Cannabinoid induced degranulation of rabbit neutrophils

We have examined the effects of various cannabinoids on the degranulation of rabbit peritoneal neutrophils. Several cannabinoids were found to cause a dose-dependent and noncytotoxic release of lysosomal enzymes from the neutrophils. The degranulation induced by cannabidiol is rapid ($\text{t}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 2.3 min}$), and enhanced by extracellular calcium and cytochalasin B. In addition to their intrinsic activity, cannabinoids also modulate the neutrophils' responses to the chemotactic peptide formyl-methionyl-leucyl-phenylalanine. This investigation represents the initial step toward the characterization of the effect of cannabinoids on the excitation-activation coupling sequence of hormonally responsive cells.     

Reduced chemotactic peptide binding in juvenile periodontitis: a model for neutrophil function

Chemotactic peptide binding sites from peripheral blood neutrophils of Localized Juvenile Periodontitis patients and normal controls were quantitated using tritiated N-formylmethionylleucyl-phenylalanine and a rapid filtration assay. It was found that there is a significant reduction in the number of binding sites per cell on neutrophils from the patient group whereas binding affinity remained the same as control values. A direct correlation between $\text{in}\text{vitro}$ neutrophil chemotactic response and density of binding sites was found. Since these patients present little clinical illness, localized juvenile periodontitis can be a valuable model for the study of human neutrophil function.     

The biosynthesis of ubiquitin by parathyroid gland

Data are presented on the isolation, biosynthesis, and identification of a small peptide (H) from parathyroid gland. Under our experimental conditions this peptide (H) represents, in addition to secretory protein-I and proparathyroid hormone, the other major protein which is rapidly synthesized during shorterm incubations of tissue slices. N-terminal sequence analysis was performed on samples of peptide H and the resulting data used to conduct a search of the sequence data bank. The search established the identity of peptide H as ubiquitin. These findings establish parathyroid gland as another system which rapidly produces ubiquitin $\text{in}$$\text{vitro}$, in addition to the systems employing hypothalamus and pituitary where ubiquitin biosynthesis was initially observed by Seidah $\text{et}$$\text{al}$ and Scherrer $\text{et}$$\text{al}$.     

Effects of oligomycin and quercetin on the hydrolytic activities of the (Na+ + K+)-dependent ATPase

Quercetin inhibited a dog kidney ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ preparation without affecting $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for ATP or $\text{K}{}_{0.5}$ for cation activators, attributable to the slowly-reversible nature of its inhibition. Dimethyl sulfoxide, a selector of E₂ enzyme conformations, blocked this inhibition, while the K⁺-phosphatase activity was at least as sensitive to quercetin as the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ activity, all consistent with quercetin favoring E₁ conformations of the enzyme. Oligomycin, a rapidly-reversible inhibitor, decreased the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for ATP and the $\text{K}{}_{0.5}$ for cation activators, and its inhibition was also diminished by dimethyl sulfoxide. Although oligomycin did not inhibit the K⁺-phosphatase activity under standard assay conditions, a reaction presumably catalyzed by E₂ conformations, its effects are nevertheless accommodated by a quantitative model for that reaction depicting oligomycin as favoring E₁ conformations. The model also accounts quantitatively for effects of both dimethyl sulfoxide and oligomycin on $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$, $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ for substrate, and $\text{K}{}_{0.5}$ for K⁺, as well as for stimulation of phosphatase activity by both these reagents at low K⁺ but high Na⁺ concentrations.     

Antibacterial properties of bile acid oxazoline compounds

Chenooxazoline³ (50–100 μM) inhibited (>50%) both 7α and 7β-dehydroxylase activities in whole cells and cell extracts of $\text{Eubacterium}$ sp. V.P.I. 12708. Chenooxazoline (>50 μM) and methylchenooxazoline (>25 μM) but not lithooxazoline (≤100 μM) inhibited growing cultures of $\text{Eubacterium}$ sp. V.P.I. 12708. Chenooxazoline (100 μM) also inhibited the growth of certain members of the genera $\text{Eubacterium}$, $\text{Clostridium}$, $\text{Bacteroides}$ and $\text{Staphylococcus}$ but not $\text{Pseudomonas}$, $\text{Escherichia}$, $\text{Salmonella}$ or the eucaryotic microorganism, $\text{Saccharomyces cerevisiae}$ (_< 400 μM).     

