LC-MS characterization of N/O-glycans of α- and ß-subunits of chicken ovomucin separated by SDS-PAGE.

As the main active glycoprotein of egg white, the biological functions of chicken ovomucin α- and ß-subunit are closely related to the structure of glycans. However, the exact composition and structure of the subunit glycans are still unknown. We obtained highly pure chicken ovomucin α-subunit and ß-subunit protein bands by the strategy combined with two-step isoelectric precipitation and SDS-PAGE gel electrophoresis. The ammonia-catalyzed one-pot procedure was then used to release and capture α-and ß-subunit protein glycans with 1-phenyl- 3-Methyl-5-pyrazolone (PMP). The N/O-glycans of bis-PMP derivatives were purified and analyzed by LC-MS. More importantly, an effective dual modification was performed to accurately quantify neutral and sialylated O-glycans through methylamidation of sialic acid residues and simultaneously through carbonyl condensation reactions of reducing ends with PMP. We first showed that the α-subunit protein has only N-glycosylation modification, and the ß-subunit only O-glycosylation, a total of 22 N-glycans and 20 O-glycans were identified in the α- and ß-subunit, respectively. In addition, the complex N-glycan (47 %) and the sialylated O-glycan (77 %) are each major types of the above subunits. Such findings in this study provide a basis for studying the functional and biological activities of chicken ovomucin glycans.

Development of Heterologous Expression System and Optimization of the Method of Cholera Toxin ß-Subunit Production in E. coli.

Cholera is a deadly infection disease, which is usually associated with low hygiene levels and limited access to high-quality drinking water. An effective way to prevent cholera is the use of vaccines. Among active vaccine components there is the CtxB protein (cholera toxin ß-subunit). In the current work, we have developed a genetic system for production of the recombinant CtxB in E. coli cells and studied conditions for synthesis and purification of the target product at the laboratory scale. It has been found that the optimal algorithm for isolation of the recombinant protein is to grow E. coli culture in the synthetic M9 medium with glycerol, followed by CtxB purification out of the spent culture medium using Ni2+-chelate affinity chromatography techniques. Forty-eight hours after induction of CtxB expression, concentration of the target product could be up to 50 mg/liter in the culture medium. The CtxB protein retains its pentameric structure during expression and through purification. The latter makes it possible to consider the developed system as a promising tool for the industrial-level production of recombinant CtxB for medical and research purposes.

G-Protein ß-Subunit Gene TaGB1-B Enhances Drought and Salt Resistance in Wheat.

In the hexaploid wheat genome, there are three Gα genes, three Gß and twelve Gγ genes, but the function of Gß in wheat has not been explored. In this study, we obtained the overexpression of TaGB1 Arabidopsis plants through inflorescence infection, and the overexpression of wheat lines was obtained by gene bombardment. The results showed that under drought and NaCl treatment, the survival rate of Arabidopsis seedlings' overexpression of TaGB1-B was higher than that of the wild type, while the survival rate of the related mutant agb1-2 was lower than that of the wild type. The survival rate of wheat seedlings with TaGB1-B overexpression was higher than that of the control. In addition, under drought and salt stress, the levels of superoxide dismutase (SOD) and proline (Pro) in the wheat overexpression of TaGB1-B were higher than that of the control, and the concentration of malondialdehyde (MDA) was lower than that of the control. This indicates that TaGB1-B could improve the drought resistance and salt tolerance of Arabidopsis and wheat by scavenging active oxygen. Overall, this work provides a theoretical basis for wheat G-protein ß-subunits in a further study, and new genetic resources for the cultivation of drought-tolerant and salt-tolerant wheat varieties.

Does Background Matter? A Comparative Characterization of Mouse Models of Autosomal Retinitis Pigmentosa rd1 and Pde6b-KO.

