Binding characteristics of KNI-272 to plasma proteins, a new potent tripeptide HIV protease inhibitor

The binding characteristics of KNI-272, a potent and selective human immunodeficiency virus (HIV) protease inhibitor, were evaluated in rat and human plasma, and in solutions of human alpha 1-acid glycoprotein (AAG) and human serum albumin (HSA). The unbound fractions (Fu) of KNI-272 were 12.13 and 2.24% in rat and human plasma, respectively, at the drug concentration of 1.0 microgram mL-1. Although KNI-272 binds to both AAG and HSA, the Fu of KNI-272 in AAG solution was 1.83%, and only one-quarter of that in HSA solution (Fu = 6.78%). Binding displacing agents, such as disopyramide, warfarin, diazepam, and digitoxin, were used to determine the binding site of KNI-272 on these plasma proteins. The Fu of KNI-272 in AAG solution increased 14-fold when disopyramide was added to the AAG solution. In addition, warfarin, diazepam, and digitoxin were added to HSA solution as representative drugs bound to distinct binding sites on HSA, namely sites I, II, and III, respectively. The Fu values of KNI-272 in HSA solution significantly increased when warfarin and diazepam were added. In particular, with the addition of warfarin to HSA solution, the Fu of KNI-272 increased to 16%. The modified Scatchard plots of KNI-272 binding to AAG and HSA both showed biphasic curves, and the KNI-272 binding sites at low concentration range on AAG and HSA disappeared with the addition of disopyramide and warfarin, respectively. Therefore, it is considered that KNI-272 binds to the identical site as disopyramide on AAG and site I on HSA in the low KNI-272 concentration range. By comparing the KNI-272 binding parameters obtained in human plasma and these protein solutions, we can assume that KNI-272 binding at low concentration in human plasma is mainly concerned with the binding on AAG. As KNI-272 concentration in plasma increases, HSA becomes concerned with KNI-272 binding.     

Observation of initial deposition process of electroless nickel plating by quartz crystal microbalance method and microscopy

The initial deposition process of electroless nickel plating was investigated by combining a quartz crystal microbalance (QCM) method with microscopy. The authors found an anomalous deposition rate in the initial deposition and four stages were noted: an induction period before the initiation of deposition, an acceleration period with an increase in the deposition rate, a deceleration period with a decrease in the deposition rate and a stationary period at a constant deposition rate. The practical surface area of the deposits increased until the deposits became continuous and reached a constant value. On the other hand, the deposition rate per unit practical surface area decreased monotonously as the deposition proceeded. As a result, an anomalous initial deposition rate was observed. The four periods also appeared in the deposition when the catalyzation process was repeated 4 times. In this case, the number of grains at the initial stage was greater, and nucleation still continued until the deposits became continuous. The initial deposits, therefore, became continuous at lower thickness.     

Adenylosuccinate synthetase genes: molecular cloning and phylogenetic analysis of a highly conserved archaeal gene

Adenylosuccinate synthetase (PurA) catalyzes the first step in the de novo AMP synthesis and has been extensively studied in both Bacteria and Eukarya. We cloned the purA gene from the hyperthermophilic archaeon, Pyrococcus furiosus. The gene appears to be individually transcribed and encodes a protein of 339 amino acids. The amino acid sequence comparison with other archael PurAs found from recent genome analyses indicated that two deletions, one central and the other C-terminal, are a common feature of archaeal PurAs. None of the 21 PurA homologues analyzed from Eukarya and Bacteria exhibited this feature. Amino acid sequences of PurAs in Archaea showed 64% average identities which were significantly higher than the 50% and 55% calculated for Bacteria and Eukarya, respectively. Several residues conserved in PurAs of both Eukarya and Bacteria and shown to be of catalytic importance are missing in the archaeal PurAs. Phylogenetic analysis using PurA as the marker grouped life into 3 domains, hence it was consistent with results derived from 16-18S ribosomal RNA sequences. The topology within the three domains, in general, portrayed the hitherto accepted evolutionary relationship among the organisms utilized. PurA can, thus, serve as an additional marker to evaluate phylogenetic inferences drawn from sequence data from rRNA and other conserved genes. The presence of two unique deletions in both euryarchaeal and crenarchaeal PurAs, but not in those of Bacteria and Eukarya, is a strong evidence confirming the common lineage of these two subdomains of Archaea.     

