Heritable trait variation is that due to genetic variation. Heritability refers to the proportion of trait variation that can be attributed to genetic factors. Genetic correlation refers to the proportion of total genetic variation in two traits that is shared. Sexual selection refers to a mode of natural selection in which certain alleles are favored over others because of their effects on acquiring

mates rather than survival. Alleles are alternative versions of genetic variants at a given locus. Mutation load refers to an individual’s aggregate burden of deleterious mutations (rare alleles) across the genome, which is heritable IDH assay across generations. Good gene indicators are traits that reflect underlying genetic fitness, for example low mutation load. Pleiotropic genes influence more than one trait. Cross-trait assortative mating occurs when two different Selleck Ku-0059436 traits correlate across mates, for example males of above-average height mating with females of above-average intelligence. Extended twin-family designs take advantage of the genetic relatedness between multiple

family members, for example, twins, their spouses, and their parents, in order to investigate the importance of environmental and genetic influences on one or multiple traits. Sexual dimorphism refers to the difference between male and female phenotypes. Fisher’s Fundamental Theorem states that ‘the rate of increase in fitness of any organism at any time is equal to its genetic variance in fitness at that time.’ It has often been interpreted to mean that

additive genetic variation should be low in traits related to fitness. Phenotypes are observable characteristics or traits of an organism. Recessive/additive/dominant refer to how likely an allele is to be expressed in the phenotype. At a diallelic locus, a fully recessive allele will not be expressed unless both copies are present, while the fully dominant allele will be fully expressed with only one copy. Many dominance relationships are partial rather than full, yielding a spectrum of dominance or recessivity. Additivity is intermediate between fully recessive and fully dominant. SNP (single-nucleotide polymorphism) is a type of allele where a single-nucleotide PtdIns(3,4)P2 position is variable in the population. Often, the term ‘SNP’ is used for loci where the minor allele frequency is >1% and ‘mutation’ when the minor allele frequency is <1%. Homozygosity occurs when two copies of the same allele are present at a locus, as opposed to heterozygosity, in which the two alleles at a locus are different. Runs of homozygosity are stretches of contiguous SNPs (e.g. 60+) that are consistently homozygous along some stretch of an individual’s genome. Linkage studies test for coinheritance of alleles and traits within families.

Blood was obtained with informed consent. From six subjects, plasma was also prepared from blood in heparin or citrate vials (Becton Dickinson) and peripheral blood mononuclear cells (PBMC) were obtained by Ficoll separation of heparinized blood

samples. PBMC (3 × 106 cells/ml) supernatants were collected after 2 days culture in AIM-V serum free medium (Gibco) at 37 °C in 5% CO2. Animal samples used were rhesus and cynomolgus macaque plasma (from the Swedish Center for Disease Control, Solna Sweden), and serum from cow and horse (Gibco), mouse and rat (Sigma) and goat (Jackson ImmunoResearch, Suffolk, UK). All samples were stored at − 20 °C until tested. For analysis in ELISA, samples were used as such or were treated with 1 M HCl (50 μl acid/100 μl plasma or serum; 20 μl acid/100 μl PBMC supernatant) Selleckchem PFT�� at RT for 10 min followed by addition of 1 M NaOH with 0.5 M Hepes (50 μl for plasma or serum; 20 μl for PBMC supernatant). The relationship between observed Seliciclib concentration levels of analytes in the LAP and TGF-β1 ELISA was evaluated by Spearman rank correlation (Analyse-it Software Ltd., Leeds, UK). Twenty mAbs obtained from mice immunized with Latent TGF-β1 all reacted with LAP1 and Latent TGF-β1 but

not TGF-β1 in indirect ELISA. Combinations of all mAbs were evaluated in capture ELISA. MAb MT593 together with MT517-biotin yielded the best detection of LAP1 and Latent TGF-β1 with no reactivity with TGF-β1 (Fig. 2A). A TGF-β1 ELISA used in parallel displayed the opposite reactivity pattern recognizing only TGF-β1 (Fig. 2B). CHO-K1 cells were transfected with plasmids encoding LAP isoforms and a GFP reporter. In flow cytometry, all plasmids yielded GFP + transfected cells. Expression of LAP was confirmed using a mAb to the C-terminal His6-tag in all LAP isoforms. The frequency of Interleukin-2 receptor GFP + His6+ cells ranged from 8 to 16% with a background < 1% in mock transfectants (Fig. 3A). A similar frequency of LAP1 + transfected cells was found in ELISpot, utilizing

