Mu- vs. delta-opioid influence on respiratory and sleep behavior during development.

The role of mu- vs. delta-opioid receptors in modulating cardiorespiratory and sleep/wake behavior was studied in sixteen 4- to 11-, and 26- to 33-day-old chronically instrumented piglets. Each underwent 1.

Exogenous antigens internalized through transferrin receptors activate CD4+ T cells.

The role of endosomes in exogenous Ag processing was investigated by targeting Ag into the endosomal transport pathway via transferrin receptors. The Ag, pigeon cytochrome c and chicken OVA, were coupled to human ferric transferrin by a heteroligation technique. The conjugates were significantly more efficient than native Ag in stimulating Ag-specific CD4+ T cells, when the APC expressed transferrin receptors.

N-isopropyliodoacetamide in the reduction and alkylation of proteins: use in microsequence analysis.

A new reagent, N-isopropyliodoacetamide (NIPIA), for alkylation of sulfhydryl groups on proteins for microdigestion and microsequencing is described. The utility of this reagent in both of these procedures has been demonstrated. NIPIA was shown to be especially useful in microsequence analysis, where it yields high sensitivity in detection of Cys residues.

Role of antigen-specific T cell help in the generation of in vivo antibody responses. I. Antigen-specific T cell help is required to generate a polyclonal IgG1 response in anti-IgD antibody-injected mice.

A system in which injection of mice with an antibody to mouse IgD that they recognize as foreign stimulates a large, T cell-dependent IgG response was used to study whether Ag-specific T cell help is required to stimulate polyclonal (non-Ag-specific) IgG production in vivo. Igha x Ighb allotype heterozygous mice were injected with a conjugate of a foreign Ag coupled to a mAb specific for one of the two IgD allotypes expressed in these mice. This conjugate cross-links mIgD on B cells that express the recognized allotype.

Failure of signaling through a chimeric class I-immunoglobulin molecule expressed on the surface of transfected B lymphoma cells and cells of transgenic mice.

To test the possibility that the crosslinkage of molecules expressing a transmembrane region derived from the membrane form of the mu immunoglobulin heavy chain would be sufficient for signal transduction in B cells, a chimeric gene (Kk-mu) consisting of extracellular exons of the class I gene H-2Kk and the transmembrane and cytosolic exons of the mu constant region gene was introduced into WEHI-231 B lymphoma cells and into mouse blastocysts. A protein consistent with the predicted product of the Kk-mu gene was expressed in a transfected cell clone (S18) and in transgenic mice. Crosslinkage of Kk-mu protein with soluble, Sepharose-bound, or dextran-conjugated anti-H-2Kk antibodies failed to induce the accumulation of inositol phosphates or to elevate intracellular calcium concentrations in either S18 cells or B lymphocytes from transgenic mice.

Membrane IgM-mediated signaling of human B cells. Effect of increased ligand binding site valency on the affinity and concentration requirements for inducing diverse stages of activation.

The potential for ligand-initiated signal transduction through B cell membrane IgM is assessed in terms of ligand concentration, binding site valency, and binding site affinity for membrane Ig. Estimates of the physicochemical requirements for achieving G0* enhancement of class II MHC expression, G1 entry, and S phase entry in human B cells were made by comparing the stimulatory effects of three affinity-diverse anti-Cmu2 mAb when in bivalent (unconjugated) form, or as mAb-dextran conjugates with low binding site valency (oligovalent ligands) or high binding site valency (multivalent ligands). An increase in binding site number (and concomitant molecular mass) caused a profound reduction in both the minimal concentration and affinity requisites for B cell activation.

Biochemical analysis of the immune B cell defect in xid mice.

Previous studies have shown that B cells from xid immune defective CBA/N mice that are unresponsive do not proliferate after stimulation with unconjugated anti-Ig. The experiments in this manuscript demonstrate that dextran-anti-Ig conjugates, which induce extensive and prolonged sIg cross-linking, are able to stimulate proliferation of xid B cells. The ability of these conjugates to stimulate proliferation of xid B cells is not related to their ability to stimulate higher levels of PIP2 breakdown.

Synthesis of N alpha-(tert-butoxycarbonyl)-N epsilon-[N-(bromoacetyl)-beta-alanyl]-L-lysine: its use in peptide synthesis for placing a bromoacetyl cross-linking function at any desired sequence position.

A new amino acid derivative, N alpha-(tert-butoxycarbonyl)-N epsilon-[N-(bromoacetyl)-beta-alanyl]-L-lysine (BBAL), has been synthesized as a reagent to be used in solid-phase peptide synthesis for introducing a side-chain bromoacetyl group at any desired position in a peptide sequence. The bromoacetyl group subsequently serves as a sulfhydryl-selective cross-linking function for the preparation of cyclic peptides, peptide conjugates, and polymers. BBAL is synthesized by condensation of N-bromoacetyl-beta-alanine with N alpha-Boc-L-lysine and is a white powder which is readily stored, weighed, and used with a peptide synthesizer, programmed for N alpha-Boc amino acid derivatives.

