Development of tissue plasminogen activator specific "on demand cleavable" (odc) linkers for radioimmunotherapy by screening one-bead-one-compound combinatorial peptide libraries

New strategies are needed to protect normal organs from radiation in cancer radioimmunotherapy (RIT). This can be achieved by rapid clearance of radiometal in the circulation after accumulation of radioimmunoconjugates (RIC) in the tumor. Our strategy is to place highly efficient and specific cleavable linkers between radiometal chelates and the tumor targeting agents. Such linkers must be resistant to cleavage by enzymes present in the plasma and the tumor. After radiotargeting agents have accumulated in the tumor, a cleaving agent can be administered "on demand" to cleave a specific linker, resulting in the release of radiometal from the circulating RIC in a form that will have rapid renal clearance. We have selected TNKase, a thrombolytic agent approved for patient use, as our model on-demand cleaving agent. To identify TNKase-specific linkers, we screened fluorescent-quenched random "one-bead-one-compound" (OBOC) combinatorial peptide libraries. d-Amino acid containing peptides that were specific for TNKase but were resistant to cleavage by plasma and tumor-associated proteases were identified. One of these peptide substrates (rqYKYkf) was used to link the DOTA chelate to ChL6, a monoclonal antibody known to target breast cancer. This antibody conjugate was stable in plasma for 7 days while preserving the immunoreactivity to intact tumor cells. The addition of TNKase at clinical achievable plasma level (10 mug/mL) resulted in the release of 28% of the radiometal from the radioimmunoconjugate within 72 h. This lead linker, after further optimization to increase its response to TNKase, may be useful in the development of more effective radioimmunotherapeutic and radioimaging agents.     

L-Amino acid oxidase from Naja naja oxiana venom

A new l-amino acid oxidase (LAAO) was isolated from the Central Asian cobra Naja naja oxiana venom by size exclusion, ion exchange and hydrophobic chromatography. The N-terminal sequence and the internal peptide sequences share high similarity with other snake venom l-amino acid oxidases, especially with those isolated from elapid venoms. The enzyme is stable at low temperatures (− 20 °C, − 70 °C) and loses its activity by heating at 70 °C. Specific substrates for the isolated protein are l-phenylalanine, l-tryptophan, l-methionine and l-leucine. The enzyme has antibacterial activity inhibiting the growth of Gram-positive (Bacillus subtilis) and Gram-negative (Escherichia coli) bacteria. N. naja oxiana LAAO dose-dependently inhibited ADP- or collagen-induced platelet aggregation with IC₅₀ of 0.094 μM and 0.036 μM, respectively. The antibacterial and anti-aggregating activity was abolished by catalase.     

Enhancement of taxane production in hairy root culture of Taxus x media var. Hicksii

This study assessed the effect of two precursors (l-phenylalanine and p-amino benzoic acid) used alone or in combination with methyl jasmonate, on the growth and accumulation of paclitaxel, baccatin III and 10-deacetylbaccatin III in hairy root cultures of Taxus x media var. Hicksii. The greatest increase in dry biomass was observed after 4 weeks of culturing hairy roots in medium supplemented with 1 μM of l-phenylalanine (6.2 g L⁻¹). Addition of 1 μM of l-phenylalanine to the medium also resulted in the greatest 10-deacetylbaccatin III accumulation (422.7 μg L⁻¹), which was not detected in the untreated control culture. Supplementation with 100 μM of l-phenylalanine together with 100 μM of methyl jasmonate resulted in the enhancement of paclitaxel production from 40.3 μg L⁻¹ (control untreated culture) to 568.2 μg L⁻¹, the highest paclitaxel content detected in the study. The effect of p-amino benzoic acid on taxane production was less pronounced, and the highest yield of paclitaxel (221.8 μg L⁻¹) was observed when the medium was supplemented with 100 μM of the precursor in combination with methyl jasmonate.Baccatin III was not detected under the conditions used in this experiment and the investigated taxanes were not excreted into the medium.     

