S-adenosyl-L-methionine (SAMe) halts the autoimmune response in patients with primary biliary cholangitis (PBC) via antioxidant and S-glutathionylation processes in cholangiocytes.

S-adenosyl-L-methionine is an endogenous molecule with hepato-protective properties linked to redox regulation and methylation. Here, the potential therapeutic value of SAMe was tested in 17 patients with PBC, a cholestatic disease with autoimmune phenomena targeting small bile ducts. Nine patients responded to SAMe (SAMe responders) with increased serum protein S-glutathionylation. That posttranslational protein modification was associated with reduction of serum anti-mitochondrial autoantibodies (AMA-M2) titers and improvement of liver biochemistry. Clinically, SAMe responders were younger at diagnosis, had longer duration of the disease and lower level of serum S-glutathionylated proteins at entry. SAMe treatment was associated with negative correlation between protein S-glutathionylation and TNFα. Furthermore, AMA-M2 titers correlated positively with INFγ and FGF-19 while negatively with TGFß. Additionally, cirrhotic PBC livers showed reduced levels of glutathionylated proteins, glutaredoxine-1 (Grx-1) and GSH synthase (GS). The effect of SAMe was also analyzed in vitro. In human cholangiocytes overexpressing miR-506, which induces PBC-like features, SAMe increased total protein S-glutathionylation and the level of γ-glutamylcysteine ligase (GCLC), whereas reduced Grx-1 level. Moreover, SAMe protected primary human cholangiocytes against mitochondrial oxidative stress induced by tBHQ (tert-Butylhydroquinone) via raising the level of Nrf2 and HO-1. Finally, SAMe reduced apoptosis (cleaved-caspase3) and PDC-E2 (antigen responsible of the AMA-M2) induced experimentally by glycochenodeoxycholic acid (GCDC). These data suggest that SAMe may inhibit autoimmune events in patients with PBC via its antioxidant and S-glutathionylation properties. These findings provide new insights into the molecular events promoting progression of PBC and suggest potential therapeutic application of SAMe in PBC.

The effect of short term treatment with probiotic VSL#3 on various clinical and biochemical parameters in patients with liver cirrhosis.

The evidence is mounting that alterations of innate immunity and gut microbiota contribute to chronic liver disease and its complications. Modulation of intestinal microbiota is an emerging therapeutic strategy in hepatology. Probiotics through modulation of intestinal milieu have the potential to affect the course of liver disease. The data concerning the influence of probiotics on various plasma molecules and compounds involved in the pathogenesis of hyperdynamic circulatory state in liver cirrhosis is still not confluent and require further evaluation. In our study twenty patients with compensated and decompensated liver cirrhosis and ten healthy controls received probiotic VSL#3 daily for 28 days. Plasma levels of interleukin 6 (IL-6), vascular endothelial growth factor (VEGF), plasminogen activator inhibitor (PAI), macrophage inflammatory protein 3/α (MIP-3 α/CCL20), monocyte chemotactic protein-1α (MCP-1/CCL2), human myeloperoxidase (MPO), nitric oxide (NO), prostaglandins, thromboxane (TXB2) and big-endothelin were measured at baseline, day 14 and 28 of probiotic administration. The incidence of hepatic encephalopathy was assessed with critical flicker frequency. Changes in clinical, biochemical and microbiological parameters were evaluated. The stage of liver cirrhosis correlated with an increase in plasma levels of pro-inflammatory cytokines (IL-6) and chemotactic chemokines involved in immune cell trafficking (MIP-3α/CCL20). Probiotic administration in patients with liver cirrhosis led to modulation of plasma levels of several molecules and compounds measured (MIP-3α/CCL20, NO, big-endothelin, TXB2 and MPO). The grade of encephalopathy during the course of probiotic supplementation remained unaffected in both groups of patients. VSL#3 treatment was well tolerated and safe in patients with liver disease. In patients with compensated and decompensated liver cirrhosis, VSL#3 manipulates selected plasma molecules and compounds involved in hyperdynamic circulatory dysfunction. Short term VSL#3 administration affects several clinical and biochemical parameters commonly altered in liver cirrhosis.

