Risperidone effects on heterochromatin: the role of kinase signaling.

Epigenetic effects of anti-psychotic medications are poorly understood. We have appropriated a model whereby heterochromatin is established through 24- or 48-h lipopolysaccharide (LPS) treatment, and tested the epigenetic effects of risperidone along the adenylyl cyclase/protein kinase A (AC/PKA) pathway in human liposarcoma cells that express the LPS-sensitive Toll-like receptor (TLR)-4. Human SW872 cells were cultured with LPS and mRNA expression levels and epigenetic modifications of dimethylated lysine 9 of histone 2 (H3K9me2), geterochromatin protein 1γ (HP1γ) and phospho-H3S10 at promoters of interleukin (IL)-6, tumor necrosis factor (TNF)-α and IL1ß were measured. Pharmacological manipulation of the AC/PKA pathway was achieved through treatment with a PKA inhibitor (H89), mitogen- and stress-activated kinase 1 (MSK1) inhibitor (SB-747651A) or forskolin. Twenty-four and 48-h LPS treatment establishes heterochromatin at selected promoters, corresponding to decreased mRNA expression. Concurrent risperidone treatment with LPS treatment can both 'block' and 'reverse' heterochromatin formation. Forskolin treatment resulted in a similar disassembling effect on heterochromatin. Conversely, inhibition of PKA by H89 or MSK1 both blocked 'normalizing' effects of risperidone on LPS-induced heterochromatin. Our results demonstrate that risperidone can disassemble heterochromatin, exerting this effect along the G-protein/AC/PKA pathway. This approach can also be utilized to investigate functional outcomes of single or combined pharmacological treatments on chromatin assemblies in human cells.

Tuning of the surface plasmon resonance of aluminum nanoshell near-infrared regimes.

The natural abundance of aluminum is far better than those of noble metals like silver and gold. Its plasmonic properties have not been explored in detail, as they generally lie in the ultraviolet region of the electromagnetic (EM) spectrum. Nevertheless, recent advanced research work in the literature predicts aluminum to be the next and best plasmonic material suitable for exciting cost-effective applications. Inspired by this, we have analyzed the optical properties of aluminum metal nanoshells using semi-analytical and numerical finite difference time domain models. In the semi-analytical model, we have discussed an electrostatic approach for nanoshell geometry and derived the exact Frolich condition and corresponding plasmon resonance peak position. These plasmonic peaks and their tunability in different regimes of the electromagnetic spectrum via the shell thickness are the main thrust of this work. For proof of concept, we have compared simulated and modeled results in different parameter domains and observed that both sets of results are in agreement with each other. We have also visualized the distribution of the electric field intensity near the aluminum nanoshell surface under SPR conditions, which covers a broad range of applications.

Nonlinear laser pulse response in a crystalline lens.

The propagation characteristics of a spatial Gaussian laser pulse have been studied inside a gradient-index structured crystalline lens with constant-density plasma generated by the laser-tissue interaction. The propagation of the laser pulse is affected by the nonlinearities introduced by the generated plasma inside the crystalline lens. Owing to the movement of plasma species from a higher- to a lower-temperature region, an increase in the refractive index occurs that causes the focusing of the laser pulse. In this study, extended paraxial approximation has been applied to take into account the evolution of the radial profile of the Gaussian laser pulse. To examine the propagation characteristics, variation of the beam width parameter has been observed as a function of the laser power and initial beam radius. The cavitation bubble formation, which plays an important role in the restoration of the elasticity of the crystalline lens, has been investigated.

Optical forces and torques on realistic plasmonic nanostructures: a surface integral approach.

We develop a novel formalism to calculate the optical forces and torques on complex and realistic nanostructures by combining the surface integral equation (SIE) technique with Maxwell's stress tensor. The optical force is calculated directly on the scatterer surface from the currents obtained from the SIE, which does not require an additional surface to evaluate Maxwell's stress tensor; this is especially useful for intricate geometries such as plasmonic antennas. SIE enables direct evaluation of forces from the surface currents very efficiently and accurately for complex systems. As a proof of concept, we establish the accuracy of the model by comparing the results with the calculations from the Mie theory. The flexibility of the method is demonstrated by simulating a realistic plasmonic system with intricate geometry.

