Synthesis, chemical, radiochemical and radiobiological evaluation of a new 99mTc-labelled bombesin-like peptide.

A new pentadecapeptide bombesin analogue was prepared by Fmoc synthesis, purified by HPLC and identified by electron ionization mass spectrometry. The biological activity of the new peptide was tested on isolated human colonic muscle cells and compared to native bombesin. Labelling of the new biomolecule with Tc-99m yielded a single radioactive species which remained stable at room temperature for eight hours. In a binding assay, the radiolabelled peptide showed high affinity for oat-cell carcinoma (Kd = 9.8 nM) and colorectal adenocarcinoma (Kd = 27.2 nM). Biodistribution studies, performed in normal rodents, indicated uptake by organs that normally express bombesin receptors, such as liver, intestines and kidneys. Scintigraphic studies, performed in nude mice transplanted with small cell lung carcinoma and colon cancer cells, showed significant tumor uptake two hours p.i. The new synthetic pentadecapeptide appears to have promise for several malignancies, including oat-cell lung carcinoma, colorectal cancer and gastroenteropancreatic (GEP) tumors.

Technetium labeled bombesin-like peptide: preliminary report on breast cancer uptake in patients.

Bombesin-like peptides are neurotransmitters and cancer growth factors. Several tumors, breast cancer among them, show one or more than one of the three known bombesin receptors. We have synthesized and labeled with technetium 99m a new pentadecapeptide, analogue to the leu13 amphibian bombesin (99mTc BN). Labeling yield was 83 +/- 4%. Prone Scintimammography was performed on five patients affected by breast cancers (T categorization: two T1b and three T1c), after injecting 0.7 mg, 185 to 296 MBq (5 to 8 mCi) of the peptide. Total body scan did not show free technetium biodistribution. No adverse reaction was observed. Prone Scintimammography with 99mTc Sestamibi (99mTc SM) was also performed few days later. 99mTc BN detected all 5 cancers, whereas 99mTc SM only four: all the T1c and one T1b cancer. Two of them showed axillary node invasion that was detected by both the radiotracers. A fibroadenoma present on contralateral breast to the one with cancer, was not detected neither by 99mTc SM nor by 99mTc BN. Tumor/breast normal tissue ratio (T/B) was constantly higher with 99mTc BN than with 99mTc SM. Maximal T/B was measured as 1.79 with 99mTc SM and 2.25 with 99mTc BN 5 min after fast i.v. administration. In conclusion our 99mTc BN is taken up by primary breast cancer showing higher T/B than 99mTc SM (p < 0.01). In our limited scale, 99mTc BN appears to be safe and, in our limited scale, even more accurate than 99mTc SM for detecting breast cancer.

Effect of thymosin peptides on the chick chorioallantoic membrane angiogenesis model.

The effect of alpha- and beta-thymosin peptides, namely prothymosin alpha (ProT(alpha)), thymosin alpha(1) (T(alpha)1), parathymosin alpha (ParaT(alpha)), thymosin beta(4) (Tbeta4), thymosin beta(10) (Tbeta10), and thymosin beta(9) (Tbeta9), on the angiogenesis process was investigated using the chick chorioallantoic membrane as an in vivo angiogenesis model. The thymosin peptides tested were applied in 10 microl aliquots containing 0.01-4 nmoles of Tbeta4, Tbeta10 or Tbeta9, 0.016-6.66 nmoles of T(alpha)1, 4.1 pmoles-1.66 nmoles of ProT(alpha), and 4.4 pmoles-1.76 nmoles of ParaT(alpha). Phorbol 12-myristate 13-acetate and hydrocortisone were also used as positive and negative control, respectively. Tbeta4, ProT(alpha) and T(alpha)1 were found to enhance angiogenesis, while Tbeta10, Tbeta9 and ParaT(alpha) exhibited an inhibitory effect on the angiogenesis process. When mixtures of Tbeta4 and Tbeta10 containing active amounts of the two peptides at different proportions were applied, the promoting effect of Tbeta4 on angiogenesis was reversed in the presence of increasing concentrations of Tbeta10 and vice versa. The effect of Tbeta10, Tbeta9, ProT(alpha) and ParaT(alpha), in parallel with Tbeta4 and T(alpha)1, on the angiogenesis process was investigated for the first time as far as we know and the results of this study offer more insight into the biological regulatory roles of thymosin peptides, and provide helpful information about their therapeutic potential. Whether these agents could be used either as inhibitors of angiogenesis in disease states where uncontrolled angiogenesis is involved, e.g. in carcinogenesis, or as angiogenesis promoters that could be useful in wound healing, fracture repair, peptic ulcers etc., remains to be further studied.

