Study of granule reappearance and histamine synthesis in rat mast cells maintained in short-term cultures

A single cutaneous application of components of poison oak or ivy urushiol oils to mice results in contact sensitivity with properties of delayed-type hypersensitivity. The compounds, 3-heptadecylcatechol (HDC, from poison oak urushiol) and 3-pentadecylcatechol (PDC, from poison ivy urushiol) are completely cross-reactive. Covalent bond formation between the o-quinone intermediate of PDC and nucleophilic functionalities such as those found on proteins is known to occur in a regiospecific manner. Amino nucleophiles preferentially attack the 5-position on the catechol ring while thiol nucleophiles attack the 6-position. The present paper describes the immunological properties of the three possible ring monomethylated analogs of PDC. When mice were treated with a single epicutaneous painting with these analogs, only the 5-methyl compound (5-Me-PDC) was found to be an ineffective sensitizer. The 5-Me-PDC analog, however, was capable of inducing cellular proliferation in draining lymph nodes. Furthermore, epicutaneous pretreatment with 5-Me-PDC suppressed the subsequent induction of contact sensitivity to PDC and HDC in an antigen-specific manner. Equivalent treatment with the 6-Me-PDC analog (a good sensitizing agent) resulted in a less consistent and weaker suppressive effect, while the 4-Me-PDC analog did not display any suppressive activity. The suppressive activity could be demonstrated up to 15 days following primary painting with 5-Me-PDC. Lymph node cells obtained from mice 10 days after a single painting with 5-Me-PDC could transfer the suppressive effect. Under certain circumstances 5-Me-PDC could also sensitize, indicating that the analog retains some sensitizing abilities. Hence, blocking the 5-position of the catechol ring results in a major alteration in the type of immune response elicited. Since 5-Me-PDC is specifically blocked in terms of nucleophilic attack by amino groups, it is suggested that attack by amino nucleophiles is of primary importance in the peripheral metabolic processing of these catechols, which in turn determines the outcome of the immune response.     

Simultaneous isolation of major viral proteins in one step.

We have developed a rapid and simple technique for the simultaneous isolation of all the major viral proteins from RNA tumor viruses. The basis for this procedure is analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Using dansylated virus as internal marker it is possible to follow the migration of unlabeled viral proteins since dansylation does not change the mobility of labeled proteins (8). The method results in approximately 80% recovery of starting protein and is very reproducible. Using radioimmunoassay no alteration of the purified proteins is detectable.     

Alkyl alkanethiolsulfonate sulfhydryl reagents: β-Sulfhydryl-modified derivatives of l-cysteine as substrates for trypsin and α-chymotrypsin

Derivatives of l-cysteine and the A chain of bovine insulin have been chemically modified at the cysteinyl β-sulfhydryl by certain sulfhydryl-specific alkyl alkanethiolsulfonate reagents. The alkanethiolation products possess mixed-disulfide side chains structurally similar to the side chains of lysine and phenylalanine and hence were studied here as substrates for trypsin and α-chymotrypsin, respectively. Kinetic parameters were obtained for the enzyme-catalyzed hydrolyses of the modified l-cysteine analogs and of specific reference amino acids which were derivatized analogously at both the α-amino and α-carboxyl groups and assayed identically. For both enzymes it was found that the specificity constants, $\text{k}{}_{\mathrm{<mtext>cat</mtext>}}\text{K}{}_{\mathrm{m}}$, for analog esters compare favorably with those for specific reference esters, whereas specificity constants for analog amides compare much less favorably with those for specific reference amides. This discrepancy is largely a consequence of the k_cat values for the analog amides being relatively much lower than the corresponding values for the reference amides. Consistent with this trend, no detectable enzyme-catalyzed hydrolysis of the amide bonds at the sites of modified cysteine residues in the A chain of bovine insulin was observed. It is proposed that the predominant kinetic consequence of the mixed-disulfide side chains of the alkanethiolated cysteine moieties is a decrease in the acylation rate constants, k₂, arising from an increase in the transition-state free energies of acylation.     

Collagen in the egg shell membranes of the hen

Collagen-like proteins have been found in the egg shell membranes of the hen. Materials similar to types I and V collagens were detected in each of the two layers of this membrane, the thick outer membrane and the thin inner membrane. Collagen was extracted by acid-pepsin digestion and isolated by differential salt precipitation. Identification of type-specific collagen-like material was established by coelectrophoresis on SDS-polyacrylamide gels using known collagen standards. These bands were susceptible to digestion by bacterial collagenase. From differential staining of the gels it was estimated that the ratio of collagen types I:V was approximately 100:1. Further confirmation of these biochemical results was obtained with immunofluorescence microscopy using type-specific antisera against chicken types I and V collagen with the indirect sandwich technique. Both the inner and outer shell membranes contained the two types of collagen. Within each membrane, the large, coarse 2.5-micron fibers contained predominantly type I collagen-like material, while type V collagen was mainly associated with the delicate narrower fibers of approximately 0.6-micron diameter. These tended to be concentrated in the inner membrane. At the electron microscopic level, both types of fibers were coated with glycoproteins that stained positively with ruthenium red. The deposition of these collagen-like substances by the hen oviduct on to the surface of the developing egg is an additional example of interstitial-type collagen synthesis and secretion by epithelial rather than by mesenchymal cells.     

