Immunohistochemical localization of inhibin/activin alpha, beta(A) and beta(B) subunits and follistatin in bovine oocytes during in vitro maturation and fertilization

The aim of this study was to evaluate the distribution of inhibin/activin alpha, beta(A) and beta(B) subunits and follistatin in immature oocytes and in matured oocytes before and after IVF. Denuded oocytes were submitted to a whole-mount immunofluorescence procedure. Specimens were imaged and fluorescent intensities quantified by scanning laser confocal microscopy. Immunoreactivity for inhibin alpha subunit (both alpha(C) and pro-alpha. regions), abundant in the ooplasm of immature oocytes, decreased after maturation (a 68% and 88% decrease, respectively; P < 0.001), but increased after IVF by 2- and 5.7-fold, respectively (P < 0.01). Intense staining for PA was detected in immature oocytes (predominantly in the outer ooplasm and zona pellucida) but after maturation and fertilization it was localized mainly in the zona pellucida, perivitelline space and oolemma. Immunoreactivity for RA in the ooplasm decreased by 58% after maturation (P < 0.001) but increased again by 75% after fertilization (P < 0.01). Immunoreactivity for beta(B) was localized mainly in the zona pellucida and did not change after maturation. However, immurloreactivity for beta(B) was not detected in the zona pellucida after fertilization, but remained unchanged in unfertilized oocytes. Immunoreactivity for follistatin was detected in the ooplasm and zona pellucida of immature oocytes but decreased progressively in the ooplasm after maturation (a 63% decrease; P < 0.001) and did not change after IVF. Examination of partially denuded cumulus-oocyte complexes confirmed abundant expression of alpha(C), pro-alpha, beta(A) and follistatin immunoreactivity in cumulus cells, whereas beta(B) subunit staining was weak or absent in cumulus cells, but intense in the zona pellucida. In conclusion, the present study shows that qualitative and quantitative changes in the distribution of inhibin/activin subunits and follistatin accompany oocyte maturation and fertilization. The possibility, indicated by these observations, that activin A and activin B may play distinct roles in bovine oocyte maturation and fertilization warrants further study.

Single-particle tracking uncovers dynamics of glutamate-induced retrograde transport of NF-κB p65 in living neurons

Retrograde transport of NF-κB from the synapse to the nucleus in neurons is mediated by the dynein/dynactin motor complex and can be triggered by synaptic activation. The calibre of axons is highly variable ranging down to 100 nm, aggravating the investigation of transport processes in neurites of living neurons using conventional light microscopy. In this study we quantified for the first time the transport of the NF-κB subunit p65 using high-density single-particle tracking in combination with photoactivatable fluorescent proteins in living mouse hippocampal neurons. We detected an increase of the mean diffusion coefficient (Dmean) in neurites from 0.12 ± 0.05 µm2/s to 0.61 ± 0.03 µm2/s after stimulation with glutamate. We further observed that the relative amount of retrogradely transported p65 molecules is increased after stimulation. Glutamate treatment resulted in an increase of the mean retrograde velocity from 10.9 ± 1.9 to 15 ± 4.9 µm/s, whereas a velocity increase from 9 ± 1.3 to 14 ± 3 µm/s was observed for anterogradely transported p65. This study demonstrates for the first time that glutamate stimulation leads to an increased mobility of single NF-κB p65 molecules in neurites of living hippocampal neurons.

Distinctive Immunomodulatory and Inflammatory Properties of the Escherichia coli Type II Heat-Labile Enterotoxin LT-IIa and Its B Pentamer following Intradermal Administration

