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Phenobarbital-inducible cytochrome P450 variants have a higher affinity (app. Ks = 0.6 microM). The difference in affinity has enabled us to quantitate, by metyrapone-induced difference spectral analysis, phenobarbital-inducible cytochromes P450 in liver microsomal membranes. The result obtained by this simple, rapid technique compare well with those found by more sophisticated techniques such as radioimmunoassay and immunoprecipitation.

Hydrolysis of human C-reactive protein (CRP) at pH 4.5 and pH 7.4 with neutrophil-derived lysosomal enzymes yielded 10% trichloroacetic acid soluble peptides (Mr less than 14,000). These peptides inhibited neutrophil superoxide production, chemotaxis, degranulation and phagocytosis at 2 micrograms/ml. This inhibition was not observed with native CRP or intermediate peptides (Mr greater than 14,000). CRP peptides (Mr less than 14,000) also caused a dose-related inhibition of Quin-2 fluorescence indicating interference with intracellular calcium movements during cell activation. These results point to a potential regulatory role for CRP-derived degradation products on neutrophils during inflammation.

A total of 301 Black patients without rheumatic diseases was studied to estimate the prevalence and causes of positive rheumatoid factor. The latex slide test was positive in 25% of patients and 21% had a 'diagnostic' titre of 80 or more. A significant relationship between gamma-globulin levels and rheumatoid factor positivity was demonstrated. IgG appeared to be the most important in determining this relationship. The most common ailments among sero-positive patients were cirrhosis and tuberculosis.

The effect of specific antigen challenge on the lung function of eight allergic asthmatic patients after placebo and indomethacin pretreatment has been investigated. Plasma levels of thromboxane B2(TxB2), metabolite of thromboxane A2, 6-keto-PGF1 alpha, metabolite of epoprostenol, (prostacyclin, PGI2) and beta-thromboglobulin (beta TBG) following antigen challenge in these eight patients have also been measured after placebo and indomethacin pretreatment. Each patient underwent two antigen inhalations 1 week apart. One challenge took place after 4 days pretreatment with indomethacin capsules 25 mg four times daily, and the other took place following 4 days placebo pretreatment, one matched capsule four times daily. The order of administration was random but balanced and blind with respect to the patient. Following placebo pretreatment two patients presented with an early antigen response only, four presented with a biphasic antigen response and two presented with a delayed antigen response only. The asthmatic response for each patient was consistent on re-exposure. Indomethacin pretreatment suppressed the delayed antigen induced asthmatic response. This suppression was reproducible. There was a rise in plasma TxB2 levels on antigen challenge following placebo pretreatment but not following indomethacin pretreatment, whereas there was a rise in 6-keto-PGF1 alpha after antigen challenge following indomethacin but not placebo pretreatment. No significant change in plasma beta TBG or platelet counts from control values was observed following antigen challenge with either placebo or indomethacin pretreatment. It is suggested that the production of PGI2 and suppression of TxA2 by indomethacin pretreatment contribute to the suppression of the delayed antigen induced asthmatic response and that platelets play a minimal role in this process.

Of 50 patients in whom Morton's metatarsalgia was diagnosed during an 18-year period, 12 (24%) had rheumatoid arthritis at the time of presentation. After conservative management had failed, 20 patients had subtotal excision of the intermetatarsophalangeal bursa and associated digital nerve; in two patients, only the nerve was excised. During the follow-up period ranging from two months to 15 years, an additional eight patients developed sero-positive rheumatoid arthritis. Thus, a total of 20 patients (40%) presenting with Morton's metatarsalgia had rheumatoid disease at initial presentation or later developed this disease. Histological changes in the intermetatarsophalangeal bursa consistent with rheumatoid arthritis were found in ten patients. Of these, two were known already to have rheumatoid arthritis, three subsequently developed rheumatoid arthritis, and five do not yet have other evidence of the disease. The evidence suggests that Morton's metatarsalgia is associated with rheumatoid arthritis and is the basic etiology in a significant number of patients.