Live birth after artificial oocyte activation using a ready-to-use ionophore: a prospective multicentre study.

Artificial oocyte activation has been proposed as a suitable means to overcome the problem of failed or impaired fertilization after intracytoplasmic sperm injection (ICSI). In a multicentre setting artificial oocyte activation was applied to 101 patients who were diagnosed with fertilization abnormalities (e.g. less than 50% fertilized oocytes) in a previous conventional ICSI cycle. Female gametes were activated for 15 min immediately after ICSI using a ready-to-use Ca(2+)-ionophore solution (A23187). Fertilization, pregnancy and live birth rates were compared with the preceding cycle without activation. The fertilization rate of 48% in the study cycles was significantly higher compared with the 25% in the control cycles (P < 0.001). Further splitting of the historical control group into failed (0%), low (1-30%) and moderate fertilization rate (31-50%) showed that all groups significantly benefitted (P < 0.001) in the ionophore cycle. Fewer patients had their embryo transfer cancelled compared with their previous treatments (1/101 versus 15/101). In total, 99% of the patients had an improved outcome with A23187 application resulting in a 28% live birth rate (35 babies). These data suggest that artificial oocyte activation using a ready-to-use compound is an efficient method.

Relationship between pretransplantation liver status and health-related quality of life after grafting: a single-center prospective study.

BACKGROUND: Severity of liver disease evaluated with Model for End-Stage Liver Disease (MELD)/Child-Pugh-Turcotte (CPT) score is of importance in liver transplantation (LTx) assessment. The Medical Outcomes Study Short Form (SF-36) is a widely used generic questionnaire of health-related quality of life (HRQoL). This study was a prospective analysis of the effect of pretransplantation liver status on HRQoL after the procedure. MATERIALS AND METHODS: One hundred and seven (62 male, 45 female, median age 52 years) consecutive patients were included. MELD/CPT score and diabetes status were evaluated during LTx assessment. Patients were divided into 3 groups depending on the period after LTx: 6 to 12 months (group I), 13 to 36 months (group II), and >37 months (group III). They also were divided into 2 groups depending on the age at LTx: group I (<50 years) and group II (>50 years). SF-36 was used in the assessment of HRQoL. RESULTS: Correlation between pretransplantation MELD/CPT score and HRQoL was only seen in the general health domain of the SF-36 in patients from group I (r = 0.64; P = .004 and r = 0.61; P = .02, respectively). Diabetes exerted a significant effect on the physical component summary (P = .02), again in group I. No significant correlation was observed between MELD/CPT score and the presence of diabetes in groups II and III. Regarding age at LTx, no significant correlation between MELD/CPT score and HRQoL was seen. CONCLUSIONS: Liver status assessed with MELD and CPT scores before transplantation has a minor effect on HRQoL after LTx and exerts no significant effect in patients evaluated >12 months after LTx. Patients with diabetes seem to have worse quality of life early after surgery; however, diabetic and nondiabetic patients had comparable HRQoL scores later on after LTx.

Synthetic study on cystinyl peptides using solution and solid phase methodology: human IgG1 hinge region.

Synthetic study on cystinyl peptides using solution and solid phase methodology was carried out with the central hinge region of immunoglobulin IgG1. In the solid phase synthesis of hexadecapeptide 1c, the time necessary for the formation of disulfide bonds between linear precursors was shortened four times by the action of pure oxygen in buffered solution, in comparison with air oxidation. The product was thus obtained devoid of impurities from side reactions. In the preparation of the shortened bis-cystinyl analogs 2k and 3d of the natural hexadecapeptide 1c, both the classical and polyethylene glycol (PEG6000) solution methods were utilized using a disulfide synthon (Boc-Cys-OPfp)2 to obtain peptide chains in a natural parallel alignment. In the PEG6000 strategy, lysine as a linker on both sides of the polymer was attached to enhance the loading capacity. The leucine residue, instead of proline one, was introduced to the carboxy terminus to facilitate a specific enzymatic cleavage of the peptides from PEG6000 by thermolysine.

Upper gastrointestinal endosonography in patients evaluated for liver transplantation.

