Effects of calcium and chelating agents on the ability of various agonists to increase cyclic GMP in pancreatic acinar cells

In dispersed acinar cells from guinea pig pancreas we found that chelating extracellular calcium with EDTA did not alter cellular cyclic GMP but caused a 50% reduction in the increase in cyclic GMP caused by the synthetic C-terminal octapeptide of porcine cholecystokinin (cholecystokinin octapeptide). This effect was maximal within 2 min and preincubating the cells with EDTA for as long as 30 min caused no further reduction in the action of cholecystokinin octapeptide. In acinar cells preincubated without calcium, adding calcium caused a time dependent increase in the action of cholecystokinin octapeptide and this increase was maximal after 10 min of incubation. An effect of extracellular calcium on the action of cholecystokinin octapeptide could be detected with 0.5 mM calcium and was maximal with 2.0 mM calcium. Magnesium alone or with calcium did not alter the action of cholecystokinin octapeptide. Extracellular calcium did not alter the time course or the configuration of the dose vs. response curve for the action of cholecystokinin octapeptide on cellular cyclic GMP. Low concentrations of EGTA (0.1 mM) decreased the effect of cholecystokinin octapeptide on cellular cyclic GMP to the same extent as did EDTA or preincubating acinar cells without calcium. Increasing EGTA above 0.1 mM caused progressive augmentation of the action of cholecystokinin octapeptide on cellular cyclic GMP and this augmentation did not require extracellular calcium or magnesium. Results similar to those obtained with cholecystokinin octapeptide were also obtained with bombesin, carbamylcholine, litorin and eledoisin. In contrast, the action of sodium nitroprusside on cyclic GMP in pancreatic acinar cells was not altered by adding EDTA or EGTA.These results indicate that the ability of extracellular calcium to influence the action of cholecystokinin octapeptide and other agents on cyclic GMP results from changes in cellular calcium and not from effects of extracellular calcium per se. The action of low concentrations of EGTA on the increase in cyclic GMP caused by various agents reflects the ability of EGTA to chelate extracellular calcium. The actions of high concentrations of EGTA were independent of extracellular calcium or magnesium and appear to reflect a direct action of EGTA on pancreatic acinar cells.     

Colony-forming B cells in F1 hybrid and transplanted CBA/N mice.

The conditions for evaluation of suppressor cell regulation of the pokeweed mitogen (PWM)-induced plaque-forming cell (PFC) responses of peripheral blood (PB) B cells in normal individuals using allogeneic cocultures is described. In 14 separate experiments, after preincubation with concanavalin A (Con A) for 2 days, PB cells suppressed the PWM-induced anti-sheep erythrocyte (SRBC) PFC response of fresh allogeneic PB cells to 17% of the expected PFC response (P < 0.05). In addition, control cells incubated for 2 days in the absence of Con A suppressed the PWM- induced PFC response of allogeneic cells in 6 of 14 experiments to the same extent as did the Con A-generated cells (P < 0.01). It was found that unstimulated control cells (without Con A activation) from normal subjects who themselves were nonresponders to PWM stimulation (< 50 PFC/10⁶ cells) usually suppressed the PFC response of allogeneic cells (P < 0.05), while control cells from normal subjects who consistently had a good PFC response to PWM stimulation (> 75 PFC/10⁶ cells) did not suppress the PFC response of allogeneic cells. The spontaneously occurring suppressor cell in nonresponder PB cell suspensions was sensitive to 3000-R irradiation, and the nonresponder state was not associated with a decreased blastogenic response to PWM. Thus, some normal subjects who themselves had a poor PWM-induced PFC response had irradiation-sensitive, spontaneously occurring suppressor cells which were capable of suppressing the PWM-induced PFC response of normal responders. The majority of normal subjects (90%) were good PFC responders to PWM stimulation and did not spontaneously suppress the PFC response of allogeneic cells to PWM, but did have PB cells which were capable of being activated by Con A to suppress.     