We have investigated the ability of camphor, menthol, pinene, limonene and myrcene to induce in rats members of a cytochrome P-450 sub-family termed PB P-450. These proteins have recently been designated as members of the P450IIB sub-family. None of these naturally occurring terpenoids significantly changed the total content of cytochromes P-450 or cytochrome b5. Radioimmunoassay results showed that PB P-450 was induced 6-fold by camphor and to a lesser extent by menthol and pinene. The induction was confirmed by Western blotting. It was shown by nucleic acid hybridization that induction of PB P-450 by terpenoids was mediated by an increase in the amount of the corresponding mRNA. Analysis of the denaturation of mRNA-cDNA hybrids demonstrated that the mRNA induced by the terpenoids was encoded by a member of the P450IIB sub-family. None of the terpenoids had an effect on the amount of mRNA coding for P450IA2 (a cytochrome P-450 inducible by beta-naphthoflavone and isosafrole). The results indicate that cytochromes P-450 induced by a synthetic compound, phenobarbital, may have originally evolved in response to terpenoid compounds normally present in the environment.

Carbonic anhydrase III (CA III) was identified in the products of rat soleus muscle RNA translation in vitro by both a radioimmunoassay and a specific immunoprecipitation technique followed by SDS--polyacrylamide gel electrophoresis analysis of the precipitated antigen. The primary translation product has the same Mr-value as the native isoenzyme. CA III mRNA was found to represent approximately 0.55% of the total mRNA present in rat soleus muscle.

A compound consisting of a beta-stimulant, salbutamol (Ventolin; Allen & Hanburys) (100 micrograms/puff), and a steroid, beclomethasone dipropionate (Becotide; Allen & Hanburys) (50 micrograms/puff), was studied to test the hypothesis that the corticosteroid could enhance the bronchodilator properties of the beta-stimulant during chronic asthma and simulated acute attacks (antigen challenge). Conventional doses (200 micrograms and 100 micrograms of salbutamol and beclomethasone respectively) were compared using a schedule which included a second administration 1 hour later. The results obtained on the baseline bronchial responsiveness of chronic asthmatics and during the delayed asthmatic response (simulated acute asthma) were similar. The compound was as effective as salbutamol alone but not more so. A significantly greater bronchodilator response was recorded in all patients after the second administration of both the compound and salbutamol alone. The practical advantages of having one rather than two inhalers are evident, but the appropriate application of this compound agent, probably in a prophylactic role, must be defined.

Conditions for solubilizing and iodinating the heterobifunctional thiol-cleavable photoreactive crosslinking reagent sulfosuccinimidyl-2-(p-azidosalicylamido)-1,3'-dithiopropionate which leave the ester moiety, disulfide bond, and azido group reactive are described. Iodination was performed in a mixture of dimethyl sulfoxide and bicarbonate, pH 9.0 (1:20, v/v), as solubilizing agent and Iodogen as oxidant. The lectin phytohemagglutinin was derivatized with the iodinated crosslinker and the interaction between phytohemagglutinin and mononuclear cells was chosen as the model system to monitor the efficiency of sulfosuccinimidyl-2-(p-azidosalicylamido)-1,3'-dithiopropionate as a crosslinking reagent. Transfer of 125I to the biologically significant T11 lymphocyte receptor in addition to 125I labeling of other membrane proteins to which the lectin binds was detected by polyacrylamide gel electrophoresis under reducing conditions.

The uptake of C-reactive protein (CRP)-pneumococcal C-polysaccharide (CPS) complexes by neutrophils was studied. A specific CRP dependent mechanism of uptake was demonstrated. This promoted CPS (complexed to CRP) clearance which was further enhanced by additional complement activation. Physiological concentrations of low density lipoprotein inhibited entry of complexed CPS into neutrophils but had no effect on entry of CRP alone. Pure human CRP was shown to have no effect on neutrophil chemotaxis and oxidative metabolism.

The representation of mRNA sequences in normal and SV40-transformed WI-38 human diploid fibroblasts was examined. Variations in the hybridization of polysomal RNAs to 3H-labeled DNAs complementary to poly A+ mRNA of normal and SV40-transformed cells suggest differences in the mRNA populations. a comparison of the representation of histone RNA sequences of normal and SV40-transformed human diploid cells did not reveal significant differences.

NADPH-cytochrome c (P-450) reductase from liver microsomes of phenobarbital-treated rats has been purified in a single step by affinity chromatography on agarose-hexane-adenosine 2',5'-diphosphate. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, enzyme assay, and radioimmunoassay the protein obtained by this single step procedure is as pure as that isolated by multicolumn procedures.