the LAP ELISA mAbs, whereas the other transfectants were negative (data not shown). Purified LAP1 migrated as an 80 kD homodimer in SDS-PAGE and could be reduced to monomers (Fig. 3B). An additional LAP-reactive mAb obtained, MT324, yielded similar results in Western blot (Fig. 3B). Analysis of cell supernatants (Fig. 3C) and lysates (Fig. 3D) from LAP1, -2, − 3 and mock transfectants in the LAP ELISA confirmed a specificity restricted to LAP1. Also the individual reactivity of the LAP ELISA mAbs and mAb MT324, the only mAb functional in Western blot, with LAP1, -2 and − 3 CHO cell supernatants was analyzed. All three mAbs were specific for LAP1 with MT517 displaying the strongest, and MT324 the weakest, reactivity (Fig. 4).

This training was generally once off, with little in-service training, refresher training

or course updates provided [29], [32] and [33]. In relation to the content of the training, a client centred problem management approach, historically characterized training for HCT in South Africa [35]. More recently, there has been training in behaviour change counselling (BCC) to reduce risk behaviour and selleck compound improve adherence, using variations of the Information, Motivation and Behavioural Skills (IMB) model [26], [35], [36], [44], [45], [46], [47], [48], [49], [50] and [54]. The need for training to be expanded beyond HCT and BCC to include screening and counselling for mental disorders, especially depression was identified by a number of studies [39][29], [32] and [33]. The inclusion of stress reduction techniques and coping skills to help lay counsellors manage job stressors was identified by one study [27]. Several studies reveal that support and supervision of lay counsellors in routine care is generally poor [29], [32], [33], [34], [38] and [39]. Two independent reviews over a decade apart [38] and [39] found that anywhere from a quarter [38] to one third [39] of organizations reviewed provide any form of structured supervision and support. Where supervision and support is provided, there also appears to

be little distinction between supervision and debriefing [39]. Given the tendency for lay counsellors to EPZ5676 cell line resort to advice giving, regular supervision in micro-counselling skills (attending behaviour and basic skills that facilitate listening and exploration to achieve understanding of a problem) was suggested by one study [37]. Given the stressors associated with counselling, a number of studies recommend the need for psychological support structures to improve quality and prevent burn-out [29], [33] and [34]. Poor role definition and lack of clear pathways for advancement for lay counsellors emerged from a number of studies [31], [32], [33] and [40]. Lay counsellors feel excluded from the professional hierarchy and are often Inositol monophosphatase 1 treated as an extra resource at primary health facilities, being expected to perform

multiple tasks over and above their counselling duties [33], wherever there is a need. These tasks include administration, taking vital signs, doing home visits [33], as well as tasks that should be the responsibility of the professional nurse, e.g., conducting CD4 counts, providing feedback about the results, and issuing medication [32] and [40]. This poor role definition impacts negatively on how lay counsellors are perceived by other health care staff, as well as their own self-perception. Several studies found that lay counsellors do not feel appreciated or accepted as part of the health care team by other health care staff [29], [31] and [33] and also held a negative perception of their own roles [31] and [33] resulting in poor work engagement and burn-out [27].

Guangzhou is Y-27632 cell line the largest city of southern China and the third largest Chinese city. As of 2010, the city’s administrative area had a population of 12.8 million, making Guangzhou the most populous southern city. Not established until 1979, when it was no more than a market town situated on the border with the then British colony of Hong Kong, Shenzhen has become one of the largest cities in the Pearl River delta as well

as the largest manufacturing base in the world. Today, this Special Economic Zone (SEZ) is the 10th most populous city in China with some 10.4 million residents. Some estimates place the population surrounding the Pearl River Delta Economic Zone, which can today be referred to as a Mega City – the world’s first megalopolis – at 40 million including Shenzhen (>10 million), Dongguan (>8 million), Foshan (>7 million), Jiangmen (>4 million) and Zhongshan (>3 million). In 2008, Guangzhou alone was identified as a Beta World City by the Globalization and World Cities Research Network. If, therefore, Guangzhou itself, sitting at the head of the Pearl’s estuary, is included we can estimate a delta-wide urban population of some 150 million people. This