BACKGROUND: Endosonography (EUS), which merges endoscopic and ultrasound examinations, is a useful modality to display abnormal vessels that develop in the intrinsic circulation, frequently called "deep" varices. If these pathological veins exceed of 5 mm diameter, they significantly increase the risk of bleeding among patients with cirrhosis. In the most recent pilot study EUS proved useful to assess children for orthotopic liver transplantation (OLT). AIM: We performed a cross-sectional study of EUS on 33 (22 males and 11 females) adult cirrhotic subjects being assessed for OLT. MATERIALS/METHODS: We used an echoendoscope at 7.5 MHz/12 MHz/20 MHz to evaluate the esophagus and stomach, including "deep" periesophageal/perigastric varices (adjacent to the muscularis propria) and paraesophageal/paragastric varices (outside the muscularis propria). "Deep" varices were considered to be large if >5 mm. RESULTS: On endoscopy, 26 (79%) patients showed esophageal varices (EV), including 11 (33%) with large (>5 mm) varices. Gastric varices (GV) were observed in 13 (39%) subjects, with 3 patients displaying large (>5 mm) varices. On EUS large "deep" EV (both para and periesophageal) were observed in 12 (36%) subjects, among whom 5 (42%) did not have large varices on endoscopy. Large "deep" GV were found on EUS in 12 (36%) subjects. On endoscopy 4 of them (33%) showed no varices and 3 (25%) had small GV. CONCLUSIONS: EUS offers a precise evaluation of portal hypertension in OLT candidates. "Deep" potentially dangerous varices, which are undetected with routine endoscopy, were noted in a significant proportion of patients. The role of EUS in prioritizing subjects for OLT must be evaluated in a prospective study.

Assessment of a modified Child-Pugh-Turcotte score to predict early mortality after liver transplantation.

OBJECTIVE: The Model for End-Stage Liver Disease (MELD) predicts mortality on the transplant list; however, it has not been of much use to predict posttransplant outcomes. Several prognostic models have been tested among patients with cirrhosis; nevertheless, their predictive value has not been established in the posttransplant setting. We recently modified the Child-Pugh-Turcotte (CPT) score by adding creatinine levels (CPT + Cr), which has proven useful for patients with alcoholic cirrhosis. This retrospective analysis sought to predict early (1 month) mortality using CPT + Cr versus 5 other prognostic models in patients who underwent orthotopic liver transplantation (OLT) at our center. MATERIALS AND METHODS: We included 48 consecutive patients (30 males, 18 females, median age 51 years). The predictive values of CPT + Cr were compared with CPT scores without or with the Huo modification, CPT + Na, MELD, and MESO, which is the MELD to serum Na ratio. Pearson correlations and ROC curves as evidenced by the area under the curve (AUC) were determined for each index. P < .05 was considered to be significant. RESULTS: CPT + Cr showed the highest correlation with the risk of death (r = .368, P = .01); MELD and MESO were the lowest (r = .204, P = NS; and r = .254, P = NS, respectively). ROC analysis showed the best predictive value of CPT and CPT-Crea with AUC of 0.758 (P = .010) and 0.748 (P = .011) respectively, as compared to 0.689 for MESO and 0.659 for MELD (both NS). CONCLUSIONS: A modified CPT score with creatinine levels may be of value to predict early death after OLT. Its usefulness must be validated in a prospective study of a large patient cohort.

Modified Charlson Comorbidity Index in predicting early mortality after liver transplantation.

BACKGROUND: The Charlson Comorbidity Index for orthotopic liver transplantation (CCI-OLT) is a modified clinical score recently proposed to be useful for the assessment of long-term survival after OLT. It includes 9 associated conditions selected upon a multivariate analysis of a large cohort of transplant recipients. Its role in predicting early mortality after OLT has not yet been investigated. We sought to CCI-OLT as a potential predictor of 1-month mortality after OLT. MATERIALS/METHODS: One hundred ninety-seven OLT were performed in our center between March 2002 and February 2009. After exclusion of patients who underwent transplantation for fulminant hepatic failure or those who underwent regrafting, we included a group of 169 patients. Viral (39%) and alcohol-induced (23%) cirrhosis were the most common indications for OLT. The CCI-OLT index was assessed in all patients. RESULTS: In total, 146 (86%) subjects survived and 23 (14%) died within 1 month after LT. Fifty-one (30%) patients suffered at least 1 comorbidity that was included in the CCI-OLT. Direct comparison between survivor versus nonsurvivor groups showed no significant difference in terms of the total frequency of comorbidities (30.1% vs 30.4%; P > .99) or the number or the type of comorbidity. The most commonly associated condition in both groups was diabetes mellitus. CONCLUSION: Unlike the case of long-term survival, CCI-OLT did not seem to predict early (1-month) mortality after OLT.

