Sequence analysis of sub-genotype D hepatitis B surface antigens isolated from Jeddah,Saudi Arabia

Little is known about the prevalence of HBV genotypes/sub-genotypes in Jeddah province, although the hepatitis B virus (HBV) was identified as the most predominant type of hepatitis in Saudi Arabia. To characterize HBV genotypes/sub-genotypes, serum samples from 15 patients with chronic HBV were collected and subjected to HBsAg gene amplification and sequence analysis. Phylogenetic analysis of the HBsAg gene sequences revealed that 11 (48%) isolates belonged to HBV/D while 4 (18%) were associated with HBV/C. Notably, a HBV/D sub-genotype phylogenetic tree identified that eight current isolates (72%) belonged to HBV/D1, whereas three isolates (28%) appeared to be more closely related to HBV/D5, although they formed a novel cluster supported by a branch with 99% bootstrap value. Isolates belonging to D1 were grouped in one branch and seemed to be more closely related to various strains isolated from different countries. For further determination of whether the three current isolates belonged to HBV/D5 or represented a novel sub-genotype, HBV/DA, whole HBV genome sequences would be required. In the present study, we verified that HBV/D1 is the most prevalent HBV sub-genotype in Jeddah, and identified novel variant mutations suggesting that an additional sub-genotype designated HBV/DA should be proposed. Overall, the results of the present HBsAg sequence analyses provide us with insights regarding the nucleotide differences between the present HBsAg/D isolates identified in the populace of Jeddah, Saudi Arabia and those previously isolated worldwide. Additional studies with large numbers of subjects in other areas might lead to the discovery of the specific HBV strain genotypes or even additional new sub-genotypes that are circulating in Saudi Arabia.     

Elevated ovarian expression and serum concentration of α inhibin in the luteal phase during follicular development in the squirrel monkey (Saimiri boliviensis) compared to the human

The goal of the present investigation was to determine in the squirrel monkey the source and pattern of inhibin, a hormone known to effect reproductive steroid levels via pituitary and ovarian mechanisms. Since this seasonally polyestrous species is known to have elevated serum levels of reproductive steroids compared to other primates, the levels of ovarian alpha subunit mRNA expression and serum total alpha inhibin, estradiol, progesterone, and luteinizing hormone were measured and compared to human levels. Expression of the alpha subunit was robust in monkey luteal tissue compared to expression in human luteal tissue. Squirrel monkey serum inhibin peaked 4 days after the luteinizing hormone surge and correlated with progesterone changes. These luteal serum levels of inhibin were greater than 12 times higher than the human levels yet bio‐LH activities were less than in the human during the luteal phase. Inhibin concentrations during the non‐breeding season were generally half the levels measured in the breeding season and undetectable in ovariectomized animals. However, exogenous FSH stimulation induced a marked rise in inhibin, which correlated with an estradiol rise. In conclusion, abundant alpha inhibin subunit expression in the luteal ovary of the squirrel monkey and loss of serum delectability in ovariectomized animals indicates that the principle source of inhibin in the squirrel monkey is the ovary. Elevated serum inhibin levels during the luteal phase concurrent with ovulatory‐size follicular development is unique among species studied thus far. Possible simultaneous inhibin production from both follicular and luteal tissue may be responsible for the exceptionally high inhibin levels. Am. J. Primatol. 47:165–179, 1999. © 1999 Wiley‐Liss, Inc.     

Toxicity of Artemisia annua (Asteraceae) essential oil on the tea mealy bug,Pseudococcus viburni Sigornet (Hemiptera: Pseudococcidae)