Interaction between pivaloylcarnitine and l-carnitine transport into L6 cells overexpressing hOCTN2

Patients ingesting pivalic acid containing prodrugs develop hypocarnitinemia. Pivalic acid is cleaved from such drugs and excreted renally as pivaloylcarnitine. Plasma concentrations (reflecting the concentration in the glomerular filtrate entering the proxinmal tubule) in patients treated with cefditoren pivoxil are approximately 5 μM for pivaloylcarnitine and 10 μM for carnitine. Kinetic studies were performed using L6 cells overexpressing the human kidney carnitine transporter (hOCTN2) to assess the mechanisms leading to hypocarnitinemia in such patients. l-carnitine transport showed saturation kinetics (K_m 6.3 μM) and could be inhibited competitively by pivaloylcarnitine (K_i 70 μM). Pivaloylcarnitine was also transported by OCTN2 (K_m 212 μM) and its transport could be inhibited competitively by l-carnitine (K_i 7.8 μM). Haldane and Eadie-Hofstee plots were linear for both carnitine and pivaloylcarnitine. Our data indicate that both carnitine and pivaloylcarnitine bind to OCTN2 at a single, identical site. Considering the low plasma and tubular pivaloylcarnitine concentration, the high K_m of pivaloylcarnitine regarding OCTN2 and the inhibition of pivaloylcarnitine transport by carnitine, pivaloylcarnitine is unlikely to be reabsorbed under these conditions. On the other hand, our data indicate that the renal reabsorption of carnitine is not impaired in patients treated with pivalic acid containing prodrugs. Hypocarnitinemia in such patients therefore develops due to massive renal losses of pivaloylcarnitine and not due to inhibition of carnitine reabsorption by pivaloylcarnitine.     

Inhibitors of bacterial N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) and demonstration of in vitro antimicrobial activity

The dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) is a critical bacterial enzyme for the construction of the bacterial cell wall. A screen biased toward compounds containing zinc-binding groups (ZBG’s) including thiols, carboxylic acids, boronic acids, phosphonates and hydroxamates has delivered a number of micromolar inhibitors of DapE from Haemophilus influenzae, including the low micromolar inhibitor l-captopril (IC₅₀ = 3.3 μM, K_i = 1.8 μM). In vitro antimicrobial activity was demonstrated for l-captopril against Escherichia coli.     

Evaluation of l-glutamine for cryopreservation of boar spermatozoa

The aim of the present study was to evaluate the protective effect of l-glutamine (l-Gln) against cryopreservation injuries on boar sperm. In Experiment 1, l-Gln from 20 to 80 mM was evaluated as a supplement for a standard freezing extender (egg yolk – EY – 20%, and glycerol 3%). No significant improvement (P > 0.05) was obtained for any post-thaw sperm parameter assessed (objective sperm motility – CASA system – and flow cytometric analysis of plasma and acrosomal membrane integrity −SYBR14/PI/PE-PNA− and plasma membrane stability −M540/YoPro1−). In Experiment 2, l-Gln was evaluated as a partial glycerol substitute in the freezing extender. Significant (P < 0.05) enhancement of post-thaw sperm motion parameters was achieved in sperm frozen in the presence of 2% glycerol and 80 mM l-Gln compared to control (3% glycerol). In Experiment 3, l-Gln was evaluated as an EY substitute in the freezing extender, and no functional sperm were recovered after thawing sperm frozen in the presence of l-Gln and the absence of EY. In conclusion, l-Gln has the ability to cryoprotect boar sperm when it is used as a partial glycerol substitute in the freezing extender.     

Development of an interference-free biosensor for l-glutamate using a bienzyme salicylate hydroxylase/l-glutamate dehydrogenase system

An amperometric biosensor was developed for the interference-free determination of l-glutamate with a bienzyme-based Clark electrode. This sensor is based on the specific dehydrogenation by l-glutamate dehydrogenase (GLDH, EC 1.4.1.3) in combination with salicylate hydroxylase (SHL, EC 1.14.13.1). The enzymes were entrapped by a poly(carbamoyl) sulfonate (PCS) hydrogel on a Teflon membrane. The principle of the determination scheme is as follows: the specific detecting enzyme, GLDH, catalyses the specific dehydrogenation of l-glutamate consuming NAD⁺. The product, NADH, initiates the irreversible decarboxylation and the hydroxylation of salicylate by SHL in the presence of oxygen. This results in a detectable signal due to the SHL-enzymatic consumptions of dissolved oxygen in the measurement of l-glutamate. The sensor has a fast steady-state measuring time of 20 s with a quick response (1 s) and a short recovery (1 min). It shows a linear detection range between 10 μM and 1.5 mM l-glutamate with a detection limit of 3.0 μM. A Teflon membrane, which is used to fabricate the sensor, makes the determination to avoid interferences from other amino acids and electroactive substances.     

