A photoaffinity label for the thromboxane A2/prostaglandin H2 receptor in human blood platelets

A photoactive iodoarylazide derivative (I-APA-PhN3) of the competitive thromboxane A2/prostaglandin H2 (TXA2/PGH2) antagonist 13-azaprostanoic acid is evaluated. Upon photoactivation, the compound was found to inhibit specifically and irreversibly human platelet aggregation induced by the TXA2/PGH2 mimetic U46619. In receptor-binding studies using [3H]U46619, I-APA-PhN3 exhibited an IC50 of 300 nM for inhibition of U46619 binding. Photoactivation of I-APA-PhN3 resulted in an irreversible 58% reduction in specific binding of U46619. This compound and its corresponding ratio-iodinated form will prove to be useful tools for the isolation and purification of the TXA2/PGH2-binding protein in human platelets.     

Trifluoperazine Activates and Releases Latent ATP-Generating Enzymes Associated with the Synaptic Plasma Membrane

Neurone-specific enolase (NSE) and the brain form of creatine phosphokinase (CPK-BB) were previously found to be present in rat synaptosomal plasma membranes (SPM) using two-dimensional gel (2-D gel) and peptide analysis; enzymatic activities of these and of pyruvate kinase (PK), all involved in ATP generation, were shown to be "cryptic" unless the SPM were treated with Triton X-100. We now show that enzymatic activation also occurs when the SPM are treated with trifluoperazine (TFP). TFP activation occurred even when the enzymes were membrane associated, showing that solubilization was not responsible for "unmasking" the enzyme activities. When TFP treatment was performed at alkaline instead of neutral pH, NSE and CPK-BB were released as well as PK, nonneuronal enolase, and aldolase which were identified by 2-D gel and tryptic peptide analysis. Other proteins released included calmodulin, actin, and the 70-kilodalton heat-shock cognate protein. Tubulin, synapsin I, and a 35-kilodalton basic protein were largely unaffected. The latter was identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase on the basis of 2-D gel and peptide analyses and subsequent partial sequencing of a rat brain cDNA coding for the same protein. TFP treatment is thus useful for activating latent enzymes as well as for distinguishing enzymes that have a different disposition on the membrane.     

Detection of luteinizing hormone beta messenger ribonucleic acid (RNA) in individual gonadotropes after castration: use of a new in situ hybridization method with a photobiotinylated complementary RNA probe

Patterns of gonadotropin storage in individual gonadotropes change with alterations in the physiological state. After castration in the male rat, there is a 2.5-fold increase in the percentage of gonadotropes and an increase in the proportion of gonadotropes storing both LH and FSH. In addition, there are 6- to 8-fold increases in the pituitary concentrations of LH beta subunit mRNAs. In order to determine whether these changes are due to increases in the number of gonadotropes containing subunit mRNA, or the amount of mRNA per cell or both, an in situ hybridization technique using a photobiotinylated rat LH beta cRNA probe (bio-LH beta-cRNA) was applied to detect LH beta mRNA in fixed whole rat pituitary cells from intact or castrated rats. After hybridization, the bio-LH beta-cRNA was localized with either avidin-biotin peroxidase complex or the fluorescent streptavidin phycoprobe methods. The cells containing LH beta mRNA were then counted and the amount of mRNA per cell was measured by video microdensitometry. Ten percent of the anterior pituitary cells from intact animals contained LH beta mRNA. After castration (2-4 weeks) this percentage rose to 19-24.5%. Image and microdensitometric analyses showed that castration produced a 1.9-fold increase in the amount of LH beta mRNA per cell, and a 2.2-fold increase in the area of cells containing LH beta mRNA. Hence, castration resulted in an increase in the level of LH beta mRNA per cell as well as the number of LH beta mRNA-containing cells. When in situ hybridization was followed by immunocytochemistry in cells from intact rats, 83% of gonadotropes that stained for LH beta and 80% of gonadotropes that stained for FSH beta contained LH beta mRNA whereas after castration 99% of LH-storing and 93% of FSH-storing cells contained LH beta mRNA. This new in situ hybridization protocol is rapid and allows quantification of mRNA within individual gonadotropes. In addition, since the hybridization protocol does not apparently alter the gonadotropin antigens, the hormone content of the same gonadotrope may be defined by immunocytochemistry.     

Influence of thyroidal status on pituitary content of thyrotropin beta- and alpha-subunit, growth hormone, and prolactin messenger ribonucleic acids

Thyroidectomized rats were used to study the effects of a single injection of T3 on pituitary mRNA synthesis and hormone secretion. T3 was injected ip at doses of 0, 0.2, 1, or 5 micrograms/100 g body weight, and and animals were killed 24 h later. T3 caused a significant decrease in serum TSH, but caused no significant change in either serum GH or PRL. Pituitary mRNA was quantified by slot blot hybridization with cDNA probes specific for alpha-TSH, beta-TSH, PRL, and GH. We found that both the alpha and beta mRNA subunits decreased, that PRL mRNA remained relatively unchanged, and that GH mRNA increased with increasing T3 dose. The data show that a single dose of T3 can profoundly influence mRNA levels in the anterior pituitary; the lowest dose of T3 caused maximum inhibition of alpha-TSH mRNA while beta-TSH mRNA declined further in a dose-dependent manner.     

