Large Lung Volumes Delay the Onset of the Physiological Breaking Point During Simulated Diving.

During breath holding after face immersion there develops an urge to breathe. The point that would initiate the termination of the breath hold, the "physiological breaking point," is thought to be primarily due to changes in blood gases. However, we theorized that other factors, such as lung volume, also contributes significantly to terminating breath holds during face immersion. Accordingly, nine naïve subjects (controls) and seven underwater hockey players (divers) voluntarily initiated face immersions in room temperature water at Total Lung Capacity (TLC) and Functional Residual Capacity (FRC) after pre-breathing air, 100% O2, 15% O2 / 85% N2, or 5% CO2 / 95% O2. Heart rate (HR), arterial blood pressure (BP), end-tidal CO2 (etCO2), and breath hold durations (BHD) were monitored during all face immersions. The decrease in HR and increase in BP were not significantly different at the two lung volumes, although the increase in BP was usually greater at FRC. BHD was significantly longer at TLC (54 ± 2 s) than at FRC (30 ± 2 s). Also, with each pre-breathed gas BHD was always longer at TLC. We found no consistent etCO2 at which the breath holding terminated. BDHs were significantly longer in divers than in controls. We suggest that during breath holding with face immersion high lung volume acts directly within the brainstem to actively delay the attainment of the physiological breaking point, rather than acting indirectly as a sink to produce a slower build-up of PCO2.

Effect of chronic autoimmune nerve growth factor deprivation on sympathetic neuroaxonal dystrophy in rats.

Nerve growth factor (NGF) deficiency has been proposed as a possible pathogenetic mechanism underlying the sympathetic autonomic neuropathy which develops in clinical and experimental diabetes and aging. To determine if long-term NGF deficiency alone would reproduce the distinctive sympathetic neuropathology of streptozocin-induced diabetes or aging in rats, nondiabetic animals were deprived of NGF for 12 months using an autoimmune paradigm. Neuroaxonal dystrophy (NAD), the neuropathologic hallmark of experimental sympathetic diabetic neuropathy and aging, was not increased in frequency in prevertebral superior mesenteric or paravertebral superior cervical ganglia in comparison to age-matched controls. Residual neurons in chronically NGF deprived sympathetic ganglia did not show significant atrophy, chromatolysis, active neuronal degeneration or intraganglionic debris. Postganglionic noradrenergic axons in ileal mesenteric nerves also failed to develop NAD in chronic autoimmune NGF-deprived rats as they would have in animals diabetic for the same duration. These results suggest that simple, isolated NGF deficiency maintained for long periods of time in nondiabetic animals is not sufficient to produce NAD in the pattern of experimental rat diabetes and aging.

Fine structure of presynaptic axonal terminals in sympathetic autonomic ganglia of aging and diabetic human subjects.

The neuropathologic changes that may underlie autonomic nervous system dysfunction in nondiabetic elderly human subjects or as a complication of diabetes have been systematically examined in sympathetic ganglia of a series of autopsied human subjects. As in animal models of aging and diabetes, enormously swollen terminal axons were found closely apposed to the perikarya of principal sympathetic neurons in prevertebral superior mesenteric sympathetic ganglia of aged and diabetic human subjects. Dystrophic axons consisted of two stereotyped forms: the first was composed of large numbers of misaligned aggregates of neurofilaments surrounded by variable numbers of small dense core vesicles; the second was characterized by large numbers of mitochondria, vacuoles, and dense and multivesicular bodies. The fine structural characteristics of neuroaxonal dystrophy, its predilection for prevertebral rather than paravertebral sympathetic ganglia, and the tendency for multiple dystrophic axons to cluster preferentially around selected neurons were identical in aged and diabetic human ganglia and were similar to changes seen in animal models of aging and diabetes. Neither diabetic nor aging ganglia demonstrated evidence of neuronal degeneration. Such structural changes may represent a degenerative influence of diabetes and aging on the normal remodeling of nerve terminals in autonomic ganglia, i.e., the continually ongoing process of turnover and replacement of axonal terminals. Similarity of lesions in human diabetes and aging suggests the possibility of pathogenetic mechanisms that are common to diabetes and the aging process. The substantial parallels between humans and animal models provide support for the validity of testing some proposed pathogenetic mechanisms directly in animal models.

Insulin bound to the insulin receptor of IM-9 lymphocytes is more accessible to antiinsulin antibodies after treatment with dithiothreitol: bound insulin is deeply buried in the receptor-binding site.