Analytical techniques for determining biotin.

Biotin is a vitamin of the B-complex, which plays an important biochemical role in every living cell. In the recent years, the interest in this vitamin has been rekindled, mainly due to its association with serious human disorders, such as the inherited syndrome multiple carboxylase deficiency, which can be successfully treated with biotin administration. Diagnosis of biotin deficiency as well as monitoring of biotin levels in biological fluids of patients receiving biotin treatment is crucial. Equally important is the determination of biotin levels in pharmaceutical preparations as well as in food and food supplement products, which constitute the main source of biotin in humans. Several analytical methods for measuring biotin in various samples, e.g. human fluids, pharmaceutical formulations, food material etc., have been reported in the literature. In this review, the most representative of these methods are presented, and their characteristics are evaluated.

Investigation of the epitopic structure of thymosin beta10 by epitope mapping experiments.

We present here a study on the epitopic structure and the immunochemical characteristics of thymosin beta10 (Tbeta10), a 43 aminoacid peptide involved in important cellular mechanisms, by using the epitope mapping Multipin method. Octapeptides overlapping by one amino acid so as to represent the whole sequence of Tbeta10 were synthesized on polystyrene pins and screened, using an ELISA method, with a polyclonal antiserum raised against intact recombinant Tbeta10. The octapeptides were also tested with anti-peptide oligoclonal antisera raised against the synthetic fragments Tbeta10[1-16] and Tbeta10[31-43], with polyclonal antisera raised against natural thymosin gamma4 (Tbeta4) or thymosin beta9 (Tbeta9), and with anti-peptide oligoclonal antisera raised against various fragments of Tbeta4 (i.e. Tbeta4[1-11], Tbeta4[30-43] and Tbeta4[16-38]). Four distinct epitopic fragments were revealed, namely the sequences 1-13, 19-30, 29-40 and 36-43. Among them, the sequence 36-43 appears to offer unique immunochemical characteristics to the Tbeta10 molecule.

Direct ELISA method for the specific determination of prothymosin alpha in human specimens.

An enzyme linked immunosorbent assay, specific for prothymosin alpha (ProT alpha) was developed using an antibody against the synthetic C-terminal peptide ProT alpha[101-109] and isolated bovine ProT alpha for the preparation of standard solutions and immunoplates. Due to the antibody used, the ELISA developed was capable of fully discriminating between ProT alpha, the naturally occuring and partially homologous peptide parathymosin alpha (ParaT alpha) and the peptide thymosin alpha1 (T alpha1), whose sequence is identical to the [1-28] sequence of ProT alpha, and its in vivo occurrence is under question. Moreover, due to its improved sensitivity, the ELISA was capable of directly determining ProT alpha concentration in human serum and tissue extracts, without any pretreatment of the samples. ProT alpha levels were directly measured in sera obtained from 48 apparently healthy individuals and 27 patients with diagnosed breast cancer and found to range from 0.67 to 2.34 microg/ml (mean value 1.27 +/- 0.49 microg/ml) and from 0.47 to 1.74 microg/ml (mean value 1.02 +/- 0.29 microg/ml), respectively. ProT alpha levels were also measured in four breast tumor and adjacent normal breast tissue extracts and found to be elevated in the tumor extracts.

Indirect enzyme-linked method for determining biotin in human serum.

An indirect enzyme-linked assay was developed for quantifying biotin concentrations in human sera. Biotin standard solutions or unknown samples are preincubated with streptavidin-conjugated horseradish peroxidase (streptavidin-HRP) and added to plates coated with biotinylated bovine IgG (B-IgGb). The concentration of the streptavidin-HRP is such that the streptavidin binding sites are sufficient to bind apparently all the biotin present in samples, whereas, the remaining sites are inversely proportional to the amount of biotin in analysed sample. These sites could subsequently interact with the immobilized B-IgGb providing signal. The assay demonstrated dynamic range 5 to 640 ng/L, detection limit 2 ng/L, intra- and interassay C.V., 1.6-3.9% and 3.7-7.2% respectively, recovery 100-114% and linear recovery 90-117%. Serum biotin determined: healthy individuals 66 to 600 ng/L, pregnant women (> or = 36 weeks) 60 to 360 ng/L, and patients under chronic haemodialysis 0.56 to 1.62 micrograms/L. The method described is among those few which have been experimentally evaluated for their capabilitity of assessing biotin in human sera.