Spread and persistence of a rugulosin-producing endophyte in Picea glauca seedlings

We have studied Picea glauca (white spruce) endophyte colonization and its affect on the growth of Choristoneura fumiferana (spruce budworm). Here we examine the spread and persistence of a rugulosin-producing endophyte and rugulosin in needles from trees maintained in the nursery, as well as in trees planted in a test field site. Additionally, we report toxicity of rugulosin against three P. glauca needle herbivores: C. fumiferana, Lambdina fiscellaria (hemlock looper) and Zeiraphera canadensis (spruce budmoth). Reduction in body weight for both the C. fumiferana and L. fiscellaria were observed at 25 and 50 μm, respectively, and head capsules were reduced at 100 and 150 μm. Z. canadensis larvae did not perform as well in tests due to an Aspergillus fumigatus infection, but were shown to be lighter when tested with 100 and 150 μm compared with controls. The endophyte and its toxin were shown to spread throughout the nursery-grown seedlings. After 3.5 and 4.5 y post-inoculation (one and two years in the test site), the inoculated endophyte and its toxin had remained present with an average rugulosin concentration of 1 μg g⁻¹.     

Growth inhibitory activity of extracts and compounds from Cimicifuga species on human breast cancer cells

The purpose of this report is to explore the growth inhibitory effect of extracts and compounds from black cohosh and related Cimicifuga species on human breast cancer cells and to determine the nature of the active components. Black cohosh fractions enriched for triterpene glycosides and purified components from black cohosh and related Asian species were tested for growth inhibition of the ER⁻ Her2 overexpressing human breast cancer cell line MDA-MB-453. Growth inhibitory activity was assayed using the Coulter Counter, MTT and colony formation assays.Results suggested that the growth inhibitory activity of black cohosh extracts appears to be related to their triterpene glycoside composition. The most potent Cimicifuga component tested was 25-acetyl-7,8-didehydrocimigenol 3-O-β-d-xylopyranoside, which has an acetyl group at position C-25. It had an IC₅₀ of 3.2 μg/ml (5 μM) compared to 7.2 μg/ml (12.1 μM) for the parent compound 7,8-didehydrocimigenol 3-O-β-d-xylopyranoside. Thus, the acetyl group at position C-25 enhances growth inhibitory activity.The purified triterpene glycoside actein (β-d-xylopyranoside), with an IC₅₀ equal to 5.7 μg/ml (8.4 μM), exhibited activity comparable to cimigenol 3-O-β-d-xyloside. MCF7 (ER⁺Her2 low) cells transfected for Her2 are more sensitive than the parental MCF7 cells to the growth inhibitory effects of actein from black cohosh, indicating that Her2 plays a role in the action of actein. The effect of actein on Her2 overexpressing MDA-MB-453 and MCF7 (ER⁺Her2 low) human breast cancer cells was examined by fluorescent microscopy. Treatment with actein altered the distribution of actin filaments and induced apoptosis in these cells.These findings, coupled with our previous evidence that treatment with the triterpene glycoside actein induced a stress response and apoptosis in human breast cancer cells, suggest that compounds from Cimicifuga species may be useful in the prevention and treatment of human breast cancer.     

Gene cloning, purification, and characterization of α-methylserine aldolase from Bosea sp. AJ110407 and its applicability for the enzymatic synthesis of α-methyl-l-serine and α-ethyl-l-serine

The gene encoding α-methylserine aldolase was isolated from Bosea sp. AJ110407. Sequence analysis revealed that the predicted amino acid sequence encoded by the 1320-bp open reading frame was 65.0% similar to the corresponding sequence of the enzyme isolated from Ralstonia sp. AJ110405. The gene was expressed in Escherichia coli, and the recombinant enzyme was purified. Gel filtration revealed the molecular mass of the purified enzyme to be approximately 78 kDa, suggesting that the enzyme is a homodimer. The enzyme exhibited a specific peak at 429 nm in the spectrum and contained 1 mol pyridoxal 5′-phosphate per mole of the subunit. The V_max value was 1.40 μmol min⁻¹ mg⁻¹, and the K_m value was 1.5 mM for the reaction wherein formaldehyde was released from α-methyl-l-serine. This enzyme could also catalyze the reverse reaction, i.e., the synthesis of α-methyl-l-serine from l-alanine and formaldehyde. This activity was inhibited in the excess of formaldehyde; however, α-methyl-l-serine was efficiently produced from l-alanine in the presence of formaldehyde. This method was also applicable for producing α-ethyl-l-serine from l-2-aminobutyric acid.     

