Simultaneous and rapid monitoring of biomass and biopolymer production by Sphingomonas paucimobilis using Fourier transform-near infrared spectroscopy

The application of Fourier Transform near infrared spectroscopy (FT-NIRS) to near real-time monitoring of polysaccharide and biomass concentration was investigated using a gellan-producing strain of Sphingomonas paucimobilis grown in a stirred tank reactor. Successful models for both biomass and gellan were constructed despite the physichochemical complexity of the viscous process fluid. Modelling of biomass proved more challenging than for gellan, partly because of the low range of biomass concentration but a model with a good correlation coefficient (0.94) was formulated based on second derivative spectra. The gellan model was highly satisfactory, with an excellent correlation coefficient (0.98), again based on second derivative spectra. No sample pre-treatment was required and all spectral scanning was carried out on whole broth. Additionally, both models should be robust in practice since both were formulated using low numbers of factors. Thus, the near real time simultaneous monitoring of gellan and biomass in this highly complex matrix using FT-NIRS potentially opens the way to greatly improved process control strategies.     

Amperometric cytochrome c3-based biosensor for chromate determination

The chromate reductase activity of cytochrome c(3) (Cyt c(3), M(r) 13000), isolated from the sulfate-reducing bacterium Desulfomicrobium norvegicum, was used to develop an amperometric biosensor to measure chromate (CrO(4)(2-)) bioavailability. The performance of various biosensor configurations for qualitative and quantitative determination of Cr(VI) was studied. Biosensor properties depend on the technique used to immobilize the enzyme on the electrode (glassy carbon electrode). Immobilization of Cyt c(3) by entrapment in poly 3,4-ethylenedioxythiophene films denatured the enzyme, while application of an adsorption technique did not affect enzyme activity but the detection range was limited. The best results were obtained with dialysis membranes, which allowed the determination of Cr(VI) from 0.20 to 6.84 mg l(-1) (3.85-132 microM) with a sensitivity of 35 nA mg(-1) l (1.82 nA microM(-1)). No interference was observed with As(V), As(III) and Fe(III). Only a small amount of Cyt c(3) (372 ng of protein) was needed for this biosensor.     

Corticotropin-releasing hormone activates protein kinase C in an isoenzyme-specific manner

Protein kinase C (PKC) has recently emerged as mediator of corticotropin-releasing hormone (CRH) effects. Aim of the present study was to study the effects of CRH on each PKC isoenzyme. As a model we have used the PC12 rat pheochromocytoma cell line, expressing the CRH type 1 receptor (CRHR1). Our data were as follows: (a) CRH-induced rapid phosphorylation of conventional PKCalpha and PKCbeta, accompanied by parallel increase of their concentration within nucleus. (b) CRH suppressed the phosphorylation of novel PKCdelta and PKCtheta;, which remained in the cytosol. (c) CRH-induced transient phosphorylation of atypical PKClambda and had no effect on PKCmu. (d) The effect of CRH on each PKC isoenzyme was blocked by a CRHR1 antagonist. (e) Blockade of conventional PKC phosphorylation inhibited CRH-induced calcium ion mobilization from intracellular stores as well as the CRH-induced apoptosis and Fas ligand production. In conclusion, our findings suggest that CRH via its CRHR1 receptor differentially regulates PKC-isoenzyme phosphorylation, an apparently physiologically relevant effect since blockade of conventional PKC phosphorylation abolished the biological effect of CRH.     

Wild type and <Emphasis Type="Italic">H43Y</Emphasis> variant of human <Emphasis Type="Italic">TRIM5α</Emphasis> show similar anti-human immunodeficiency virus type 1 activity both in vivo and in vitro

Polymorphisms in human genes have been shown to affect the rate of disease progression to acquired immune deficiency syndrome in human immunodeficiency virus type 1 (HIV-1)-infected individuals. Recently, tripartite motif 5α (TRIM5α) was identified as a factor that confers resistance to HIV-1 infection in Old World monkey cells. Subsequently, Sawyer et al. (Curr Biol 16:95–100, 2006) reported a single nucleotide polymorphism (H43Y) in the human TRIM5α gene and TRIM5α protein with 43Y was found to lose its ability to restrict HIV-1. In the present study, we reevaluated effects of this allele on in vitro anti-HIV-1 activity as well as on HIV-1 disease progression in European and Asian cohorts of HIV-1-infected individuals. Our epidemiological and molecular biological findings clearly indicate H43Y has a very minor effect on anti-HIV-1 activity of TRIM5α, suggesting that this allele is immaterial, at least in HIV-1-infected Europeans and Asians.     

