Synthesis of peptides by the solid-phase method. IV. Des-Arg(9)-bradykinin and analogs

We have synthesized a series of 19 analogs of the octapeptide fragment of bradykinin (BK), des-Arg 9-bradykinin, in order to perform a structure-activity study of this peptide on the newly discovered B1 receptor of bradykinin. The first time, each residue of the octapeptide was replaced by L-alanine to pinpoint biologically important residues. Thereafter, both phenylalanine residues in positions 5 and 8 were substituted by L-tyrosine methyl ether, L-cyclohexylalanine, D-phenylalanine, and L-leucine. This paper describes the synthesis of the analogs by the solid phase method. A Beckman peptide synthesizer was used to assemble the peptides on the resin support. Couplings were performed by the symmetrical anhydribe procedure. After cleavage with liquid HF, the peptides were purified by ion-exchange chromatography on carboxymethyl-cellulose and by gel filtration on Bio-Gel P2 resin. The purity of the octapeptides was then checked by tic, paper electrophoresis, amino acid analysis, and elemental analysis. The new peptides were tested on the rabbit aorta in order to evaluate their kinin-like activities and to see if they act as antagonist. The results of the biological assays are discussed in terms of structure-activity relationships.     

Molecular biology of PCSK9: its role in LDL metabolism

Proprotein convertase subtilisin-like kexin type 9 (PCSK9) is a newly discovered serine protease that destroys low density lipoprotein (LDL) receptors in liver and thereby controls the level of LDL in plasma. Mutations that increase PCSK9 activity cause hypercholesterolemia and coronary heart disease (CHD); mutations that inactivate PCSK9 have the opposite effect, lowering LDL levels and reducing CHD. Although the mechanism of PCSK9 action is not yet clear, the protease provides a new therapeutic target to lower plasma levels of LDL and prevent CHD.     

Localization and characterization of aminopeptidase P in bovine adrenal medulla.

Aminopeptidase P (EC 3.4.11.9) is demonstrated for the first time in the cytosolic fraction of chromaffin cells of the bovine adrenal medulla. The enzyme is inhibited by metal chelators and by sulfhydryl-reactive agents, which suggests that both a tightly bound metal ion and a cysteine residue are necessary for enzymatic activity. Aminopeptidase P might be important for the modulation of the biological activity of neuropeptides. Its occurrence in the adrenal chromaffin cells provides a useful tool for studying the function of this unique proline-specific peptidase in neuropeptide processing and secretion.     

Localization and ultrastructure of pulmonary dendritic cells during delayed-type hypersensitivity reactions in mice

Dendritic cells (DCs) are widely distributed in the airways and can serve as potent antigen-presenting cells. To clarify their involvement in the cell-mediated immune responses of the lung, we immunohistochemically investigated their distribution and kinetics during pulmonary delayed-type hypersensitivity (DTH) reactions induced in sensitized mice by intratracheal instillation of hapten. Cellular infiltrate appeared around the bronchiole and its accompanying blood vessel at 12 h after elicitation and progressively expanded by 48 h. As quantitated by computer-assisted morphometry, I-A(+) DCs and CD4(+) Th cells significantly increased in number around the bronchiole to a maximum at 24 h, whereas F4/80(+) macrophages were predominantly accumulated around the accompanying vessel with a peak at 48 h. Serial-section analysis revealed that DCs were colocalized with Th cells in the inflamed peribronchiolar tissue. Immunoelectron microscopy demonstrated that DCs found inside and around the capillaries and venules of peribronchiolar interstitium displayed round forms, indicating their emigration from here, while those situated far from the microvessels were elongated, often in close apposition to the lymphocytes. Mitosis of DCs was rarely seen. The present results suggest that peribronchiolar accumulation of DCs resulting from accelerated influx of blood-borne immature DCs and the interaction with T cells at the application site may play inducing roles in the development of pulmonary DTH reactions by enhancing the recruitment of macrophages.     

Depressor action of bradykinin agonists relative to metabolism by angiotensin-converting enzyme, carboxypeptidase N, and aminopeptidase P.