Deoxyguanosine toxicity in a mouse T lymphoma: relationship to purine nucleoside phosphorylase-associated immune dysfunction.

The absence of either of the enzymes adenosine deaminase (ADA) or purine nucleoside phosphorylase is associated with an immunodeficiency disease. Because all four nucleoside substrates of the enzyme purine nucleoside phosphorylase accumulate in the urine of patients who lack this enzyme (Cohen et al., 1976), we examined the toxicity of each of the four substrates using a mouse T cell lymphoma (S49) in continuous culture. Of the four substrates (inosine, deoxyinosine, guanosine and deoxyguanosine), only deoxyguanosine is cytotoxic at concentrations lower than 100 μM; furthermore, only deoxyguanosine is directly phosphorylated in S49 cells. Mutant S49 cells lacking deoxycytidine kinase (EC 2.7.1.74) are resistant to the toxic effects of deoxyguanosine, and these same mutants do not phosphorylate deoxyguanosine. Thus the cytotoxicity of exogenous deoxyguanosine correlates with the intracellular concentration of accumulated deoxyGTP.The addition of deoxyguanosine results in the depletion of deoxyCTP in S49 cells, indicating that deoxyGTP is an inhibitor of ribonucleotide reductase. Furthermore, the addition of deoxycytidine prevents the toxic effects of deoxyguanosine. Thus a therapy for purine nucleoside phosphorylase-deficient patients might include deoxycytidine to alleviate the proposed deoxyCTP starvation in those tissues capable of phosphorylating deoxyguanosine.     

Anion binding to oxidized type 2 depleted and native laccase: a spectroscopically effective model for exogenous ligand binding to the type 3--type 2 active site

Xanthine oxidase, a mammalian nitroreductase, catalyzed the binding of [³H]1-nitropyrene to DNA. The binding was dependent on the presence of hypoxanthine and was inhibited by allopurinol, a specific xanthine oxidase inhibitor. These data support the hypothesis that nitroreduction is a necessary step in the metabolic activation of 1-nitropyrene to a bacterial mutagen.     

A theoretical model for immobilized whole cell enzyme

A method of estimating effectiveness factor for immobilized whole cells is developed by considering microbial cells as microspheres containing enzyme activity dispersed in the gel phase of the support matrix. The proper model equations describing the system are solved and the corresponding effectiveness factors calculated for various bead sizes, and numbers and activities of cells. The cell wall resistance (permeability) is found to be one of most important variables in the system. The model is applied in predicting the experimental data of other investigators.     

Inhibition by somatostatin of the proliferation of T-lymphocytes and Molt-4 lymphoblasts

The proliferation of Molt-4 lymphoblasts and phytohemagglutinin-stimulated human T lymphocytes in vitro was inhibited significantly by 10(-12) to 10(-10) M to 10(-13) to 10(-9) M somatostatin, as assessed by the uptake of [3H]thymidine and [3H]leucine, respectively. The inhibitory effect of somatostatin was not attributable to cytotoxicity and was associated with a mean degradation of 52 and over 95% of immunoreactive somatostatin, respectively, after 3 and 24 hr of incubation at 37 degrees C. The specific suppression of Molt-4 lymphoblasts and T lymphocytes by somatostatin represents a distinct mechanism for the specific regulation of immunological responses by neuropeptides of the peripheral nervous system and gastrointestinal tract.     

Evidence for morphine and morphine-like alkaloid responses resistant to naloxone blockade in the rat vas deferens.

The application of morphine or surrogates to the isolated rat vas deferens maintained at 37° C in Tyrode solution, produced an increase in the electrically induced muscular twitch. In contrast, leucine enkephalin or D-alanine₂methionine enkephalinamide produced a dose-dependent inhibition of the muscular twitch. The effect of morphine and derivatives was not antagonized by naloxone, but the depression caused by the opiate pentapeptides or β-Endorphin was readily antagonized and reversed by naloxone. Tolerance developed to the $\text{in vitro}$ effect of morphine; vasa deferentia obtained from tolerant-dependent rats were about six times less sensitive to the effect of morphine and about five times less sensitive to the depression caused by leucine enkephalin as compared to their respective paired, placebo implanted control rats.     

Preparation and characterization of a stable half met derivative of type 2 depleted Rhus laccase: Exogenous ligand binding to the type 3 site

We report the preparation and characterization of a stable half met (Cu(II)Cu(I)) type 2 copper depleted derivative of $\text{Rhus}$ laccase. Anion binding studies to this mixed valent type 3 protein form indicate no tight binding of anions nor group 1 - group 2 ligand behavior. This suggests that, in contrast to the well-characterized hemocyanins and tyrosinase coupled binuclear sites, exogenous ligands do $\text{not}$ appear to bridge the type 3 binuclear copper ions in laccase.     