A combination of bioassay and biochemical approaches were used to determine toxicity of Artemisia annua essential oil (AaEO) Pseudococcus viburni. AaEO via leaf dipping bioassay showed LC₅₀ values of 0.693 and 0.419% after two time exposures. Different concentrations of AaEO caused deterrence index between 28.58 to 86.26% by the calculated ED₅₀ of 0.4%. Although, α-esterase activity using α-naphtyl acetate increased in the treated nymphs by AaEO after 24 hours but it showed the lower activity in the treated nymphs using β-naphtyl acetate. Glutathione S-transferase assayed by CDNB showed the higher activity in the treated nymphs than control after 24 hours while the adverse results gained not only after 48 hours but also after 24 hours by using DCNB. No significant differences were found in the activity of alanine aminotransferase versus control, but aspartate aminotransferase and γ-glutamyl transferase showed the statistically higher activities in the treated nymphs in comparison with control. Activities of aldolase and lactate dehydrogenase were significantly lower than those of control. Only acid phosphatase showed the significantly altered activity in the treated nymphs in comparison with control after 24 hours. Results of our study indicated significant toxicity, deterrence and physiological effects of AaEO on P. viburni.     

Release Mechanisms Behind Polysaccharides-Based Famotidine Controlled Release Matrix Tablets

Polysaccharides, which have been explored to possess gelling properties and a wide margin of safety, were used to formulate single-unit floating matrix tablets by a direct compression technique. This work has the aim to allow continuous slow release of famotidine above its site of absorption. The floating approach was achieved by the use of the low density polypropylene foam powder. Polysaccharides (κ-carrageenan, gellan gum, xyloglucan, and pectin) and blends of polysaccharides (κ-carrageenan and gellan gum) and cellulose ethers (hydroxypropylmethyl cellulose, hydroxypropylcellulose, sodium carboxymethyl cellulose) were tried to modulate the release characteristics. The prepared floating tablets were evaluated for their floating behavior, matrix integrity, swelling studies, in vitro drug release studies, and kinetic analysis of the release data. The differential scanning calorimetry and Fourier transform infrared spectroscopy studies revealed that changing the polymer matrix system by formulation of polymers blends resulted in formation of molecular interactions which may have implications on drug release characteristics. This was obvious from the retardation in drug release and change in its mechanistics.     

Demonstration of Proton-coupled Electron Transfer in the Copper-containing Nitrite Reductases