The type I and type II heat-labile enterotoxins (LT-I and LT-II) are strong mucosal adjuvants when they are coadministered with soluble antigens. Nonetheless, data on the parenteral adjuvant activities of LT-II are still limited. Particularly, no previous study has evaluated the adjuvant effects and induced inflammatory reactions of LT-II holotoxins or their B pentameric subunits after delivery via the intradermal (i.d.) route to mice. In the present report, the adjuvant and local skin inflammatory effects of LT-IIa and its B subunit pentamer (LT-IIaB(5)) were determined. When coadministered with ovalbumin (OVA), LT-IIa and, to a lesser extent, LT-IIaB(5) exhibited serum IgG adjuvant effects. In addition, LT-IIa but not LT-IIaB(5) induced T cell-specific anti-OVA responses, particularly in respect to induction of antigen-specific cytotoxic CD8(+) T cell responses. LT-IIa and LT-IIaB(5) induced differential tissue permeability and local inflammatory reactions after i.d. injection. Of particular interest was the reduced or complete lack of local reactions, such as edema and tissue induration, in mice i.d. inoculated with LT-IIa and LT-IIaB(5), respectively, compared with mice immunized with LT-I. In conclusion, the present results show that LT-IIa and, to a lesser extent, LT-IIaB(5) exert adjuvant effects when they are delivered via the i.d. route. In addition, the low inflammatory effects of LT-IIa and LT-IIaB(5) in comparison to those of LT-I support the usefulness of LT-IIa and LT-IIaB(5) as parenterally delivered vaccine adjuvants.

A DNA Vaccine Encoding the Enterohemorragic Escherichia coli Shiga-Like Toxin 2 A(2) and B Subunits Confers Protective Immunity to Shiga Toxin Challenge in the Murine Model

Production of verocytotoxin or Shiga-like toxin (Stx), particularly Stx2, is the basis of hemolytic uremic syndrome, a frequently lethal outcome for subjects infected with Stx2-producing enterohemorrhagic Escherichia coli (EHEC) strains. The toxin is formed by a single A subunit, which promotes protein synthesis inhibition in eukaryotic cells, and five B subunits, which bind to globotriaosylceramide at the surface of host cells. Host enzymes cleave the A subunit into the A(1) peptide, endowed with N-glycosidase activity to the 28S rRNA, and the A(2) peptide, which confers stability to the B pentamer. We report the construction of a DNA vaccine (pStx2 Delta AB) that expresses a nontoxic Stx2 mutated form consisting of the last 32 amino acids of the A(2) sequence and the complete B subunit as two nonfused polypeptides. Immunization trials carried out with the DNA vaccine in BALB/c mice, alone or in combination with another DNA vaccine encoding granulocyte-macrophage colony-stimulating factor, resulted in systemic Stx-specific antibody responses targeting both A and B subunits of the native Stx2. Moreover, anti-Stx2 antibodies raised in mice immunized with pStx2 Delta AB showed toxin neutralization activity in vitro and, more importantly, conferred partial protection to Stx2 challenge in vivo. The present vector represents the second DNA vaccine so far reported to induce protective immunity to Stx2 and may contribute, either alone or in combination with other procedures, to the development of prophylactic or therapeutic interventions aiming to ameliorate EHEC infection-associated sequelae.

Inhibin B and the ovarian follicular reserve

Inhibins are dimeric glycoproteins composed of an α-subunit and a βA-subunit (inhibin A) or βB-subunit (inhibin B), which inhibits pituitary gonadotropin secretion of FSH. The inhibin B is a product of the cohort of antral follicles. The ovarian follicle number decrease steadily as a function of increasing age, with consequent falls in the levels of inhibin B and increase of FSH levels. It is sufficient to maintain ovulatory function and continued secretion of estradiol. Elevated FSH levels seem to occur late in the sequence of events associated with ovarian failure. The inhibin B, produced by granulosa cells, is the earliest marker of the decline in ovarian follicular reserve across reproductive aging.

