Abstract: A 34‐amino acid synthetic peptide was derived from the third domain of human alpha‐fetoprotein, and the peptide was shown to inhibit estrogen‐stimulated growth. Under certain conditions, however, the peptide lost growth‐inhibitory activity. A biophysical study of the peptide was undertaken with a goal of obtaining completely reliable preparations. The peptide was studied using gel‐filtration column chromatography as a function of peptide concentration and age of solution, and was found to exhibit complex aggregation behaviors. During the early period (0–3 h) after dissolving lyophilized peptide into pH 7.4 buffer, solutions were composed mostly of trimers. At higher peptide concentrations (≥ 3.0 g/L), the trimers aggregated extensively to a large aggregate (minimum size ≈ 102 peptides). At 5.0–8.0 g/L, these large aggregates increased in size (up to ≈ 146 peptides) until trimers were largely exhausted from solution. During the later times (> 3 h) after sample preparation, the trimeric oligomer of the peptide dissociated slowly to form dimers for samples at 0.10–3.0 g/L. After their build‐up, a very small number of dimers associated to form hexamers. Disulfide bonds stabilized the dimers as indicated by the conversion of dimers to trimers upon the addition of a reducing agent, and the failure of dimers to form in the presence of reducing agent. Reducing agent did not affect trimer or large aggregate formation. Trimers were found to be active in an assay monitoring inhibition of estrogen‐stimulated growth, whereas dimers and large aggregates were inactive. The two cysteines in the peptide were modified to either S‐ methylcysteine or S‐ (2‐aminoethyl)cysteine, and both derivatives showed significant growth‐inhibition activity. A serine analog in which both cysteines were replaced had very different aggregation behavior than the cysteine peptide and lacked its growth inhibitory ability. Peptide aggregation is critically important in establishing the ability of the peptide to inhibit growth and have anticancer activity, but the state of its two cysteines is of little influence.
Proteolytically active forms of thrombin ( alpha- and gamma-thrombin) and thrombin receptor peptides inhibited the release of nitrite, a stable endproduct of nitric oxide, evoked by interleukin-1 beta (IL-1 beta ) in cultured vascular smooth muscle cells while proteolytically inactive forms [D-Phe-Pro-Arg chloromethyl ketone-alpha-thrombin (PPACK-alpha-thrombin) and diisopropylphosphoryl-alpha-thrombin (DIP-alpha-thrombin)] had either no or only minimal inhibitory effects. Under bioassay conditions, perfusates from columns containing IL-1 beta-activated vascular smooth muscle cells or cells treated with IL-1 beta plus PPACK-alpha-thrombin relaxed detector blood vessels. These relaxations were abolished by the inhibitor of nitric oxide synthesis, NG-nitro-L-arginine. No relaxations were obtained with untreated cells or IL-1 beta-treated cells in the presence of alpha-thrombin. The expression of inducible nitric oxide synthase mRNA and protein in vascular smooth muscle cells by IL-1 beta was impaired by alpha-thrombin. These results demonstrate that thrombin regulates the expression of the inducible nitric oxide synthase at a transcriptional level via the proteolytic activation of the thrombin receptor in vascular smooth muscle cells.
Porcine follicular fluid contains several factors capable of inhibiting the binding, in vitro, of follicle-stimulating hormone (FSH) to receptor, including an agonist and an antagonist of FSH biological activity in vitro. FSH receptor-binding inhibitory activity (FSH-BI) was determined with assays using radioligand (125iodide-human FSH) receptor (calf-testes membrane); in vitro biological assays (cultured immature rat Sertoli cells) were used to determine antagonist/agonist activity. FSH antagonist activity is due to a low (less than 5000) molecular weight FSH-BI that is soluble in acidic acetone and insoluble in diethyl ether allowing preparative scale isolation. Additional purification was achieved by anion-exchange and reverse-phase high-performance liquid chromatography. Highly purified, biologically active FSH-BI contained the amino acids Ser, Gly, Arg, Thr, Ala, Pro, Val, and Lys; hexoses (phenol-sulfuric acid-positive reaction); and ethanolamine. Thus, this FSH antagonist appears to be a complex glycopeptide--possibly derived from membrane components, as suggested by the presence of ethanolamine and carbohydrate residues. Porcine follicular fluid, therefore, contains a low molecular weight FSH antagonist that, along with the high molecular weight FSH agonist previously identified, may regulate gonadal responsiveness to FSH through interactions with the FSH receptor.
To investigate the levels of interleukin (IL)-23 in patients with early rheumatoid arthritis (eRA) and the effect of anti-tumour necrosis factor (anti-TNF)-α treatment on IL-23 levels.Treatment-naïve eRA patients from the OPERA cohort were included (n = 151). Patients were randomized to methotrexate (MTX) plus adalimumab (ADA; n = 75) or MTX plus placebo-ADA (PLA; n = 76). Plasma samples were obtained at baseline and at months 3, 6, and 12 together with values for C-reactive protein (CRP), the 28-joint Disease Activity Score based on CRP (DAS28CRP), scores on the Clinical Disease Activity Index (CDAI) and the Simplified Disease Activity Index (SDAI), visual analogue scale (VAS) for pain/fatigue/physician global and total Sharp/van der Heijde score (TSS). IL-23 was measured at each time point.IL-23 levels decreased significantly in the ADA group from 20.6 pg/mL (IQR 13.1-32.7 pg/mL) at baseline to 18 pg/mL (IQR 7.2-25.0 pg/mL) at 12 months (p < 0.01). No significant decrease in IL-23 level was observed in the PLA group. No associations between baseline IL-23 levels and measures of disease activity (DAS28CRP, CRP, CDAI, or SDAI) at 12 or 24 months were present in the treatment groups. Baseline IL-23 correlated inversely with changes in TSS and symptom duration before diagnosis.Our data show increased baseline levels and a significant decrease in IL-23 levels in eRA patients treated with anti-TNF-α. The inverse correlation with duration of symptoms before diagnosis supports the importance of IL-23 in the preclinical disease development of RA.