Poor responder-high responder: the importance of soluble vascular endothelial growth factor receptor 1 in ovarian stimulation protocols.

This study was designed to detect vascular endothelial growth factor (VEGF) and its soluble receptor (sVEGFR-1) in follicular fluid specimens and to evaluate the importance of sVEGFR-1 with respect to ovarian response to gonadotrophin stimulation. A total of 69 patients was treated for IVF with recombinant human follicle stimulating hormone (FSH). Concentrations of VEGF and sVEGFR-1 were quantified in follicular fluids from oocyte retrievals. Patients were designated to three groups with respect to the number of harvested oocytes: group A, 1-5 oocytes; group B, 6-10 oocytes; group C, >10 oocytes. In group A, 1133 +/- 870 pg VEGF/ml follicular fluid per oocyte were quantified, in group B 426 +/- 262 pg VEGF/ml per oocyte, and in group C 274 +/- 179 pg VEGF/ml per oocyte. Soluble VEGFR-1 concentrations resulted in 1200 +/- 523 pg/ml follicular fluid per oocyte in group A, 255 +/- 193 pg/ml per oocyte in group B, and 79 +/- 69 pg/ml per oocyte in group C. No free sVEGFR-1 could be detected in any follicular fluid. An index to estimate the biological activity of VEGF by dividing VEGF/sVEGFR-1 revealed an increasing availability of VEGF with higher ovarian response to gonadotrophin therapy. In group A this index was 1.03, in group B 1.71, and in group C 3.21. A delicate balance between VEGF and sVEGFR-1 is necessary to allow an adequate ovarian reaction to gonadotrophin therapy. Excess of bio-active VEGF increases the risk for ovarian hyperstimulation syndrome. Excess of sVEGFR-1 results in poor response and goes in parallel with reduced chances for conception.

The peptides of alpha-aminosuberic acid II. Synthesis of deamino-dicarba-eel-calcitonin sequence 1-9.

The paper describes the synthesis of Asu6-octapeptide derivatives by condensing two alternative pentapeptide fragments with Asu-containing tripeptides. After partial deprotection these linear peptides compounds are subject to cyclization experiments aimed to give the N-terminal [1-9] sequence of deamino-dicarba-eel calcitonin. This is a key substance for the semi-synthesis of the respective analogues of eel calcitonin.

The peptides of alpha-aminosuberic acid I. New intermediates for synthetic studies.

The paper describes the synthesis of alpha-aminosuberic acid derivatives suitable for the synthesis of peptides. These include Z-, Boc- and Fmoc-protection on the alpha-amino group, benzyl ester, Boc-hydrazide and Z-hydrazide as well as the free carboxylic function in the side chain, and methyl ester, benzyl ester or free alpha-carboxylic group. Their use is demonstrated on the synthesis of the respective derivatives of Asu-Val-Leu. The enzyme catalyzed reaction was successfully used both as a route to L-Asu from the D,L-compound as well as for the direct synthesis of the optically active tripeptide derivative from the Z-D,L-Asu-OH.

Enzymatic semisynthesis of dicarba analogs of calcitonin.