The reduction of nitrite (NO₂⁻) into nitric oxide (NO), catalyzed by nitrite reductase, is an important reaction in the denitrification pathway. In this study, the catalytic mechanism of the copper-containing nitrite reductase from Alcaligenes xylosoxidans (AxNiR) has been studied using single and multiple turnover experiments at pH 7.0 and is shown to involve two protons. A novel steady-state assay was developed, in which deoxyhemoglobin was employed as an NO scavenger. A moderate solvent kinetic isotope effect (SKIE) of 1.3 ± 0.1 indicated the involvement of one protonation to the rate-limiting catalytic step. Laser photoexcitation experiments have been used to obtain single turnover data in H₂O and D₂O, which report on steps kinetically linked to inter-copper electron transfer (ET). In the absence of nitrite, a normal SKIE of ∼1.33 ± 0.05 was obtained, suggesting a protonation event that is kinetically linked to ET in substrate-free AxNiR. A nitrite titration gave a normal hyperbolic behavior for the deuterated sample. However, in H₂O an unusual decrease in rate was observed at low nitrite concentrations followed by a subsequent acceleration in rate at nitrite concentrations of >10 mm. As a consequence, the observed ET process was faster in D₂O than in H₂O above 0.1 mm nitrite, resulting in an inverted SKIE, which featured a significant dependence on the substrate concentration with a minimum value of ∼0.61 ± 0.02 between 3 and 10 mm. Our work provides the first experimental demonstration of proton-coupled electron transfer in both the resting and substrate-bound AxNiR, and two protons were found to be involved in turnover.Denitrification is an anaerobic respiration pathway found in bacteria, archaea, and fungi, in which ATP synthesis is coupled to the sequential reduction of nitrate (NO₃⁻) and nitrite (NO₂⁻) (NO₃⁻ → NO₂⁻ → NO → N₂O → N₂) (1–3).³ The first committed step in this reaction cascade is the formation of gaseous NO by nitrite reductase (NiR), the key enzyme of this pathway. Two distinct classes of periplasmic NiR are found in denitrifying bacteria, one containing cd₁ hemes as prosthetic groups (4–6) and the other utilizing two copper centers to catalyze the one-electron reduction of nitrite (7). Copper-containing NiRs are divided into two main groups according to the color of their oxidized type 1 copper center (T1Cu), with shades ranging from blue to green (3, 7). NiR from Alcaligenes xylosoxidans subsp. xylosoxidans (NCIMB 11015, AxNiR), which is analyzed in this study, is a member of the blue CuNiR group. The blue and green subclasses show a high degree of sequence similarity (70%) (8) and have similar trimeric structures with each monomer (∼36.5 kDa in AxNiR) consisting of two greek key β-barrel cupredoxin-like motifs as well as one long and two short α-helical regions (7, 9).Each NiR monomer contains two copper-binding sites per catalytic unit. One is a T1Cu center, which receives electrons from a physiological redox partner protein and is buried 7 Å beneath the protein surface (10), and the other copper is a type 2 center (T2Cu), constituting the catalytically active substrate-binding site (11). The physiological electron donor for the blue NiRs are the small copper protein azurin (14 kDa) (7) and cytochrome c₅₅₁ (7, 12, 13). The T1Cu, which is responsible for the color of NiR, serves as the electron delivery center and is coordinated by two histidine residues as well as one cysteine and one methionine residue. The catalytic T2Cu, which like all T2Cu centers has very weak optical bands, is ligated to three His residues and an H₂O/OH⁻ ligand in the resting state. This H₂O/OH⁻ ligand is held in place by hydrogen bonds to the active site residues, Asp-92 (AxNiR numbering) and His-249, and gets displaced by the substrate during catalytic turnover (14). The T2Cu is located at the base of a 13–14-Å substrate access channel at the interface of two monomers with one of the three His residues being part of the adjacent subunit (15, 16). The two copper centers are connected by a 12.6-Å covalent bridge provided by the T1Cu-coordinating Cys and by one of the T2Cu His ligands (17, 18). This linkage has been suggested to constitute the electron transfer (ET) pathway from the T1Cu center to the catalytically active T2Cu center via 11 covalent bonds (19).Intramolecular ET from T1- to T2Cu has been extensively examined using pulse radiolysis studies (7, 19–24). In a variety of NiR species, ET could be measured, both in the presence and absence of substrate, with observed ET rate constants (k_ET(obs)) ranging from ∼150 to ∼2000 s⁻¹. According to the Marcus semi-classical ET theory (25), the redox potentials (E′₀, redox midpoint potential at pH 7.0) of the copper centers affect both the thermodynamic equilibrium and the ET kinetics. In the absence of substrate, the difference in the redox potentials has been found to be insignificant at pH 7 (E′₀ (T1Cu) ∼240 mV and E′₀ (T2Cu) ∼230 mV (20)), implying a thermodynamically equal electron distribution between the two metal centers. From an enzymatic point of view, however, approaching this equilibrium position on such a fast time scale (≥150 s⁻¹) is unfavorable in the absence of substrate, as NiR has been shown to form an inactive species with a reduced T2Cu that is devoid of the H₂O/OH⁻ ligand and unable to bind nitrite (26, 27). Substrate binding has been proposed to induce a favorable shift in the T2Cu redox potential, which would be expected to result in an accelerated ET compared with the substrate-free reaction (7, 16, 25, 27–30). However, k_ET(obs) values in AxgNiR (GIFU1051) have been demonstrated to be lower in the nitrite-bound than in the substrate-free enzyme between pH 7.7 and 5.5 (21). Below pH 5.5, the ET rate constants were observed to be similar in the nitrite-free and -bound enzyme (21).In addition to changes in the redox potentials and thus in the driving force of the ET reaction, several structural changes in the redox centers have been reported as a result of substrate binding, which may also influence the inter-copper ET rate by changing the reorganization energy (16, 25, 30, 31). These rearrangements include subtle changes in the Cys-His bridge linking T1- and T2Cu (32) and conformational transitions of the catalytically relevant active site residue Asp-92 (see below and Ref. 29). Moreover, the presence of nitrite has been postulated to be relayed to the T1Cu site via the so-called substrate sensor loop (via His-94, Asp-92, and His-89 in AxNiR), thereby triggering ET to the T2Cu (19, 27, 29, 32). The tight coupling of ET to the presence of substrate has been argued to prevent the formation of a deactivated enzyme species with a prematurely reduced T2Cu (14, 16, 19, 26, 27, 33). In accordance with such a feedback mechanism, in a combined crystallographic and single-crystal spectroscopic study, inter-copper ET could only be detected in crystals where nitrite was bound to the T2Cu site, whereas in the absence of substrate no such ET was observed (34). This finding, however, contradicts the pulse radiolysis results at room temperature (see above), and the apparent discrepancy between solution studies and x-ray crystallographic data collected at cryogenic temperature remains to be resolved.The one-electron reduction of nitrite to NO involves two protons according to the chemical net equation NO₂⁻ + 2H⁺ + e⁻ → NO + H₂O, if the T2Cu is ligated by an H₂O molecule in the resting state rather than an OH⁻ ion. Although the exact enzymatic mechanism is still somewhat controversial (35, 36), one suggested reaction sequence is given in Scheme 1. The potential participation of active site residues in catalyzing the proton transfer (PT) steps has been investigated by studying the pH dependence of NiR under steady-state conditions as well as by pulse radiolysis. The trends obtained for k_cat and k_ET(obs), are similar with pH optima between 5.2 and 6, indicating the involvement of two amino acid residues (21, 22, 37). Asp-92 and His-249 have been proposed as acid-base catalysts (18, 21, 22, 28, 38), and the abrupt drop in rates at increasing pH may indicate that OH⁻ can act as a competitive inhibitor for nitrite (39). The relevance of these active site residues, however, as well as the timing of the two protonation steps is still a matter of debate (35, 40, 41).⁴Open in a separate windowSCHEME 1.A potential reaction mechanism proposed for CuNiRs. Adapted from Ref. 36. Nitrite is shown to bind to the oxidized T2Cu as nitrous acid, thus involving the first protonation step. It coordinates to the oxidized T2Cu center in a bidentate fashion. Following inter-copper ET yielding a reduced T2Cu center, the initially deprotonated Asp-92 accepts a proton, which is subsequently transferred to the substrate. His-249 may be a potential source of this second proton. PT and ET reactions may be reversible and they may be concerted rather than sequential as suggested by the arrows. See text for further information.There are no experimental studies that have been aimed at directly examining the kinetic coupling of PT and ET steps in AxNiR. In this study of the blue AxNiR, our aims were to gain further insight into the mechanism of nitrite reduction by combining multiple turnover experiments with laser photoexcitation studies to measure the (single turnover) inter-copper ET. An extensive analysis of the solvent kinetic isotope effect (SKIE) has been employed as a means of determining whether solvent-exchangeable protons and/or water molecules play a rate-limiting role in the catalytic turnover and/or in inter-copper ET.     

