Chemotactic factor causes rapid decreases in phosphatidylinositol,4,5-bisphosphate and phosphatidylinositol 4-monophosphate in rabbit neutrophils

Stimulation of rabbit neutrophils prelabeled with 32P by the synthetic chemotactic peptide f-Met-Leu-Phe induces a rapid decrease in the radioactivity in both phosphatidylinositol, 4,5 bis phosphate and phosphatidylinositol 4-monophosphate. The mean +/- standard error of the mean values of the maximum decrease in phosphatidylinositol, 4,5 bis phosphate occurred at 10 seconds following stimulation and is equal to 19 +/- 3% of the control value. The corresponding value for phosphatidylinositol 4-monophosphate occurred at 60 seconds following stimulation and is equal to 37 +/- 7% of the control value. On the other hand, the radioactivity in phosphatidic acid and lysophospholipids increased continuously with time following stimulation. The relationship of these changes to calcium release and neutrophil activation is discussed.     

Streptozotocin diabetes results in increased responsiveness of adipocyte lipolysis to glucagon

Adipocytes from streptozotocin-diabetic rats are approximately 50-times more sensitive to the lipolytic action of glucagon. This change is only perceived in the presence of a small quantity of adenosine deaminase which itself has little effect on basal lipolysis. Insulin treatment restores glucagon sensitivity to normal.     

Selective generation of leukotriene B4 by tracheal epithelial cells from dogs

The incubation of suspensions of canine tracheal epithelial cells of greater than 95% purity with arachidonic acid (25-200 micrograms/ml) for 60-120 min resulted in the generation of a maximum of 36.2 +/- 9.1 picomoles of leukotriene B4/10(6) cells, less than 2.0 picomoles of leukotrienes C4, D4, and E4/10(6) cells, and 1030 +/- 463, 767 +/- 500, and 324 +/- 100 picomoles/10(6) cells of 15-, 12-, and 5-hydroxy-eicosatetraenoic acids, respectively (mean +/- SEM, n = 8). The identity of leukotriene B4 was established by chromatographic and spectral properties, by reactivity with mono-specific anti-plasma, and by the chemotactic activity for neutrophils. Thus, the epithelium may be an important source of mediators of inflammation and hypersensitivity of pulmonary airways.     

Low infectivity of vesicular stomatitis virus (VSV) particles released from interferon-treated cells is related to glycoprotein deficiency

We have investigated the mechanism for the low infectivity of vesicular stomatitis virus (VSV) released from interferon (IFN) -treated cells. With 10-30 units/ml of IFN there was an approximately 5-30 fold reduction in the production of virus particles, as measured by VSV proteins; however, the infectivity of the VSV released from IFN-treated mouse LB, JLS-V9R, or human GM2504 was drastically reduced (2 to 4 logs). The low infectivity of VSV was directly related to a deficiency in virion glycoprotein (G). IFN treatment did not change the specific infectivity of the VSV particles released by HeLa cells; their G protein was also not reduced. A further effect of IFN to reduce the amount of virion M protein appeared to be secondary and was probably not related to the reduced infectivity of VSV.     

A system for coupled multiple-column separation of proteins

Purification of proteins is commonly a multiple-step process involving size exclusion, ion exchange, affinity, hydrophobic, and other modes of chromatography. In an effort to circumvent the laborious process of collecting the solutes from each column and reintroducing them onto a second column, a valving system is described that directs the samples eluted from a high-performance liquid chromatographic column through a detector with a high-pressure cell into either a second column or into storage loops of a multiloop value. This multiloop value is referred to as a high-pressure fraction collector. After development of the first column is complete, a second solvent can be directed to the second column or high-pressure fraction collector to elute the solutes back through the detector and onto any other column in the system. The process of eluting a sample from a column through a single detector and directing it to the high-pressure fraction collector or any other column in the system may be repeated a number of times. Such valving systems make it possible to chromatograph a single protein component on two or three columns in a short time.     

High-performance liquid affinity chromatography on silica-bound alcohol dehydrogenase

Horse liver alcohol dehydrogenase was immobilized on glycerylpropyl-silica (10 micron, 1000-A pores) activated with 2,2,2-trifluoroethanesulfonyl chloride (tresyl chloride). The coupling and activity yield was almost 100%. The coenzyme-binding sites were equivalent and virtually unaffected by the immobilization process, as judged from Scatchard plots and active-site titrations. The silica-bound enzyme, packed in steel columns, was integrated with HPLC equipment and then successfully used for chromatography of adenine nucleosides, adenine nucleotides, and triazine dyes. Dissociation constants were calculated from chromatographic data and found to correspond well with literature values. The dissociation constants for a number of nucleotide derivatives with potential application in affinity chromatography were also determined. The spaces were found to affect the binding strength of the nucleotides in a qualitatively predictable way. Theoretical plate heights were calculated and found to be in the range 0.01 to 0.1 cm. Attempts to correlate peak widths with the rate constants for the binary complexes involved were only partially successful.     