does not, however, include either the gambling city of Macau, until 1999 a former Portuguese colony, with a resident population of ∼0.5 million (but a much greater transient one) nor, until 1997, the former British see more colony of Hong Kong, which with a population of >7.2 million people (and a transient one of >126 million), is classified as an Alpha + City. With a land area of only 1104 km2, the Special Administrative Region of Hong Kong’s lack of space has created the world’s most vertical city. Kowloon, with a population density of ∼44,000 km2 ranks as one of, if not the, most dense human conurbations ever known. The Pearl River’s delta 6-phosphogluconolactonase today, therefore,

is probably home to, conservatively, over 160 million people but growth has not yet ended. Regional goals for 2020 include the development of two or three new cities, the expansion of road, rail, seaport and airport infrastructures and the construction of the 50 km long Hong Kong–Zhuhai–Macau Bridge – across the Pearl currently traversed by thousands of ferries each day. Since the end of the last ice age, sea levels have risen in southern China by over 10 m (but were even higher in the Early Holocene) so that the Pearl River’s delta contains hundreds of little islands (former mountain tops), there being some 235 within Hong Kong’s 1650 km2 territorial waters alone. And, because of the vast amounts of silt deposited by the river, estimated at ∼86 million tonnes each year, the estuary’s flanks are bordered (or used to be) everywhere by mangrove stands – these lie close to the northern limits of the component species’ ranges.

3B). There is also a direct link between ER stress and TNF-α. Silencing of ATF4 and CHOP prevented the upregulation of TNF-α in cells [7]. Similarly, the induction of TNF-α was observed in human bronchial epithelial cells after exposure to titanium dioxide nanoparticles [44]. ZnO-NPs induced the expression of TNF-α in human keratinocytes. The up-regulation of TNF-α was dependent on the activation of the extracellular signal-regulated kinase (ERK) of MAPK pathways

[24]. TNF-α belongs to the group of proinflammatory cytokines involved in the pathogenesis of several diseases including cancer [32], rheumatoid arthritis, diabetes and inflammatory bowel disease [45]. TNFα is known as an endogenous tumor promoter [19]. Therefore, chronic human HER2 inhibitor exposure to SiO2-NPs may ultimately result Fulvestrant in vivo in adverse effects on human health. Our data further corroborate on previous results the induction of ER stress by SiO2-NPs [12]. We therefore hypothesise that ER stress and up-regulation of UPR may be considered as a more general effect induced by nanoparticles. Chronic and severe ER stress results in the activation of apoptotic pathways. Expression of CHOP, an important proapoptotic marker gene, is induced by ATF-4. CHOP itself induces the expression of the apoptotic genes BIM (member of the Bcl-2 family) and p53 upregulated modulator of apoptosis (PUMA). The IRE1 pathway may induce apoptosis by the

activation of the apoptosis signalling kinase 1 (ASK1) and through interaction with tumor necrosis factor-associated factor 2 (TRAF2). Therefore, Vasopressin Receptor SiO2-NPs may show hepatotoxic activity through ER stress and induction of UPR. Another important gene transcript up-regulated in response to ER stress is Noxa [36], which induces apoptosis by the Usp9x-Mcl-1 pathway [47]. This could also contribute to the hepatotoxic action of SiO2-NPs. Constant ER stress contributes to the development of the metabolic syndrome, is linked with hepatic steatosis and ER stress also inhibits hepatic lipoprotein secretion [18], [27] and [34]. UPR activation including eIF2α phosphorylation and splicing of XBP-1 mRNA was detected during adipogenesis. [40]. Additionally, the UPR plays

also a role in cancer development. Activation of ATF-4 is critical for tumor cell proliferation and tumor growth [48]. The IRE1α-XBP-1 pathway is important for tumor cell survival and growth [5]. Therefore, it is conceivable that chronic exposure to SiO2-NPs may result in the induction of these alterations in the liver. P53 is important for apoptosis, genomic stability, DNA repair, inhibition of angiogenesis and inhibition of growth by stopping the cells cycle in the G1/S phase. In case of irreversible DNA damage, p53 leads to induction of apoptosis [2]. In more than 50% cancers the p53 protein is either absent or non-functional due to various other reasons [16]. We found a significant down-regulation of p53 in Huh7 cells after exposure to SiO2-NPs ( Fig. 4B).