Epileptogenesis: A model for the involvement of slow membrane events and extracellular potassium

A computer model was developed, based in part on voltage-clamp data from Aplysia bursting pacemaker cells. This model was used to explore the interaction of slow subthreshold membrane properties with the synaptic events and the accumulation of extracellular potassium which are thought to occur in experimental epileptic foci. Results of the model suggest a new role for potassium ions and subthreshold membrane events in epileptogenesis—namely, that an increase in extracellular potassium may transform a group of stable cells into unstable, oscillatory cells, independent of synaptic activity. The model also suggests that a population of cells may be more susceptible than single cells to this transformation.     

A new discontinuous buffer system for the electrophoresis of cationic proteins at near-neutral pH

A simple, discontinuous buffer system for polyacrylamide gel electrophoresis near neutral pH is described. The buffer is MOPS (3-[N-morpholine]propanesulfonic acid), the leading ion K⁺ and the trailing ion histidine. The system offers improved resolution of cationic proteins.     

Developmental appearance of myosin heavy and light chain isoforms in vivo and in vitro in chicken skeletal muscle

Three myosin heavy chain isoforms with unique peptide maps appear sequentially in the development of the chicken pectoralis major muscle. An embryonic isoform is expressed early and throughout development in the embryo. A second isoform appears just after hatching and predominates by 10 days ex ovo. A third isoform, indistinguishable from adult myosin heavy chain, predominates by 8 weeks after hatching. This sequence of myosin isoform change does not, however, appear during myogenesis in vitro. In cultures prepared from embryonic myoblasts only embryonic myosin heavy chain is expressed. This is true even in cultures maintained for 30 days. Myosin light chain expression also changes in vivo with a progressive increase in fast light chain 3 accumulation. In vitro, however, this shift to increasing fast light chain 3 accumulation does not occur. The results indicate that the myosin heavy chain and light chain pattern observed in vitro is identical to that of the embryonic muscle and that the conditions necessary for the shift in expression to a more mature myosin phenotype are not present in myogenic cultures. These cultures are therefore potentially of great value in probing further the neural and humoral determinants of muscle fiber maturation and growth.     

An NADH-coupled assay for femtogram or nanogram quantities of chymotrypsin

Chymotrypsin can be determined with an NADH-coupled assay. Hydrolysis of the substrate benzoyltyrosine ethyl ester is monitored by coupling the liberation of ethanol to the production of NADH and determining the NADH spectrophotometrically or fluorometrically. Nanogram quantities of chymotrypsin can be determined in milliliter volumes. With these microfluorescence methods this assay can be performed in a final volume of less than a nanoliter, allowing determination of femtogram quantities of chymotrypsin, the amount present in an individual zymogen granule.     

Intermediate filament protein synthesis in preimplantation murine embryos

The synthesis of two extraembryonic endodermal cytoskeletal proteins (Endo B, Mr = 50,000; Endo A, Mr = 55,000) was detected by immunoprecipitation at the 4- to 8-cell stage of preimplantation mouse development. The first detectable synthesis of both proteins occurs at about the same time as the earliest allocation of cells to the trophectodermal lineage. Both Endo A and B were identified in the two-dimensional gel pattern of blastocyst cytoskeletal proteins prepared by nonionic detergent and high-salt extraction. Endo A and B were identified as the y and x blastocyst cytoskeletal proteins, respectively, previously described by other investigators. Antibodies to Endo B are shown to react with intermediate filaments at the electron microscopic level, confirming that Endo B is an authentic intermediate filament protein. Previously, the TROMA 1 monoclonal antibody prepared by other investigators was shown to react specifically with Endo A and to decorate trophoblast cytoskeletons but did not react with the inner cell mass of blastocysts. Endo B antibodies are now also shown to decorate trophoblast cytoskeletons.     

Characteristics of an SV40-plasmid recombinant and its movement into and out of the genome of a murine cell

A bacterial plasmid carrying the early region of SV40 (pOT) has been stably established in high molecular weight (hmw) DNA of mouse L cells by selection for the herpes virus thymidine kinase (tk) gene. DNA blotting has demonstrated that most cell lines contain multiple discrete copies of pOT, generally with an intact SV40 early region. No free copies of pOT have been detected. Both pOT and tk sequences may be amplified up to 20–200 copies of the SV40 early region. In contrast to the uniform staining pattern normally observed in SV40-transformed lines, indirect immunofluorescence using antiserum to the SV40 T antigen has demonstrated that the expression of the early region is heterogeneous in these cell lines. This fraction expressing T is characteristic of a given cell line, and varies from 0 to 99% positive. Several pOT cell lines have been fused to simian cells, and replicating low molecular weight DNAs were isolated from the heterokaryons. Transformation of E. coli with this DNA demonstrates that pOT can be rescued from hmw DNA in L cells and reestablished as a plasmid in E. coli. Excision is generally precise when pOT is introduced to the murine cells as a supercoiled molecule, and imprecise when pOT is introduced in linear form.