A novel pathway of rapid TLR-triggered activation of integrin-dependent leukocyte adhesion that requires Rap1 GTPase

Rapid β2-integrin activation is indispensable for leukocyte adhesion and recruitment to sites of infection and is mediated by chemokine- or P-selectin glycoprotein ligand-1–induced inside-out signaling. Here we uncovered a novel pathway for rapid activation of integrin-dependent leukocyte adhesion, triggered by toll-like receptor (TLR)–mediated signaling. TLR2 or TLR5 ligation rapidly activated integrin-dependent leukocyte adhesion to immobilized ICAM-1 and fibronectin. Consistently, in vivo administration of the TLR2-ligand Pam3CSK4 increased integrin-dependent slow rolling and adhesion to endothelium within minutes, as identified by intravital microscopy in the cremaster model. TLR2 and TLR5 ligation increased β2-integrin affinity, as assessed by the detection of activation-dependent neoepitopes. TLR2- and TLR5-triggered integrin activation in leukocytes required enhanced Rap1 GTPase activity, which was mediated by Rac1 activation and NADPH oxidase-2–dependent reactive oxygen species production. This novel direct pathway linking initial pathogen recognition by TLRs to rapid β2-integrin activation may critically regulate acute leukocyte infiltration to sites of pathogen invasion.     

Mutations in PRDM5 in brittle cornea syndrome identify a pathway regulating extracellular matrix development and maintenance

Extreme corneal fragility and thinning, which have a high risk of catastrophic spontaneous rupture, are the cardinal features of brittle cornea syndrome (BCS), an autosomal-recessive generalized connective tissue disorder. Enucleation is frequently the only management option for this condition, resulting in blindness and psychosocial distress. Even when the cornea remains grossly intact, visual function could also be impaired by a high degree of myopia and keratoconus. Deafness is another common feature and results in combined sensory deprivation. Using autozygosity mapping, we identified mutations in PRDM5 in families with BCS. We demonstrate that regulation of expression of extracellular matrix components, particularly fibrillar collagens, by PRDM5 is a key molecular mechanism that underlies corneal fragility in BCS and controls normal corneal development and maintenance. ZNF469, encoding a zinc finger protein of hitherto undefined function, has been identified as a quantitative trait locus for central corneal thickness, and mutations in this gene have been demonstrated in Tunisian Jewish and Palestinian kindreds with BCS. We show that ZNF469 and PRDM5, two genes that when mutated cause BCS, participate in the same regulatory pathway.     

Exercise as a model to study redox homeostasis in blood: the effect of protocol and sampling point

Twenty males ran either on a level treadmill (nonmuscle-damaging condition) or on a downhill treadmill (muscle-damaging condition). Blood and urine samples were collected before and after exercise (immediately after, 1h, 4h, 24h, 48h, and 96h). The following assays were performed: F(2)-isoprostanes in urine, protein carbonyls in plasma, glutathione, superoxide dismutase, glutathione peroxidase, and catalase in erythrocytes. The main finding was that monophasic redox responses were detected after nonmuscle-damaging exercise compared to the biphasic responses detected after muscle-damaging exercise. Based on these findings, muscle-damaging exercise may be a more appropriate experimental model to induce physiological oxidative stress.     