Many retinal degenerative diseases result in vision impairment or permanent blindness due to photoreceptor loss or dysfunction. It has been observed that Pde6brd1 mice (rd1), which carry a spontaneous nonsense mutation in the pde6b gene, have a strong phenotypic similarity to patients suffering from autosomal recessive retinitis pigmentosa. In this study, we present a novel mouse model of retinitis pigmentosa generated through pde6b gene knockout using CRISPR/Cas9 technology. We compare this Pde6b-KO mouse model to the rd1 mouse model to gain insights into the progression of retinal degeneration. The functional assessment of the mouse retina and the tracking of degeneration dynamics were performed using electrophysiological methods, while retinal morphology was analyzed through histology techniques. Interestingly, the Pde6b-KO mouse model demonstrated a higher amplitude of photoresponse than the rd1 model of the same age. At postnatal day 12, the thickness of the photoreceptor layer in both mouse models did not significantly differ from that of control animals; however, by day 15, a substantial reduction was observed. Notably, the decline in the number of photoreceptors in the rd1 model occurred at a significantly faster rate. These findings suggest that the C3H background may play a significant role in the early stages of retinal degeneration.

Flexible functional interactions between G-protein subunits contribute to the specificity of plant responses.

Plants being sessile integrate information from a variety of endogenous and external cues simultaneously to optimize growth and development. This necessitates the signaling networks in plants to be highly dynamic and flexible. One such network involves heterotrimeric G-proteins comprised of Gα, Gß, and Gγ subunits, which influence many aspects of growth, development, and stress response pathways. In plants such as Arabidopsis, a relatively simple repertoire of G-proteins comprised of one canonical and three extra-large Gα, one Gß and three Gγ subunits exists. Because the Gß and Gγ proteins form obligate dimers, the phenotypes of plants lacking the sole Gß or all Gγ genes are similar, as expected. However, Gα proteins can exist either as monomers or in a complex with Gßγ, and the details of combinatorial genetic and physiological interactions of different Gα proteins with the sole Gß remain unexplored. To evaluate such flexible, signal-dependent interactions and their contribution toward eliciting a specific response, we have generated Arabidopsis mutants lacking specific combinations of Gα and Gß genes, performed extensive phenotypic analysis, and evaluated the results in the context of subunit usage and interaction specificity. Our data show that multiple mechanistic modes, and in some cases complex epistatic relationships, exist depending on the signal-dependent interactions between the Gα and Gß proteins. This suggests that, despite their limited numbers, the inherent flexibility of plant G-protein networks provides for the adaptability needed to survive under continuously changing environments.

A novel nonsense mutation in the ß-subunit of the epithelial sodium channel causing Liddle syndrome.

PURPOSE: Liddle syndrome is a hereditary form of arterial hypertension caused by mutations in the genes coding of the epithelial sodium channel - SCNN1A, SCNN1B and SCNN1G. It is characterised by early onset of hypertension and variable biochemical features such as hypokalaemia and low plasma concentrations of renin and aldosterone. Phenotypic variability is large and, therefore, LS is probably underdiagnosed. Our objective was to examine a family suspected from Liddle syndrome including genetic testing and evaluate clinical and biochemical features of affected family members. MATERIALS AND METHODS: Thirteen probands from the Czech family, related by blood, underwent physical examination, laboratory tests, and genetic testing. Alleles of SCNN1B and SCNN1G genes were examined by PCR amplification and Sanger sequencing of amplicons. RESULTS: We identified a novel mutation in the ß-subunit of an epithelial sodium channel coded by the SCNN1B gene, causing the nonsense mutation in the protein sequence p.Tyr604*. This mutation was detected in 7 members of the family. The mutation carriers differed in the severity of hypertension and hypokalaemia which appeared only after diuretics in most of them; low aldosterone level (< 0.12 nmol/l) was, however, present in all. CONCLUSIONS: This finding expands the spectrum of known mutations causing Liddle syndrome. Hypoaldosteronemia was 100% sensitive sign in the mutation carriers. Low levels are observed especially in the Caucasian population reaching 96% sensitivity. Assessment of plasma aldosterone concentration is helpful for differential diagnosis of arterial hypertension. CONDENSED ABSTRACT: Liddle syndrome is a hereditary form of arterial hypertension caused by mutations in the genes encoding the epithelial sodium channel's α-, ß- and γ-subunit. It is usually manifested by early onset of hypertension accompanied by low potassium and aldosterone levels. We performed a physical examination, laboratory tests and genetic screening in 13 members of a Czech family. We found a new mutation of the SCNN1B gene which encodes the ß-subunit of the epithelial sodium channel. We describe the variability of each family member phenotype and point out the relevance of using aldosterone levels as a high sensitivity marker of Liddle syndrome in Caucasians.