Effect of multiphoton ionization on performance of crystalline lens.

This Letter presents a model for propagation of a laser pulse in a human crystalline lens. The model contains a transverse beam diffraction effect, laser-induced optical breakdown for the creation of plasma via a multiphoton ionization process, and the gradient index (GRIN) structure. Plasma introduces the nonlinearity in the crystalline lens which affects the propagation of the beam. The multiphoton ionization process generates plasma that changes the refractive index and hence leads to the defocusing of the laser beam. The Letter also points out the relevance of the present investigation to cavitation bubble formation for restoring the elasticity of the eyes.

Toward the identification of peripheral epigenetic biomarkers of schizophrenia.

Schizophrenia (SZ) is a heritable, nonmendelian, neurodevelopmental disorder in which epigenetic dysregulation of the brain genome plays a fundamental role in mediating the clinical manifestations and course of the disease. The authors recently reported that two enzymes that belong to the dynamic DNA methylation/demethylation network-DNMT (DNA methyltransferase) and TET (ten-eleven translocase; 5-hydroxycytosine translocator)-are abnormally increased in corticolimbic structures of SZ postmortem brain, suggesting a causal relationship between clinical manifestations of SZ and changes in DNA methylation and in the expression of SZ candidate genes (e.g., brain-derived neurotrophic factor [BDNF], glucocorticoid receptor [GCR], glutamic acid decarboxylase 67 [GAD67], reelin). Because the clinical manifestations of SZ typically begin with a prodrome followed by a first episode in adolescence with subsequent deterioration, it is obvious that the natural history of this disease cannot be studied only in postmortem brain. Hence, the focus is currently shifting towards the feasibility of studying epigenetic molecular signatures of SZ in blood cells. Initial studies show a significant enrichment of epigenetic changes in lymphocytes in gene networks directly relevant to psychiatric disorders. Furthermore, the expression of DNA-methylating/demethylating enzymes and SZ candidate genes such as BDNF and GCR are altered in the same direction in both brain and blood lymphocytes. The coincidence of these changes in lymphocytes and brain supports the hypothesis that common environmental or genetic risk factors are operative in altering the epigenetic components involved in orchestrating transcription of specific genes in brain and peripheral tissues. The identification of DNA methylation signatures for SZ in peripheral blood cells of subjects with genetic and clinical high risk would clearly have potential for the diagnosis of SZ early in its course and would be invaluable for initiating early intervention and individualized treatment plans.

Heterochromatin as an incubator for pathology and treatment non-response: implication for neuropsychiatric illness.

Heterochromatin is a higher order assembly that is characterized by a genome-wide distribution, gene-repression, durability and potential to spread. In this light, it is an appealing mechanism to interpret the neurobiology of complex brain disorders such as schizophrenia where downregulation of expression appears to be the norm. H3K9 methylation (H3K9me) can initiate the seeding of a heterochromatin assembly on an inactive or poorly coordinated promoter as a consequence of a decline in transactivators either from disuse or from misuse. H3K9me can extend its influence by spatial spreading through the mechanism of recursively recruiting adapters, such as heterochromatin protein 1 (HP1) homodimers. HP1 itself serves as a platform for other repressive proteins such as DNA methyltransferases. In full color, heterochromatin can occupy genome-wide gene networks, tissue specific ontologies and even rearrange the nuclear architecture. Heterochromatin in the brain is modified by small molecule pharmacology and serves a physiological role in the functioning of dopamine neurons and the construction of memory. From a therapeutic perspective, the durable nature of heterochromatin implies that it may require disassembly before the full genomic-potential of standard pharmacotherapies is achieved, especially in treatment resistant patients.

Polymorphism in the prolactin promoter and its association with growth traits in chickens.