Isso permitiu‐lhes escolher uma metodologia mais correta para análise dos resultados e chegar a conclusões diferentes do trabalho de Arena e de Mederacke et al.18 and 19. Tal como os 2 autores anteriormente referidos, Caetano et al. verificaram um aumento muito ligeiro da dureza

hepática 30‐60 minutos após a ingestão de uma refeição standard. Mas o impacto na decisão clínica foi Forskolin clinical trial nulo, pois as diferenças pré e pós‐prandiais não fizeram variar o estádio da fibrose como demonstraram na tabela 3. Quanto ao trabalho de Arena et al. 18 foram estudados doentes com hepatite C crónica submetidos a biopsia hepática, mas não foi incluído um grupo controlo. É um estudo multicêntrico, realizado por numerosos operadores de diferentes hospitais, o que é um fator de enviesamento que pode diminuir a qualidade dos exames. Verificou‐se mais uma vez a preferência pela utilização de testes não paramétricos para avaliação da distribuição dos resultados Z VAD FMK no mesmo indivíduo, desconhecendo‐se o porquê desta opção. Neste trabalho a variabilidade pré e pós‐ingestão de alimentos foi proporcional ao estádio da fibrose, sendo mais acentuada nos doentes com cirrose hepática. Contudo, as diferenças não foram suficientes para avaliar o grau de gravidade da cirrose, nomeadamente

predizer a presença ou não de varizes esofágicas. Os resultados de Arena et al. 18 foram discrepantes dos de Mederacke et al. 19, já que estes não encontraram variabilidade pós‐prandial da elasticidade hepática nos doentes com valores > 10 kPa. Contudo, particularmente nos doentes com cirrose hepática, a sua avaliação mais detalhada poderá ter um valor potencial na avaliação do prognóstico da cirrose. Contudo, para uma correta avaliação do seu valor potencial são necessários mais estudos, uma vez que os trabalhos atualmente publicados têm múltiplas

Thalidomide limitações que vão desde o poder da amostra, ausência de biopsia hepática, ausência de informação sobre o tipo de sonda utilizada na elastografia, escolha de diferentes pontes de corte na diferenciação dos diferentes estádios de fibrose, omissão da variabilidade intra e interobservador, não utilização da IQR/M na análise dos resultados, metodologia inadequada e ausência de informação sobre a influência de terapêutica concomitante que interfira na dinâmica da circulação portal. Podemos assim concluir que a avaliação da elasticidade hepática é um processo dinâmico que obriga a uma análise individual clínica e laboratorial concomitante, de modo a valorizar a influência de outros fatores no resultado deste exame. A elasticidade hepática pode diminuir após a ingestão de alimentos, mas o seu impacto na prática clínica ainda não está cientificamente provado para recomendar que o FS© seja efetuado em jejum a todos os doentes.

Comparison of the fluorescence of oil-in-water emulsion with light scattering was a significant aspect of this research (see Figure 5). The fluorescence intensity is always R428 less than the intensity of scattered radiation, even in ultraviolet spectral areas, where fluorescence is the most significant. While fluorescence, though less intensive, is comparable with the scattering of ultraviolet radiation, the difference between the intensities of these phenomena is more

than an order of magnitude for light of wavelength longer than 400 nm and increases with increasing wavelength for any kind of oil. Consequently, fluorescence makes only a small contribution to the scattering flux of the visible light coming from an emulsion. The above remarks refer to the situation where the intensity of the illuminating radiation of any wavelength is equal – this follows directly from the fact that the intensity of light scattered or fluoresced by an emulsion is

measured in relation to the intensity of the illuminating radiation. These remarks are all the more valid for emulsion illuminated by solar light. Taking into consideration the spectrum of the solar radiation reaching the Earth’s surface (Dera 2003), one can assume that fluorescence plays a negligible part in the radiation Olaparib scattered in an emulsion. The possible quenching of fluorescence by dissolved oxygen does not change this conclusion. Oxygen is natural component of seawater, and the saturation of subsurface water often

exceeds 100% and is greater than the saturation of the samples tested. The results are limited to scattering 3-mercaptopyruvate sulfurtransferase at right angles, but this does not alter the above conclusion. An oil-in-water emulsion is an isotropic medium and its fluorescence does not depend on the angle of illumination, in contrast to scattering. The index of scattering reaches a minimum at 90° and observations at an angle other than 90° will cause fluorescence to be even less than the scattered radiation. These comments refer to the scattering of unpolarized light. Illumination of an emulsion with polarized radiation causes the scattering-to-fluorescence ratio to be different. The results can be summed up as follows: • Emulsions fluoresce in the spectral region from 260 to > 400 nm; the range of fluorescence and shapes of the spectra depend on the kind of oil. These investigations lead to the following conclusions concerning natural seawater containing emulsified petroleum: 1. The measurement and modelling of ultraviolet radiation scattering require the fluorescence of an emulsion to be taken into consideration. “
“A – characteristic area of plume cross-section Effluent transport phenomena in the aquatic environment are interdisciplinary problems (Fischer et al. 1979).