Simultaneous determination of cysteine sulfinic acid and cysteic acid in rat brain by high-performance liquid chromatography

A sensitive and specific assay method for cysteine sulfinic acid (CSA) and cysteic acid (CA) using high-performance liquid chromatography has been developed. The method includes post-column derivatization of various amino acids with o-phthalaldehyde in the presence of 2-mercaptoethanol. The column packed with cation-exchange resin ($\text{ISC-07}\text{S1504}$, Shimadzu Sci entific instruments, Inc., Kyoto, Japan) was used for obtaining general separation of amino acids except CSA and CA, while the separation of CSA and CA was achieved using a strong-base anion exchange ($\text{ISA-07}\text{S2504}$, Shimadzu Scientific Instruments) column. The fluorescence peak area for CSA was linear between 20 pmol and 5 nmol, whereas that for CA was 10 pmol to 5 nmol. The regional distribution of CSA, CA, and other amino acids in the rat brain was studied using this new assay method.     

Multiple Molecular Forms of Enkephalins in the Guinea Pig Hippocampus

Abstract: The combined techniques of HPLC and radioimmunoassay were used to identify and quantitate enkephalin-related peptides in the guinea pig hippocampus. Both met- and leu-enkephalin were identified, in approximately a 2:1 ratio, as well as a third enkephalin-like molecule that is neither met- nor leu-enkephalin. The third enkephalin elutes earlier than met- or leu-enkephalin from a reversed-phase column, has a molecular weight similar to the other enkephalins, and is as active as these enkephalins are in inhibiting binding of labeled opiates to rat brain membranes. All regions of the hippocampus (dentate gyrus, CA1–2, CA3–4, and subiculum) contain all three immunoreactive peptides. Immunocytochemical techniques, using antisera raised against met-enkephalin, show with one antiserum immunoreactivity in the granule cell-mossy fiber system, and with the other scattered immunoreactive cells mostly in the CA2 region. Enkephalins are not confined to the mossy fiber system, as previously suggested, but may be a component of another hippocampal innervation.     

Biological effects of incorporation of O6-methyldeoxyguanosine into Chinese hamster V79 cells

Analysis of the biological effects of specific DNA alkylations by simple alkylating agents is complicated by the variety of sites involved. It is, therefore, of value to be able to incorporate into cellular DNA nucleosides alkylated in a single position, e.g., O⁶-methyldeoxyguanosine. Such cellular incorporation is particularly difficult to achieve because this nucleoside is rapidly demethylated by adenosine deaminase. We have attempted to achieve such incorporation into the DNA of V79 cells by using coformycin, an inhibitor of adenosine deaminase, and by forcing the cells to depend on exogenous purines by the use of medium containing aminopterin. The DNA of V79 cells exposed to O⁶-methyl-[8-³H]deoxyguanosine (2.4 μM, sp. act. 14 500 Ci/mole) showed an incorporation level of 4 × 10⁻⁸ nucleotides. When 1000-fold higher concentrations were employed (3–15 mM, sp. act. 1.6 Ci/mole), significant cytotoxicity and inhibition of DNA synthesis was observed. However, because it was not economically feasible to administer high specific activity O⁶-methyldeoxyguanosine to the cells at these concentrations, we could not determine the amount of labeled nucleoside incorporated into DNA. Examination of the frequency of 6-thioguanine-resistant cells in these treated populations showed no significant increase above the background level. Comparison of the cytotoxic effect of O⁶-methyldeoxyguanosine with deoxyadenosine showed that the toxicity induced by O⁶-methyldeoxyguanosine could have resulted from mimicry of deoxyadenosine, rather than by incorporation of the alkylated nucleoside itself.     

Pro-opiomelanocortin peptides in the human fetal pituitary

Levels of immunoreactive pro-opiomelanocortin (POMC) peptides (N- and C-terminal ACTH, N- and C-terminal LPH and α-MSH) have been measured in pituitary extracts from human fetuses of 12–22 weeks gestation. The levels of ACTH were 30–200 times higher than α-MSH in all fetuses studied. Sephadex G-75 and G-25 chromatography of 8 extracts showed peaks of 34 kilodaltons (K) POMC, 22K ACTH, β-LPH, γ-LPH, β-endorphin, approximately 8K ACTH, 1–39 ACTH, α-MSH and CLIP. The 8K and 22K forms of ACTH are both partly glycosylated.In vitro culture of pituitaries from 2 fetuses (22 and 26 weeks gestation) gave a detectable basal output of ACTH but not of α-MSH. Stimulation of these pituitary cells with human fetal and rat hypothalamic extracts and with synthetic ovine CRF-41 produced a significant increase in ACTH release, and either small or undetectable amounts of α-MSH.These results demonstrate the presence of POMC-related peptides in early gestation human fetal pituitaries and suggest that ACTH, and not α-MSH, is the major corticotrophic hormone at this stage of gestation.