Mapping the receptor binding regions of human chorionic gonadotropin (hCG) using disulfide peptides of its beta-subunit: possible involvement of the disulfide bonds Cys(9)-Cys(57) and Cys(23)-Cys(72) in receptor binding of the hormone.

Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone essential for the establishment and maintenance of pregnancy. The alpha- and beta-subunits of hCG are highly cross-linked internally by disulfide bonds which seem to stabilize the tertiary structures required for the noncovalent association of the subunits to generate hormonal activity. The purpose of this study was to delineate the role of the disulfide bonds of hCGbeta in receptor binding of the hormone. Six disulfide peptides incorporating each of the six disulfide bonds of hCGbeta were synthesized and screened, along with their linear counterparts, for their ability to competitively inhibit the binding of [125I] hCG to sheep ovarian corpora luteal LH/CG receptor. Disulfide peptide Cys (9-57) was found to be approximately 4-fold more potent than the most active of its linear counterparts in inhibiting radiolabeled hCG from binding to its receptor. Similarly, disulfide peptide Cys (23-72) exhibited receptor binding inhibition activity, whereas the constituent linear peptides were found to be inactive. The results suggest the involvement of the disulfide bonds Cys(9)-Cys(57) and Cys(23)-Cys(72) of the beta-subunit of hCG in receptor binding of the hormone. This study is the first of its kind to use disulfide peptides rather than linear peptides to map the receptor binding regions of hCG.     

Forecasting the flight activity of Lobesia botrana (Denis & Schiffermüller) (Lepidoptera,Tortricidae) in Southwestern Spain

This study aimed at elaborating a forecasting tool of the phenology of the serious pest Lobesia botrana in Southwestern Spanish vineyards, by analysing data on male catches in sex pheromone traps recorded over a 12‐year period. Our data confirmed the minor importance of the first generation which appears during flowering time, both in terms of male trap catches and damage of L. botrana to the inflorescences. Therefore, data related to the first flight were not further processed, although they were considered for the computation of degree‐days of the following generations. The outcome of the elaboration of temperature accumulations and data on male captures for the second and third flights was a statistically acceptable linear behaviour obtained by properly transforming the variables. The models established proved to be efficient and may represent a useful tool to improve the efficacy of integrated pest management strategies targeting L. botrana in the studied region.     

Prenatal growth of long bones in rhesus and squirrel monkeys (Macaca mulatta and Saimiri sciureus)

Fetal long bone growth was studied from 59 radiographs representing 35 macaques and 79 radiographs representing 16 squirrel monkeys. From lateral abdominal radiographs of pregnant females total lengths of long bone shadows were measured to the nearest millimeter with a sliding caliper. A linear regression line was fitted to the data for each species. The high correlation coefficients (min. = 0.92) indicated not only that over 80% of the variance in longbone length was associated with the regression but also that linear regression was an acceptable model. It was determined that the macaque long bones grew more rapidly prenatally than the squirrel monkey long bones. Saimiri long bones grew at a faster rate during the second half of gestation than would be expected considering that the macaque long bones were approximately twice the length of Saimiri long bones at birth and that the duration of their gestation was about the same. Thus squirrel monkeys must achieve a greater percentage of their birth size during the second half of gestation.     

Facile entry to (-)-(R)- and (+)-(S)-mexiletine

The title compounds, 1a and 1b, have been synthesized in a three-step sequence starting from (-)-(S) and (+)-(R)-propylene oxide, respectively, in acceptable overall yields. The enantiomeric excess values for 1a and 1b were 96% and 93% respectively, as assessed by HPLC analysis on a chiral stationary phase of the corresponding N-acetyl derivatives. The synthetic route herein presented may represent a facile entry to highly enriched mexiletine enantiomers, alternative to those previously reported in the literature.     

Transport and Partitioning of Lead in Indian Mustard (Brassica juncea) and Wheat (Triticum aestivum)

Lead (Pb) contamination in soils is a serious concern because it can be taken up by crops and then transferred through the food chain, posing a potential risk to human health. Indian mustard (Brassica juncea) and wheat (Triticum aestivum) are important crop species known to accumulate heavy metals in their tissues. This study aimed at understanding the transport and accumulation of Pb in these two species and the risk associated with consumption of these foods, which would help us in mitigating accumulation of Pb in edible tissues. The plants were grown at different Pb concentrations for the entire life cycle, and the partitioning of the metal to different tissues was examined. The results showed that plant species differ widely in their ability to transport and accumulate Pb in different tissues. In B. juncea, there was significant accumulation of Pb in both siliques and seeds, whereas most of the Pb in wheat was concentrated in the vegetative tissues and less to the flag leaf and reproductive tissues. In both species, although seed Pb concentrations exceeded acceptable limits, dietary intake did not exceed acceptable limits in most treatments, indicating that more studies on Pb transport and redistribution in crop species is necessary.     