The current study investigated whether trait ratings of the speakers’ body movements are coupled to the amount of applause or hecklings the speakers received throughout their entire speech. We thus intended to demonstrate that people make sense of parsimonious nonverbal cues and that judgments based on such cues can serve

as predictors of behavioral outcomes in a real life setting of high ecological validity. Other “thin slices” studies have already linked job performances or election results to certain behaviors or the appearance of a person. Such variables, however, provide no insight into the direct impact of nonverbal cues on human communication. In contrast to that our research not only focused on body motion but also examined its relationship to behavioral responses that occur in

a direct interaction between an audience KU-57788 mw and a speaker. We provide evidence that motion cues, Vorinostat chemical structure indeed, reflect socially relevant information that affects behavioral responses arising in interpersonal communication processes. To sum up, by using trait ratings as predictors of real life outcomes (i.e., audience reactions) we show that people not only read meaning into body motion but also infer relevant social information from it. We randomly selected 60 speeches (30 male and 30 female) from three parliamentary sessions of the German parliament. From these speeches, we extracted brief, randomly chosen video segments with an average length of 15 s. To create stick-figure movies of the speakers’ performances, we used the computer program SpeechAnalyzer that enabled us to run through a movie frame by frame and to position landmarks on the speakers’ major joints and their Ureohydrolase heads (Koppensteiner, 2013 and Koppensteiner and Grammer, 2010). To capture body movements these landmarks were repositioned according to the position shifts of a speaker’s body. Thus, landmark positions were translated into time series of two dimensional coordinates on which basis we created

stick figure movies we used for our rating experiments. At locations throughout the University of Vienna we recruited 60 persons (33 females and 27 males; age M = 22.5 years, SD = 3.7) for the stick figure rating experiment. Participants performed the rating task on their own using a computer-controlled interface. Stimuli were presented on the left-hand side of the user interface; rating scales with the items dominant, trustworthy, and competent and items from a German version of a brief questionnaire measuring the Big Five personality domains (i.e., Ten-Item Personality Inventory, TIPI) were presented on the right hand side ( Gosling et al., 2003 and Muck et al., 2007). The scales were divided into 200 subunits with 0 indicating strongly disagree and 200 strongly agree. Each participant rated a subset of 20 randomly selected stick figure animations.

, 2001). Gallic acid ester derivatives, such as octyl and dodecyl gallates, showed an inhibitory potency on protein kinase activity, which results in a 50–250

times greater apoptosis induction than that of its precursor gallic acid for various human cell lines tested, indicating a selectivity for fast-growing cells. These findings support the study of octyl and dodecyl gallates as potential anticancer agents. It was shown that octyl gallate induces apoptosis with DNA fragmentation in rat and human hepatocytes (Inoue et al., 1994 and Nakagawa et al., 1997) and in other types of human tumor cells (Serrano et al., 1998). Dodecyl gallate disrupts the mitochondrial membrane potential, promotes the efflux of cytochrome c to the cytosol, activates the caspase learn more cascade and Stem Cell Compound Library induces oligonucleosomal breakdown of DNA on a mouse lymphoma cell line ( Roy et al., 2000). It was also demonstrated that dodecyl gallate not only prevents the formation of chemically induced skin tumors in mice but is also able to kill selectively tumor cells in established tumors ( Ortega et al., 2003). A screening of the cytotoxic activity of gallic acid and its n-alkyl esters derivatives in the B16F10 murine melanoma cell line was performed