Binding of insulin to its receptor followed by covalent cross-linking with disuccinimidyl suberate (DSS) dramatically impairs the ability of antiinsulin antibodies (both polyclonal and monoclonal) to bind to the insulin moiety. We have used dithiotheitol, which has major effects on the oligomeric structure of the insulin receptor, to determine if this decreased antibody recognition is due to alteration in the conformation of insulin itself or to steric factors. Treatment of the covalently cross-linked insulin-receptor complex with dithiothreitol (DTT) increased the ability of the polyclonal and two monoclonal antiinsulin antibodies to immunoprecipitate the insulin-receptor complex. This treatment decreased immunoprecipitation by antireceptor antibodies. The effect of DTT may have been due to a reversal of either a binding-induced conformational change in the insulin moiety or an alteration in the conformation of the insulin-receptor complex, thereby decreasing steric hindrance. In an effort to choose between these two possible explanations, we prepared a biotinylated derivative of insulin which was cross-linked to the receptor. Since the biotin moiety is relatively rigid, it seemed improbable that binding to the receptor would alter the conformation of the epitope recognized by antibiotin antibodies and that the change would be reversed by DTT. Treatment of the cross-linked biotin-insulin-receptor complex with DTT did increase the ability of both antiinsulin and antibiotin antibodies to immunoprecipitate the cross-linked receptor complex. Identification of the cross-linked receptor on reduced sodium dodecyl sulfate-polyacrylamide gels confirms that the DTT treatment alters the distribution of the oligomeric forms of the receptor. These studies favor the hypothesis that when bound to its receptor, most of the insulin molecule is sequestered within the receptor-binding site such that there is steric hindrance to the approach of antiinsulin antibodies. Moreover, DTT alters the conformation of the cross-linked insulin-receptor complex so as to decrease this steric hindrance.

Binding of insulin to its receptor impairs recognition by monoclonal anti-insulin antibodies.

The interaction between insulin and its receptor was investigated using both monoclonal and polyclonal anti-insulin antibodies. After covalent cross-linking of 125I-insulin to the insulin receptor on cultured human lymphocytes (IM-9 cells) using disuccinimidyl suberate, we inquired whether the insulin-receptor complex could be immunoprecipitated with anti-insulin antibodies. While a polyclonal guinea pig anti-insulin antiserum succeeded in immunoprecipitating receptor-bound 125I-insulin, binding to the receptor decreased the avidity of the antiserum for the insulin moiety by a factor of approximately 1000-fold. Sixteen distinct monoclonal murine anti-insulin antibodies were employed to immunoprecipitate receptor-bound 125I-insulin. Of these 16 monoclonal antibodies, only one (antibody 5.9F4) could be shown to recognize receptor-bound 125I-insulin. Moreover, even with antibody 5.9F4, binding of 125I-insulin to its receptor reduced the affinity of the antibody by a factor of 10- to 100-fold. These data strongly suggest that, when insulin binds to its receptor, the majority of the insulin molecule is unavailable for binding by anti-insulin antibodies. It seems likely that the hormone binding site on the receptor may be very large, thereby allowing for sequestration of the majority of the insulin molecule with relatively little of the hormone remaining exposed.

Fine specificity of cloned insulin-specific T cell hybridomas: evidence supporting a role for tertiary conformation.

Two groups of cloned insulin-specific T cell hybridomas were derived from the fusion of (BALB/c X A/J)F1 T cells with the BW5147 tumor line. Group I responded only to the immunogen pork insulin and failed to respond to rat insulin, which differs only at amino acids A4 and B3. The second group of T cell hybridomas exhibited a broader pattern of reactivity to heterologous insulins because they could be stimulated with the B chain-identical pork, beef, sheep, and human insulins, as well as by isolated oxidized B chain. These hybridomas, however, displayed a much greater reactivity to pork insulin than to beef insulin or to the oxidized B chain. Comparison of the reactivity profiles of these two groups of hybridomas with the three-dimensional structure of the insulin molecule strongly suggested that group I cells were recognizing an immunogenic moiety composed of residues A4 and/or B3 in association with A chain loop determinants and that group II hybrids recognized a moiety composed of the amino acid sequence of the B chain and the A chain loop. Both groups of hybrids were restricted to the high-responder I-Ad allele but could be distinguished by their pattern of alloreactivity. We were unable to show any effect of a panel of monoclonal anti-insulin antibodies on the induction of interleukin 2 secretion by these hybridomas or to demonstrate the presence of idiotypic determinants on these T hybrids by using a panel of anti-idiotypic reagents raised against the monoclonal anti-insulin antibodies.

Mapping epitopes on the insulin molecule using monoclonal antibodies.