S--vinyl homocysteine, an analog of ethionine that is highly mutagenic for S. typhimurium TA100.

Two types of bovine pituitary gland polyprenols were resolved by silica gel chromatography; i. e., the high molecular weight dolichols of 17 to 23 isoprene units with the OH-terminal isoprene residue saturated, and a fully unsaturated decaprenol. The latter compound was found to be a mixture of molecules differing in the proportion of $\text{cis}$- and $\text{trans}$- isoprene units.     

Tuftsin: A hormone-like tetrapeptide with antimicrobial and antitumor activities

A specific fraction of immunoglobulin G binds to polymorphonuclear neutrophils and stimulates their phagocytic activity. This phagocytosis-stimulating activity resides solely in a small peptide termed tuftsin, of the sequence Thr-Lys-Pro-Arg, which has been isolated from the leukophilic immunoglobulin G fraction. The physiological significance of tuftsin has been demonstrated in splenectomized patients and patients with a congenital tuftsin abnormality, in whom the low levels of tuftsin in sera (measurable by radioimmunoassay) coincides with a high incidence of infection. Tuftsin has also been shown to enhance bactericidal activity in addition to phagocytosis. Its biological activities appear to be mediated via specific tuftsin receptors which have been found on macrophages, monocytes and granulocytes. In addition, tuftsin possesses chemotactic, migration-enhancing and mitogenic properties for leukocytes and has recently been shown to enhance their anti-tumor activity $\text{in}$$\text{vitro}$ as well as $\text{in}$$\text{vivo}$. Other known activities of tuftsin include effects on the activity of the hexose monophosphate shunt, on the concentrations of intracellular cyclic nucleotides and on the efflux of Ca²⁺ in leukocytes. Tuftsin has been chemically synthesized in various laboratories using different procedures and also is available commercially. The above features of tuftsin plus the expected low toxicity of this peptide make tuftsin a very attractive agent for immunotherapy against infection and cancer. However, a great deal of caution needs to be exercised when using tuftsin due to inhibitory contaminants found in certain commercial preparations.     

Absorption of α-MSH from subcutaneous and intraperitoneal sites in the rat

Pentobarbitone anesthetized rats were injected with 30 nmol (50 μg) α-MSH administered intraperitoneally (IP) and subcutaneously (SC) in an acid-saline vehicle, or SC in a zinc phosphate vehicle. Concentrations of α-MSH in plasma were measured by radioimmunoassay. The pharmacokinetic parameters for the three modes of administration were determined by fitting a one-compartment open model to the plasma level data. The $\text{t}\text{1}\text{2}$ for absorption using the saline vehicle was 7.3 and 5.6 min from the IP and SC sites, respectively. The $\text{t}\text{1}\text{2}$ for absorption from the zinc phosphate complex of 17.7 min was significantly longer. Five percent of the IP dose was absorbed into the systemic circulation giving a peak plasma level of 14.1 nmol/l. The absorption of 2–3 percent was significantly lower following SC administration; peak plasma levels were 8.3 and 4.8 nmol/l for the saline and zinc phosphate vehicles, respectively. The low percentage absorption values indicated a high degree of metabolism of the peptide by peripheral tissues on its passage from the injection sites into the circulation.     

Selective alterations in hepatic enzyme response after reduction of nuclear triiodothyronine receptor sites by partial hepatectomy and starvation.