in previous studies in our laboratory using the MTT viability test. In that study, the gallates that induced cell death by apoptosis with an IC50 value below 50 μM after 24 h of incubation were selected. The mechanistic studies with these gallates in B16F10 very cells showed that octyl gallate induces free radical generation, decyl and dodecyl gallates activate the transcription factor NF-κB and tetradecyl gallate promotes the inhibition of cell adhesion ( Locatelli et al., 2009). Based on the mechanisms suggested above and on the size of the lateral chain of the gallic acid ester derivatives, we selected octyl and dodecyl gallates for further studies to determine their influence on apoptosis signaling in B16F10 cells. The cell culture media and fetal calf serum were

purchased from Cultilab (São Paulo, Brazil). The antibiotics (penicillin/streptomycin) were purchased from GIBCO (Grand Island, NY, USA), the DEVD-AMC fluorogenic substrate for caspase-3 from Biomol International (Plymouth Meeting, PA, USA), the JC-1 probe (5,5′,6′,6-tetrachloro-1,1′,3,3′-tetraethylbenzymidazolcarbocianyne iodide) and DCFH2-DA (2′,7′-dichlorofluorescein diacetate) from Invitrogen (Carlsbad, CA, USA) and the solvent dimethyl sulfoxide (DMSO) from Merck (Darmstadt, Germany). The specific antibodies were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA) or Cell Signaling Technology Inc. (Danvers, MA, USA), as indicated below, and all other reagents were purchased from Sigma–Aldrich (St. Louis, MO, USA). Stock solutions of gallic acid (GA), octyl gallate (G8) and dodecyl gallate (G12), at a final concentration of 20 mM, were prepared in 100% DMSO and diluted in cell culture medium to a maximum final concentration of 0.5% of the solvent.

On the other hand, knowledge regarding the composition of Crotalus oreganus abyssus venom is scarce [24]. From previous experiments in our laboratory in which we have studied the effects of peptides isolated from C. o. abyssus venom, we showed the presence of a natriuretic peptide in its venom (now called Coa_NP1) that produced hypotensive and vasorelaxation effects [5]. The aim of the present study was to identify and investigate the systemic and vascular effects of a new natriuretic peptide isolated from C. o. abyssus venom (Coa_NP2). All reagents were purchased from Aldrich

or Sigma Co. (USA). C. o. abyssus (Coa) venom was obtained from The National Natural Toxins Research Center (NNTRC) of Texas A&M University-Kingsville (Kingsville, TX, USA). C. o. abyssus (Coa) whole venom was submitted to Proteasome inhibitor an FPLC molecular exclusion chromatographic column packed with Sephadex 75 (Akta Primer, GE, USA), exactly as described in [5]. In this separation, five peaks were observed (I–V) ( Fig. 1A). Only fraction V presented a hypotensive effect. To perform a better study and for separation

of peptides and proteins, fraction Thiazovivin V was then submitted to ultra-filtration using the MidJet apparatus (Ge Healthcare, USA), equipped with the UFP-10-C-MM01A cartridge, and a superficial area of 26 cm2, cut off: 10,000 Da (Ge Healthcare, USA). The filtrate presented hypotensive effects and was lyophilized and stored at −20 °C, until use. The filtrate was subjected to reverse phase HPLC (model 2010, Shimadzu, Japan) using an analytical C5 column (Supelco, 250 mm × 4.6 mm), which was previously equilibrated with buffer A (0.1% TFA). The filtrate (10 mg) were dissolved completely in buffer A (0.1% TFA), centrifuged at 5000 × g and then loaded onto a reverse-phase column. The peptides were purified using a linear gradient of buffer B concentration (66% acetonitrile in buffer A) and the chromatographic UV monitoring was carried out at 216 nm [6] and [7]. For electrophoresis, Tricine PAGE-SDS Farnesyltransferase was used for characterization of low molecular weight proteins

and peptides [32]. Each peak or peak group was tested for its action on blood pressure and Coa_NP2 showed positive results. Two milligrams of the purified peptide were dissolved in 200 μl of a 6 mol/l guanidine chloride solution containing 0.4 mol/l of Tris–HCl and 2 mmol/l EDTA (pH 8.15). Nitrogen was blown over the top of the protein solution for 15 min, before reducing with DTT (6 M, 200 μl). This solution was incubated in the dark at 37 °C for 1 h and desalted using a Sephadex G25 column (0.7 cm × 12 cm) with 1 mol/l acetic acid buffer. The reduced peptide was sequenced using an automatic peptide sequencer (890C automatic sequencer, Beckman, USA). The phenylthyoidantoin (PTH) amino acids were identified by comparing their retention times to the 20 PTH amino acid standards [2].