Arabidopsis cryptochrome 1 promotes stomatal development through repression of AGB1 inhibition of SPEECHLESS DNA-binding activity.

Cryptochromes are blue light photoreceptors that mediate various light responses in plants and mammals. The heterotrimeric G-protein is known to regulate various physiological processes in plants and mammals. In Arabidopsis, cryptochrome 1 (CRY1) and the G-protein ß subunit AGB1 act antagonistically to regulate stomatal development. The molecular mechanism by which CRY1 and AGB1 regulate this process remains unknown. Here, we show that Arabidopsis CRY1 acts partially through AGB1, and AGB1 acts through SPEECHLESS (SPCH), a master transcription factor that drives stomatal initiation and proliferation, to regulate stomatal development. We demonstrate that AGB1 physically interacts with SPCH to block the bHLH DNA-binding domain of SPCH and inhibit its DNA-binding activity. Moreover, we demonstrate that photoexcited CRY1 represses the interaction of AGB1 with SPCH to release AGB1 inhibition of SPCH DNA-binding activity, leading to the expression of SPCH-target genes promoting stomatal development. Taken together, our results suggest that the mechanism by which CRY1 promotes stomatal development involves positive regulation of the DNA-binding activity of SPCH mediated by CRY1 inhibition of the AGB1-SPCH interaction. We propose that the antagonistic regulation of SPCH DNA-binding activity by CRY1 and AGB1 may allow plants to balance light and G-protein signaling and optimize stomatal density and pattern.

FSH ß-subunit mutations in two sisters: the first report from the Indian sub-continent and review of previous cases.

Isolated FSH deficiency due to mutations in the gene for ß-subunit of FSH is an extremely rare autosomal recessive disease of which only eleven cases have been reported so far. The clinical features include absent breast development and primary amenorrhea in females and azoospermia with normal testosterone levels in males. In this study we report two Kashmiri sisters born to native Kashmiri consanguineous parents with failure of onset of puberty. Hormonal evaluation revealed undetectable serum FSH and estradiol and high LH. Genetic analysis of FSH ß-gene identified one nonsense mutation (c.343C > T:p. Arg115Stop) in exon 3. The two sisters were homozygous for this nonsense mutation while the parents were heterozygous. Incorporation of a stop codon at 115 codon position is predicted to result in the formation of truncated FSH ß protein, lacking 14 amino acid from the carboxy-terminus (p.Arg115Stop). Very recently, this same mutation was reported for the first time in a Chinese male. Ours is the first ever report of any FSH ß-subunit mutation from the Indian sub-continent and this particular mutation in any female from anywhere in the world. We conclude and emphasize that this diagnosis should be considered in girls with delayed puberty and selective deficiency of FSH.

A Model for the Homotypic Interaction between Na+,K+-ATPase ß1 Subunits Reveals the Role of Extracellular Residues 221-229 in Its Ig-Like Domain.

The Na+, K+-ATPase transports Na+ and K+ across the membrane of all animal cells. In addition to its ion transporting function, the Na+, K+-ATPase acts as a homotypic epithelial cell adhesion molecule via its ß1 subunit. The extracellular region of the Na+, K+-ATPase ß1 subunit includes a single globular immunoglobulin-like domain. We performed Molecular Dynamics simulations of the ectodomain of the ß1 subunit and a refined protein-protein docking prediction. Our results show that the ß1 subunit Ig-like domain maintains an independent structure and dimerizes in an antiparallel fashion. Analysis of the putative interface identified segment Lys221-Tyr229. We generated triple mutations on YFP-ß1 subunit fusion proteins to assess the contribution of these residues. CHO fibroblasts transfected with mutant ß1 subunits showed a significantly decreased cell-cell adhesion. Association of ß1 subunits in vitro was also reduced, as determined by pull-down assays. Altogether, we conclude that two Na+, K+-ATPase molecules recognize each other by a large interface spanning residues 221-229 and 198-207 on their ß1 subunits.

Transcripts of Kv7.1 and MinK channels and slow delayed rectifier K+ current (IKs) are expressed in zebrafish (Danio rerio) heart.