Bradykinin (BK) receptor agonists and antagonists contain modifications that confer resistance to specific peptidases. In control studies, rat plasma degraded BK (10.3 +/- 0.3 nmol/min/ml) via angiotensin-converting enzyme (ACE; EC 3.4.15.1; 5.2 +/- 0.3 nmol/min/ml), carboxypeptidase N (CPN; EC 3.4.17.3; 3.2 +/- 0.4 nmol/min/ml), aminopeptidase P (APP; EC 3.4.11.9; 0.6 +/- 0.2 nmol/min/ml), and other (unidentified) activity (2.1 +/- 0.6 nmol/min/ml). In contrast, BK agonist analogs were hydrolyzed more slowly due to selective resistance to these plasma peptidases. In addition to Lys-Lys-BK (B1087), which is partially resistant to ACE, [Hyp3,Phe8-r-Arg9]BK (B7642) was completely resistant to ACE, CPN, and the unidentified plasma activity (1.9 +/- 0.3 nmol/min/ml), and D-Arg0[Hyp3,Phe8-r-Arg9]BK (B7644) was resistant to all plasma hydrolysis, including APP (less than 0.2 nmol/min/ml). In vivo ACE-resistant B1087 exhibited a depressor potency and duration of action greater than BK and equivalent to that of BK in the presence of the ACE inhibitor enalapril. Although the B7642 and B7644 agonists were also more potent and longer acting than BK, the increases were no more than that seen for B1087, despite their additional resistance to CPN (B7642) and CPN and APP (B7644). The duration of action of these analogs was, however, increased after renal ligation. These data demonstrate the importance of ACE to the metabolism of circulating BK and BK analogs. In contrast, resistance to CPN and APP are not associated with further potentiation. Beyond ACE resistance, it is likely that the development of more potent, longer-acting BK agonists and antagonists will relate to other factors, such as renal processing independent of CPN and APP.     

Cerebral metabolism in male patients with schizophrenia who have seriously and dangerously violently offended: a 31P magnetic resonance spectroscopy study

There is biochemical evidence to suggest that membrane phospholipid metabolism may be impaired in some patients with schizophrenia. The aim of this study was to test the hypothesis that patients with schizophrenia who have violently offended while psychotic suffer from changes in cerebral phospholipid metabolism. Cerebral 31-phosphorus magnetic resonance spectroscopy was carried out in 15 male patients with schizophrenia who had violently offended (homicide, attempted murder, or wounding with intent to cause grievous bodily harm) while psychotic and in a control group of 13 age-matched healthy male control subjects. Spectra were obtained from 70x70x70mm(3) voxels in the brain using an image-selected in vivo spectroscopy pulse sequence. betaNTP was lower (P < 0.04) and gammaNTP was higher (P < 0.04) in the patient group compared with the normal control group. Our results are suggestive of increased cerebral energy metabolism taking place in the forensic patients.     

Creatine kinase isoenzyme BB in serum of patients undergoing chronic hemodialysis and with kidney transplant.

The activity of creatine kinase isoenzyme BB (CK-BB) was determined in serum of healthy adults and in patients undergoing maintenance hemodialysis and with kidney transplant. In the healthy adults examined, an activity of 0.56 +/- 0.16 U/l (mean +/- SD) was found. The arithmetic mean of CK-BB activity in patients with renal insufficiency under hemodialysis was 1.42 +/- 0.87 U/l and differed from that of the healthy group. The CK-BB activity in patients with kidney transplant was not different from that of the control group. The occurrence of CK-BB in serum is discussed from diagnostic and methodological point of view.     

Iron metabolism in Trypanosoma lewisi infection: serum iron and serum iron-binding capacity.

Progressive changes in iron levels, total iron binding capacity and hematocrit values in sera of rats infected with Trypanosoma lewisi are described. The host dietary group were: (1) complete or full complement; (2) iron-deficient, and (3) pair-fed or calorically restricted. The hematocrit values of T. lewisi-infected rats given the various diets were not significantly different from those of the controls. The decrease in total iron binding capacity (TIBC) of rats inoculated with T. lewisi and fed complete and pair-fed diets ranged up to 15% over uninfected controls. TIBC levels in rats fed an iron-deficient diet and inoculated with T. lewisi ranged up to 32% over uninfected controls. TIBC levels of deficient infected rats were significantly different from the controls from day 90 to infection to the end of the observation period. Serum iron (SI) values of non-infected rats regardless of dietary regimen showed significantly higher values than T. lewisi-infected animals between days 95 and 120. The average SI value, for this period, in adequately fed control rats was 204 +/- 7 microgram/100 ml as compared to 172 +/- 5 microgram/100 for trypanosome-infected rats. SI levels of rats on a pair-fed diet and infected with T. lewisi decreased to 17% over uninfected controls. SI levels of animals on an iron-deficient diet and infected with T. lewisi decreased up to 76% over uninfected controls.     