Mutations affecting disulphide bonds contribute to a fairly common prevalence of F13B gene defects: results of a genetic study in 14 families with factor XIII B deficiency

Severe factor XIII (FXIII) deficiency is a rare autosomal recessive coagulation disorder affecting one in two million individuals. The aim of the present study was to screen for and analyse F13B gene defects in the German population. A total of 150 patients presenting with suspected FXIII deficiency and one patient with severe (homozygous) FXIII deficiency were screened for mutations in F13A and F13B genes. Twenty-five individuals presented with detectable heterozygous mutations, 12 of them in the F13A gene and 13 of them in the F13B gene. We report on the genotype-phenotype correlations of the individuals showing defects in the F13B gene. Direct sequencing revealed 12 unique mutations including seven missense mutations (Cys5Arg, Ile81Asn, Leu116Phe, Val217Ile, Cys316Phe, Val401Glu, Pro428Ser), two splice site mutations (IVS2-1G>C, IVS3-1G>C), two insertions (c.1155_1158dupACTT, c.1959insT) and one in-frame deletion (c.471-473delATT). Two of the missense mutations (Cys5Arg, Cys316Phe) eliminated disulphide bonds (Cys5-Cys56, Cys316-Cys358). Another three missense mutations, (Leu116Phe, Val401Glu, Pro428Ser) were located proximal to other cysteine disulphide bonds, therefore indicating that the region in and around these disulphide bonds is prone to functionally relevant mutations in the FXIII-B subunit. The present study reports on a fairly common prevalence of F13B gene defects in the German population. The regions in and around the cysteine disulphide bonds in the FXIII-B protein may be regions prone to frequent mutations.

Characterisation of six novel A-subunit mutations leading to congenital factor XIII deficiency and molecular analysis of the first diagnosed patient with this rare bleeding disorder

In 1960, the first case report on factor XIII deficiency was published describing a seven-year-old Swiss boy with a so far unknown bleeding disorder. Today, more than 60 mutations in the factor XIIIA- and B-subunit genes are known leading to congenital factor XIII deficiency. In the present study, we describe six novel mutations in the factor XIII A-subunit gene. Additionally, we present the molecular characterisation of the first described patient with congenital factor XIII deficiency. The six novel mutations include a small deletion, Glu202 delG, leading to a premature stop codon and truncation of the protein, and a splice site mutation at the exon 10/intron 10 boundary, +1G/A, giving rise to an incorrect spliced mRNA lacking exons 10 and 11. The remaining four mutations are characterised by the single amino acid changes Met159Arg, Gly215Arg, Trp375Cys, and His716Arg, and were expressed in COS-1 cells. Antigen levels and activity of the mutants were significantly reduced compared to the wild-type. The patient described in 1960 also shows a single amino acid change, Arg77Cys. Structural analysis of all mutant enzymes suggests several mechanisms leading to destabilisation of the protein.

The vacuolar-ATPase B1 subunit in distal tubular acidosis: novel mutations and mechanisms for dysfunction

Mutations in the B1 subunit of the multisubunit vacuolar ATPase cause autosomal-recessive distal renal tubular acidosis and sensorineural deafness. Here, we report a novel frameshift mutation that truncates the C-terminus of the human B1 subunit. This mutant protein failed to assemble with other subunits in the cytosol to form the complex that can be targeted to vesicular structures in mammalian cells. Loss of proton pump activity was demonstrated in a functional complementation assay in B-subunit null yeast. The mutation caused loss of a discreet C-terminal region critical for subunit interaction not related to the C-terminal PDZ motif. Co-expression studies failed to demonstrate dominant negative effects of this truncated mutant over wild-type B1. Analysis of 12 reported B1 subunit missense mutations showed one polymorphic allele had intact pump function, two point mutants had intact assembly but defective proton pumping, and the remaining nine had disrupted assembly with no pump function. One presumed polymorphic allele was actually an inactivating mutation. Our study shows that multiple mechanisms of pump dysfunction result from B1 subunit mutations with a common outcome being defective assembly. Polymorphisms of the B1 subunit in the general population may affect renal acidification and urinary chemistry.

Potent immunogenicity of the B subunits of Escherichia coli heat-labile enterotoxin: receptor binding is essential and induces differential modulation of lymphocyte subsets.