Zebrafish are increasingly used as a model for human cardiac electrophysiology, arrhythmias, and drug screening. However, K+ ion channels of the zebrafish heart, which determine the rate of repolarization and duration of cardiac action potential (AP) are still incompletely known and characterized. Here, we provide the first evidence for the presence of the slow component of the delayed rectifier K+channels in the zebrafish heart and characterize electrophysiological properties of the slow component of the delayed rectifier K+current, IKs. Zebrafish atrium and ventricle showed strong transcript expression of the kcnq1 gene, which encodes the Kv7.1 α-subunit of the slow delayed rectifier K+ channel. In contrast, the kcne1 gene, encoding the MinK ß-subunit of the delayed rectifier, was expressed at 21 and 17 times lower level in ventricle and atrium, respectively, in comparison to the kcnq1. IKs was observed in 62% of ventricular myocytes with mean (± SEM) density of 1.23 ± 0.37 pA/pF at + 30 mV. Activation rate of IKs was 38% faster (τ50 = 1248 ± 215 ms) than kcnq1:kcne1 channels (1725 ± 792 ms) expressed in 3:1 ratio in Chinese hamster ovary cells. Microelectrode experiments demonstrated the functional relevance of IKs in the zebrafish heart, since 100 µM chromanol 293B produced a significant prolongation of AP in zebrafish ventricle. We conclude that AP repolarization in zebrafish ventricle is contributed by IKs, which is mainly generated by homotetrameric Kv7.1 channels not coupled to MinK ancillary ß-subunits. This is a clear difference to the human heart, where MinK is an essential component of the slow delayed rectifier K+channel.

An update on the biophysical character of the human eukaryotic elongation factor 1 beta: Perspectives from interaction with elongation factor 1 gamma.

The ß-subunit of the human eukaryotic elongation factor 1 complex (heEF1ß) plays a central role in the elongation step in eukaryotic protein biosynthesis, which essentially involves interaction with the α- and γ-subunits (eEF1γ). To biophysically characterize heEF1ß, we constructed 3 Escherichia coli expression vector systems for recombinant expression of the full length (FL-heEF1ß), N-terminus (NT-heEF1ß), and the C-terminus (CT-heEF1ß) regions of the protein. Our results suggest that heEF1ß is predominantly alpha-helical and possesses an accessible hydrophobic cavity in the CT-heEF1ß. Both FL-heEF1ß and NT-heEF1ß form dimers of size 62 and 30 kDa, respectively, but the CT-heEF1ß is monomeric. FL-heEF1ß interacts with the N-terminus glutathione transferase-like domain of heEF1γ (NT-heEF1γ) to form a 195-kDa complex or a 230-kDa complex in the presence of oxidized glutathione. On the other hand, NT-heEF1ß forms a 170-kDa complex with NT-heEF1γ and a high molecular weight aggregate of size greater than 670 kDa. Surface plasmon resonance analysis confirmed that (by fitting the Langmuir 1:1 model) FL-heEF1ß associated with monomeric or dimeric NT-heEF1γ at a rapid rate and slowly dissociated, suggesting strong functional affinity (KD  = 9.6 nM for monomeric or 11.3 nM for dimeric NT-heEF1γ). We postulate that the N-terminus region of heEF1ß may be responsible for its dimerization and the C-terminus region of heEF1ß modulates the formation of an ordered heEF1ß-γ oligomer, a structure that may be essential in the elongation step of eukaryotic protein biosynthesis.

Transcriptional regulation of follicle-stimulating hormone ß-subunit in marmoset by an alternate distal promoter.

Follicle-stimulating hormone (FSH) is essential for mammalian folliculogenesis and spermatogenesis. Common marmoset (Callithrix jacchus) is a New World primate which exhibits an unusual FSH profile across the ovarian cycle with a mid-follicular FSH peak that is not observed in Catarrhini primates like humans. Since transcription of FSH ß-subunit gene (FSHß) is a rate-limiting step in the production of mature FSH, this study aimed to investigate the regulation of marmoset FSHß gene expression in comparison to human. In silico analysis of the FSHß promoter sequences identified a TATA box element upstream of the conventional TATA box element in marmoset but not in human sequence. FSHß mRNA transcript longer than the conventional transcript was detected in marmoset pituitary implying presence of a distal transcription start site. In luciferase reporter assays, the marmoset putative distal promoter had higher activity than the corresponding human region even in absence of the conventional proximal promoter. Indeed higher affinity binding of TATA box-binding protein to the putative distal TATA box element was obtained in electrophoretic mobility shift assay. This suggests existence of a differential regulation of FSHß transcription in marmoset compared to humans.