Cholera-toxin-dependent ADP-ribosylation of the adenylate cyclase regulatory protein in turkey erythrocyte membranes

Incubation of turkey erythrocyte membranes with cholera toxin and [³²P]NAD caused toxin-dependent incorporation of ³²P into a 42,000 M_r peptide which could be distinguished from toxin-independent ³²P incorporation into other membrane proteins. The radiolabeled 42,000 M_r peptide could be extracted from the membranes using Lubrol PX. When toxin-treated membranes were incubated with isoproterenol and GMP before detergent solubilization, the 42,000 M_r labeled peptide was adsorbed by GTP-γ-agarose which, with the same conditions, adsorbed the adenylate cyclase guanine nucleotide regulatory protein. The labeled peptide and guanine nucleotide regulatory protein activity were coeluted from the affinity matrix by guanylyl-β,γ-imidodiphosphate, GDP, and GMP. Guanosine 5′-O-(2-thiodiphosphate), an analog of GDP which blocks guanine nucleotide- and fluoride-stimulated adenylate cyclase activity, caused elution of labeled peptide which exhibited no regulatory protein activity. Our data support the view that the 42,000 M_r peptide is part of the adenylate cyclase guanine nucleotide regulatory protein. The labeled peptide allows identification of both active and inactive regulatory protein and should be useful in monitoring the purification of the regulatory protein from turkey erythrocytes.     

Radioimmunoassay for pig angiotensin I converting enzyme: a comparison of immunologic with enzymatic activity.

Angiotensin I converting enzyme in body fluids and extracts of various pig tissues was measured by radioimmunoassay and by enzymic assay. Based on the ratios of enzymic to immunologic activity, the extracts could be separated into two groups. One group, with ratios around 4 U/mg, included urine and extracts from the adrenal, choroid plexus, epididymis, gall bladder, heart, liver, retina, spleen, and testis. The other group, with ratios around 12 U/mg, contained serum and extracts from lung and kidney. Explanations are offered for why one group had a lower enzymic to immunologic ratio than the other.     

Use of the Airfuge for analysis and preparation of receptors incorporated into liposomes: Studies with the receptor for immunoglobulin E

A Beckman Airfuge has been employed for studying the interaction between lipids and the receptor for immunoglobulin E (IgE). For analytic experiments, samples were applied underneath a discontinuous sucrose gradient. After a 30-min centrifugation in a fixed-angle rotor, liposomes floated toward the top of the gradient whereas unincorporated receptor-IgE complexes remained at the bottom of the tube. Liposomes with incorporated receptors were also efficiently separated in the ACR-90 preparative rotor. These methods of "Airfuge flotation" can provide useful adjuncts to more traditional methods for density-gradient centrifugation especially when rapid analysis of small samples is desired.     

Activation of human B lymphocytes. XVII. Synergy between nonspecific and specific signals in the antigen-specific responses of human B cells

The incubation of human peripheral blood lymphocytes (PBL) with the natural killer (NK)-sensitive MOLT-4 cell line results in PBL-target cell conjugate formation by certain lymphocyte subpopulations. Following velocity sedimentation, the PBL depleted of these conjugate-forming subpopulations are markedly diminished in the ability to mediate either antibody-dependent cellular cytotoxicity (ADCC) or NK activity. The immediate testing of highly pure PBL subpopulations isolated from the NK target conjugates does not reveal the expected recovery of augmented ADCC or NK levels. Following in vitro incubation, however, the PBL NK target-binding subpopulations do manifest augmented levels of both NK and ADCC, whereas the depleted PBL continue to display diminished NK and ADCC levels. In addition, the degree of augmented NK and ADCC levels recovered by the NK target-binding PBL subpopulations appears dependent on both the time and the temperature of in vitro incubation. Moreover, the ADCC recovery patterns are identical to those observed for NK activity regardless of the time and temperature of in vitro incubation. These results directly demonstrate that the PBL subpopulations isolated from certain NK target cells are functionally enriched in the ability to mediate from ADCC and NK activity.     

Preparation and spectroscopic characterization of molecular species of brain phosphatidylserines

This study describes the first preparation and spectroscopic characterization of naturally occurring phospholipids separated according to degree of unsaturation. Phosphatidylserines (PS) have been prepared from bovine brain and shown to be pure by extensive thin layer chromatographic analysis as well as by infrared spectroscopy and fatty acid analysis. The PS has been separated according to degree of unsaturation and prepared using AgNO3-impregnated silica gel H thin-layer chromatography. Fatty acid analysis of the two principal PS subfractions indicates that they are enriched in the molecular species 1-octadecanoyl-2-docosahexaenoyl-sn-glycero-3-phosphorylserine and 1-octadecanoyl-2-octadecenoyl-sn-glycero-3-phosphorylserine. The identity of the two PS subfractions was further verified by rechromatographing on several thin layer systems and by infrared spectroscopy. With the use of a 100 MHz Fourier transform nuclear magnetic resonance (NMR) spectrometer, the spectra of bovine whole brain, white matter, gray matter, monoenoic, and hexaenoic PS were obtained. Distinct proton resonances were assigned to double bond protons, protons adjacent to a double bond, and protons between two double bonds, using fatty acid methyl ester standards. The various PS preparations gave different intensities of the various proton resonances which correlated with differences in fatty acid composition. The method provides a convenient, non-destructive spectroscopic method for distinguishing monoenoic and polyunsaturated species of intact phospholipids. Electron spin resonance studies of nitroxide-labelled cholestane in sonicated PS vesicles showed greater probe motion as the unsaturation of the acyl chains was increased. The hexaenoic PS vesicles were more fluid than monoenoic PS vesicles at all temperatures in the range 10-55 degrees C. These results suggest that neuronal membranes are more fluid than myelin membranes as neuronal membranes contain more hexaenoic phospholipids.     