Introduction: Procollagen type 1 N-terminal Propeptide (P1NP) is a precursor protein of collagen type 1, a major component of cardiac extracellular matrix. The association of P1NP to clinical outcome in patients with suspected acute coronary syndrome (ACS) has so far not been investigated. The objective of the present study was to assess the predictive value of P1NP concerning adverse clinical outcome in patients admitted for suspected ACS. Methods: Plasma P1NP was measured with an enzyme immune assay in samples taken upon admission from patients enrolled in the Risk markers in Acute Coronary Syndrome (RACS) study (NCT00521976), a Norwegian single center study including patients with suspected ACS from November 2002 to September 2003. The primary endpoint was a composite of all-cause mortality, myocardial infarction (MI), or stroke within 1 year. Additional analyzes included the composite and its individual components at 1, 2, and 7 years, and cardiac death at 1 and 2 years. Associations between quartiles of P1NP and the endpoints were assessed in both univariate analyzes and in stepwise multivariable Cox regression analyzes fitted with significant clinical variables, past medical history, medication, TnT, BNP, and eGFR before P1NP was introduced. Results: Samples were available from 813 patients (Mean age 69.6 years, 61.4 % males, 52.9% elevated TnT). Mean P1NP levels was 46.2 ng/mL (CI 44.8-47.6). In multivariable Cox analyzes, P1NP was found to be significantly associated with the composite of all-cause mortality, MI, or stroke at 1 year (HR [Q4vsQ1] 1.82, CI 1.12-2.98; p=0.017). This association did not remain significant beyond 1 year. No association was seen for any of the individual components, or cardiac death, at any timepoint, or in patients with elevated TNT (p>0.05 for all). Conclusions: This first study on P1NP in suspected ACS revealed a significant association with adverse clinical outcome (all-cause death, MI, or stroke) at 1 year. However, no predictive value was found concerning outcome for the individual components of the primary endpoint, or cardiac death, at any timepoint, or in patients with elevated TNT.
Monoclonal antibodies (mabs) to human (h) FSH were utilized to probe epitopes of the β-subunit of hFSH (hFSHβ). These mabs had an average approximate affinity constant (K8 of 108M-1 for hFSHβ and 1O7M-1 for heterodimeric hFSH. Hormone specificity of mabs for hFSHβ was demonstrated by a lack of cross-reactivity with hCGα, FSHα, or LHα. Epitope specificity of each mab was initially assessed by determining whether solid phase mab could bind to [125I]hFSH already bound to mabs in liquid phase. In addition, it was determined whether [125I]mab could bind to hFSH already bound to solid-phase mabs. Both epitope cross-matching protocols indicated that all mabs bound to the same epitopes on hFSHβ. Next, synthetic peptides corresponding to the sequence of hFSHβ were used in an enzyme-linked immunosorbent assay to map this epitope. All mabs bound to peptides 7-19, 1-20, 33-53, and 66-85 but did not bind or bound weakly to peptides 81-100, 95-103, and 103-110. Titration experiments were performed using different concentrations of peptide (0.3-41 nmol) and one mab 3G3 (500 ng-25 ng) in the enzyme-linked immunosorbent assay. The product of the lowest mass of both peptide and antibody which gave a positive result was used to rank the peptides for their binding with mab 3G3. Peptides were ranked in the following descending order of potency: 33-53, 49-67, 66-85>>>16-36,1-20, 95-103, 52-65, 81-100, and 103-110. Ability of the mabs to inhibit binding of [125I]hFSH to bovine testis membrane receptor (Rec) was also studied. When [125I]hFSH was preincubated with increments of each mab for 2 h at 25 C before adding Rec with further incubation for 16 h, all mabs inhibited [l25I]hFSH binding to Rec. The data suggest that most of the hFSHβ molecule has a conformation enabling all antibody recognizable regions to be in close proximity to each other. The present study provides evidence for an assembled epitope comprising in part, amino acids 33-53, which has been previously shown to be involved in receptor binding. Peptide sequences 49-67 and 66-85 are neighboring sequences in this assembled epitope which contains the determinants for receptor binding.
Treatment of peptides with excess HgO in the presence of alkaline cyanide leads to cleavage of the peptides at glycine residues. The reaction appears to involve both C- and N-mercuration with subsequent release of 2mol mercury per mol of glycine. An intermediate glyoxylic acid residue in Schiff base linkage is postulated. Treatment of the heptapeptide Phe-Ala-Lys-Gly-Leu-Asp-Val with alkaline HgO and KCN for 6 h at 25° resulted in greater than 90% cleavage, and the resultant reaction products were separated by reverse phase chromatography and identified by amino acid analysis. N-terminal products were approximately equimolar Phe-Ala-Lys, Phe-Ala-Lys-Gly, and Phe-Ala-Lys-amide. C-terminal products were predominantly Leu-Asp-Val (63%), plus Gly-Leu-Asp-Val (9%), and oxalyl-Leu-Asp-Val (8%). This method may be useful for cleavage of peptides or proteins containing glycine residues.