The semisynthesis of eel[L-alpha-aminosuberic acid]calcitonin (elcatonin) was accomplished by alpha-chymotrypsin-catalyzed coupling of two peptide segments in a single reaction without the protection of any functional group. The eel calcitonin-(10-32)-peptide was prepared by a gene manipulation. The esters of cyclic desamino nonapeptide (segment 1-9) were synthesized by the conventional solution method including a thermolysin-mediated resolution of DL-alpha-aminosuberic acid via one-step tripeptide synthesis leading to the 7-9 sequence. The main aim of this work was to determine the conditions for protease-catalyzed segment condensation while avoiding a concurrent cleavage of other proteolytically labile peptide bonds in the hormone. The alpha-chymotrypsin condensation strategy under usual conditions led to a complicated mixture of split products with an insignificant amount of the required peptide. When the coupling reaction was carried out at 0 degrees C, the reaction resulted in a satisfactory yield of elcatonin with the complete conversion of the acyl donor (1-9 segment) accompanied by negligible concurrent peptide bond digestion. The same strategy was employed for the preparation of analogous dicarba salmon calcitonin using a synthetic elcatonin-(10-32)-peptide. Both calcitonin analogs exhibited hypocalcemic activity corresponding to the international standard of elcatonin. We demonstrate in this work a peptide synthesis based on the combination of genetic engineering, chemical synthesis and proteinase-catalyzed segment condensation. This approach enables effective incorporation of an unnatural amino acid into calcitonins without the side-chain protection.

Effect of 17-norleucine-VIP on gastroduodenal motility relative to serum VIP concentration and blockade of NOS.

Mechanical and electrical activity in the antrum, pylorus, and duodenum was evaluated in the conscious dog, instrumented with seven strain gauges and five platinum electrodes. 17-Norleucine-vasoactive intestinal peptide (17-N-Leu-VIP) or 17-N-Leu-VIP plus NG-nitro-L-arginine methyl ester (L-NAME) was injected intra-arterially close to the pylorus to identify influences of nitric oxide (NO) on effects of VIP. VIP concentration was measured by radioimmunoassay in serum samples collected from the cubital and portal veins before and up to 2 h after VIP injection. VIP (0.004-0.006 mg.kg-1.10 min-1) abolished phasic contractions in the interdigestive state for 16.8 min and in the digestive state for 14.4 min, whereas whole serum VIP concentration rose above 42.4 +/- 13 pmol/l. Administration of L-NAME did not significantly influence the effects of VIP. Aftereffects of VIP, consisting of a reduced motility index, lasted 33 +/- 10.6 min in the interdigestive state and 44.5 +/- 42 min in the digestive state. This VIP aftereffect in the interdigestive state was shortened in time by the addition of L-NAME. The results overall suggest that NO release is a factor only in the aftereffects of VIP.

Effect of human gastrin-17 with and without acid suppression on human esophageal motility.

The putative role of gastrin for the regulation of esophageal motility is a matter of debate. Accordingly it was the aim of this study a) to examine if physiological postprandial plasma levels of human gastrin-17 (hG-17) can affect esophageal motility, especially the pressure of the lower esophageal sphincter (LESP), and b) to assess the contribution of augmented acid secretion during gastrin infusion. In a first series of experiments postprandial plasma gastrin levels were determined in 8 healthy volunteers following the ingestion of a mixed meal. Gastrin rose from a baseline of 21 +/- 2 pg/ml to 67 +/- 8 pg/ml and returned almost to basal levels within 120 minutes. In a second experimental series the effect of i.v. synthetic human gastrin-17 (hG-17) was studied in 17 volunteers. At a lower dose of 0.75 ng/kg/min hG-17 increased plasma gastrin to 62 +/- 7 pg/ml while a higher dose of 1.5 ng/kg min elicited a supraphysiological increase to 119 +/- 11 pg/ml. Infusion of hG-17 caused a significant increase of the LESP from 19.0 to 25.8 mmHg (p < 0.05, low dose) and from 18.5 mmHg to 23.3 mmHg (p < 0.05, high dose) when compared to the effect of i.v. saline. To exclude effects of augmented acid secretion during hG-17 infusion the experiments were repeated after complete blockade of acid secretion with famotidine 40 mg i.v. After famotidine pretreatment hG-17 caused a similar increase of LESP from 20.1 to 25.9 mmHg (low dose) and from 19.9 to 24.1 mmHg (high dose).(ABSTRACT TRUNCATED AT 250 WORDS)

In man histamine and muscarinergic mechanisms are essential mediators of acid secretion in response to synthetic human gastrin (1-17).

