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%.     

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.     

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.     

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.     

Distribution of various nickel compounds in rat organs after oral administration

In this study, eight kinds of nickel (Ni) compounds were orally administered to Wistar male rats and the distribution of each compound was investigated 24 h after the administration. The Ni compounds used in this experiment were nickel metal [Ni−M], nickel oxide (green) [NiO(G)], nickel oxide (black) [NiO(B)], nickel subsulfide [Ni₃S₂], nickel sulfide [NiS], nickel sulfate [NiSO₄], nickel chloride [NiCl₂], and nickel nitrate [Ni(NO₃)₂]. The solubilities of the nickel compounds in saline solution were in the following order; [Ni(NO₃)₂>NiCl₂>NiSO₄]≫[NiS>Ni₃S₂]>[NiO(B)>Ni−M>NiO(G)]. The Ni level in the visceral organs was higher in the rats given soluble Ni compounds; Ni(NO₃)₂, NiCl₂, NiSO₄, than that in the rats receiving other compounds. In the rats to which soluble Ni compounds were administered, 80–90% of the recovered Ni amounts in the examined organs was detected in the kidneys. On the other hand, the Ni concentration in organs administered scarcely soluble Ni compounds; NiO(B), NiO(G), and Ni−M were very low. The estimated absorbed fraction of each Ni compounds was increased with the increase of the solubility. These results suggest that the kinetic behavior of Ni compounds administered orally is closely related with the solubility of Ni compounds, and that the solubility of Ni compounds is one of the important factors for determining the health effect of Ni compounds.     

Synthesis of shrimp red pigment-concentrating hormone analogs and their biological activity in locusts

Two analogs of the red pigment-concentrating hormone (RPCH) have been synthesized by the solid-phase method: [Thr6]-RPCH (I) and [Tyr4, Thr6]-RCPH (II). Analog I has the same amino acid composition as the second adipokinetic hormone (AKH-II) isolated from locust corpora cardiaca. Bioassay for lipid-mobilizing activity in adult male locusts gave the following increases in hemolymph lipid content: AKH-I, 3.5; I, 2.4; II, 2.9. The biological response shown by I lends support to the conclusion that its sequence is that of the presumptive AKH-II. Replacement of Phe in position 4 by Tyr does not reduce the adipokinetic response.     

beta-Endorphin: structure-activity relationships in the guinea pig ileum and opiate receptor binding assays.

The opiate activities of some derivatives and enzymatic digests of camel and human β-endorphin were determined in the guinea pig ileum and rat brain opiate receptor binding assays. Derivatives of β-endorphins altered within the amino-terminal five residues showed pronounced losses in activity. Anisylation of the C-terminal glutamic acid residue of β_h-endorphin produced only small reductions in activity. Chymotryptic digestion greatly weakened the opiate activities of β_h-endorphin, whereas carboxypeptidase A, tryptic and leucine aminopeptidase digests showed only small losses in potency. The C-terminus of β-endorphin appears to contribute little directly to opiate activity. Amino acid analysis and assay of the leucine aminopeptidase digests suggest that the larger potency of β-endorphin relative to Met-enkephalin may be a consequence of its greater resistance to exopeptidase attack.     

Ligand-dependent reactivity of (Na+ + K+)-ATPase with showdomycin

Showdomycin inhibited pig brain ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ with pseudo first-order kinetics. The rate of inhibition by showdomycin was examined in the presence of 16 combinations of four ligands, i.e., Na⁺, K⁺, Mg²⁺ and ATP, and was found to depend on the ligands added. Combinations of ligands were divided into five groups in terms of the magnitude of the rate constant; in the order of decreasing rate constants these were: (1)$\text{Na}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{+}\text{ATP}$, (2) $\text{Mg}{}^{\mathrm{2+}}$, $\text{Mg}{}^{\mathrm{2+}}\text{+}\text{K}{}^{\mathrm{+}}$, $\text{K}{}^{\mathrm{+}}$ and none, (3) $\text{Na}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{,}\text{Na}{}^{\mathrm{+}}$, $\text{K}{}^{\mathrm{+}}\text{+}\text{Na}{}^{\mathrm{+}}$ and $\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}$, (4) $\text{Mg}{}^{\mathrm{2+}}\text{+}\text{K}{}^{\mathrm{+}}\text{+}\text{ATP}\text{,}\text{K}{}^{\mathrm{+}}\text{+}\text{ATP}$ and $\text{Mg}{}^{\mathrm{2+}}\text{+}\text{ATP}\text{, (5)}\text{K}{}^{\mathrm{+}}\text{+}\text{Na}{}^{\mathrm{+}}\text{+}\text{ATP}$, $\text{Na}{}^{\mathrm{+}}\text{+}\text{ATP}$, $\text{Na}{}^{\mathrm{+}}\text{+}\text{ATP}$, $\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{+}\text{Mg}{}^{\mathrm{2+}}\text{+}\text{ATP}$ and ATP. The highest rate was obtained in the presence of Na⁺, Mg²⁺ and ATP. The apparent concentrations of Na⁺, Mg²⁺ and ATP for half-maximum stimulation of inhibition ($\text{K}{}_{0.5}{}^{\mathrm{<mtext>s</mtext>}}$) were 3 mM, 0.13 mM and 4μM, respectively. The rate was unchanged upon further increase in Na⁺ concentration from 140 to 1000 mM. The rates of inhibition could be explained on the basis of the enzyme forms present, including E₁, E₂, ES, E₁-P and E₂-P, i.e., E₂ has higher reactivity with showdomycin than E₁, while E₂-P has almost the same reactivity as E₁-P. We conclude that the reaction of ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ proceeds via at least four kinds of enzyme form (E₁, E₂, E₁ · nucleotide and EP), which all have different conformations.     

Total work rate of breathing optimization in CO-2 inhalation and exercise.

The hypothesis that respiratory frequency and the relative durations of inspiration and expiration are regulated according to a total cycle work rate minimization criterion was explored. Effects of negative work performed by the respiratory muscles and dead space variation as a function of tidal volume were included in a formulation which yielded a theoretically predictable optimal frequency and relative duration of inspiration and expiration at all levels of ventilation. Predicted cycle characteristics based on measured mechanical parameters were compared with data taken during CO-2 inhalation (3 and 5%) and moderate exercise (MRR = 3 and 6) in three normal human subjects. No major difference in breathing pattern was observed between CO-2 inhalation and exercise. Results suggest that conditions for minimization of total cycle work rate are achieved asympototically as the level of ventilation rises above the resting level. At rest and at low levels of hyperpnea complete work rate optimization is not achieved.