New segment synthesis of alpha-inhibin-92 by the acyl disulfide method

The thiocarboxyl group reacts with diaryl disulfides to give an unsymmetrical acyl disulfide in dimethylformamide (DMF) and a symmetrical diacyl disulfide in aqueous DMF. Both acyl disulfides react with the alpha-amino group to form the peptide bond. The method was used in a new segment synthesis of alpha-inhibin-92 (alpha-IB-92) with use of 2,2'-dipyridyl disulfide as activator. Thiocarboxyl peptides were synthesized by the solid-phase method on 4-[alpha-(Boc-Gly-S)benzyl]phenoxyacetamidomethyl-resin. The segments alpha-IB-92-(1-34)SH (I), Msc-alpha-IB-92-(35-65)SH (II), Msc-alpha-IB-92(66-92)OH (III), and Msc-alpha-IB-92-(35-92)OH (VI) were prepared in yields of 33, 36, 41, and 25%, respectively, with use of crystalline symmetrical anhydrides in double and triple coupling protocols. Segments I, II, and III were used in a 3-segment synthesis of alpha-IB-92 with an overall yield based on starting resin of about 8% while a 2-segment synthesis using I and IV gave 11%. An all stepwise synthesis of alpha-IB-92 gave 4.5%.     

Lung hyperinflation in isolated dog lungs during high-frequency oscillation

To study the phenomenon of lung hyperinflation (LHI), i.e., an increase in lung volume without a concomitant rise in airway pressure, we measured lung volume changes in isolated dog lungs during high-frequency oscillation (HFO) with air, He, and SF6 and with mean tracheal pressure controlled at 2.5, 5.0, and 7.5 cmH2O. The tidal volume and frequency used were 1.5 ml/kg body wt and 20 Hz, respectively. LHI was observed during HFO in all cases except for a few trials with He. The degree of LHI was inversely related to mean tracheal pressure and varied directly with gas density. Maximum expiratory flow rate (Vmax) was measured during forced expiration induced by a vacuum source (-150 cmH2O) at the trachea. Vmax was consistently higher than the peak oscillatory flow rate (Vosc) during HFO, demonstrating that overall expiratory flow limitation did not cause LHI in isolated dog lungs. Asymmetry of inspiratory and expiratory impedances seems to be one cause of LHI, although other factors are involved.     

The receptors responsible for heat production in brown adipose tissue in the young rabbit

It is known that adrenergic agonists stimulate thermogenesis in the brown fat of the young rabbit but the receptors responsible for mediating the response have not been identified. The infusion of either noradrenaline or isoproterenol (1-2 micrograms . kg-1 X min-1) produced an increase in subcutaneous temperature (0.93 +/- 0.15 and 1.22 +/- 0.10 degrees C, respectively over the interscapular brown fat. At low doses (0.4 microgram . kg-1 X min-1) only isoproterenol was effective. The thermogenic response to isoproterenol was blocked by atenolol, a beta 1-adrenergic antagonist. Neither salbutamol or terbutaline, both beta 2-agonists, produced a temperature increase. Collectively, these data suggest that stimulation of beta 1-adrenoceptor is primarily responsible for the thermogenic activity of brown fat in the rabbit. However, it was found that 53% of the increase in temperature could be blocked by prazosin, an alpha 1-antagonist. Phentolamine was not effective as a blocker. Although a maximal brown fat thermogenic response can be achieved by stimulating the beta-adrenoceptors, the alpha-adrenoceptors appears to play at least an auxiliary role in young rabbit.     

The noradrenaline content and innervation of brown adipose tissue in the young rabbit

This work examined the noradrenaline content of brown adipose tissue, the metabolic response to endogenous noradrenaline released during tyramine infusion, and the innervation of brown fat at the electron microscopic level in the young rabbit. The noradrenaline content (ng/g) of the interscapular and cervical fat deposits ranged from 256 +/- 51 to 343 +/- 59 and 399 +/- 18 to 694 +/- 92, respectively, in four groups of rabbits (1-2, 7-8, 12-13, and 25-27 days of age). There was considerable variation amongst animals in each age group, but no evidence of a major increase or decrease in noradrenaline content during the first 4 weeks of life. Intravenous infusion of tyramine (100 micrograms X kg-1 X min-1) increased plasma noradrenaline concentration, oxygen consumption, and blood flow to brown fat. Thus noradrenaline released from endogenous sites, as well as injected noradrenaline, will initiate the thermogenic response of brown fat. Ultrastructurally, unmyelinated axons that were not organized in a fascicle were observed adjacent to the adipocytes in the late gestation fetus. By 1 week of age of axons were surrounded by Schwann cell cytoplasm which formed a fascicle. However, no evidence of myelination was found up to 21 days of age. Collectively, the data indicate that the brown adipocyte is fully responsive at 1-2 days of age even though myelination of the nerves is incomplete, and that the incomplete development of the sympathetic nerves at birth is not a factor in the synthesis of noradrenaline in the very young rabbit. In addition, brown fat of the newborn rabbit is not as thermogenically active as the brown fat of the cold-acclimated rat.     