HuR Mediates Changes in the Stability of AChR ß-Subunit mRNAs after Skeletal Muscle Denervation.

Acetylcholine receptors (AChRs) are heteromeric membrane proteins essential for neurotransmission at the neuromuscular junction. Previous work showed that muscle denervation increases expression of AChR mRNAs due to transcriptional activation of AChR subunit genes. However, it remains possible that post-transcriptional mechanisms are also involved in controlling the levels of AChR mRNAs following denervation. We examined whether post-transcriptional events indeed regulate AChR ß-subunit mRNAs in response to denervation. First, in vitro stability assays revealed that the half-life of AChR ß-subunit mRNAs was increased in the presence of denervated muscle protein extracts. A bioinformatics analysis revealed the existence of a conserved AU-rich element (ARE) in the 3'-untranslated region (UTR) of AChR ß-subunit mRNA. Furthermore, denervation of mouse muscle injected with a luciferase reporter construct containing the AChR ß-subunit 3'UTR, caused an increase in luciferase activity. By contrast, mutation of this ARE prevented this increase. We also observed that denervation increased expression of the RNA-binding protein human antigen R (HuR) and induced its translocation to the cytoplasm. Importantly, HuR binds to endogenous AChR ß-subunit transcripts in cultured myotubes and in vivo, and this binding is increased in denervated versus innervated muscles. Finally, p38 MAPK, a pathway known to activate HuR, was induced following denervation as a result of MKK3/6 activation and a decrease in MKP-1 expression, thereby leading to an increase in the stability of AChR ß-subunit transcripts. Together, these results demonstrate the important contribution of post-transcriptional events in regulating AChR ß-subunit mRNAs and point toward a central role for HuR in mediating synaptic gene expression. SIGNIFICANCE STATEMENT: Muscle denervation is a convenient model to examine expression of genes encoding proteins of the neuromuscular junction, especially acetylcholine receptors (AChRs). Despite the accepted model of AChR regulation, which implicates transcriptional mechanisms, it remains plausible that such events cannot fully account for changes in AChR expression following denervation. We show that denervation increases expression of the RNA-binding protein HuR, which in turn, causes an increase in the stability of AChR ß-subunit mRNAs in denervated muscle. Our findings demonstrate for the first time the contribution of post-transcriptional events in controlling AChR expression in skeletal muscle, and points toward a central role for HuR in mediating synaptic development while also paving the way for developing RNA-based therapeutics for neuromuscular diseases.

Comparison of marmoset and human FSH using synthetic peptides of the ß-subunit L2 loop region and anti-peptide antibodies.

Follicle stimulating hormone (FSH) is a glycoprotein hormone required for female and male gametogenesis in vertebrates. Common marmoset (Callithrix jacchus) is a New World primate monkey, used as animal model in biomedical research. Observations like, requirement of extremely high dose of human FSH in marmosets for superovulation compared to other primates and generation of antibodies in marmoset against human FSH after repeated superovulation cycles, point towards the possibility that FSH-FSH receptor (FSHR) interaction in marmosets might be different than in the humans. In this study we attempted to understand some of these structural differences using FSH peptides and anti-peptide antibody approach. Based on sequence alignment, in silico modeling and docking studies, L2 loop of FSH ß-subunit (L2ß) was found to be different between marmoset and human. Hence, peptides corresponding to region 32-50 of marmoset and human L2ß loop were synthesized, purified and characterized. The peptides displayed dissimilarity in terms of molecular mass, predicted isoelectric point, predicted charge and in the ability to inhibit hormone-receptor interaction. Polyclonal antibodies generated against both the peptides were found to exhibit specific binding for the corresponding peptide and parent FSH in ELISA and Western blotting respectively and exhibited negligible reactivity to cross-species peptide and FSH in ELISA. The anti-peptide antibody against marmoset FSH was also able to detect native FSH in marmoset plasma samples and pituitary sections. In summary, the L2ß loop of marmoset and human FSH has distinct receptor interaction ability and immunoreactivity indicating possibility of subtle conformational and biochemical differences between the two regions which may affect the FSH-FSHR interaction in these two primates. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Growth differentiation factor 9 (GDF9) forms an incoherent feed-forward loop modulating follicle-stimulating hormone ß-subunit (FSHß) gene expression.

