavage fluid (BALF) from all of the MP-infected rats (n = 4) while they were all negative in BALF from the control animals (n = 4, P < 0.05) and in BALF from those rats both infected with MP and, at the same time, treated with erythromycin (n = 4, P < 0.05). Bacterial cultures of the bronchial and lung tissue were negative in all three groups. The observation under a transmission electron microscope indicated that the interalveolar septa were widened with increased amount of collagen in the MP-infected rats while there were no obvious abnormalities in the other two groups. (2) Strong positive expression of PDGF-B chain mRNA was found in the plasma of monocytes and macrophages located in the locally widened interalveolar septa and alveolar spaces in the lung tissue from the MP-infected animals with the integral optical densities being 37.42 +/- 9.05 (n = 4) which was significantly higher than the values of control group (0.42 +/- 0.08, n = 4, P < 0.01) and of the group with MP-infection plus erythromycin treatment (1.62 +/- 0.40, n = 4, P < 0.01). These results suggest that PDGF-BB may be involved in the process of the development of pulmonary interstitial fibrosis caused by the repeated MP-infection. It may be an important growth factor for mediating the roles of monocytes and macrophages to promote the aggregation and proliferation of fibroblasts which can then secrete collagen in large quantity in the pulmonary interstitium.

Animals↗
PubMed2004

Activation of TLR3 and TLR5 in channel catfish exposed to virulent Edwardsiella ictaluri.

Two toll-like receptors (TLR3 and TLR5) were identified from a catfish cDNA fry library based on sequence similarity to other vertebrate TLR genes. Expression (using real-time PCR) of TLR3 and TLR5 was measured for two strains of channel catfish in previously non-exposed fish 2, 5, 8, and 21 days after experimental Edwardsiella ictaluri challenge to determine if TLRs are associated with host response to E. ictaluri infection. Expression of TLR5 was higher than TLR3 (P<0.0001). TLR3 expression in kidney was elevated in Norris strain (P=0.480) and differed over time in spleen (P=0.0134). Fold induction of TLR5 compared to non-exposed fish increased on days 5 (Norris; 154.72+/-62.12 fold induction) and 8 (USDA103; 164.65+/-50.56) post-exposure in liver and was slightly increased on day 5 (Norris; 10.17+/-24.73, USDA103; 42.56+/-24.73) in kidney. Upregulation of TLR3 suggests a more widespread function in primitive fish. TLR5 was highly expressed in liver tissue, which may be due to macrophage aggregation during ESC infection. This suggests that toll-like receptors are an important component of the innate immune system of catfish.

Animals↗
PubMed1997

Human breast carcinoma cells synthesize a protein immunorelated to platelet glycoprotein-Ib alpha with different functional properties.

Although tumor cell-induced platelet aggregation is thought to mediate an early step in the metastatic process, little is known about tumor adhesive receptors responsible for the initial platelet-tumor attachments. Because our preliminary work demonstrated that a platelet-immunorelated glycoprotein Ib alpha (GPIb alpha) receptor expressed by the human breast carcinoma cell line MCF-7 participates in tumor-induced platelet aggregation, we examined the synthesis and functional characteristics of this MCF-7-immunorelated GPIb alpha. When 35S-cysteine-labeled, digitonin-lysed MCF-7 cells were immunoprecipitated with platelet-specific monoclonal antibodies (mAbs) to GPIb alpha, major radioactive bands were observed. Northern blots showed MCF-7 transcripts for GPIb alpha under both high- and low-stringency hybridization conditions. In the presence of purified human iodine 125-labeled von Willebrand factor (125I-labeled vWf) with or without the addition of ristocetin, unlabeled vWf was observed to competitively bind to fixed MCF-7 cells (50% inhibitory concentration = 10 microg/ml, dissociation constant = approximately 3.8 +/- 1.9 nmol/L, 2.7 x 106 + 445,000 binding sites/cell) in which non-GPIb alpha vWf binding sites were blocked. 125I-vWf binding to blocked MCF-7 cells could be selectively and completely inhibited by mAbs specific for the vWf binding domain of GPIb alpha but not by mAbs against the GPIX subunit, the GPIb alpha subunit, or alternate GPIb alpha epitopes other than the vWf-binding domain. Finally, when whole blood substrate was incubated with a mAb specific for the GPIb binding epitope of vWf, MCF-7-induced platelet aggregation was virtually abolished in comparison with control specimens (N = 8; p < 0.0009). These findings (1) confirm the synthesis and expression of an MCF-7 protein with homology to platelet GPIb alpha, (2) confirm that the functional activity of this MCF-7-immunorelated GPIb alpha differs from that of platelet GPIb alpha, and (3) suggest that MCF-7-immunorelated GPIb alpha in its adhesive interactions with plasma vWf may constitute an initial event in MCF-7-induced platelet aggregation.

Anti-Bacterial Agents↗
PubMed1969

Alternative developmental pathways determined by environmental conditions in the cellular slime mold Dictyostelium discoideum.

The cellular slime mold Dictyostelium discoideum grows in the soil as a population of independent, uninucleate amoebae. Upon entrance to the stationary phase, the amoebae collect in multicellular aggregates to form organized fruiting bodies composed of spores and stalk cells. Depending upon environmental conditions, the developing aggregate either constructs the fruiting body at the site of aggregation or transforms into a structure that can migrate to a more favorable location. Environmental conditions that favor migration are (i) the accumulation of metabolite(s) produced by the aggregate and (ii) a low ionic strength in the substratum. Conditions that prevent migration or that stop a migrating slug are (i) the presence of buffer and (ii) illumination by overhead light.

Amoeba↗