Influence of polymer architecture and molecular weight of poly(2-(dimethylamino)ethyl methacrylate) polycations on transfection efficiency and cell viability in gene delivery

Nonviral gene delivery with the help of polycations has raised considerable interest in the scientific community over the past decades. Herein, we present a systematic study on the influence of the molecular weight and architecture of poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) on the transfection efficiency and the cytotoxicity in CHO-K1 cells. A library of well-defined homopolymers with a linear and star-shaped topology (3- and 5-arm stars) was synthesized via atom transfer radical polymerization (ATRP). The molecular weights of the polycations ranged from 16 to 158 kDa. We found that the cytotoxicity at a given molecular weight decreased with increasing number of arms. For a successful transfection a minimum molecular weight was necessary, since the polymers with a number-average molecular weight, M(n), below 20 kDa showed negligible transfection efficiency at any of the tested polyelectrolyte complex compositions. From the combined analysis of cytotoxicity and transfection data, we propose that polymers with a branched architecture and an intermediate molecular weight are the most promising candidates for efficient gene delivery, since they combine low cytotoxicity with acceptable transfection results.     

Identification of linear plasmid pAM1 in the flavonoid degrading strain Actinoplanes missouriensis(T) (DSM 43046)

By pulsed-field gel electrophoresis, a linear DNA element of about 100 kb was identified in Actinoplanes missouriensis(T) DSM 43046, which grows on the flavonoids hesperidin, rutin and quercetin, and which contains a CO forming quercetinase. Among six Actinoplanes species and strains tested, including A. globisporus(T) DSM 43857, A. philippinensis(T) DSM 43019, A. brasiliensis(T) DSM 43805, A. auranticolor(T) DSM 43031, and A. utahensis(T) DSM 43147, only the A. missouriensis strain exhibited such a genetic element. The linear plasmid, named pAM1, has proteins covalently attached to its 5'-ends like other linear replicons of actinomycetes. Attempts to cure pAM1 failed, however a mutant with reduced plasmid content was obtained, which showed reduced ability to degrade the flavonoid rutinosides rutin and hesperidin. Plasmid pAM1 is the first extrachromosomal genetic element identified in an Actinoplanes species and may be useful to develop genetic tools for biotechnologically important Actinoplanes strains.     

Evacuation of pelleted feed and the suitability of titanium(IV) oxide as a feed marker for gut kinetics in Nile tilapia

The present study assessed the suitability of titanium(IV) oxide, TiO₂, as a digesta passage marker in Nile tilapia Oreochromis niloticus and studied the shape of the evacuation curve in this species. In three separate trials, fish were given one dose of either 0·5, 0·25 or 0·1% of their body mass (% BME) of feed marked with 1% TiO₂ or 0·5% BME of the same feed without marker. The fish were serially slaughtered at intervals after feeding and the stomach contents analysed for dry mass and marker content. The data for individual trials were analysed with the linear, square root, surface area and exponential evacuation models and parameter comparisons showed that, although the marker interfered slightly with the evacuation process, true meal size could be predicted more accurately from the marker data. The results of an analysis of the combined data sets suggested that stomach evacuation in this species is dependent more on food particle surface area (surface area model) than on stomach content mass (exponential model) as is generally assumed. On the basis of these results, it was concluded that TiO₂ at an inclusion level of 1% is an acceptable marker for quantifying evacuation with a view to predicting food consumption but should be used with caution in digestibility studies.     

In vitro biotransformations of the prostaglandin D2 (DP) antagonist MK-0524 and synthesis of metabolites

Metabolites of the potent DP antagonist, MK-0524, were generated using in vitro systems including hepatic microsomes and hepatocytes. Four metabolites (two hydroxylated diastereomers, a ketone and an acyl glucuronide) were characterized by LC-MS/MS and 1H NMR. Larger quantities of these metabolites were prepared by either organic synthesis or biosynthetically to be used as standards in other studies. The propensity for covalent binding was assessed and was found to be acceptable (<50 pmol-equiv/mg protein).     