The importance of receptor binding in the potent immunogenicity of Escherichia coli heat-labile enterotoxin B subunit (EtxB) was tested by comparing its immunogical properties with those of a receptor binding mutant, EtxB(G33D). Subcutaneous immunization of EtxB(G33D) resulted in 160-fold reduction in antibody titer compared with wild-type EtxB, whereas its oral delivery failed to provoke any detectable secretory or serum anti-B subunit responses. Moreover, the two proteins induced strikingly different effects on lymphocyte cultures in vitro. EtxB, in comparison with EtxB(G33D), caused an increase in the proportion of B cells, many of which were activated (CD25+); the complete depletion of CD8+ T cells; an increase in the activation of CD4+ T cells; and an increase in interleukin 2 and a decrease in interferon gamma. These data indicate that EtxB exerts profound effects on immune cells, suggesting that its potent immunogenicity is dependent not only on efficient receptor-mediated uptake, but also on direct receptor-mediated immunomodulation of lymphocyte subsets.

Immuno-potentiating activity of surfactant vesicles in DNA and subunit vaccination

Enhanced immune responses for DNA and subunit vaccines potentiated by surfactant vesicle based delivery systems outlined in the present study, provides proof of principle for the beneficial aspects of vesicle mediated vaccination. The dehydration-rehydration technique was used to entrap plasmid DNA or subunit antigens into lipid-based (liposomes) or non-ionic surfactant-based (niosomes) dehydration-rehydration vesicles (DRV). Using this procedure, it was shown that both these types of antigens can be effectively entrapped in DRV liposomes and DRV niosomes. The vesicle size of DRV niosomes was shown to be twice the diameter (~2µm) of that of their liposome counterparts. Incorporation of cryoprotectants such as sucrose in the DRV procedure resulted in reduced vesicle sizes while retaining high DNA incorporation efficiency (~95%). Transfection studies in COS 7 cells demonstrated that the choice of cationic lipid, the helper lipid, and the method of preparation, all influenced transfection efficiency indicating a strong interdependency of these factors. This phenomenon has been further reinforced when 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): cholesteryl 3b- [N-(N’ ,N’ -dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol)/DNA complexes were supplemented with non-ionic surfactants. Morphological analysis of these complexes using transmission electron microscopy and environmental scanning electron microscopy (ESEM) revealed the presence of heterogeneous structures which may be essential for an efficient transfection in addition to the fusogenic properties of DOPE. In vivo evaluation of these DNA incorporated vesicle systems in BALB/c mice showed weak antibody and cell-mediated immune (CMI) responses. Subsequent mock challenge with hepatitis B antigen demonstrated that, 1-monopalmitoyl glycerol (MP) based DRV, is a more promising DNA vaccine adjuvant. Studying these DRV systems as adjuvants for the Hepatitis B subunit antigen (HBsAg) revealed a balanced antibody/CMI response profile on the basis of the HBsAg specific antibody and cytokine responses which were higher than unadjuvated antigen. The effect of addition of MP, cholesterol and trehalose 6,6’-dibehenate (TDB) on the stability and immuno-efficacy of dimethyldioctadecylammonium bromide (DDA) vesicles was investigated. Differential scanning calorimetry showed a reduction in transition temperature of DDA vesicles by ~12°C when incorporated with surfactants. ESEM of MP based DRV system indicated an increased vesicle stability upon incorporation of antigen. Adjuvant activity of these systems tested in C57BL/6j mice against three subunit antigens i.e., mycobacterial fusion protein- Ag85B-ESAT-6, and two malarial antigens - merozoite surface protein-1, (MSP1), and glutamate rich protein, (GLURP) revealed that while MP and DDA based systems induced comparable antibody responses, DDA based systems induced powerful CMI responses.