The pituitary hormone prolactin has a wide variety of functions involving growth, behavioral, and ovarian activities in chickens. The objectives of the present study were to identify polymorphisms in the prolactin promoter and estimate their effects on growth traits in White Leghorn chickens. Among 28 haplotypes found, the h1 haplotype was predominant. Body weight at 16 and 64 weeks and age at sexual maturity were significantly associated with haplotype combinations (P < 0.05). The h1/h1 haplogroup showed the highest body weight at 16 weeks of age, and h1/h7 was the highest at 64 weeks. The lowest age at sexual maturity was found in birds with the h1/h6 haplotype combination, and mRNA expression of prolactin was lowest in h1/h4 birds and highest in h1/h5 birds. The prolactin promoter was highly polymorphic and had significant associations with growth traits in White Leghorn chickens.

Identification of haplotypes in promoter of prolactin gene and their effect on egg production and quality traits in layer chicken.

Expression of prolactin hormone is a crucial event in regulating egg production in chickens for which promoter plays the vital role in expressing the prolactin gene. The objective of the present study was to identify haplotypes in the prolactin promoter and their effects on egg production and egg quality traits in White Leghorn chicken. Single stranded conformation polymorphism followed by sequencing was conducted to explore polymorphism at 561 bp promoter of prolactin gene. The effect of haplotype combinations on egg production and quality traits were estimated following general linear model technique. The expression of prolactin by different haplogroups was quantified by qPCR. Total 28 haplotypes were found in White Leghorn chicken of which h1 haplotype possessed the highest frequency of 0.46 and h8, h14, h16, h25, h26, and h28 haplotypes had the lowest frequency (0.1%). The egg production up to 52 and 64 weeks of age were found to be significantly (p < 0.05) associated with haplotype combinations where the highest 52-w (52 weeks) egg production was found in animals with h1/h22 combination and the lowest production was observed in the birds with h1/h2 haplogroup. The haplotype combinations had the significant effect (p < 0.05) on Haugh Unit, yolk index and albumen weight at 40 weeks of age; Haugh Unit and albumen weight at 52 weeks of age and Haugh unit, yolk weight and yolk percentage at 64 weeks of age. The prolactin expression in h1/h22 birds was found to be the lowest and in h1/h5 birds to be the highest. The prolactin expression showed significant effect on 52-w egg production and albumin weight at 52 weeks age. In conclusion, it may be stated that the prolactin promoter was highly polymorphic and had the significant association with egg production and quality traits in White Leghorn chicken.

Association of polymorphism in the prolactin promoter and egg quality traits in laying hens.

Single strand conformation polymorphism analysis and DNA sequencing was performed in White Leghorn hens to explore the polymorphisms present in the promoter of the prolactin gene. The effects of different genotypes on egg production and quality traits were determined, and expression of the prolactin gene in different genotypes was quantified by real time-PCR. Five genotypes and four alleles at each of two Fragments of the promoter were found, of which the FG genotype in Fragment 1 and the PQ genotype in Fragment 2 were the most predominant genotypes. The genotypes of Fragment 1 had significant effects (P < 0·05) on Haugh unit, albumen weight, albumen percentage and shell percentage at 40 weeks of age; egg weight and yolk index at 52 weeks of age; and egg weight at 64 weeks of age. Prolactin expression in the genotypes of Fragment 1 differed significantly and GH genotyped birds had the highest level of expression. The genotypes of Fragment 2 did not show any significant differences of expression. It was concluded that the prolactin gene promoter was highly polymorphic, and had significant effects on egg quality traits in White Leghorn hens.

Genetic analysis of juvenile growth and carcass traits in a full diallel mating in selected colored broiler lines.