Lanthanide ion catalysis of the trans-cis isomerization of trans-bis(oxalato)-diaquochromate(III) and trans-bis(malonato)diaquochromate(III) [1]

The lanthanide ion catalyzed trans-cis isomerizations of trans-bis(oxalato)diaquochromate(II) and trans-bis(malonato)diaquochromate(III) have been studied. A linear free energy relationship was found correlating the catalytic rate constants for the oxalate reaction with the corresponding formation constants of complexes formed between simple monocarboxylic acids and the light (LaGd) members of the lanthanide series. The results indicates that for this portion of the series, the reaction mechanism is related to the formation of monocarboxylate complex intermediates. When the ionic radius of the lanthanide ion decreases below a particular value (as in the latter half of the series), the metal ion remains coordinated to both carboxylates of the oxalate ion rather than simply binding to only one carboxylate. In either situation, isomerization to the cis product eventually occurs, and the lanthanide ion is released.The reaction rates associated with the trans-bis(malonato)diaquochromate(III) reaction were found to be significantly slower than those of the corresponding oxalate system. However, in the malonate system, no linear free energy relationship was found relating the catalytic rate constants with the corresponding formation constants of monocarboxylic acids. One does find a linear relationship between the catalytic rate constants for the malonate reaction and the log K₁ values for the corresponding lanthanide/malonate complexes. During the course of the trans-cis isomerization, the lanthanide ion chelates the dissociated malonate group of a pentavalent Cr(III) intermediate. In the mechanism the lanthanide ion does not aid in ring opening, and neither does it singly bond to the intermediate     

Cadmium ion-doped magnetic poly(styrene-acrylic acid) nanospheres for sensitive electrochemical immunoassay

A novel class of molecular tags, cadmium ion-doped magnetic poly(styrene-acrylic acid) nanospheres (Cd-MPSA), was first synthesized and functionalized with polyclonal rabbit anti-human luteinizing hormone antibodies (PAb(2)) for highly efficient electrochemical immunoassay of luteinizing hormone (LH). Transmission electron microscope (TEM) and Fourier transform infrared spectroscope (FTIR) were employed to characterize the prepared Cd-MPSA. By using Cd-MPSA-labeled PAb(2) as molecular tags, a novel sandwich-type immunoassay protocol was built for determination of LH on monoclonal mouse anti-human luteinizing hormone antibody (MAb(1))-functionalized gold electrode. The assay was carried out in pH 5.3 HAc-NaAc buffer solution by square wave voltammetry (SWV). The signal was obtained by the reduction of the doped cadmium ions in the Cd-MPSA. Under optimal conditions, the currents increased with the increasing LH level in the sample, and exhibited a linear range from 0.25 to 240 mIU mL(-1) with a detection limit of 0.08 mIU mL(-1) LH at 3s(B). The precision, reproducibility, and specificity were acceptable. No obvious difference was encountered in the analysis of spiking LH samples into newborn calf serum with the referenced values.     

Ternary Cu(II) complexes, Cu(H(-1)L)(ACys-) and Cu(H(-2)L)(ACys-); L = peptides, ACys- = N-acetyl-cysteinate. Analogous complexes to the intermediates in the transport of Cu(II) from Cu(H(-2)L) to cysteine

The Cu(II) in Cu(H(-2)L) has been postulated to be successively transported to cysteine (Cys) as follows; Cu(H(-2)L) <==> Cu(H(-2)L)(Cys*-) <==> Cu(H(-1)L)(Cys*-) --> Cu(H(-1)L)(Cys-), where Cys*- denotes the monodentate Cys-. N-acetyl-cysteinate (ACys-) complexes Cu(H(-2)L)(ACys-) and Cu(H(-1)L)(ACys-), having similar coordination modes to Cu(H(-2)L)(Cys*-) and Cu(H(-1)L)(Cys*-), respectively, exhibited the S --> Cu(II) charge transfer absorption at 325-355 nm and the d-d absorption at 530-610 nm. A linear interrelation existed between the energies of the CD and d-d absorptions. Cu(H(-2)L)(ACys-) were in rapid equilibrium with Cu(H(-1)L)(ACys-). Upon forming the ternary complex, pK(c2) of the parent Cu(H(-1)L) was raised to more than 1.0. The formation constants (K) of the Cu(H(-1)L)(ACys-) species from Cu(H(-1)L) were bigger than those of Cu(H(-2)L)(ACys-) from Cu(H(-2)L). The linear free-energy relationship existed between the free-energy change (deltaG) and the entropy change (deltaS) for the ternary complex formation. The rate constants (k1+) for the Cu(H(-1)L)(Cys-) formation closely correlated with the K values for Cu(H(-2)L)(ACys-). The ternary complexes containing ACys are considered to be analogous complexes to the intermediates in the transport of Cu(II) from peptides to cysteine.     