The Role of PP2A Regulatory Subunit B56 in Dictyostelium Discoideum Motility

Protein Phosphatase 2A, PP2A, is a heterotrimeric threonine/serine phosphatase system that is involved in a variety of cellular processes. This phosphatase is composed ofthree subunits: a catalytic subunit (C subunit), a scaffolding subunit (A subunit), and a regulatory subunit (B subunit). The regulatory subunit B is divided into four subclasses, B, B' (B56), B'' and B'' '. Studies showed that PP2A/B56 complexes regulate development of Dictyostelium and other metazoan cells. In addition to development, our experimental data suggest that PP2A/B56 complex also plays an important role in Dictyostelium cell motility. Cells lacking B56 was generated previously in our laboratory (Lee et al., 2008). Further studies showed that b56- cells are compromised in random cell motility compared to the wild type (AX3) cells. In contrast, b56 cells with re-introduced B56 displayed wild-type like motilities. Furthermore, one of the colleagues in our laboratory found that one of the Dictyostelium Ras species, RasG, associates with PP2A/B56 complex and RasG activation is compromised in b56- cells. Considering that Ras proteins are central in cellular motility regulation, PP2A/B56 complex may modulate cell motility through regulating Ras. We propose to determine if an introduction of constitutive active RasG proteins improves compromised b56- cell motility.

Calcium-permeable AMPA receptors mediate long-term potentiation in interneurons in the amygdala

Fear conditioning is a paradigm that has been used as a model for emotional learning in animals'. The cellular correlate of fear conditioning is thought to be associative N-methyl-D-aspartate (NMDA) receptor-dependent synaptic plasticity within the amygdala(1-3). Here we show that glutamatergic synaptic transmission to inhibitory interneurons in the basolateral amygdala is mediated solely by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. In contrast to AMPA receptors at inputs to pyramidal neurons, these receptors have an inwardly rectifying current-voltage relationship, indicative of a high permeability to calcium(4 5), Tetanic stimulation of inputs to interneurons caused an immediate and sustained increase in the efficacy of these synapses. This potentiation required a rise in postsynaptic calcium, but was independent of NMDA receptor activation. The potentiation of excitatory inputs to interneurons was reflected as an increase in the amplitude of the GABAA-mediated inhibitory synaptic current in pyramidal neurons. These results demonstrate that excitatory synapses onto interneurons within a fear conditioning circuit show NMDA-receptor independent long-term potentiation. This plasticity might underlie the increased synchronization of activity between neurons in the basolateral amygdala after fear conditioning(6).

Retrograde delivery of photosensitizer (TPPp-O-beta-GluOH)(3) selectively potentiates its photodynamic activity

Photodynamic therapy involves administration of a photosensitizing drug and its subsequent activation by visible light of the appropriate wavelength. Several approaches to increasing the specificity of photosensitizers for cancerous tissues and, in particular, through their conjugation to ligands that are directed against tumor-associated antigens have been investigated. Here, we have studied the delivery of the photocytotoxic porphyrin compound TPP(p-O-beta-D-GluOH)(3) into tumor cells that overexpress the glycosphingolipid Gb3, using the Gb3-binding nontoxic B-subunit of Shiga toxin (STxB) as a vector. To allow for site-directed chemical coupling, an STxB variant carrying a free sulfhydryl moiety at its C-terminal end has been used. Binding affinity, cellular uptake, singlet oxygen quantum yield, and phototoxicity of the conjugate have been examined. Despite some effect of coupling on both the photophysical properties of TPP(p-O-beta-D-GluOH)(3) and the affinity of STxB for its receptor, the conjugate exhibited a higher photocytotoxic activity than the photosensitizer alone and was exquisitely selective for Gb3-expressing tumor cells. Furthermore, our data strongly suggest that STxB-mediated retrograde delivery of the photosensitizer to the biosynthetic/secretory pathway is critical for optimal cytotoxic activity. In conclusion, a strong rationale for using retrograde delivery tools such as STxB in combination with photosensitizing agents for the photodynamic therapy of tumors is presented.