Serratia marcescens chitinase: One-step purification and use for the determination of chitin

The extracellular chitinase produced by Serratia marcescens was obtained in highly purified form by adsorption-digestion on chitin. After gel electrophoresis in a nondenaturing system, the purified preparation exhibited two major protein bands that coincided with enzymatic activity. A study of the enzyme properties showed its suitability for the analysis of chitin. Thus, the chitinase exhibited excellent stability, a wide pH optimum, and linear kinetics over a much greater range than similar enzymes from other sources. The major product of chitin hydrolysis was chitobiose, which was slowly converted into free N-acetylglucosamine by traces of β-N-acetylglucosaminidase present in the purified preparation. The preparation was free from other polysaccharide hydrolases. Experiments with radiolabeled yeast cell walls showed that the chitinase was able to degrade wall chitin completely and specifically.     

Structure of the scaffold in bacteriophage lambda preheads removal of the scaffold leads to a change of the prehead shell

Small angle X-ray scattering was performed on unprocessed and processed preheads, intermediates in the morphogenesis of bacteriophage λ heads. Unprocessed preheads possess an internal structure (scaffold), necessary for efficient assembly of closed shells. Processed preheads, formed after removal of the scaffold, are able to pack and cut the viral DNA in vitro. Our data show that the scaffold fills out the inside of the shell in an almost (but not completely) homogeneous fashion; structures of the scaffold with the bulk of the mass in a small core inside the shell can be excluded. Unprocessed preheads are larger than processed ones. A change in shell architecture takes place upon transition from unprocessed to processed prehead; the shell becomes roughened up. Shrinking of the shell as well as roughening up can be triggered by accidental partial degradation of the scaffold. The lattice constant of type A polyheads is in agreement with the lattice constant derived from our icosahedral models of the shell, indicating a close relationship between processed preheads and type A polyheads. This observation, together with the type of subunit clustering found, leads us to propose a simple model for the interaction of prehead shell and protein pD, which stabilizes phage DNA after packaging.     

The relationship between immunization and circulating antigen-specific plaque-forming cells

The relationship between the appearance of cells producing antibody to tetanus toxoid (TT) in the circulation and the serum titers of anti-TT IgG following booster immunization has been studied. It was found that cells producing anti-TT antibody can be detected in the circulation in a hemolytic plaque assay using sheep red blood cells (SRBC) coated with TT by the chromic chloride method. In symmetric inhibition studies using cells from TT or keyhole limpet hemocyanin (KLH)-immune donors, the homologous antigen inhibited 100% of the PFC with no cross-inhibition. Thus, the plaque-forming cells (PFC) detected in this assay are specific for the immunizing antigen. No evidence of polyclonal B-cell activation in response to TT was found, as shown by a failure to detect any PFC against unmodified or KLH or human serum albumin-treated SRBC. In addition, the increase in total Ig-secreting cells observed in a staphylococcal protein A reverse hemolytic plaque assay was always accounted for by the number of anti-TT antibody-producing cells observed. The peak number of anti-TT antibody-producing cells varied between donors, but the kinetics of their appearance was highly reproducible--none before Day 5, peak numbers between Days 6 and 8, and a sharp decline with only rare anti-TT Ig-secreting cells in the circulation by Day 15 postimmunization. Anti-TT antibody-producing cells appeared in the circulation prior to any detectable increase in serum anti-TT antibody titers, and following the disappearance of PFC from the circulation, there was no further increase in serum IgG anti-TT levels. These observations demonstrate a marked specificity of B-cell activation on boosting with a recall antigen, and a parallelism between the appearance of activated B cells in the circulation and of IgG anti-TT synthesis by the subject as a whole.     