Preliminary findings on the expression of thymosin beta-10 in human breast cancer.

Paraffin sections from 30 human breast tissue specimens were stained with a specific antibody for thymosin beta-10, ATB10(38-43). The results showed that thymosin beta-10 was detected mainly in the malignant tissue, particularly in the cancerous cells, whereas the normal cell population around the lesions showed very weak staining. Also, the intensity of staining in the cancerous cells was proportionally increased with the increasing grade of the lesions.

Development of specific anti-thymosin beta 10 antipeptide antibodies for application in immunochemical techniques.

We present theoretical and experimental data necessary for raising specific antibodies for thymosin beta 10, a 43-amino acid residues peptide occurring in human tissues. We postulate that thymosin beta 10 contains three major antigenic determinants (residues 2-8, 17-25, and 35-41). For antibody development, we synthesized the N-terminal fragment thymosin beta 10(1-16) as well as the C-terminal fragments thymosin beta 10(31-43) and thymosin beta 10(38-43), due to their putative antigenic properties and minimal structural similarity with the homologous peptide thymosin beta 4, which also occurs in humans. The putative antigenic determinant 17-25 is present in all beta-thymosins and was therefore not synthesized. All antisera raised against the above peptide fragments or the intact molecule of thymosin beta 10 were found capable of recognizing and binding synthetic or natural thymosin beta 10 with high specificity, showing minimal cross-reactivity with thymosin beta 4 isolated from bovine tissues or synthetic thymosin alpha 1. Due to its easy preparation and the highly specific affinity of the antibody raised against it for the intact peptide, the fragment thymosin beta 10(38-43) may be considered the antigen of choice for developing anti-thymosin beta 10 antibodies, which can eventually be applied to immunochemical studies.

Large-scale chromatofocusing-based method for isolating thymosin beta 4 and thymosin beta 9 from bovine tissues.

A large-scale method for the isolation of thymosin beta 4 (up to 120 mg) and thymosin beta 9 (up to 40 mg) from bovine lung (up to 2 kg) was developed. The isolation protocol included tissue homogenization in 0.4 M HClO4, centrifugation, solid-phase extraction through LiChroprep RP-18 material, chromatofocusing on polybuffer exchanger PBE 94-modified Sepharose and dialysis against water. The isolated products were characterized by analytical isoelectric focusing, reversed-phase HPLC, electrospray ionization mass spectrometry and amino acid analysis. The method developed is rapid and convenient, requires no expensive equipment and can be used for the isolation of thymosin beta 4 and homologous peptides from various animal tissues.

Direct colorimetric determination of solid-supported functional groups and ligands using bicinchoninic acid.

We describe new colorimetric methods for the direct determination of total solid-supported sulfydryl, aldehydo, hydrazido, and N-hydroxysuccinimido carboxylate groups as well as of immobilized cysteine, tyrosine, and thyroxin using only the commercially available bicinchoninic acid/copper protein assay reagent. The method is based on the ability of these groups to reduce Cu2+ to Cu+, which forms a chelate complex with bicinchoninic acid absorbing at 562 nm. Each assay requires only one incubation step of the solids with the reagent for 1 h at 60 degrees C. The quantitation of the different groups is finally carried out through standard curves of appropriate substances. Using the assays developed we determined the amount of the above-mentioned functional groups and ligands onto several commercially available solid supports. The values obtained were in agreement with those provided by relative literature methods and/or by the manufacturers. The assays were found to be accurate, precise (interassay CV less than 3%), and very sensitive, allowing the determination of nmol quantities of functional groups per assay tube.

High-performance liquid chromatographic separation of biotinylamide analogues used as substrates in biotinidase radioassays.

A simple, one-step protocol for synthesizing biotinylmono[125I]iodotyramine and biotinyldi[125I]iodotyramine, which are used as tracer substrates in biotinidase radioassays, is presented. This synthetic protocol uses reversed-phase HPLC for the isolation of the biotinylamide analogues from the reaction mixture. The HPLC method developed can potentially be applied to a scaled-up synthetic protocol for non-radioactive biotinylmono- and diiodotyramine. In addition, it can be used for the identification of the above-mentioned compounds.

Colorimetric determination of reactive solid-supported primary and secondary amino groups.