Barley embryo 5S rRNA hybridizes efficiently with barley embryo 18S rRNA but not with 26S rRNA. Mouse sarcoma 5S rRNA also selectively hybridizes, to a smaller extent, with mouse sarcoma 18S rRNA. The barley embryo 5S–18S rRNA complex has a sharp melting profile and a “Tm” of $\text{ca}$. 59° in 0.1 $\text{M}$ NaCl. The mouse sarcoma 5S–18S rRNA complex has a broader transition breadth and a “Tm” of $\text{ca}$ 52°. The conditions used for hybridization lead to very specific reconstitution of the “natural” complex between 5.8S and 26S–28S rRNA since both the $\text{in}\text{vivo}\text{and}\text{in}\text{vitro}$ complexes between 5.8S and 26S–28S rRNA from barley embryos and mouse sarcoma have equally sharp melting profiles and a “Tm” of $\text{ca}$. 52° in 0.1 $\text{M}$ NaCl.     

Degradation of hemin IX and synthetic hemins to α-bilirubins and their conjugates in the isolated perfused rat liver

Hemin IX was perfused through rat liver of a normal, untreated animal. Its degradation products, collected in the bile fluid over a period of 90 min, were found to consist of the bilirubin IX-α diglucuronide (56%), the mixture of bilirubin IX-α monoglucuronides (42%), and free bilirubin IX-α (2%). When the synthetic hemin XIII $\text{2}$ was perfused with the same technique, it was found to be degraded in the same way. The bile fluid contained the diglucuronide of bilirubin XIII-α $\text{10}$ (55%), the monoglucuronide of bilirubin XIII-α $\text{9}$ (43%) and the free bilirubin XIII-α 8 (2%). Similar results were obtained when the iron 1,4-di(β-hydroxyethyl)-2,3,5,8-tetramethyl-6,7-di(β-carboxyethyl) porphyrin $\text{3}$ was perfused; the diglucuronide of the α-bilirubin $\text{11}$ comprised 65% of the excreted bile bilirubins, the monoglucuronide was 25% of the total and the free α-bilirubin $\text{11}$ 10% of the total. Perfusion of hematohemin gave 58% of the diglucuronide of α-hematobilirubin, as well as 40% of the monoglucuronides, and 2% of the free α-hematobilirubin. The simultaneous perfusion of hematohemin and of hemin IX produced an inhibition of the degradation of the hemin IX, while hematohemin was degraded as described above. It was concluded that the normal rat liver is prepared to dispose of exogenously added hemins by their oxidation to α-biliverdins, reduction of the latter to the corresponding α-bilirubin and excretion of their conjugated derivatives through the bile duct.     

The preferential human mononuclear leukocyte chemotactic activity of the substituent tetrapeptides of angiotensin II

The amino- and carboxy-terminal substituent tetrapeptides of angiotensin II, Asp-Arg-Val-Tyr and Ile-His-Pro-Phe, elicit substantial human mononuclear leukocyte chemotactic responses $\text{in}\text{vitro}$ that attain maximal levels at tetrapeptide concentrations of 3 × 10^?8 M and 3 × 10^?7 M, respectively. In contrast, the angiotensin II-derived tetrapeptides evoke only marginal human neutrophil chemotactic responses. Amino acid deletions or substitutions that alter the properties of the tetrapeptides, reduce their chemotactic potency and activity. Limited proteolytic cleavage of angiotensin II thus may convert a pathway with predominantly humoral effects to a source of mediators that regulate cellular immunity and chronic inflammatory responses.     

Ornithine decarboxylase protein diversity and activity modulation in HTC cells

Ornithine decarboxylase of HTC cells was chromatographically separated into three ionically distinct but kinetically similar forms of this protein. The sequential appearance of these ornithine decarboxylase species during enzyme induction, and the accumulation of normally minor species under conditions that stabilize this enzyme, suggest that these represent modifications that are associated with the extremely rapid turnover of this protein $\text{in vivo}$. These forms may also be differentially active or unequally distributed $\text{in vivo}$ as indicated by the selective inactivation of one of the forms by short exposure to α-difluoromethylornithine.     