Prolactin-induced accumulation of casein mRNA in mouse mammary explants: A selective role of glucocorticoid

The role of glucocorticoid in the prolactin-induced accumulation of casein mRNA in mammary explants from midpregnant mice has been studied after an initial 4-day incubation to allow the level of messenger to decline to undetectable levels. Subsequent culture for 3 days: 1) with insulin and glucocorticoid did not result in detectable accumulation of messenger; 2) with insulin and prolactin resulted in a very small accumulation; 3) with insulin, glucocorticoid and prolactin elicited a 20-fold greater accumulation of casein mRNA than the system with only insulin and prolactin. Therefore, although glucocorticoids are not an absolute requirement for casein gene expression in mouse mammary tissue, they are necessary for massive accumulation of casein mRNA induced by prolactin. It appears that this dependence is not a result of either mRNA stabilization or alteration in prolactin receptors. By contrast, stimulation of total epithelial RNA synthesis by prolactin does not have this glucocorticoid dependency.     

Effects of lipid peroxidation products on the rat lens in organ culture: a possible mechanism of cataract initiation in retinal degenerative disease

Rat lenses in organ culture which are exposed to bovine rod outer segments (ROS) or to the major fatty acid of ROS, docosahexaenoic acid, are impaired in their ability to accumulate radiolabeled compounds which lenses normally accumulate by active processes. The extent of lens damage correlates well with the extent of lipid peroxidation in the culture medium as assessed by the thiobarbituric acid assay. Addition of vitamin E to the medium inhibits the effect on the lens while addition of Fe-ADP complexes potentiates the effect. Thus, the lens damage appears to be attributable to toxic species generated by peroxidation of the polyunsaturated lipid added to the culture medium. Toxic aldehyde products appear to be major mediators of the lens damage, since semi-carbazide, which avidly reacts with aldehydes, can protect lenses in this system. These findings may have relevance to the cataracts clinically associated with retinal degenerative diseases such as retinitis pigmentosa. The highly membranous photoreceptor cells are extremely rich in polyunsaturated lipid. Degeneration of these cells, which is the primary pathology in such diseases, would likely lead to peroxidation with generation of toxic products within the eye. Such products could potentially produce secondary damage to other ocular structures including the lens.     

Mitogen-induced suppressor factor(s) from human lymphocytes: effects on lymphoid and nonlymphoid cells and biophysical properties

Concanavalin A (Con A) or phytohemagglutinin activate a population of human circulating lymphocytes to exert suppressive functions. We found that supernates from the activated human lymphocytes suppress lymphocyte responses to Con A, the mixed lymphocyte reaction and pokeweed mitogen-induced IgM production. Mitogen stimulated suppressor lymphocytes, or their supernates, inhibit also the spontaneous proliferation of human retinoblastoma cells (Y-79 line) and primary cultures of human keratocytes. A correlation was always noted between the levels of inhibitory activities of the lymphocytes and their supernates. Furthermore, a good correlation was found between the levels of inhibition by the supernates of lymphocyte functions (proliferation and IgM production) and of the nonlymphoid cells' proliferation. Some of the properties of this suppressor factor(s) are: (i) produced only by the T-cell population; (ii) appears after 8 hr of Con A stimulation, peaks at 24 to 48 hr and declines later on; (iii) stable at 56 °C and labile to 70 °C; (iv) nondialyzable and present in the 40K–100K dalton fraction of a G-200 Sephadex column; (v) labile to pH 2 treatment.     

Multiple expressions of the activity of guanine nucleotide regulatory protein in human thyroid adenylate cyclase

Adenylate cyclase (ATP pyrophosphate-lyase, EC 4.6.1.1) in plasma membranes from human thyroid was highly responsive to thyrotropin. Pretreatment of thyroid plasma membranes with 5′-guanylylimidodiphosphate (Gpp(NH)p) in the presence of Mg²⁺ led to a temperature-dependent activation, which was seen neither in the absence of Mg²⁺ nor at 4 °C. By contrast, thyrotropin bound to its receptors regardless of the temperature and produced its maximal effect after 2 min of preincubation in the absence or presence of Mg²⁺. Furthermore, activation was seen after treatment with thyrotropin and Gpp(NH)p even carried out in the absence of Mg²⁺ or at 4 °C. However, the full activation by Gpp(NH)p required Mg²⁺, hormone, and elevated temperature. These observations suggest that there appears to be two types of nucleotide interaction responsible for the Gpp(NH)p activation in human thyroid membrane; one type seen in the absence of hormone may represent the system uncoupled from hormone receptor, while the fully coupled hormone-sensitive adenylate cyclase accounts for the second type of interaction which requires the presence of hormone.     