Real-time monitoring of the membrane-binding and insertion properties of the cholesterol-dependent cytolysin anthrolysin O from Bacillus anthracis

Bacillus anthracis has recently been shown to secrete a potently hemolytic/cytolytic protein that has been designated anthrolysin O (ALO). In this work, we initiated a study of this potential anthrax virulence factor in an effort to understand the membrane-binding properties of this protein. Recombinant anthrolysin O (rALO35-512) and two N-terminally truncated versions of ALO (rALO390-512 and rALO403-512) from B. anthracis were overproduced in Escherichia coli and purified to homogeneity. The role of cholesterol in the cytolytic activity of ALO was probed in cellular cholesterol depletion assays using mouse and human macrophage-like lines, and also Drosophila Schneider 2 cells. Challenging the macrophage cells with rALO35-512, but not rALO390-512 or rALO403-512, resulted in cell death by lysis, with this cytolysis being abolished by depletion of the membrane cholesterol. Drosophila cells, which contain ergosterol as their major membrane sterol, were resistant to rALO-mediated cytolysis. In order to determine the molecular mechanism of this resistance, the interaction of rALO with model membranes comprised of POPC alone, or with a variety of structurally similar sterols including ergosterol, was probed using Biacore. Both rALO35-512 and rALO403-512 demonstrated robust binding to model membranes composed of POPC and cholesterol, with amount of protein bound proportional to the cholesterol content. Ergosterol supported greatly reduced binding of both rALO35-512 and rALO403-512, whereas other sterols tested did not support binding. The rALO403-512--membrane interaction demonstrated an equilibrium dissociation constant (KD) in the low nanomolar range, whereas rALO35-512 exhibited complex kinetics likely due to the multiple events involved in pore formation. These results establish the pivotal role of cholesterol in the action of rALO. The biosensor method developed to measure ALO recognition of cholesterol in a membrane environment could be extended to provide a platform for the screening of inhibitors of other membrane-binding proteins and peptides.     