The juvenile growth and carcass traits were evaluated in a 4 × 4 full diallel crossing involving four colored broiler chicken lines viz., Naked neck (NN), Dwarf, Punjab Broiler-1 (PB-1), and Punjab Broiler-2 to study the performance and crossbreeding parameters. The data on 2,280 chicks were analyzed using least squares techniques to assess the effect of genetic group, and the significant traits were further analyzed for crossbreeding parameters. Genetic group had significant influence on the body weights and carcass traits. The cross of PB-1 × NN recorded significantly (p ≤ 0.05) higher body weight at 6 weeks of age. The data revealed that general combining ability (GCA), specific combining ability (SCA), maternal ability (MA), sex-linked effect (SE), and purebred effects significantly influenced the growth and carcass traits in the broiler crosses. The crossbred PB-1 × NN showed positive heterosis for growth as well as carcass traits. The results suggest that GCA, SCA, SE, and MA are important in the inheritance of the body weights and carcass traits indicating the presence of both additive and nonadditive genetic variation along with reciprocal effects. Therefore, pure line selection along with development of specialized sire and dam lines followed by crossing will improve the growth performance and benefit the poultry farmers. It is concluded that PB-1 as a male line and NN as a female line performed significantly higher, therefore, may be used for improving the performance of colored broilers.

Comparison of aggregated exposure to di(2-ethylhexyl) phthalate from diet and personal care products with urinary concentrations of metabolites using a PBPK model - Results from the Norwegian biomonitoring study in EuroMix.

Phthalates are widely used as plasticisers in flexible plastics and containers for food and personal care products (PCPs) and contaminates foods and PCPs. A human biomonitoring (BM) study was performed to study exposure of chemicals from foods and PCPs. For two 24-h periods, adult volunteers (n = 144) in Norway kept diaries on food eaten and usage of PCPs, and collected 24-h urine. Aggregated exposure to di(2-ethylhexyl) phthalate (DEHP) from dietary and PCPs was estimated by Monte-Carlo simulation using Oracle Crystal Ball©. Simulated urinary concentrations using physiologically based pharmacokinetic (PBPK) models were compared with measured urinary metabolites of DEHP, mono-2-ethylhexyl phthalate (MEHP), mono-2-ethyl-5-hydroxyhexyl phthalate (MEHHP), mono-2-ethyl-5-oxohexyl phthalate (MEOHP) and mono-2-ethyl 5-carboxypentyl phthalate (MECCP). DEHP exposure from food are approximately 10 times higher than exposure than from PCPs. The main contributors to dietary exposure are dairy, grain, fruits and vegetables, meat and fish. Body lotion contribute most to the exposure of DEHP from PCPs. Forward-dosimetry gives good convergence with 24-h urinary concentrations of simulated and measured BM data. The measured concentration of the MECCP metabolite correlated well with simulated high exposure, while the measured concentrations of MEHP, MEHHP and MEOHP partly overlapped with both simulated low, medium and high metabolite exposure.

Performance and bone mineralisation in broiler chicks fed on diets with different concentrations of cholecalciferol at a constant ratio of calcium to non-phytate phosphorus.

1. An experiment was conducted with broiler female chicks (720) to study the effects of graded concentrations (75, 15, 225 or 30 microg/kg) of cholecalciferol (CC) in diets containing varying levels of calcium (Ca) and non-phytate phosphorus (NPP) at a 2:1 ratio (4:2, 5:25, 6:3 or 7:35 g/kg, respectively), on the performance (2-35 d of age), bone mineralisation and mineral (Ca, P, Mn, Fe, Cu) concentration in excreta. 2. Body weight gain, food intake, tibia density and tibia ash increased, and leg abnormality score decreased with dietary increase of CC from 75 to 30 microg at 4 g Ca and 2 g NPP. However, this improvement was not comparable with the birds receiving the highest concentrations of CC, Ca and NPP (30 microg, 7 g and 3.5 g, respectively/kg diet). 3. Significant improvements in the majority of parameters noted with increasing CC up to 225 microg at 5 g Ca and 25 g NPP/kg, which was comparable to those fed the highest levels of CC, Ca and NPP. 4. Concentrations of Ca, P, Mn, Fe and Cu in excreta decreased significantly with increasing CC at all Ca:NPP ratios tested. 5. The predicted requirement of CC for most of the parameters ranged between 1625 and 25 microg/kg diet at 5 g Ca and 25 g NPP. 6. Considering the performance, bone mineralisation, and mineral concentration in excreta, it can be concluded that Ca and NPP levels in broiler diet could be reduced to 5 and 25 g, respectively, while maintaining CC at 25 microg/kg.