Protease-catalyzed dipeptide synthesis from N-protected amino acid carbamoylmethyl esters and free amino acids in frozen aqueous solutions

The kinetically controlled synthesis of N-benzyloxycarbonyl (Z)-dipeptides was investigated by the use of free amino acids as nucleophiles and a cysteine protease papain as catalyst. The coupling efficiency was significantly improved by the combined use of the carbamoylmethyl (Cam) ester of a Z-amino acid as acyl donor and frozen aqueous solution (ice, −16 or −24 °C) as reaction medium. The yield of peptide synthesis became high when both P₁- and P^′₁-positions were occupied by small non-polar amino acids (Z-Gly-Gly-OH, 76%; Z-Gly-Ala-OH, 75%; Z-Ala-Ala-OH, 72%). Similar results were observed by the use of ficin as catalyst instead of papain. Furthermore, this strategy was applied to the papain-catalyzed incorporation of a d-configured amino acid such as d-alanine into the resulting peptides. Although the coupling in aqueous solution (30 °C) afforded the desired Z-dipeptides in low yields, the freezing of reaction medium reduced significantly unfavorable hydrolysis of the acyl donors, resulting in improvement of the coupling efficiency (Z-Gly-d-Ala-OH, 80%; Z-Ala-d-Ala-OH, 45%; Z-d-Ala-Ala-OH, 22%).     

Availability of tyrosine amide for α-chymotrypsin-catalyzed synthesis of oligo-tyrosine peptides

Oligo-tyrosine peptides such as Tyr-Tyr having angiotensin I-converting enzyme (ACE) inhibitory activity could be synthesized by α-chymotrypsin-catalyzed reaction with l-tyrosine ethyl ester in aqueous media. However, peptide yield in the reaction was below 10%. Since l-tyrosine amide showed highly nucleophilic activity for the deacylation of enzyme through which a new peptide bond was made, its application to the enzymatic peptide synthesis was evaluated in this study. Addition of tyrosine amide into the reaction produced Tyr-Tyr-NH₂, of which yield exceeded 130% on the basis of tyrosine ethyl ester. Although purified Tyr-Tyr-NH₂ did not inhibit ACE activity, α-chymotrypsin could act on the dipeptide amide and convert about 40% of it to Tyr-Tyr. The use of both ester and amide forms of tyrosine is expected to be a potent procedure for α-chymotrypsin-catalyzed synthesis of antihypertensive peptides.     

Structure of the oligosaccharides isolated from Dalbergia sissoo Roxb. leaf polysaccharide

A water-soluble polysaccharide isolated from Dalbergia sissoo Roxb. leaves was purified and major homogeneous fraction obtained by GPC. Complete hydrolysis of the polysaccharide followed by paper chromatography and GLC analysis indicated the presence of l-rhamnose, d-glucuronic acid, d-galactose and d-glucose in molar ratio of 1:1:2:2.33, respectively. Partial hydrolysis of the polysaccharide furnished one tri-[I], one hepta-[II] and one nona-[III] saccharides. Hydrolysis of the oligosaccharide I, II and III followed by GLC analysis furnished d-glucose and l-rhamnose (2:1); l-rhamnose, d-galactose and d-glucuronic acid (1:3:3); and l-rhamnose, d-galactose and d-glucose (1:3:5), respectively. Methylation analysis and periodate oxidation of the oligosaccharide I indicated the presence of two non reducing glucose units linked to rhamnose by 1→2 and 1→4 linkages, respectively. Oligosaccharide II is a branched molecule with a main chain consisting of 1,3-linked β-d-galactopyranosyl (2 mol), 1,3,4 linked α-l-rhamnopyranosyl (1 mol) and 1,4,6 linked β-d-galactopyranosyl unit (1 mol) and non reducing β-d-glucuronic acid at the end along with side chains of β-d-glucouronopyranosyl units (2 mol). Oligosaccharide III is also a branched molecule with a main chain consisting of 1,3,4 linked α-l-rhamnopyranosyl (1 mol), 1,2,4 linked β-d-glucopyranosyl (1 mol), 1,3 and 1,4 linked β-d-galactopyranosyl (2 and 1 mol, respectively) having β-d-glucopyranosyl as a non reducing end.     