Studies on the role of mucus and mucosal hypersensitivity reactions during rejection of Trichostrongylus colubriformis from the intestine of immune sheep using an experimental challenge model.

Nematode-naive sheep and sheep immunised by truncated infections with Trichostrongylus colubriformis were fitted with intestinal cannulae to allow administration of challenge infection and collection of intestinal fluids. Sheep were slaughtered at various times after challenge and the distribution of larvae along the small intestine was determined. Results showed that immune sheep had significantly fewer larvae in their intestines and that some sheep could expel the challenge infection within 2 h. Mucus samples from immune sheep contained increased parasite-specific antibody, histamine and anti-parasite activity as measured by larval migration inhibition assay. Higher levels of antibody and histamine were seen in intestinal fluids of immune sheep after challenge. Immunisation of sheep by truncated infections stimulated serum IgE and resulted in significantly higher numbers of IgE-positive cells in gut tissue sections before challenge and at 2 h and 24 h after challenge. Immune sheep also had greater numbers of mucosal mast cells and globule leucocytes after challenge, compared with naive sheep. When challenge larvae were mixed with mucus from immune sheep and infused back into naive recipient sheep, there was a distinct displacement of the larval population towards the distal part of the intestine, compared with the profile of larval establishment after infusion with mucus from naive sheep. These results are further evidence for an immediate hypersensitivity reaction in the intestine of immune sheep, where challenge larvae are expelled within 2 h and confirm the direct anti-larval properties of mucus. The cannulated-sheep challenge model described here will be a useful tool to unravel the mechanism of larval rejection from immune sheep and could lead to novel therapies.     

Partial restoration by in vivo thymosin of E-rosettes and delayed-type hypersensitivity reactions in immunodeficient cancer patients

Summary Thymosin, fraction V, a partially purified extract of calf thymus, was administered to 14 patients with disseminated, far-advanced malignant disease. It increased the number of positive delayed-type hypersensitivity skin tests in 69% of the patients and the number of recall KLH responses in 85% of the subjects. Thymosin significantly increased T-lymphocyte rosettes in the group of patients with percentages originally less than 50%. Thymosin administration resulted in no toxic manifestations aside from local cutaneous hypersensitivity in one subject. Suggestive evidence of thymosin clinical activity was detected in three subjects.Thymosin's activity in these patients is compatible with and complementary to results obtained in vitro, in animals, and in children with certain primary immunodeficiency diseases. Thymosin may prove to be a major addition to the group of agents effective in the immunorestoration and immunotherapy of immunodeficient cancer patients.     

Correlation between serum osteoprotegerin and biomarkers of bone metabolism during anti-thyroid treatment in patients with Graves' disease

Bone turnover is increased in favor of resorption in hyperthyroid patients. We aimed to examine whether osteoprotegerin (OPG), which has an inhibitory effect on osteoclasts, is correlated with any biomarkers for bone turnover in Graves' disease. Twenty-one patients with Graves' disease were examined in this study, before and after treatment. Briefly, OPG, calcium, phosphorus, PTH, free T3, free T4, TSH, TSH receptor antibody and TSH-stimulating antibody were measured. Elevated serum OPG levels were decreased in accordance with anti-thyroid treatment. This change of OPG level was associated with thyroid hormone free T4 (r = 0.175, p = 0.038) but not with free T3 (r = 0.164, p = 0.052) and TSH (r = 0.046, p = 0.59). Additionally, there was a negative correlation between OPG and PTH (r = -0.37, p = 0.0001). In stepwise regression analysis, the change in serum OPG levels during anti-thyroid treatment was significantly and independently associated with PTH (F ratio = 24.4, p < 0.0001). Our findings suggest that OPG may prevent excessive bone loss in the hyperthyroid state in accordance with the change of biomarkers for bone turnover.