Evaluation of the performance and emission and spectroscopic analysis of an improved soy methyl ester.

Herein, the performance, emission and physiochemical properties of a soy methyl ester (SME) and its combination with methyl oleate (MO) in a common rail direct injection (CRDI) engine were investigated. Moreover, the performance of the engine in terms of brake power (BP), brake thermal efficiency (BTE), brake specific fuel consumption (BSFC) and NO x emission was assessed to compute the characteristics of the combination of SME with MO; the reasons for using MO in the blending process were the better ignition quality, oxidative stability and low-temperature flow properties of MO, striking a balance between oxidative stability and low flow properties. A remarkable reduction in the BSFC and an increase in the BTE were found in the blended biodiesel (S80-MO20, S70-M30, and S50-M50) as compared to the case of SME. The blended biodiesel was also characterized by Fourier transform infrared spectroscopy (FTIR), ultraviolet-visible spectroscopy (UV-vis) and nuclear magnetic resonance (NMR) spectroscopy. In the FTIR spectra, a peak was observed at 1745 cm-1, confirming the presence of a triglyceride ester linkage. Since UV-vis spectroscopy is an affordable technique, herein, it has been employed to detect the presence of conjugated dienes in the oxidized biodiesel. The linear line fitted for absorbance versus the percentage of the blended fuel at 320 nm showed a high coefficient of determination (R 2 = 0.9454). In addition, H NMR spectroscopy was employed to study the oxidative stability of the blended fuel. Different functional groups with their respective peaks (in ppm) are indicated in the NMR spectra.

Utilisation of sesame (Sesamum indicum) seed meal in broiler chicken diets.

1. Soybean meal (SBM) was replaced with sesame seed meal (SSM) at proportions of 0.00, 0.33, 0.67 and 1.00 on a protein basis in broiler diets (2 to 42 d of age). Each diet was fed to 8 replicates of 5 birds each. 2. Body weight gain, food efficiency, ready-to-cook (RTC) yield, relative weights of giblet, liver and abdominal fat were not affected by including SSM up to 0.67 proportion of SBM in starter and finisher diets (360 and 310 g/kg, respectively). The relation between level of SSM and growth performance variable (weight gain, food efficiency and RTC yield) was non-linear. 3. Weight, breaking strength and ash content of tibia decreased non-linearly with the proportion of SSM in diet. 4. The activity of alkaline phosphatase and protein concentration in serum decreased linearly with the amount of SSM in the diet.

An EMS-induced new sequence variant, TEMS5032, in the coding region of SRS3 gene leads to shorter grain length in rice (Oryza sativa L.).

Grain shape and size influence yield and consumer preferences in rice. In the present study, we characterized and mapped a short and bold grained mutant and named it as TEMS5032, as the mutant is a result of EMS-induced transition from C to T at the 5032nd bp of SRS3 gene, which is known to affect grain size in rice. The substitution led to creation of a stop codon in the motor domain of SRS3, a kinesin 13 family gene, translating into a truncated protein product. However, transcription of this gene remained unaffected in TEMS5032 compared to the wild type, N22. Further, the mutation was found to affect 13 of the 25 cell cycle-related genes as they showed differential expression with respect to N22. Based on rate of grain filling, dry matter accumulation in the endosperm and histological studies, the effect of mutation in TEMS5032 was found to be similar to a known variant, TCM758, but less severe than sar1 mutant. Sequencing of 88 rice germplasm lines in the kinesin motor domain region did not reveal the presence of this mutation, establishing it as a new variant of SRS3 gene.