Monoclonal antibodies directed against the sugar sequence of lacto-N-fucopentaose III are obtained from mice immunized with human tumors

Four hybridomas obtained from mice immunized with human adenocarcinomas of colon or stomach produce antibodies that bind specifically in solid-phase radioimmunoassay to the ceramide pentasaccharide that contains the lacto-N-fucopentaose III sequence of sugars. Binding of the antibodies to the glycolipid is inhibited by lacto-N-fucopentaose III,

but not by structurally related oligosaccharides. The antibodies bind to glycolipids of erythrocytes, granulocytes, and certain normal and malignant tissues.     

Measurement of DNA damage in unlabeled mammalian cells analyzed by alkaline elution and a fluorometric DNA assay

A fluorometric procedure is described that can be used in the alkaline elution technique for the measurement of DNA damage in cells whose DNA is not, or cannot be, radioactively labeled. The procedure can be used for the measurement of DNA single-strand breaks, DNA-protein crosslinking, and DNA interstrand crosslinking, and possibly other DNA lesions produced in unlabeled cells. Although developed for the measurement of DNA damage in tissue-cultured cells, the technique is applicable to the measurement of DNA damage in cells isolated from tissues exposed to DNA damaging agents in vivo.     

Occurrence of molybdenum in the nicotinic acid hydroxylase from Clostridium barkeri

Molybdenum, assayed by atomic absorption spectrometry, copurifies with the selenium-containing nicotinic acid hydroxylase from Clostridium barkeri. Fluorescence spectral studies on the enzyme indicate the presence, along with flavin, of another component. The fluorescence spectra of this component obtained after the aerobic denaturation of the nicotinic acid hydroxylase are similar to the fluorescence properties reported for the “pterin-like” cofactor from xanthine oxidase and several other molybdoproteins. Nicotinic acid hydroxylase from C. barkeri contains molybdenum, selenium, iron, acid-labile sulfur, and flavin with the occurrence of a “pterin-like” cofactor also a likely component.     

Increased expression of HLA-DR antigens in hydrocortisone-treated monocytes

Human peripheral blood monocytes incubated overnight with hydrocortisone had an increased expression of HLA-DR antigens. This change was noted as an increased proportion of DR-positive staining monocytes at greater fluorescence intensities as determined on a fluorescence-activated cell sorter. Hydrocortisone treatment of monocytes did not alter the expression of another Ia antigen on monocytes, HLA-DS. Neither did hydrocortisone treatment alter the expression of either Mac 120 antigen or monocyte .2 antigen on monocytes. Thus, the effect of hydrocortisone on monocyte DR antigens may be somewhat selective. Hydrocortisone also caused an increase in monocyte cell size aftr 3 to 4 days as compared to untreated controls.     

Antigen-specific human B-cell responses: modulation by immunoregulatory T-cell subsets

In vitro T-cell requirements for and modulation of human B-cell responses were studied in individuals immunized in vivo to the protein antigen keyhole limpet hemocyanin or tetanus toxoid. T cells were required for antibody synthesis in both antigen-driven and pokeweed mitogen (PWM)-driven cultures. T cells were separated into T4+ and T8+ subpopulations using monoclonal antibodies, and their modulation of antibody synthesis was studied. T4+ cells functioned as helper cells in both antigen-driven and PWM-driven cultures in a dose-dependent manner. Whereas T8+ cells suppress both total and specific immunoglobulin secretion in PWM-stimulated cultures, in antigen-stimulated cultures T8+ cells do not suppress unless activated by another cell population present in peripheral blood mononuclear cells (PBMNC). This cellular requirement was further investigated by prestimulation of cells prior to addition to optimally stimulated antigen-driven cultures of PBMNC or B cells, monocytes, and helper T cells. No suppression of these optimally stimulated cultures was seen when T8+ cells were precultured with antigen or PWM. However, after 3-5 days preculture of total T cells with PWM or antigen and then selection of T4+ cells, these cells were able to induce fresh autologous T8+ cells to suppress optimally stimulated antigen-driven cultures. Addition of a precultured mixture of T8+ cells with 20% T4+ cells also resulted in antigen-induced suppression. These data indicate that T8+ cells can suppress antigen-driven cultures but require the presence of preactivated T4+ cells for induction of this suppression of antigen-specific T-cell-dependent human B-cell responses.