Occlusion pressure response to inspiratory flow-resistive loading in anesthetized swine.

Conscious animals subjected to inspiratory flow-resistive loading augment respiratory drive [as measured by airway occlusion pressure (P100)] independently of changes in chemical drive. Past studies of anesthetized subjects, however, have failed to demonstrate this response, and investigators have concluded that its presence depends on a state of consciousness. We tested the hypothesis that respiratory depression due to anesthesia or endogenous opioids rather than unconsciousness per se was responsible for this observation.

Effects of pentobarbital on proopiomelanocortin opioid products of neonatal piglets during normoxia and hypoxia.

Abstract Endogenous opioids have been shown to suppress physiological functions in the neonate. It has been suggested that anesthesia with barbiturates might enhance this suppression by influencing opioid systems directly. To explore this possibility, naive piglets, 2.

Modulation by mu opioid antagonism of sleep and respiration in neonatal swine.

Young (3-13 days) and older (26-34 days) piglets were instrumented aseptically for chronic recording of sleep/wake states (biparietal electrocorticogram, horizontal and vertical electrooculogram, submental muscle electromyogram (EMG)), heart rate, arterial pressure, pH and gas tensions, posterior cricoarytenoid and diaphragmatic EMG (EMGpca, EMGdi). After recovery from surgery, piglets underwent 1 h daily recordings for 5 consecutive days. Experimental sessions comprised control periods followed by study periods with CTOP (10-40 micrograms/kg i.

Respiratory responses of piglets to hypercapnia during postnatal development: effects of opioids.

Resting respiratory and cardiovascular functions and the response to CO2 rebreathing were compared between 2.5 +/- 0.7 (mean +/- SE) and 34.

Rapid stimulation of large specific antibody responses with conjugates of antigen and anti-IgD antibody.

Injection of mice with goat anti-mouse IgD antibody stimulates a large IgG1 anti-goat IgG antibody response, as well as polyclonal IgG1 production. To determine if this phenomenon could be used to induce large antibody responses to other Ag, covalent conjugates were produced between BSA or other Ag and H delta a/1, a mAb specific for IgD of the a allotype, and between BSA and AF3.33, a mAb specific for IgD of the b allotype.

Ontogeny of plasma, CSF and brainstem ACTH in piglets: effects of hypoxia and anesthesia.

This study was designed to assess the postnatal maturation of adrenocorticotropin (ACTH) levels in piglets under basal conditions and in response to single, acute stressors. ACTH levels were measured by radioimmunoassay in plasma, cerebrospinal fluid (CSF) and in a dorsal medullary slice containing the nucleus tractus solitarii (dmscNTS) of young and older piglets (1.5-6 and 35-43 days old, respectively) under the following experimental conditions: (1) normoxia (both groups); (2) hypoxia, 10% O2/N2 for greater than or equal to 30 min (both groups); (3) sham anesthesia, i.

Ontogeny of sleep/wake and cardiorespiratory behavior in unanesthetized piglets.

Young and older piglets (2-15, 25-35 days old) underwent chronic recording of electrocorticogram, vertical and horizontal electrooculograms, electromyograms of submental muscles, diaphragm (EMGdi) and posterior cricoarytenoid (EMGpca), and heart rate, arterial pressure, pH and gas tensions. With age, (1) the distribution of percent time spent in various sleep-wake states differed; (2) heart rate decreased in all S/W, arterial pressure increased in wakefulness (W), transitional sleep (TS) and quiet sleep (QS); (3) respiratory frequency decreased, EMGdi and EMGpca duration and EMGpca amplitude increased in all S/W, EMGdi amplitude decreased in TS and QS and rate of rise of EMGdi and EMGpca decreased in W, TS and QS. Active sleep was characterized by smaller normalized EMGpca amplitudes in the young, short EMGpca to EMGdi intervals in both ages and predominance of prolonged diminished muscle activity (DMA) of either muscle.

A deeply recessed active site in angiotensin-converting enzyme is indicated from the binding characteristics of biotin-spacer-inhibitor reagents.

Two biotinylated derivatives of the angiotensin-converting enzyme (ACE) inhibitor, lisinopril, were synthesized. Compounds BL11 (epsilon-biotinamidocaproyl-lisinopril) and BL19 (epsilon-biotinamidocaproyl-beta-alanyl-beta-alanyl-lisinopril) have, respectively, 11 and 19 atoms of spacing structure between the biotin and the inhibitor moieties. Both compounds were found to be potent inhibitors of mouse kidney ACE, but they lost this ability in the presence of streptavidin in free solution.