Gonadotropin-releasing hormone (GnRH) is secreted in brief pulses from the hypothalamus and regulates follicle-stimulating hormone ß-subunit (FSHß) gene expression in pituitary gonadotropes in a frequency-sensitive manner. The mechanisms underlying its preferential and paradoxical induction of FSHß by low frequency GnRH pulses are incompletely understood. Here, we identify growth differentiation factor 9 (GDF9) as a GnRH-suppressed autocrine inducer of FSHß gene expression. GDF9 gene transcription and expression were preferentially decreased by high frequency GnRH pulses. GnRH regulation of GDF9 was concentration-dependent and involved ERK and PKA. GDF9 knockdown or immunoneutralization reduced FSHß mRNA expression. Conversely, exogenous GDF9 induced FSHß expression in immortalized gonadotropes and in mouse primary pituitary cells. GDF9 exposure increased FSH secretion in rat primary pituitary cells. GDF9 induced Smad2/3 phosphorylation, which was impeded by ALK5 knockdown and by activin receptor-like kinase (ALK) receptor inhibitor SB-505124, which also suppressed FSHß expression. Smad2/3 knockdown indicated that FSHß induction by GDF9 involved Smad2 and Smad3. FSHß mRNA induction by GDF9 and GnRH was synergistic. We hypothesized that GDF9 contributes to a regulatory loop that tunes the GnRH frequency-response characteristics of the FSHß gene. To test this, we determined the effects of GDF9 knockdown on FSHß induction at different GnRH pulse frequencies using a parallel perifusion system. Reduction of GDF9 shifted the characteristic pattern of GnRH pulse frequency sensitivity. These results identify GDF9 as contributing to an incoherent feed-forward loop, comprising both intracellular and secreted components, that regulates FSHß expression in response to activation of cell surface GnRH receptors.

Isolated noncatalytic and catalytic subunits of F1-ATPase exhibit similar, albeit not identical, energetic strategies for recognizing adenosine nucleotides.

The function of F1-ATPase relies critically on the intrinsic ability of its catalytic and noncatalytic subunits to interact with nucleotides. Therefore, the study of isolated subunits represents an opportunity to dissect elementary energetic contributions that drive the enzyme's rotary mechanism. In this study we have calorimetrically characterized the association of adenosine nucleotides to the isolated noncatalytic α-subunit. The resulting recognition behavior was compared with that previously reported for the isolated catalytic ß-subunit (N.O. Pulido, G. Salcedo, G. Pérez-Hernández, C. José-Núñez, A. Velázquez-Campoy, E. García-Hernández, Energetic effects of magnesium in the recognition of adenosine nucleotides by the F1-ATPase ß subunit, Biochemistry 49 (2010) 5258-5268). The two subunits exhibit nucleotide-binding thermodynamic signatures similar to each other, characterized by enthalpically-driven affinities in the µM range. Nevertheless, contrary to the catalytic subunit that recognizes MgATP and MgADP with comparable strength, the noncatalytic subunit much prefers the triphosphate nucleotide. Besides, the α-subunit depends more on Mg(II) for stabilizing the interaction with ATP, while both subunits are rather metal-independent for ADP recognition. These binding behaviors are discussed in terms of the properties that the two subunits exhibit in the whole enzyme.

The Role of Human Chorionic Gonadotropin as Tumor Marker: Biochemical and Clinical Aspects.