It is still controversial whether gastrin stimulates acid secretion by interacting with specific gastrin receptors on parietal cells or via endogenous mediators, e.g., histamine. Therefore, it was our aim to determine in healthy human volunteers (n = 14; 3 females, 11 males; age 23-28 years) the degree by which the specific histamine H2-receptor antagonist famotidine or the muscarinergic antagonist atropine block acid secretion in response to synthetic human gastrin (hG) (1-17). Famotidine was deliberately administered at a supramaximal dose (40 mg i.v. bolus) to reliably block any and all effects of endogenous histamine on the parietal cells. After an overnight fast famotidine or saline were injected i.v., and gastric secretions were collected via a nasogastric tube for the ensuing 60 min to assess basal secretion. Thereafter, hG (1-17) was infused for 60 min in randomized order at two different rates: 0.75 ng/kg/min resulting in postprandial plasma gastrin levels (55-66 pg/ml), and 1.5 ng/kg/min yielding supraphysiologic levels (110-136 pg/ml). Both rates increased basal acid secretion (meq/10 min) from 0.5 +/- 0.2 to 3.8 +/- 0.6 and 4.7 +/- 0.5, respectively. Famotidine abolished basal acid secretion and completely blocked acid and volume secretion in response to both hG (1-17) doses. After injection of famotidine both hG (1-17) doses resulted in plasma levels exceeding those in controls by 18-27 pg/ml. A similar increase (14-16 pg/ml) was observed after famotidine injection without simultaneous hG (1-17) infusion indicating that this increase was due to the release of endogenous gastrin when the acid feedback inhibition was blocked by famotidine. To study a potential additional role of cholinergic mechanisms the effect of atropine (7 micrograms/kg i.m.) on hG (1-17)-induced acid secretion was examined. Atropine reduced basal acid secretion from 0.8 +/- 0.1 to 0.1 +/- 0.08 meq/15 min. Similarly, the response to 0.75 ng/kg/min hG (1-17) was reduced by 72.9%. Basal gastrin release was not altered by atropine which, however, tended to increase serum gastrin levels during infusion of hG (1-17) by 16-24 pg/ml. We conclude that in man histamine and muscarinic mechanisms are essential mediators of gastrin-stimulated acid secretion. The present data argue against a significant direct effect of gastrin alone on human parietal cells but rather support potentiating interaction with histamine and cholinergic mechanisms.

A 1H NMR study of human calcitonin in solution.

Human calcitonin (hCT) has been investigated by NMR at 400 MHz in DMSOd6 and in an 85% DMSOd6-15% 1H2O (v/v) cryoprotective mixture. All backbone and side-chain resonances have been assigned, and the secondary structure has been determined in both solvents. In DMSOd6, the simultaneous presence of d alpha N, dNN, and some specific weak medium-range nuclear Overhauser effects, together with the amide temperature coefficients and the analysis of the NH-alpha CH spin-spin coupling constants, indicates that hCT is highly flexible but with three domains (comprising segments Asn3-Gly10, Gln14-Thr21, and Thr25-Ala31) in extended conformations which dynamically transform into isolated beta turns in the N- and C-terminal regions and into adjacent tight turns, resembling a 3(10) helix structure, in the central part. The DMSO-water mixture rigidifies the polypeptide chain, favoring an ordered, extended conformation. NOESY data indicate the presence of a short double-stranded antiparallel beta sheet in the central region made by residues 16-21 and connected by a two-residue hairpin loop formed by residues 18 and 19. Two tight turns, formed by residues 3-6 and 28-31, were also identified. The central beta sheet does not favor an amphipathic distribution of the residues as found for salmon calcitonin [Motta, A., Castiglione Morelli, M. A., Goud, N., & Temussi, P. A. (1989) Biochemistry 28, 7998-8002]. This is in agreement with the smaller tendency of hCT to form the amphipathic alpha helix, postulated to be responsible for the interaction of hCT with lipids. The possible role of the cis-trans isomerism of Pro is discussed.

Fully synthetic immunogens. Part III. Synthesis of hinge-peptide/gastrin conjugates and their immunological properties.