Purification and Characterization of a Substrate-Size-Recognizing Metalloendopeptidase from Streptococcus cremoris H61

During the ripening of Gouda-type cheese, two kinds of endopeptidases were found to participate in the degradation of αs1-CN(f1-23), a specific product from αs1-casein hydrolyzed by chymosin. One of the endopeptidases, lactic acid bacteria endopeptidase (LEP-II), which can recognize the size of its substrates, has already been purified and characterized (T. R. Yan, N. Azuma, S. Kaminogawa, and K. Yamauchi, Eur. J. Biochem. 163:259-265, 1987). The other endopeptidase, LEP-I, was purified to homogeneity by conventional chromatographic techniques from Streptococcus cremoris H61. The enzyme appeared to be monomeric, with an apparent molecular weight of 98,000, and its isoelectric point was 5.1. For the hydrolysis of αs1-CN(f1-23), the enzyme had an optimum pH and temperature of 7.0 to 7.5 and 40°C, respectively. Its activity was inhibited by such chelating agents as EDTA and 1,10-phenanthrolin, and it could be fully reactivated by Mn²⁺. Inhibitors specific for serine and thiol proteases had no effect on the protease activity. The enzyme showed a high affinity toward the Glu-Asn peptide bond of αs1-CN(f1-23) and αs1-CN(f91-100) but showed no hydrolysis activity toward αs1-CN(f1-52), αs1-CN(61-122), αs1-CN(136-196), αs1-casein, β-casein, κ-casein, α-lactalbumin, and β-lactoglobulin. The K_m and V_max of LEP-I for αs1-CN(f1-23) were 14.2 pM and 139 U, respectively.     

Natural killer cell-target interactions: the role of 4F2 antigen in the human system

The manner by which natural killer cells discriminate between target and nontarget cells is a subject of intense investigation. Recently, the antigen recognized by the 4F2 monoclonal antibody has been implicated as the target recognition molecule of cloned human natural killer cells. Here we report the results of our studies on the possible role of 4F2 antigen in target recognition by natural killer cells present in the peripheral blood. In our hands, the 4F2 antibody only weakly blocked the killing of K562 leukemia cells by human natural killer cells. Furthermore, no correlation was observed between the level of cell surface 4F2 antigen and the natural killer susceptibility of several tumor cell lines, the ability of these cells to bind to natural killer cells, or the ability of these cell lines to compete with K562 cells in a natural killer assay. Therefore, the 4F2 antigen does not appear to be the target recognition molecule of most peripheral blood natural killer cells.     

Feedback optimization of fed-batch fermentation

The problem of feedback optimization of the feed rate for fed-batch fermentation processes is formulated in the framework of singular control theory and switching hypersurfaces. Using four differential balance equations that describe a general class of fedbatch processes and a general objective function to be minimized, it is shown that under certain restrictions the feedback optimization of the feed rate can be realized as a nonlinear function of the state variables, such as the concentrations of cell mass, substrate and product, and the fermentor volume. The restrictions on the initial conditions, the fermentation kinetics and the objective function, that are needed for realization of the feedback optimization, are provided. Fed-batch fermentation models of lysine and alcohol are used to construct switching curves and to illustrate the feedback optimization of the feed flow rates.     

Purification and characterization of a novel metalloendopeptidase from Streptococcus cremoris H61. A metalloendopeptidase that recognizes the size of its substrate

An endopeptidase (LEP-II), which has a unique substrate specificity, was purified to homogeneity by conventional chromatographic techniques from Streptococcus cremoris H61. The enzyme was a metalloendopeptidase since it was inhibited by EDTA and 1,10-phenanthroline; the metal-depleted enzyme could be fully reactivated by micromolar levels of Zn2+ and was not inhibited by specific inhibitors for serine or thiol protease. The molecular mass of the enzyme was estimated to be 80 kDa by Sephacryl S-300 gel filtration and high-performance liquid chromatography with a TSK-G3000SW column. The enzyme consisted of two identical subunits and the N-terminal sequence of LEP-II was determined up to the 19th residue. Although the enzyme had a broad substrate specificity it specifically hydrolyzed the peptide bonds involving the amino groups of hydrophobic amino acid residues. Various small polypeptides, such as alpha s1-CN(f1-23), alpha s1-CN(f91-100), oxidized insulin B chain, glucagon and some biologically active peptides were hydrolyzed. However, a variety of larger polypeptides or proteins, such as alpha s1-CN(f1-54), alpha s1-CN(f61-123), alpha s1-CN(f136-196), alpha s1-casein, beta-casein, and kappa-casein were not hydrolyzed. LEP-II recognized the size of its substrates, which were limited below a molecular mass of about 3.5 kDa.