N-Acetylhexosamine triad in one molecule: Chemoenzymatic introduction of 2-acetamido-2-deoxy-β-d-galactopyranosyluronic acid residue into a complex oligosaccharide

A complex trisaccharide β-d-GalpNAcA-(1 → 4)-β-d-GlcpNAc-(1 → 4)-d-ManpNAc (3) was prepared in a good yield (35%) in a transglycosylation reaction catalyzed by β-N-acetylhexosaminidase from Talaromyces flavus using p-nitrophenyl 2-acetamido-2-deoxy-β-d-galacto-hexodialdo-1,5-pyranoside (1) as a donor followed by the in situ oxidation of the aldehyde functionality by NaClO₂. The disaccharide β-d-GlcpNAc-(1 → 4)-d-ManpNAc (2) was used as galactosyl acceptor. A disaccharide β-d-GalpNAcA-(1 → 4)-d-GlcpNAc (4; 39%) originated as a by-product in the reaction. Oligosaccharides comprising a carboxy moiety at C-6 are shown to be very efficient ligands to natural killer cell activation receptors, particularly to human receptor CD69. Thus, oxidized trisaccharide 3 is the best-known oligosaccharidic ligand to this receptor, with IC₅₀ = 2.5 × 10⁻⁹ M. The presented method of introducing a β-d-GalpNAcA moiety into carbohydrate structures is versatile and can be applied in the synthesis of other complex oligosaccharides.     

Morphological and structural characterization of a polysaccharide from Gynostemma pentaphyllum Makino and its anti-exercise fatigue activity

The crude polysaccharide was obtained from Gynostemma pentaphyllum Makino by water extraction followed by ethanol precipitation. The polysaccharide was successively purified by chromatography on DEAE-52 and SephadexG-150 column, and three polysaccharide fractions were obtained and termed GPP1-a, GPP2-b, and GPP3-a, respectively. The administration with GPP1-a markedly prolonged exhaustive exercise time of the mice. Structural features of GPP1-a were investigated by a combination of instrumental and chemical analyses, including atomic force microscope (AFM), scanning electron microscope (SEM), partial acid hydrolysis, periodate oxidation, Smith degradation, methylation analysis, gas chromatography–mass spectrometry (GC–MS) analysis and NMR spectroscopy. The results indicate that GPP1-a has a backbone of (1 → 4)-linked α-d-Glucose residues, which occasionally branches at O-6. The branches are mainly composed of (1 → 6)-linked α-d-Glucose, (1 → 3)-linked β-d-Galactose and (1 → 6)-linked α-d-Galactose residues, and terminated with β-d-Galactose residues and β-l-Arabinose residues.     

Screening of microorganisms producing α-methylserine hydroxymethyltransferase, purification of the enzyme, gene cloning, and application to the enzymatic synthesis of α-methyl-l-serine

Through the screening of microorganisms capable of utilizing α-methylserine, three representative strains belonging to the bacterial genera Paracoccus, Aminobacter, and Ensifer were selected as potent producers of α-methylserine hydroxymethyltransferase, an enzyme that catalyzes the interconversion between α-methyl-l-serine and d-alanine via tetrahydrofolate. Among these strains, Paracoccus sp. AJ110402 was selected as the strain exhibiting the highest α-methylserine hydroxymethyltransferase activity. The enzyme was purified to homogeneity from a cell-free extract of this strain. The native enzyme is a homodimer with apparent molecular mass of 85 kDa and contains 1 mol of pyridoxal-5′-phosphate per mol of the subunit. The K_m for α-methyl-l-serine and tetrahydrofolate was 0.54 mM and 73 μM, respectively. The gene from Paracoccus sp. AJ110402 encoding α-methylserine hydroxymethyltransferase was cloned and expressed in Escherichia coli. Sequence analysis revealed an open reading frame of 1278 bp, encoding a polypeptide with a calculated molecular mass of 46.0 kDa. Using E. coli cells as whole-cell catalysts, 9.7 mmol of α-methyl-l-serine was stereoselectively obtained from 15 mmol of d-alanine and 13.2 mmol of formaldehyde.     

Mechanisms of catalysis and inhibition operative in the arginine deiminase from the human pathogen Giardia lamblia