Differential adjustment in gill Na(+)/K(+)- and V-ATPase activities and transporter mRNA expression during osmoregulatory acclimation in the cinnamon shrimp Macrobrachium amazonicum (Decapoda, Palaemonidae)

We evaluate osmotic and chloride (Cl(-)) regulatory capability in the diadromous shrimp Macrobrachium amazonicum, and the accompanying alterations in hemolymph osmolality and [Cl(-)], gill Na(+)/K(+)-ATPase activity, and expression of gill Na(+)/K(+)-ATPase alpha-subunit and V-ATPase B subunit mRNA during salinity (S) acclimation. We also characterize V-ATPase kinetics and the organization of transport-related membrane systems in the gill epithelium. Macrobrachium amazonicum strongly hyper-regulates hemolymph osmolality and [Cl(-)] in freshwater and in salinities up to 25 parts per thousand S. During a 10-day acclimation period to 25 parts per thousand S, hemolymph became isosmotic and hypo-chloremic after 5 days, [Cl(-)] alone remaining hyporegulated thereafter. Gill Na(+)/K(+)-ATPase alpha-subunit mRNA expression increased 6.5 times initial values after 1 h, then decreased to 3 to 4 times initial values by 24 h and to 1.5 times initial values after 10 days at 25 parts per thousand S. This increased expression was accompanied by a sharp decrease at 5 h then recovery of initial Na(+)/K(+)-ATPase activity within 24 h, declining again after 5 days, which suggests transient Cl(-) secretion. V-ATPase B-subunit mRNA expression increased 1.5-fold within 1 h, then reduced sharply to 0.3 times initial values by 5 h, and remained unchanged for the remainder of the 10-day period. V-ATPase activity dropped sharply and was negligible after a 10-day acclimation period to 21 parts per thousand S, revealing a marked downregulation of ion uptake mechanisms. The gill epithelium consists of thick, apical pillar cell flanges, the perikarya of which are coupled to an intralamellar septum. These two cell types respectively exhibit extensive apical evaginations and deep membrane invaginations, both of which are associated with numerous mitochondria, characterizing an ion transporting epithelium. These changes in Na(+)/K(+)- and V-ATPase activities and in mRNA expression during salinity acclimation appear to underpin ion uptake and Cl(-) secretion by the palaemonid shrimp gill.

Pyrrolidine dithiocarbamate down-regulates vascular matrix metalloproteinases and ameliorates vascular dysfunction and remodelling in renovascular hypertension

BACKGROUND AND PURPOSE Mounting evidence implicates matrix metalloproteinase (MMP) in the vascular dysfunction and remodelling associated with hypertension. We tested the hypothesis that treatment with pyrrolidine dithiocarbamate (PDTC), which interferes with NF-kappa B-induced MMPs gene transcription, could exert antihypertensive effects, prevent MMP-2 and MMP-9 up-regulation, and protect against the functional alterations and vascular remodelling of two-kidney, one clip (2K1C) hypertension. EXPERIMENTAL APPROACH Sham-operated or hypertensive rats were treated with vehicle or PDTC (100 mg.Kg(-1).day(-1)) by gavage for 8 weeks. Systolic blood pressure (SBP) was monitored weekly. Aortic rings were isolated to assess endothelium-dependent relaxations. Quantitative morphometry of structural alterations of the aortic wall was carried out in haematoxylin/eosin sections. Formation of vascular reactive oxygen species (ROS), and inducible (i) NOS and phosphorylated-p65 NF-kappa B subunit expression were measured in the aortas. MMP-2 and MMP-9 aortic levels and gelatinolytic activity were determined by gelatin and in situ zymography and by immunofluorescence. KEY RESULTS Treatment with PDTC attenuated the increases in SBP and prevented the endothelial dysfunction associated with 2K1C hypertension. Moreover, PDTC reversed the vascular aortic remodelling, the increases in aortic ROS levels and in iNOS and phosphorylated-p65 NF-kappa B expression found in 2K1C rats. These effects were associated with attenuation of 2K1C up-regulation of aortic MMP-2 and MMP-9 levels and gelatinolytic activity. CONCLUSION AND IMPLICATIONS These findings suggest that PDTC down-regulates vascular MMPs and ameliorates vascular dysfunction and remodelling in renovascular hypertension, thus providing evidence supporting the suggestion that PDTC is probably a good candidate to be used to treat hypertension.