Tryptic peptide map analysis of the major human blood platelet membrane glycoproteins separated by two-dimensional polyacrylamide gel electrophoresis

Washed platelets were surface-labelled by lactoperoxidase catalyzed iodination and either the platelets or membranes were solubilized in detergent and applied to a wheat germ agglutinin-Sepharose column and a Lens culinaris lectin Sepharose column coupled sequentially. The glycoproteins eluted from the lectin columns were separated by two-dimensional gel electrophoresis. Alternatively, labelled whole platelets or membranes were solubilized and then directly separated by two-dimensional polyacrylamide gel electrophoresis. Spots corresponding to specific glycoproteins identified by apparent isoelectric point ($\text{p}\text{I}$), apparent molecular weight ($\text{M}{}_{\mathrm{<mtext>r</mtext>}}$), staining and labelling characteristics were cut from the gels and analyzed by tryptic peptide mapping. The maps of the individual glycoproteins (GP) Ia, Ib, IIa, IIb, GP_132–135^4–4.5 IIIa, IIIb and IIIc were all different. Glycoproteins with the same $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ but different $\text{p}\text{I}$ were distinct with the exception of regions of GP Ib. There were minor differences in the maps of glycoproteins separated in the reduced or non-reduced state. Tryptic peptide maps provide a valuable additional parameter for the identification and characterization of platelet glycoproteins.     

Spondyloepiphyseal dysplasia, chondroitin sulfate type: a possible defect of PAPS--chondroitin sulfate sulfotransferase in humans

Four patients with an unusual form of spondyloepiphyseal dysplasia excreted in the urine undersulfated chondroitin 6-sulfate (Biochem. Med. $\text{7}$, 415–423, 1973). The sera of these patients show a low activity of PAPS — chondroitin sulfate sulfotransferase, while the undersulfated chondroitin sulfate present in their urine is a much better acceptor of ${}^{35}\text{SO}{}_4$ than standard chondroitin sulfate when they are incubated with [${}^{35}\text{S}$]PAPS and normal sulfotransferases. These results suggest that in these patients the skeletal lesions are secondary to a defect in the synthesis of chondroitin sulfate involving specifically the sulfotransferase activity.     

Mechanism of corticotropin action in rat adrenal cells I. The effects of inhibitors of protein synthesis and of microfilament formation on corticosterone synthesis

The contribution of protein synthesis and formation of microtubules and microfilaments to corticotropin-stimulated steriodogenesis in rat adrenal cell suspensions has been assessed by use of a series of inhibitors to each function. Five inhibitors of protein synthesis (cycloheximide, puromycin, blastocidin S, anisomycin, and trichodermin) each exhibited time-dependent inhibition of corticotropin-stimulated steroidogenesis. For the first 30 min, steroidegenesis was more extensively inhibited than protein synthesis, after which the effectiveness of the inhibitors diminished on steroidegenesis but not on protein synthesis. The reversal effects was not observed at high levels of inhibitors. One inhibitor of microfilament fromation (cytochalasin B) and four inhinitors of microtubule formation (colchicine, podophyllotoxin, vinblastine sulfate and griseofulvin) inhibited steroidogenesis without inhibiting protein synthesis and without any reversal effect with prolonged incubation. The actions of all ten inhinitors were shown to be fully reversible. Cell superfusion of adrenal cells showed that the decay of steroidogenesis upon addition of all the protein synthesis inhibitors was similar to decay upon removal of corticotropin from the medium ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 4–6}\text{min}$). Recoveries from inhibition upon removal of the inhibitors were similar to each other and comparable to initial corticotropin stimulation of the cells (lag of 3–5 min, $\text{f}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 7–9}\text{min}$). Similar kinetics of inhibition and recovery were observed for vinblastine sulfate while a direct inhbition of cytochrome P-450_sec by an aminoglutethimide was complete within 1 min and was rapidly reversed.Injection of each inhibitors (all classes) into hypophysectomized rats inhibited the elevation of plasma corticosterone by corticotropin. The extent of cholesterol combination with cytochrome P-450_sec in adrenal mitochondria isolated from these rats was also decreased by all inhbitors. Decreases in plasma corticosterone correlated directly with decreases in cholesterol combination with cytochrome P-450_sec (r = 0.94).It is concluded that protein synthesis and steroidogenesis must be intimately coupled propbably due to the requirement of a labile protein for cholesterol transport to cytochrome P-450_sec. An involvement of microtubules and microfilaments in this process is clearly indicated.     