A simple and sensitive method for the quantitative determination of solid-supported primary and/or secondary amino groups using commercially available reagents is described. The solid supports are treated in an aqueous environment with either 2-iminothiolane (ITL) or sulpho-succinimidyl-3-(4-hydroxyphenyl)propionate (sulpho-SHPP), which introduce one sulphydryl or one hydroxyphenyl group per amino group reacted, respectively. These groups are capable of reducing Cu2+ to Cu+ in alkaline medium. Thus, after removal of the excess reagents through washing, subsequent incubation of the solids with 2,2'-bicinchoninic acid (BCA) copper protein reagent results in production of Cu+ in the solution, which forms a chelate complex with BCA absorbing at 562 nm. The quantitation of the groups introduced on the surfaces, and therefore of the reacted amino groups, is carried out through standard curves of cysteine solutions for ITL, or tyrosine solutions for sulpho-SHPP-treated solids. Using ITL, only the primary amino groups are determined, whereas sulpho-SHPP provided the primary and secondary reactive amino groups. The method is versatile and can be used for the estimation of amino groups onto several biomedical solid matrices, and should provide useful information for the covalent immobilization of ligands (e.g. drugs, antibodies).

Determination of serum biotinidase activity with biotinyl derivatives of lodotyramines as substrates.

We synthesized biotinylated mono- and di-iodotyramine and their radioactive counterparts and used these substances as substrates to estimate serum biotinidase activity in a radioassay system. The Km values determined for mono- and di-iodobiotinyl derivatives were 15.8 and 25.9 microM, respectively, whereas, the maximum velocities of the enzymatic reaction were 27.0 and 8.7 nmol.min-1.mL-1, respectively. Both substrates competed with biocytin for the same active site of the enzyme and the Ki values were 7.30 and 9.56 microM for the mono- and di-iodinated substrate, respectively. Higher assay sensitivity was obtained using [125I]biotinyl-monoiodotyramine as substrate, and the values obtained were directly related with those determined with the well-established colorimetric method (r = 0.9377, n = 31). However, for routine use, the assay may be accomplished by diluting the radiotracer with biocytin instead of its "cold" counterpart, because it is a commercially available reagent. The values obtained in this case were very well correlated with those determined by the colorimetric assay as well (r = 0.9289, n = 31).

A 125I-radioimmunoassay for diethylstilbestrol in serum of patients with prostatic cancer treated with stilphostrol.

A new radioimmunoassay for determining diethylstilbestrol in serum using N-(4'-OH-[3'-125I]iodophenethyl)-6-(4-O-diethylstilbestryl)-hex anamide as a radiotracer and a double antibody as a separation reagent is described. The radiotracer is prepared by synthesizing 6-(4-O-diethylstilbestryl)-hexanoic acid and coupling its succinimidyl ester with mono-[125I]tyramine in tetrahydrofuran (16 h, 20-22 degrees C). The standard curve is linear (semi-log transformation) and the assay is sensitive (< 0.022 pmol/tube), reproducible (intra- and interassay coefficient of variation values, 5.3 and 8.1%, respectively), and accurate (recovery values, 95-101%), with a non-specific binding less than 3.2%. Diethylstilbestrol concentrations measured in sera of nine patients with prostatic cancer by the proposed assay ranged from 0.170 to 2.517 mumol/l, which corresponded to an only three-fold dosage variation. In all cases tested, dosing was adequate to retain markers of prostatic cancer in serum within accepted limits; nevertheless, individualization of dosing may be necessary to minimize toxicity.

A thymosin beta 4 ELISA using an antibody against the N terminal fragment thymosin beta 4 [1-14].

A thymosin beta 4 ELISA was developed in which thymosin beta 4, absorbed on microwells, competed with thymosin beta 4 in solution for the binding sites of an anti-thymosin beta 4 antibody. The antibody molecules finally immobilized on the microwells were detected using a goat anti-rabbit immunoglobulin/horseradish peroxidase conjugate in combination with the substrate 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt, and measuring the relevant optical density values. Anti-thymosin beta 4 antibodies were raised in rabbits against intact thymosin beta 4 as well as against selected fragments of the peptide, i.e., the N terminal fragments thymosin beta 4[1-14] and thymosin beta 4[1-11]. The antibody against thymosin beta 4[1-14] was used in the thymosin beta 4 ELISA, because it showed minimal cross-reactivity (0.1%) with the highly homologous peptide thymosin beta 9 as well as exhibiting the highest titre. The ELISA procedure developed, apart from showing a minimal cross-reaction with thymosin beta 9, was fast, easy to perform and exhibited good assay characteristics.