Detection of Neu1 Sialidase Activity in Regulating TOLL-like Receptor Activation

Mammalian Toll-like receptors (TLRs) are a family of receptors that recognize pathogen-associated molecular patterns. Not only are TLRs crucial sensors of microbial (e.g., viruses, bacteria and parasite) infections, they also play an important role in the pathophysiology of infectious diseases, inflammatory diseases, and possibly in autoimmune diseases. Thus, the intensity and duration of TLR responses against infectious diseases must be tightly controlled. It follows that understanding the structural integrity of sensor receptors, their ligand interactions and signaling components is essential for subsequent immunological protection. It would also provide important opportunities for disease modification through sensor manipulation. Although the signaling pathways of TLR sensors are well characterized, the parameters controlling interactions between the sensors and their ligands still remain poorly defined. We have recently identified a novel mechanism of TLR activation by its natural ligand, which has not been previously observed ^1,2. It suggests that ligand-induced TLR activation is tightly controlled by Neu1 sialidase activation. We have also reported that Neu1 tightly regulates neurotrophin receptors like TrkA and TrkB ³, which involve Neu1 and matrix metalloproteinase-9 (MMP-9) cross-talk in complex with the receptors ⁴. The sialidase assay has been initially use to find a novel ligand, thymoquinone, in the activation of Neu4 sialidase on the cell surface of macrophages, dendritic cells and fibroblast cells via GPCR Gαi proteins and MMP-9 ⁵. For TLR receptors, our data indicate that Neu1 sialidase is already in complex with TLR-2, -3 and -4 receptors, and is induced upon ligand binding to either receptor. Activated Neu1 sialidase hydrolyzes sialyl α-2,3-linked β-galactosyl residues distant from ligand binding to remove steric hinderance to TLR-4 dimerization, MyD88/TLR4 complex recruitment, NFkB activation and pro-inflammatory cell responses. In a collaborative report, Neu1 sialidase has been shown to regulate phagocytosis in macrophage cells ⁶. Taken together, the sialidase assay has provided us with powerful insights to the molecular mechanisms of ligand-induced receptor activation. Although the precise relationship between Neu1 sialidase and the activation of TLR, Trk receptors has yet to be fully elucidated, it would represent a new or pioneering approach to cell regulation pathways.Download video file.^{(57M, mov)}     

Characterization of fertility alteration and marker validation for male sterility genes in novel PTGMS lines hybrid rice

Photoperiod and thermosensitive genetic male sterile (PTGMS) lines have become one of the main sources of global rice production increasing. This study was conducted to evaluate the fertility alteration and validate the male sterility genes using validation markers in novel Egyptian Indica and Japonica PTGMS lines under natural conditions. The study revealed that the new genetic male sterile lines belong to the type of photo–thermosensitive genetic male sterility (PTGMS). The fertility alteration of these lines has influenced by photoperiod and temperature interaction. The new PTGMS lines have three sensitive periods of fertility alteration; transformation, sterility, and fertility period. Furthermore, the sensitive stage of fertility transformation might be from secondary branch primordial to pollen mother cells (PMC) meiosis. Under the natural Sakha condition, the new PTGMS lines were stable sterile under the condition of day length upper 13,75 h and temperature over 25 °C, while its convert to fertile under day length under 13 h, and temperature lower than 24 °C. The co-dominant markers identified the pms3 and tms5 genes in the new PTGMS lines, indicated that the fertility alteration in these lines controlled by photoperiod and thermosensitive stages.