Comparison between the G-banded karyotype of the aoudad (Ammotragus lervia) and sheep (Ovis aries)

Karyotypes of the aoudad and sheep were compared on the basis of G-banded chromosomes at the 450 band level. The common G-banded karyotype showed the homology of all aoudad chromosomes (2n=58) with sheep chromosomes (2n=54) or sheep chromosome arms. The results of cytogenetic investigations suggest that in this case karyotype evolution has led to reduction in chromosome number as a result of centric fusions. The formation of the first metacentric chromosome occurred in the aoudad. The homology of the G-banding pattern in sheep and aoudad suggests the conservation in linear arrangement of genetic material. Thus comparative cytogenetics can be a useful tool in gene mapping.     

Chondrogenic properties of human periosteum-derived progenitor cells (PDPCs) embedded in a thermoreversible gelation polymer (TGP)

Periosteum-derived progenitor cells (PDPCs) were isolated using a fluorescence-activated cell sorter and their chondrogenic potential in biomaterials was investigated for the treatment of defective articular cartilage as a cell therapy. The chondrogenesis of PDPCs was conducted in a thermoreversible gelation polymer (TGP), which is a block copolymer composed of temperature-responsive polymer blocks such as poly(N-isopropylacrylamide) and of hydrophilic polymer blocks such as polyethylene oxide, and a defined medium that contained transforming growth factor-β3 (TGF-β3). The PDPCs exhibited chondrogenic potential when cultured in TGP. As the PDPCs-TGP is an acceptable biocompatible complex appropriate for injection into humans, this product might be readily applied to minimize invasion in a defected knee.     

Assessing the efficiency of urban waste biocomposting by analytical pyrolysis (Py-GC/MS)

Analytical pyrolysis (Py-GC/MS) was used to study complex composting processes. The technique was first validated for reproducibility and finally applied to assess the efficiency of a microbial bio-accelerator product (CBB) in composting organic residues with different composition. Fresh lignocellulosic and urban wastes were treated with CBB and the composting kinetics studied to investigate the transformations undergone in the course of biocomposting. Our findings demonstrate that these changes, as well as the efficiency of CBB, can be monitored through the molecular characterization of the released pyrolysis products. The CBB bacterial product effectively seems to favour composting accelerating the process and shorten composting time. Analytical pyrolysis was informative in assessing to which extent compost transformation reached an acceptable stabilization point. The technique could be also developed into a semi-quantitative tool to monitor changes of main organic matter components (polysaccharides, proteins, lignin, lipids, etc.) as composting proceeds.     

Discovery of N-benzyl-N'-(4-pipyridinyl)urea CCR5 antagonists as anti-HIV-1 agents (II): modification of the acyl portion

Modification of the acyl moiety in the CCR5 lead molecule 2 led to identification of several new classes of CCR5 antagonists. Antiviral activity and pharmacokinetic properties of the synthesized compounds were evaluated. Structure-activity relationship (SAR) derived from these studies further guided the optimization efforts, ultimately leading to the discovery of 36 with an acceptable drug-like profile.     

Right- and left-handed (Z) helical conformations of the hairpin d(C-G)5T4(C-G)5 monomer and dimer

The partial self-complementary 24-mer oligodeoxynucleotide d(C-G)5T4(C-G)5 forms a hairpin which can be enzymatically dimerized to a dumbbell structure. The blunt-ended nature of the hairpin is indicated by its ability to inhibit the T4 DNA ligase catalyzed joining of phi X174 HaeIII fragments. The hairpin monomer and dimer (dumbbell) undergo a reversible B to Z transition as shown by ultraviolet, circular dichroism, and 31P NMR spectroscopy. The Z form of the hairpin monomer and dimer is supported by monovalent ions (Na+), divalent ions (Mg2+ but not Mn2+), and dehydrating (ethanol) conditions. The conformational transition of d(C-G)5T4(C-G)5 monomer requires higher ionic or dehydrating conditions than those necessary for the corresponding linear oligomer d(C-G)5. The contribution of the loop (-T4-) of the hairpin to the apparent free energy change for the B to Z conformational transition at the midpoint was calculated to be 3.8 kJ mol-1.