The uptake of multilamellar liposomes into human leukocytes in whole blood in vitro was evaluated on the basis of the cellular association of liposomal markers (3H-labelled cholesterol, lipid phase; [14C]inulin, aqueous phase). The entry of liposomes into human blood leukocytes was linear for 60 min and was mediated by a saturable mechanism displaying affinity constants of 0.28 +/- 0.17 and 0.16 +/- 0.05 mM liposomal lipid (means +/- S.E.) for liposomal lipid and aqueous phase markers, respectively. Amicon filtration analysis of incubation mixtures containing blood and liposomes (phosphatidylcholine:dicetyl phosphate:cholesterol, 70:20:10) showed that 34% of [14C]inulin was lost (neither liposome-associated nor cell-associated) after 60 min. By preincorporating sphingomyelin (35 mol%) into multilamellar liposomes, the leakage of the model aqueous phase marker inulin was reduced to 8% after 60 min, thus enhancing the drug carrier potential of liposomes in blood. As a consequence of their interaction with liposomes, the polymorphonuclear leukocytes in whole blood decreased in apparent buoyant density, while maintaining their viability. These results indicate that blood leukocytes in their natural milieu of whole blood are capable of interacting with, and taking up multilamellar liposomes.

To ascertain the activity and substrate specificity of nuclear protein kinases during various stages of the cell cycle of HeLa S3 cells, a nuclear phospho-protein-enriched sample was extracted from synchronised cells and assayed in vitro in the presence of homologous substrates. The nuclear protein kinases increased in activity during S and G2 phase to a level that was twice that of kinases from early S phase cells. The activity was reduced during mitosis but increased again in G1 phase. When the phosphoproteins were separated into five fractions by cellulose-phosphate chromatography each fraction, though not homogenous, exhibited differences in activity. Variations in the activity of the protein kinase fractions were observed during the cell cycle, similar to those observed for the unfractionated kinases. Sodium dodecyl sulfate polyacrylamide gel electrophoretic analysis of the proteins phosphorylated by each of the five kinase fractions demonstrated a substrate specificity. The fractions also exhibited some cell cycle stage-specific preference for substrates; kinases from G1 cells phosphorylated mainly high molecular weight polypeptides, whereas lower molecular weight species were phosphorylated by kinases from the S, G2 and mitotic stages of the cell cycle. Inhibition of DNA and histone synthesis by cytosine arabinoside had no effect on the activity or substrate specificity of S phase kinases. Some kinase fractions phosphorylated histones as well as non-histone chromosomal proteins and this phosphorylation was also cell cycle stage dependent. The presence of histones in the in vitro assay influenced the ability of some fractions to phosphorylate particular non-histone polypeptides; non-histone proteins also appeared to affect the in vitro phosphorylation of histones.

We have previously shown that the 43-fold induction by phenobarbital of the major phenobarbital-inducible cytochrome P-450 of rat liver microsomal membranes (PB P-450) is mediated by a 20-fold increase in the amount of its mRNA in the cytoplasm. Here we demonstrate that the induction of the mRNA can be almost entirely accounted for by an increase in the rate of transcription of genes coding for PB P-450, and involves little or no change in the rates of processing, transport or degradation of the mRNA. Phenobarbital treatment resulted in no amplification or rearrangement of PB P-450 genes.

1. After conventional fractionation of rat liver homogenates in 0.88m-sucrose the mitochondrial fraction was subjected to short-term water lysis followed by separation of the resulting membrane preparations. 2. Phosphatidate formation was measured in all subcellular fractions and subfractions and was compared with the distribution of succinate dehydrogenase, monoamine oxidase, rotenone-insensitive NADH cytochrome c reductase, arylsulphatase, urate oxidase, arylesterase and glucose 6-phosphatase. 3. The results obtained indicated that mitochondria were capable of synthesizing phosphatidate, though this activity was only about one-third of the total homogenate activity. 4. Mitochondrial phosphatidate formation was located predominantly in the outer mitochondrial membrane. Although this membrane preparation was found to be significantly contaminated by the microsomal fraction, this contamination was estimated to account for not more than about 20% of the total phosphatidate formation observed in preparations of outer mitochondrial membrane.