Synthesis of a peptide with full somatostatin activity

A peptide has been synthesized according to the structure proposed for somatostatin by the solid phase method. The synthetic product was assayed and found to possess full somatostatin activity as compared with the natural material.     

Applications of fractal analysis to physiology

This review describes approaches to the analysis of fractal properties of physiological observations. Fractals are useful to describe the natural irregularity of physiological systems because their irregularity is not truly random and can be demonstrated to have spatial or temporal correlation. The concepts of fractal analysis are introduced from intuitive, visual, and mathematical perspectives. The regional heterogeneities of pulmonary and myocardial flows are discussed as applications of spatial fractal analysis, and methods for estimating a fractal dimension from physiological data are presented. Although the methods used for fractal analyses of physiological data are still under development and will require additional validation, they appear to have great potential for the study of physiology at scales of resolution ranging from the microcirculation to the intact organism.     

Kinetics of alpha 2-macroglobulin endocytosis and degradation in mutant and wild-type Chinese hamster ovary cells

The production of Chinese hamster ovary (CHO) cell mutants which are defective in endocytosis has led to a greater understanding of the process by which cells sort ligands and their receptors. Robbins and coworkers have obtained CHO mutants which are resistant to diphtheria toxin, defective in the delivery of endocytosed lysosomal enzymes to lysosomes, and have a decreased uptake of iron from transferrin (Robbins et al.: J. Cell Biol. 96:1064-1071, 1983). We have previously shown that these CHO mutants are markedly deficient in the acidification of early endocytic compartments (Yamashiro and Maxfield: J. Cell Biol. 105:2713-2721, 1987). In this study we examined the endocytosis of alpha 2-macroglobulin (alpha 2M) to determine whether the defects in early endosome acidification would alter the processing of this ligand. We found that the CHO mutants DTG 1-5-4 and DTF 1-5-1 bind, internalize, and degrade 125I-alpha 2M in a manner similar to the wild-type cells. We also found that the CHO mutants retain the ability to recycle the receptors for alpha 2M. Since the binding of alpha 2M is greatly reduced at mildly acidic pH (approximately 6.8), only slight acidification of the endosomal compartment should be sufficient to achieve sorting of alpha 2M from its receptor. In contrast, lysosomal enzymes require more acidic conditions (pH less than 6.0) for dissociation. The different behavior of the two ligands provides biochemical evidence for a partial (but not complete) defect in early endosome acidification in the mutants. The data also indicate that pH regulation in a relatively narrow range can achieve differential sorting of various ligands.     

Epstein-Barr virus infection induces expression in B lymphocytes of a novel gene encoding an evolutionarily conserved 55-kilodalton actin-bundling protein.

A novel human mRNA whose expression is induced over 200-fold in B lymphocytes by latent Epstein-Barr virus (EBV) infection was reverse transcribed, cloned, and sequenced. The mRNA is predicted to encode a protein containing four peptides which precisely match amino acid sequences from a previously identified 55-kDa actin-bundling protein, p55. In vitro translation of the cDNA results in a 55-kDa protein which binds to actin filaments in the presence of purified p55 from HeLa cells. The p55 mRNA is undetectable in non-EBV-infected B- and T-cell lines or in a myelomonocytic cell line (U937). Newly infected primary human B lymphocytes, EBV-transformed B-cell lines, latently infected Burkitt tumor cells expressing EBNA2 and LMP1, a chronic myelogenous leukemia cell line (K562), and an osteosarcoma cell line (TK143) contain high levels of p55 mRNA or protein. In EBV-transformed B cells, p55 localizes to perinuclear cytoplasm and to cell surface processes that resemble filopodia. The p55 mRNA is detected at high levels in spleen and brain tissues, at moderate levels in lung and placenta tissues, and at low levels in skeletal muscle, liver, and tonsil tissues and is undetectable in heart, kidney, pancreas, and bone marrow tissues. Immunohistochemical staining of human brain tissue demonstrates p55 localization to the perinuclear cytoplasm and dendritic processes of many, but not all, types of cortical or cerebellar neurons, to glial cells, and to capillary endothelial cells. In cultured primary rat neurons, p55 is distributed throughout the perinuclear cytoplasm and in subcortical filamentous structures of dendrites and growth cones. p55 is highly evolutionarily conserved since it shows 40% amino acid sequence identity to the Drosophila singed gene product and 37% identity to fascin, an echinoderm actin-bundling protein. The evolutionary conservation of p55 and its lack of extensive homology to other actin-binding proteins suggest that p55 has specific microfilament-associated functions in cells in which it is differentially expressed, including neural cells and EBV-transformed B lymphocytes.     

Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.