As core molecule for the multiple attachment of antigenic peptides we have selected the human IgG1 hinge fragment 225-232/225'-232'. Two types of conjugates of this double-chain bis-cystinyl hinge-peptide were prepared i) by linking its C-termini to [NIe15]-human-little-gastrin-[2,17] and ii) by elongating the resulting hinge-peptide/[NIe15]-little-gastrin-[2-17] conjugate at the two N-termini with the human big-gastrin sequence 1-14 to produce the big-gastrin-[1-14]/hinge-peptide/little-gastrin-[2-17] conjugate. For the synthesis of these peptide structures both the route via the preformed double-chain bis-cystinyl peptide and the route via suitably protected monomeric bis-cysteinyl peptides were used. For the latter approach advantage was taken of the previous observation about the preferred oxidation of the bis-cysteinyl hinge-peptide 225-232 to the dimer in parallel alignment. Both synthetic routes led to identical products. Immunization experiments in guinea pigs with the synthetic hybrids led to surprisingly strong immune responses with anti-little-gastrin antibody titers comparable to those induced by the iso-1-cytochrome c/little-gastrin-[2-17] conjugate as carrier-hapten system. These findings show that the two gastrin constructs are fully competent immunogens. Additionally, the gastrin receptor-like specificity of the antibodies indicates that both the synthetic hybrids and the cytochrome c conjugate allow for expression of a little-gastrin-specific conformational epitope similar to the bioactive structure of this hormone. The usefulness of such synthetic hybrids is further confirmed by the observation that the bivalent immunogen, containing both the little-gastrin 2-17 and the big-gastrin 1-14 sequence, is capable of inducing an immune response against both antigenic sequences, although with different efficiency. These results fully confirm our expectations.

Fully synthetic immunogens. Part II. Studies on parallel dimerization of the human IgG1 hinge-fragment 225-232 with N- or C-terminal gastrin related extensions.

The bis-cysteinyl hinge-fragment 225-232 of human IgG1 has been extended at the N- or C-terminus with Nle15-desamido-human-little-gastrin-[5-17] and Nle15-human-little-gastrin-[5-17]-NH2, respectively. Thermodynamically controlled air oxidation of the resulting bis-cysteinyl-peptides led to the predominant formation of the corresponding dimers in parallel alignment despite the incorporation of the immunoglobulin-unrelated gastrin-sequences. These surprising results confirm the high degree of structural information inherent in the hinge-sequence and its intrinsic tendency to fold into the correct structure in terms of cysteine pairings. This protein subdomain-the hinge-peptide-is therefore well suited as core molecule for the design of fully synthetic immunogens with multiple attachment of antigenic determinants.

Cyclic hexapeptides related to somatostatin. Synthesis and biological testing.

As a continuation of our program to study the structure-function relationship of the peptide hormone somatostatin, we report the synthesis and biological potencies of a series of cyclic hexapeptide analogs related to somatostatin. The parent peptide of this series was designed by Veber and coworkers, c[-Pro6-Phe7-D-Trp8-Lys9-Thr10-Phe11-], and has been reported to be superactive in the inhibition of the release of growth hormone. (The superscript numbers refer to the positions of residues in native somatostatin). The series of analogs has been designed to examine the role of the so-called bridging region, Phe11-Pro6, which has been postulated to be important in maintaining the proper conformation of the biologically active tetrapeptide, Phe-D-Trp-Lys-Thr. We have incorporated peptidomimetics and the retro-inverso modification into the bridging region of the molecule, with the aim of affecting the conformational preferences found in the parent peptide. Results from the biological assay--in vitro inhibition of growth hormone--and the conformational analysis (adjoining paper) of our analogs will provide insight into the relationship between structure and biological activity of somatostatin.

Side products in the synthesis of secretin.

Analytical studies on side products in the synthesis of secretin led to the discovery of a further side reaction, involving conversion of the carboxamide function of glutamine to glutamic acid gamma-methyl ester. The chemical structure of the side products was determined by NMR spectroscopy upon their tryptic digestion and isolation of the modified secretin fragments. Possible pathways for the observed side reaction are discussed.