Surface immunoglobulin-mediated B-cell activation in the absence of detectable elevations in intracellular ionized calcium: a model for T-cell-independent B-cell activation.

We recently showed that anti-immunoglobulin conjugated to high molecular weight dextran is 1000-fold more mitogenic for B cells than unconjugated anti-immunoglobulin. This system serves as a model for T-cell-independent type 2 antigens such as haptenated Ficoll, dextran, and bacterial polysaccharides, which can also stimulate B-cell proliferation and antibody production at low concentrations. We show here that conjugated anti-immunoglobulin, at concentrations that stimulate significant increases in expression of major histocompatibility complex class II molecules and incorporation of thymidine into DNA, does not induce detectable modulation of surface immunoglobulin.

Neurochemicals and respiratory control during development.

During ontogeny, the central nervous system undergoes neuronal growth, regression, and remodeling. The development of neurotransmitter and modulator systems is a plastic process with individual temporal characteristics for each system. These characteristics include the synthesis, degradation, or uptake of neurochemicals and, largely independently, the appearance of their receptors.

Proopiomelanocortin opioids in brain, CSF, and plasma of piglets during hypoxia.

beta-Endorphin-like immunoreactivity (BELI), containing the biologically active beta-endorphin, its precursor beta-lipotropin (BLP), and deactivated product N-acetyl-beta-endorphin (ABE), were measured by radioimmunoassay in plasma, cerebrospinal fluid (CSF), and in a dorsal medullary slice containing the respiratory-related nucleus tractus solitarius (dmscNTS) of young and older piglets in normoxia and hypoxia. Significant increase with hypoxia occurred in the levels of BLP in the plasma and CSF and of BELI and ABE in the plasma of the young group. In the older group, such increases occurred in ABE levels of the dmscNTS, in BLP and ABE levels of the CSF, and in plasma BELI.

Posttranslational processing of opioid pro-opiomelanocortin products in piglets.

We have measured levels of beta-lipotropin, beta-endorphin, and N-acetyl-beta-endorphin in the plasma, cerebrospinal fluid (CSF), and caudal medullary brain containing the respiratory-related portion of the nucleus tractus solitarius (NTS) of 2.5 +/- 1.0- (SD) and 38.

Two-dimensional electrophoretic analysis of human milk proteins.

Identification of human milk proteins and formulation of a two-dimensional map is a first step in a project which intends to survey human milk proteins by two-dimensional electrophoresis. Thirty-four proteins have been identified using the Iso-Dalt method of separation and Western blot with immunoprobes. Identification confirms that milk is species-specific, and, therefore, breast feeding confers a decided advantage for the infant.

T cell activation by purified, soluble, class I MHC molecules. Requirement for polyvalency.

To examine the nature of the interaction of the TCR with the MHC class I Ag, we have studied the stimulation requirements of an H-2Dd-reactive T cell hybridoma, using a homogeneous, purified preparation of a molecularly engineered soluble counterpart of the class I Ag, H-2Dd/Q10b. We demonstrate that this monovalent, soluble MHC Ag is incapable of stimulating the release of IL-2 from this T cell hybridoma. However, the same preparation of the purified protein can elicit a dose-dependent response when made multivalent either by covalent coupling to soluble, high m.

Picogram quantities of anti-Ig antibodies coupled to dextran induce B cell proliferation.

To investigate the properties which enable type 2 Ag, as exemplified by dextran and Ficoll, to stimulate high levels of antibody responses in the relative absence of T cells, we conjugated anti-IgD and anti-IgM mAb to both dextran and Ficoll and examined their B cell-activating properties. Such conjugated anti-Ig antibodies stimulated both early and later stages of B cell activation at picogram concentrations, which are at least 1000-fold lower than that required for B cell stimulation by unconjugated anti-Ig antibodies, and the level of proliferation they stimulated was on average 10-fold greater. Furthermore, concentrations of anti-Ig dextran (100 pg/ml) which modulated little sIgD from the B cell surface were strong inducers of enhanced B cell expression of MHC class II molecules.

Controlled primary functionalization of agarose affinity supports by carboxymethylation and subsequent addition of spacer units.

Experimental details are given for the O-carboxymethylation of cross-linked agarose beads with chloroacetate in aqueous sodium hydroxide, formation of a tandem 2-amino-ethylamide derivative, and coupling of beta-alanine residues to the aminoethyl groups using a new reagent, N-phthalimidyl N'-ethylsulfonylethoxycarbonyl-beta-alaninate. An ideal support for attaching affinity ligands results. Extent of derivatization was controlled by the time and temperature of the first reaction.