Whole Genome Characterization of a Few EMS-Induced Mutants of Upland Rice Variety Nagina 22 Reveals a Staggeringly High Frequency of SNPs Which Show High Phenotypic Plasticity Towards the Wild-Type.

The Indian initiative, in creating mutant resources for the functional genomics in rice, has been instrumental in the development of 87,000 ethylmethanesulfonate (EMS)-induced mutants, of which 7,000 are in advanced generations. The mutants have been created in the background of Nagina 22, a popular drought- and heat-tolerant upland cultivar. As it is a pregreen revolution cultivar, as many as 573 dwarf mutants identified from this resource could be useful as an alternate source of dwarfing. A total of 541 mutants, including the macromutants and the trait-specific ones, obtained after appropriate screening, are being maintained in the mutant garden. Here, we report on the detailed characterizations of the 541 mutants based on the distinctness, uniformity, and stability (DUS) descriptors at two different locations. About 90% of the mutants were found to be similar to the wild type (WT) with high similarity index (>0.6) at both the locations. All 541 mutants were characterized for chlorophyll and epicuticular wax contents, while a subset of 84 mutants were characterized for their ionomes, namely, phosphorous, silicon, and chloride contents. Genotyping of these mutants with 54 genomewide simple sequence repeat (SSR) markers revealed 93% of the mutants to be either completely identical to WT or nearly identical with just one polymorphic locus. Whole genome resequencing (WGS) of four mutants, which have minimal differences in the SSR fingerprint pattern and DUS characters from the WT, revealed a staggeringly high number of single nucleotide polymorphisms (SNPs) on an average (16,453 per mutant) in the genic sequences. Of these, nearly 50% of the SNPs led to non-synonymous codons, while 30% resulted in synonymous codons. The number of insertions and deletions (InDels) varied from 898 to 2,595, with more than 80% of them being 1-2 bp long. Such a high number of SNPs could pose a serious challenge in identifying gene(s) governing the mutant phenotype by next generation sequencing-based mapping approaches such as Mutmap. From the WGS data of the WT and the mutants, we developed a genic resource of the WT with a novel analysis pipeline. The entire information about this resource along with the panicle architecture of the 493 mutants is made available in a mutant database EMSgardeN22 (http://14.139.229.201/EMSgardeN22).

Development and Genetic Characterization of A Novel Herbicide (Imazethapyr) Tolerant Mutant in Rice (Oryza sativa L.).

BACKGROUND: Increased water and labour scarcity in major rice growing areas warrants a shift towards direct seeded rice cultivation under which management of weeds is a major issue. Use of broad spectrum non-selective herbicides is an efficient means to manage weeds. Availability of rice genotypes with complete tolerance against broad-spectrum non-selective herbicides is a pre-requisite for advocating use of such herbicides. In the present study, we developed an EMS induced rice mutant, 'HTM-N22', exhibiting tolerance to a broad spectrum herbicide, 'Imazethapyr', and identified the mutations imparting tolerance to the herbicide. RESULTS: We identified a stable and true breeding rice mutant, HTM-N22 (HTM), tolerant to herbicide, Imazethapyr, from an EMS-mutagenized population of approximately 100,000 M2 plants of an upland rice variety, Nagina 22 (N22). Analysis of inheritance of herbicide tolerance in a cross between Pusa 1656-10-61/HTM showed that this trait is governed by a single dominant gene. To identify the causal gene for Imazethapyr tolerance, bulked segregant analysis (BSA) was followed using microsatellite markers flanking the three putative candidate genes viz., an Acetolactate Synthase (ALS) on chromosome 6 and two Acetohydroxy Acid Synthase (AHAS) genes, one on chromosomes 2 and another on chromosome 4. RM 6844 on chromosome 2 located 0.16 Mbp upstream of AHAS (LOC_Os02g30630) was found to co-segregate with herbicide tolerance. Cloning and sequencing of AHAS (LOC_Os02g30630) from the wild type, N22 and the mutant HTM and their comparison with reference Nipponbare sequence revealed several Single Nucleotide Polymorphisms (SNPs) in the mutant, of which eight resulted in non-synonymous mutations. Three of the eight amino acid substitutions were identical to Nipponbare and hence were not considered as causal changes. Of the five putative candidate SNPs, four were novel (at positions 30, 50, 81 and 152) while the remaining one, S627D was a previously reported mutant, known to result in Imidazolinone tolerance in rice. Of the novel ones, G152E was found to alter the hydrophobicty and abolish an N myristoylation site in the HTM compared to the WT, from reference based modeling and motif prediction studies. CONCLUSIONS: A novel mutant tolerant to the herbicide "Imazethapyr" was developed and characterized for genetic, sequence and protein level variations. This is a HTM in rice without any IPR (Intellectual Property Rights) infringements and hence can be used in rice breeding as a novel genetic stock by the public funded organizations in the country and elsewhere.