Giardia lamblia arginine deiminase (GlAD), the topic of this paper, belongs to the hydrolase branch of the guanidine-modifying enzyme superfamily, whose members employ Cys-mediated nucleophilic catalysis to promote deimination of l-arginine and its naturally occurring derivatives. G. lamblia is the causative agent in the human disease giardiasis. The results of RNAi/antisense RNA gene-silencing studies reported herein indicate that GlAD is essential for G. lamblia trophozoite survival and thus, a potential target for the development of therapeutic agents for the treatment of giardiasis. The homodimeric recombinant protein was prepared in Escherichia coli for in-depth biochemical characterization. The 2-domain GlAD monomer consists of a N-terminal domain that shares an active site structure (depicted by an in silico model) and kinetic properties (determined by steady-state and transient state kinetic analysis) with its bacterial AD counterparts, and a C-terminal domain of unknown fold and function. GlAD was found to be active over a wide pH range and to accept l-arginine, l-arginine ethyl ester, N^α-benzoyl-l-arginine, and N^ω-amino-l-arginine as substrates but not agmatine, l-homoarginine, N^α-benzoyl-l-arginine ethyl ester or a variety of arginine-containing peptides. The intermediacy of a Cys424–alkylthiouronium ion covalent enzyme adduct was demonstrated and the rate constants for formation (k₁ = 80 s⁻¹) and hydrolysis (k₂ = 35 s⁻¹) of the intermediate were determined. The comparatively lower value of the steady-state rate constant (k_cat = 2.6 s⁻¹), suggests that a step following citrulline formation is rate-limiting. Inhibition of GlAD using Cys directed agents was briefly explored. S-Nitroso-l-homocysteine was shown to be an active site directed, irreversible inhibitor whereas N^ω-cyano-l-arginine did not inhibit GlAD but instead proved to be an active site directed, irreversible inhibitor of the Bacillus cereus AD.     

Production and characterization of soluble human lysosomal enzyme α-iduronidase with high activity from culture media of transgenic tobacco BY-2 cells

Lysosomal storage diseases (LSDs), that collectively represent over 50 disorders, are amenable to enzyme replacement therapies. However, the current methods used to commercially produce recombinant lysosomal enzymes for this purpose, most commonly Chinese Hamster Ovary cells and human fibroblasts, are prohibitively costly. Plant bioreactors hold great promise for economic production of functional human α-l-iduronidase (hIDUA; glycosaminoglycan α-l-iduronohydrolase; EC 3.2.1.76), the enzyme deficient in the human LSD, Mucopolysaccharidosis I. We have developed and tested an expression system using transgenic tobacco BY-2 cells to produce high amounts of active hIDUA. A plant signal peptide was essential for proper expression and secretion of the 78 kDa glycosylated hIDUA into the cultured media of transgenic BY-2 cells. The yield and activity of the secreted hIDUA from long-term cultures of transgenic BY-2 cell lines were as high as 10 μg/mL media and 53,000 pmol/min/mg proteins, respectively. Thus, this transgenic BY-2 cell line presents an attractive platform for economic production and easy downstream purification of hIDUA for enzyme replacement therapy. Furthermore, this system can be used for the production and purification of other human lysosomal enzymes or pharmaceuticals.     

A novel biosensor based on l-homocysteine desulfhydrase enzyme immobilized in eggshell membrane

A novel biosensor for homocysteine determination has been developed. The biosensor was fabricated with l-homocysteine desulfhydrase immobilized on the ammonium selective electrode by means of eggshell membrane. The measurement principle is based on determination of ammonia due to the enzymatic reaction in the medium by ammonium selective electrode. The effects of enzyme loading, glutaraldehyde concentration, pH, buffer concentration, temperature, dithiotreitol (DTT) concentration and ionic strength adjustment buffer (ISA) on the biosensor response were investigated in detail. The linear detection range and limit of detection (LOD) for homocysteine were found to be 0.15–1.8 mM and 55 μM, respectively. Finally, the homocysteine biosensor has been applied to plasma samples for determination of total homocysteine contents.     

Synthesis and antitumor activity of new d-seco and d-homo androstane derivatives

Starting from 3β-hydroxy-17-oxo-16,17-secoandrost-5-ene-16-nitrile (1), the new 16,17-secoandrostane derivatives 4–9 were synthesized. On the other hand, 3β-hydroxy-17-oxa-d-homoandrost-5-ene-16-one (10) yielded the new d-homo derivatives 12, 13 and 15. In vitro antiproliferative activity of selected compounds against three tumor cell lines (human breast adenocarcinoma ER+, MCF-7, human breast adenocarcinoma ER−, MDA-MB-231, prostate cancer AR−, PC-3, and normal fetal lung fibroblasts, MRC-5) was evaluated. Compounds 3 and 12 showed strong antiproliferative activity against PC-3 cells, the IC₅₀ values being 2 μM and 0.55 μM, respectively. Compounds 6 (10 μM) and 14 (9 μM) showed moderate activity against MDA-MB-231 cells. The synthesized compounds 1–3, 5–8, 10 and 12–15 were not toxic to normal fetal lung fibroblasts cells, MRC-5.