Specific delta-opioid antagonists exert an agonist-independent inhibitory effect,similar to the agonist,on the release of GnRHIn Vitro

Summary 1. Inin vitro studies with adult male rats we have recently shown that the delta-opioid agonist DTLET inhibits the release of the Gonadotropin-Releasing Hormone (GnRH) from hypothalamic fragments containing the arcuate nucleus and the median eminence. This effect is receptor mediated and eicosanoid dependent (Gerozissiset al., 1993).2. In the present study we report that the delta-opioid antagonists with negative intrinsic activity, Diallyl-G and ICI 174864, applied under the same experimental conditions (30 min static incubations at 37°C, in a potassium rich milieu), in the absence of the agonist DTLET, also exert a similar to the agonist inhibitory effect on the release of GnRH.3. The dose-dependent inhibitory effect of Diallyl-G on GnRH release is reversed by increasing concentrations of DTLET. The mu and delta opioid antagonist, naloxone is without effect in the absence of DTLET. However, naloxone acts as an antagonist on the Diallyl-G-induced inhibition of GnRH release.4. Diallyl-G also inhibits the release of prostaglandin E₂ (PGE₂). In the presence of indomethacin or nordihydroguaiaretic acid, Diallyl-G is ineffective to further inhibit the release of GnRH. These latter observations taken together with the results of eicosanoid estimation suggest that PGE₂ but not leukotrienes participate in the agonist-independent effects of Diallyl-G on GnRH release.5. Therefore these results support the hypothesis that delta-opioid antagonists with negative intrinsic activity exert agonist-independent biological responses similar to those of the agonists.     

Structural and Biophysical Characterization of the Proteins Interacting with the Herpes Simplex Virus 1 Origin of Replication