Use of methanethiolation to investigate the catalytic role of sulphydryl groups in rabbit skeletal muscle pyruvate kinase.

Incubation of rabbit skeletal muscle pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) with methyl methanethiosulphonate resulted in the time- and inhibitor concentration-dependent loss of enzyme activity. Substrates or products of the catalytic reaction prevented the loss of activity caused by methanethiolation. Their effectiveness as protecting agents was placed in the order ADP greater than ATP greater than Mg2+ greater than phosphoenolpyruvate greater than pyruvate. The essential catalytic cation, K+, had no effect on the methanethiolation reaction. [Me-3H]Methanethiosulphonate modified all the available cysteine thiol groups which correlated to the incorporation of four SC3H3 groups per protomer. Four radioactive peptides were obtained on tryptic peptide mapping. When methanethiolation was carried out in the presence of Mg2+ alone or with Mg2+ and ATP together, then only three SC3H3 groups were incorporated into each subunit. If MgATP protected methanethiolated pyruvate kinase was reacted with iodo[2-3H]acetic acid then 1.37 +/- 0.2 groups per protomer were carboxymethylated. 70% of the radioactivity was located in a single peptide on tryptic peptide mapping. This peptide was isolated and contained the segment carboxymethyl cysteine (Glx, Asx, Ser) Arg. Collectively these data indicate that although all thiol groups are equally accessible to methyl methanethiosulphonate, only a single thiol group participates in the catalytic event. An additional role in the maintenance of structure for this thiol group was also shown in studied of reduction and thermal denaturation of the enzyme.

Modulation of the receptor binding affinity of amphiregulin by modification of its carboxyl terminal tail.

Amphiregulin (AR), a heparin-binding, epidermal growth factor (EGF) receptor ligand has homology with EGF but exhibits a lower affinity for the EGF receptor than EGF. As the mature form of AR is truncated at the C terminus and lacks a conserved leucine residue known to be essential for high affinity binding of EGF to the EGF receptor, wild-type AR (AR1-84), a C-terminally extended AR construct incorporating six residues from the predicted coding sequence of AR (AR1-90) and a similarly extended construct with a Met86 to Leu substitution (AR1-90(leu86)) were expressed as recombinant proteins in yeast, purified by heparin affinity and C18 reverse phase chromatography and their relative biological activities determined. The growth factors were tested in mitogenesis and EGF receptor autophosphorylation assays and their relative order of potencies was found to be leu86 > met86 > wt. The AR1-90(leu86) construct was found to be 50- to 100-fold more active than wild type AR1-84 consistent with previously reported studies of the role of the equivalent C-terminal leucine in EGF or TGF alpha. Significantly, the C-terminally extended form of AR, AR1-90, which utilized six residues from the predicted coding sequence, was 10-times more active than wild type AR1-84. This difference in activity of the C-terminally extended form of AR may be of biological significance since differential proteolytic processing of the AR precursor in vivo could result in production of multiple forms of the growth factor with differing affinities for the EGF receptor and hence differing biological potencies.