A panel of 18 monoclonal antibodies (mAb) delta to insulin have been prepared and used to begin to map antigenic determinants on the insulin molecule. All 18 mAb were of the IgG class, with 14 IgG1, 2 IgG2a and 2 IgG2b. The affinities of these mAb for their immunizing insulin ranged from 1 X 10(6) to 3 X 10(8) 1/M. The epitope recognized by three of the mAb, 1, 7 and 16 involves the three residues of the A chain, A 8-10, the so called A chain-loop determinant. This A chain loop is one of the most evolutionarily diverse regions of insulins from different species. Another mAb, 10, has been hypothesized to recognize a nearby epitope composed of the A chain residues, A4 and A8 and a B chain residue, B29, that are adjacent on the surface of the insulin molecule. Four of the mAb bind to synthetic B chain. The epitopes recognized by these 4 mAb and the last 10 mAb are unknown but the mAb are grouped according to their ability to bind to different species of insulin or proinsulin. The results of an 18 X 18 matrix analysis of pairs of mAb binding simultaneously to insulin indicate that, despite the finding that some mAb see similar antigenic sites on the insulin molecule, each of the mAb recognizes a unique site on the insulin molecule. Finally, a lower estimate of the number of possible antibodies made to insulin has been calculated to be greater than or equal to 115, a number only 10-fold lower than the lower limit of antibodies made to dinitrophenyl (DNP) or (4-hydroxy-5-iodo-3-nitrophenyl)acetyl (NIP), following hapten protein immunization.

Characterization of a monoclonal antibody that defines an immunoregulatory T cell subset for immunoglobulin synthesis in humans.

This study characterizes a monoclonal antibody (3A1), and partially characterizes the cell surface antigen and the functional peripheral blood T cell subset that it defines. The 3A1 antigen is present on the surface of several human T cell lines (HSB-2, CEM, MOLT-4, and others) in various amounts but is absent from the T cell line YT4E and all human B cell lines tested. Immunoprecipitation of an HSB-2 extract with 3A1 yielded one specific band with a molecular weight of approximately 40,000 in the presence of reducing agent. With directly fluoresceinated 3A1 antibody, fluorescence-activated cell sorter analysis showed that 85% of peripheral blood E-rosette-positive T cells were positive for the 3A1 antigen. After E-rosette-positive cells had been separated into 3A1+ and 3A1- cell suspensions, the 3A1+ cells helped autologous peripheral blood B cell suspensions toward pokeweed mitogen-driven proliferation and intracytoplasmic Ig production, whereas 3A1-T cells did not. Further, addition of 3A1- cells from some but not all normal subjects to cocultures of 3A1+ cells and B cells actively suppressed intracytoplasmic Ig production. However, the 3A1+T cell subset could be activated by concanavalin A to maximally suppress B cell Ig synthesis in vitro. Thus, the 3A1 antibody defines a major functional subject of peripheral blood T cells and should provide a useful marker for the study of human T cell function.

Isoelectric focusing of immunoglobulins: improved methodology.

Conditions for isoelectric focusing and detecting antibodies in thin layers of polyacrylamide have been evaluated and several improvements have been made. First, we have developed a simple method for covalently attaching the polyacrylamide gel to the glass support which improves the mechanical stability of the gel and removes the need for protein subbed plates. This in turn leads to decreased electroendosmosis and decreased background protein staining. Secondly, we have applied the methods of Nguyen and Chrambach (1977 a,b), in which amino acid solutions are used as electrodes, to focus immunoglobulins. This has eliminated cathodic drift and resulted in extremely stable pH gradients. Finally, we have found that conventional methods for detecting focused antibodies that rely on protein cross-linking before exposure to antigen often lead to distortion of the focusing patterns. Both glutaraldehyde and suberimidate destroy the antigen binding capacity of some antibodies at concentrations too low for complete fixation of protein bands. These fixation artifacts are avoided if salt-precipitated antibodies are exposed to radiolabeled antigen before fixation.

The identification of bile acid methyl esters by gas chromatography methane chemical ionization mass spectrometry.

Methane chemical ionization mass spectrometric data and gas chromatography retention parameters are reported for 31 bile acid methyl esters. The group includes both saturated and unsaturated compounds with up to three keto or hydroxyl groups. Major ions include the quasimolecular ions and fragments resulting from the loss of neutral species such as methanol, water and methane. The combination of gas-liquid chromatography retention and mass spectral data is sufficient for an unequivocal distinction among all the bile acids studied. Application of this data to the identification of bile acid metabolites is cited.