The C terminus of the herpes simplex virus type 1 origin-binding protein, UL9ct, interacts directly with the viral single-stranded DNA-binding protein ICP8. We show that a 60-amino acid C-terminal deletion mutant of ICP8 (ICP8ΔC) also binds very strongly to UL9ct. Using small angle x-ray scattering, the low resolution solution structures of UL9ct alone, in complex with ICP8ΔC, and in complex with a 15-mer double-stranded DNA containing Box I of the origin of replication are described. Size exclusion chromatography, analytical ultracentrifugation, and electrophoretic mobility shift assays, backed up by isothermal titration calorimetry measurements, are used to show that the stoichiometry of the UL9ct-dsDNA_15-mer complex is 2:1 at micromolar protein concentrations. The reaction occurs in two steps with initial binding of UL9ct to DNA (K_d ∼ 6 nm) followed by a second binding event (K_d ∼ 0.8 nm). It is also shown that the stoichiometry of the ternary UL9ct-ICP8ΔC-dsDNA_15-mer complex is 2:1:1, at the concentrations used in the different assays. Electron microscopy indicates that the complex assembled on the extended origin, oriS, rather than Box I alone, is much larger. The results are consistent with a simple model whereby a conformational switch of the UL9 DNA-binding domain upon binding to Box I allows the recruitment of a UL9-ICP8 complex by interaction between the UL9 DNA-binding domains.The initiation of DNA replication for most double-stranded DNA (dsDNA)⁶ viral genomes begins with the recognition of the origin by specific origin-binding proteins. The herpes simplex virus type 1 (HSV-1) genome encodes seven proteins required for origin-dependent DNA replication. These are the DNA polymerase (UL30) and its accessory protein (UL42), a heterotrimeric helicase-primase complex (UL5, UL8, and UL52), the single-stranded DNA-binding protein (ICP8 or UL29), and the origin-binding protein (UL9) (reviewed in Ref. 1). HSV-1 contains three functional origins, oriL and two copies of oriS. OriS, which is about 80 bp in length, consists of three UL9 recognition sites, in Boxes I, II, and III, which are arranged in two overlapping palindromes (2). Box I and Box III are part of an evolutionarily conserved palindrome that forms a stable hairpin in single-stranded DNA, which may be important in the origin rearrangement (3) during initiation of replication. Box I and II are separated by an AT-rich spacer sequence, which varies in length and nucleotide composition between the different members of the α-herpesvirus subfamily (2, 4–6).UL9 is a homodimer in solution, and EM studies, with UL9 bound to oriS, indicate the existence of a dimer or pair of dimers assembled on oriS (7). Several reports indicate that UL9 can physically interact not only with ICP8 (8) but also with other members of the HSV-1 replication complex, including UL8 (9) and UL42 (10). Thus UL9 functions as a docking protein to recruit these essential replication proteins to the viral origins. ICP8 stimulates the helicase activity of UL9 (11, 12) and binds to its C-terminal 27-aa residues (13). In the presence of ICP8, UL9 will open dsDNA containing Box I, leading to a conformational change in the origin, thus facilitating unwinding (14–16). As stated above, the changes in DNA conformation in the complete oriS may be more complex (3). Recently, it has been suggested that single-stranded oriS folds into a unique and evolutionarily conserved conformation, oriS*, which is stably bound by UL9. oriS* contains a hairpin formed by complementary base pairing between Box I and Box III in oriS (17). UL9, in the presence of the single-stranded DNA-binding protein ICP8, can convert an 80-bp double-stranded minimal oriS fragment to oriS* and form a UL9-oriS* complex. The formation of a UL9-oriS* complex requires ATP hydrolysis (18). Therefore, the UL9-oriS* complex may serve as an assembly site for the herpesvirus replisome. Macao et al. (3) proposed a model in which full-length UL9 would be required to adopt a different conformation when binding to oriS or oriS*. The implication is that UL9 partially unwinds and introduces a hairpin into the origin of replication and that the formation of oriS* is aided, in some way, by ICP8 and requires ATP hydrolysis. Macao et al. (3) suggest that the length of the single-stranded tail of the probe DNA determines the stoichiometry of the UL9-DNA complex. oriS may bind two molecules of UL9, whereas oriS* may only bind one because the hairpin formation prevents the second interaction.Photo-cross-linking studies have shown that, although the UL9 protein binds Box I as a dimer, only one of the two monomers contacts Box I, suggesting that the C terminus of UL9 undergoes a conformational change upon binding to Box I (19). The results reported here are consistent with this observation. To date there is no three-dimensional structural information available on the full-length UL9 or either of the functionally characterized (helicase and DNA binding) domains. The ability to adopt different conformations and a tendency to proteolytic degradation may be responsible for this. It has been shown that UL9 binds with very high specificity to the Box I through its DNA-binding domain, consisting of the C-terminal 317 aa (UL9ct) (20, 21). Although the importance of the binding between UL9ct and oriS for the viral life cycle is well established, the mechanism behind this interaction still remains unclear. Even though UL9ct exists as a monomer in solution, uncertainty remains as to whether one or two molecules bind to a single Box I recognition sequence. Some reports have suggested that one UL9ct molecule binds to a single copy of the sequence (22–24), whereas others have proposed that UL9ct forms a dimer when bound to DNA (25, 26). This apparent difference may well result from the different protein concentrations used in different assays/experiments, which in turn highlights the difficulty of translating in vitro equilibrium experiments into cellular nonequilibrium situations.A few years ago, the crystal structure of a 60-residue C-terminal deletion mutant of ICP8 (ICP8ΔC) was determined to 3 Å resolution (Protein Data Bank code 1URJ (27)). The structure of ICP8ΔC consists of a large N-terminal domain (aa 9–1038) and a smaller entirely helical C-terminal domain (aa 1049–1120) connected to the N-terminal domain by a disordered linker (aa 1038–1049) spanning around 18 Å in the crystal structure. ICP8 preferentially binds ssDNA over dsDNA in a nonsequence-specific and cooperative manner (28). ICP8 is a zinc metalloprotein containing one zinc atom per molecule, which is coordinated by three cysteines (Cys-499, Cys-502, and Cys-510) and a histidine (His-512) (27).In this study, we show that the 60-amino acid C-terminal deletion of ICP8 (ICP8ΔC) binds strongly to UL9ct. We present three low resolution structures in solution using small angle x-ray scattering as follows: that of the UL9ct alone, in complex with ICP8ΔC, and in complex with a 15-mer dsDNA (dsDNA_15-mer) containing the Box I sequence. Using these data and a variety of biophysical techniques, we demonstrate that the stoichiometries of the UL9ct-dsDNA_15-mer and UL9ct-ICP8ΔC-dsDNA_15-mer complexes are 2:1 and 2:1:1, respectively, at the micromolar protein concentrations used in this study. Using EM we visualize the assembly of the ICP8ΔC-UL9ct complex on oriS and estimate the size of the complex.