Fast cation flux from Torpedocalifornica membrane preparations: Implications for a functional role for acetylcholine receptor dimers

Closed membrane vesicles derived from the innervated face of $\text{Torpedo}$$\text{californica}$ electroplax respond to the cholinergic agonist carbamylcholine by a rapid efflux of cations. This response is detected by release of ²²Na enclosed within the vesicles and is considerably faster than previously reported for this $\text{in}\text{vitro}$ system. It is considered likely that the rapid response is analogous to physiological phenomena since it has the appropriate pharmacological characteristics; it desensitizes upon prolonged contact with the agonist and it has a dose-response curve in a physiological range. It is further shown that a dimeric form of the acetylcholine receptor, stabilized by chemical modification methods, is fully active in terms of the carbamylcholine elicited response.     

Degradation of cartilage proteoglycan and collagen by synovial cells: Stimulation by macrophages under activation by phagocytosis,lymphocyte factors,bacterial products or other inflammatory stimuli

When cultured together with dead ³⁵S-labelled cartilage discs or at the surface of $\text{[}{}^3\text{H]proteoglycan}\text{[}{}^{14}\text{C]collagen-coated}$ plates, synovial cells from either arthritic or normal rabbit joints digested both the proteoglycan and the collagen of the substrates after a lag-period of 1–2 days. These digestions were inversely related to the age (number of subculture passages) of the synovial cells and they could be modulated by serum components that were either inhibitory or stimulatory. They were dependent on a protein synthesis by the cells and were paralleled, in young cultures, by the release of collagenase and of a proteoglycan-degrading neutral proteinase. The co-culture of synovial cells with macrophages or their culture with macrophage-conditioned culture media caused a more rapid and more extensive degradation of collagen and proteoglycan due to the stimulation of the synovial cells by a nondialysable macrophage factor. The production of this synovial cell-activating ‘matrix regulatory monokine’ by the macrophage was enhanced by several immunological or inflammatory stimuli such as lymphocyte factors, phagocytosis, asbestos fibres, endotoxin, adjuvant muramyl dipeptide or chemotactic formyl-methionyl peptide, as well as by other membrane-active agents (phorbol myristate acetate, concanavalin A). It is presumed that these interactions are of importance in the development of cartilage destruction in rheumatoid and other chronic inflammatory arthritis.     

Preparation of copoly (γ-benzyl-l-glutamyl-N5-β-d-glucopyranosyl-l-glutamine) and its interaction with fibroblast cells

Copoly(α-amino acid)s consisting of γ-benzyl-l-glutamate and $\text{N}{}^5\text{-β-}\text{d}\text{-glucopyranosyl-}\text{l}\text{-glutamine}$ were prepared by the reaction of copoly(l-glutamate) containing succinimide ester, which served as active site for the coupling reaction with β-d-glucopyranosylamine. The α-helical conformation of these copolymers became unstable in DMF as the content of glutamine derivative increased. A dry film made from this copolymer could take a full α-helical conformation even at such a high content as 80% of the glutamine derivative, but in a wet film this ordered structure was partially disrupted by hydration. The hydraulic permeability of this copoly(α-amino acid) was clearly dependent on the molar content of glucopyranosyl groups. The attachment of fibroblast cells to these hydrated copolymer films was effectively depressed in the presence of a serum-free medium. The cells attached to the substrate were spherical in shape.