Tumor markers are biological substances that are produced/released mainly by malignant tumor cells, enter the circulation in detectable amounts and are potential indicators of the presence of a tumor. The most useful biochemical markers are the tumor-specific molecules, i.e., receptors, enzymes, hormones, growth factors or biological response modifiers that are specifically produced by tumor cells and not, or minimally, by the normal counterpart (Richard et al. Principles and practice of gynecologic oncology. Wolters Kluwer Health, Philadelphia, 2009). Based on their specificity and sensitivity in each malignancy, biomarkers are used for screening, diagnosis, disease monitoring and therapeutic response assessment in clinical management of cancer patients.This chapter is focused on human chorionic gonadotropin (hCG), a hormone with a variety of functions and widely used as a tumor biomarker in selected tumors. Indeed, hCG is expressed by both trophoblastic and non-trophoblastic human malignancies and plays a role in cell transformation, angiogenesis, metastatization, and immune escape, all process central to cancer progression. Of note, hCG testing is crucial for the clinical management of placental trophoblastic malignancies and germ cell tumors of the testis and the ovary. Furthermore, the production of hCG by tumor cells is accompanied by varying degrees of release of the free subunits into the circulation, and this is relevant for the management of cancer patients (Triozzi PL, Stevens VC, Oncol Rep 6(1):7-17, 1999).The name chorionic gonadotropin was conceived: chorion derives from the latin chordate meaning afterbirth, gonadotropin indicates that the hormone is a gonadotropic molecule, acting on the ovaries and promoting steroid production (Cole LA, Int J Endocrinol Metab 9(2):335-352, 2011). The function, the mechanism of action and the interaction between hCG and its receptor continue to be the subject of intensive investigation, even though many issues about hCG have been well documented (Tegoni M et al., J Mol Biol 289(5):1375-1385, 1999).

Biosynthesis of the phycocyanin ß-subunit in Escherichia coli BL21 and its antioxidant activity and application in the preservation of fresh-cut apples.

In this study, the biosynthesis of phycocyanin ß-subunit (CpcB) in Escherichia coli BL21 was investigated, and its antioxidant activity and application in anti-browning of fresh-cut apples was explored. Four genes (cpcB, cpeS, hox1 and pcyA) involved in the biosynthesis of CpcB were cloned and transformed into E. coli BL21 by constructing recombinant plasmid pETDuet-5. The positive transformant was screened by ampicillin resistance. The analysis of SDS-PAGE and zinc fluorescence spectrum showed that CpcB was successfully expressed in E. coli BL21 with a molecular weight of 21 kDa. The purified CpcB had a maximum absorption peak at 615 nm, and its maximum florescence emission wavelength was 640 nm. It exhibited a stronger ability to scavenge four free radicals than Vc. The color change in fresh-cut apples was obviously delayed by the CpcB treatment. These results suggest that CpcB may be used as a potential anti-browning agent for food preservation.

Structural analysis of ATP bound to the F1-ATPase ß-subunit monomer by solid-state NMR- insight into the hydrolysis mechanism in F1.

ATP-hydrolysis-associated conformational change of the ß-subunit during the rotation of F1-ATPase (F1) has been discussed using cryo-electron microscopy (cryo-EM). Since it is worthwhile to further investigate the conformation of ATP at the catalytic subunit through an alternative approach, the structure of ATP bound to the F1ß-subunit monomer (ß) was analyzed by solid-state NMR. The adenosine conformation of ATP-ß was similar to that of ATP analog in F1 crystal structures. 31P chemical shift analysis showed that the Pα and Pß conformations of ATP-ß are gauche-trans and trans-trans, respectively. The triphosphate chain is more extended in ATP-ß than in ATP analog in F1 crystals. This appears to be in the state just before ATP hydrolysis. Furthermore, the ATP-ß conformation is known to be more closed than the closed form in F1 crystal structures. In view of the cryo-EM results, ATP-ß would be a model of the most closed ß-subunit with ATP ready for hydrolysis in the hydrolysis stroke of the F1 rotation.

Development of Streptococcus gordonii-specific quantitative real-time polymerase chain reaction primers based on the nucleotide sequence of rpoB.

In this study, Streptococcus gordonii-specific quantitative real-time polymerase chain reaction (qPCR) primers, RTSgo-F2/RTSgo-R2, were developed based on the nucleotide sequences of RNA polymerase ß-subunit gene (rpoB). The specificity of the RTSgo-F2/RTSgo-R2 primers was assessed by conventional PCR on 99 strains comprising 63 oral bacterial species, including the type strain and eight clinical isolates of S. gordonii. PCR products were amplified from the genomic DNAs of only S. gordonii strains. The qPCR primers were able to detect as little as 40 fg of S. gordonii genomic DNA at a cycle threshold value of 33. These findings suggest that these qPCR primers detect S. gordonii with high specificity and sensitivity.