Variations in the molecular species of lung phosphatidylglycerol

Approximately 20% of the phosphatidylglycerol of the lung tissue of several animals was found to have both fatty acids saturated. Pulmonary washings from the lung of the rabbit and guinea pig had more saturated phosphatidylglycerol than the washed lung tissue. Lung-saturated phosphatidylglycerol was relatively low in the perinatal period, a time during which saturated phosphatidylcholine accumulated predominantly. This suggests that the metabolism of the saturated species of lung phosphatidylcholine and phosphatidylglycerol, which are considered to be the major pulmonary surfactants, may not be regulated in the same manner, at least in the perinatal lung.     

Need for and requirements for a neutron irradiation facility for fusion materials testing

The construction and operation of an intense 14-MeV neutron source is essential for the development and eventual qualification of structural materials for a fusion reactor demonstration plant (DEMO). Because of the time required for materials development and the scale-up of materials to commercial production, a decision to build a neutron source should precede engineering design activities for a DEMO by at least 20 years. The characteristic features of 14-MeV neutron damage are summarized including effects related to cascade structure, transmutation production, and dose rate. The importance of a 14-MeV neutron source for addressing fundamental radiation damage issues, alloy development activities, and the development of an engineering database is discussed. For these considerations, the basic requirements and machine parameters are derived.     

The Functional Diversity of Nitric Oxide Synthase Isoforms in Human Nose and Paranasal Sinuses: Contrasting Pathophysiological Aspects in Nasal Allergy and Chronic Rhinosinusitis

The human paranasal sinuses are the major source of intrinsic nitric oxide (NO) production in the human airway. NO plays several roles in the maintenance of physiological homeostasis and the regulation of airway inflammation through the expression of three NO synthase (NOS) isoforms. Measuring NO levels can contribute to the diagnosis and assessment of allergic rhinitis (AR) and chronic rhinosinusitis (CRS). In symptomatic AR patients, pro-inflammatory cytokines upregulate the expression of inducible NOS (iNOS) in the inferior turbinate. Excessive amounts of NO cause oxidative damage to cellular components, leading to the deposition of cytotoxic substances. CRS phenotype and endotype classifications have provided insights into modern treatment strategies. Analyses of the production of sinus NO and its metabolites revealed pathobiological diversity that can be exploited for useful biomarkers. Measuring nasal NO based on different NOS activities is a potent tool for specific interventions targeting molecular pathways underlying CRS endotype-specific inflammation. We provide a comprehensive review of the functional diversity of NOS isoforms in the human sinonasal system in relation to these two major nasal disorders’ pathologies. The regulatory mechanisms of NOS expression associated with the substrate bioavailability indicate the involvement of both type 1 and type 2 immune responses.     

Structure‐Based Mutational Study of an Archaeal DNA Ligase towards Improvement of Ligation Activity

DNA ligases catalyze the joining of strand breaks in duplex DNA. The DNA ligase of Pyrococcus furiosus (PfuLig), which architecturally resembles the human DNA ligase I (hLigI), comprises an N‐terminal DNA‐binding domain, a middle adenylylation domain, and a C‐terminal oligonucleotide‐binding (OB)‐fold domain. Here we addressed the C‐terminal helix in the OB‐fold domain of PfuLig by mutational analysis. The crystal structure of PfuLig revealed that this helix stabilizes a closed conformation of the enzyme by forming several ionic interactions with the adenylylation domain. The C‐terminal helix is oriented differently in hLigI when DNA is bound; this suggested that disruption of its interaction with the adenylylation domain might facilitate the binding of DNA substrates. We indeed identified one of its residues, Asp540, as being critical for ligation efficiency. The D540R mutation improved the overall ligation activity relative to the wild‐type enzyme, and at lower temperatures; this is relevant to applications such as ligation amplification reactions. Physical and biochemical analyses indicated that the improved ligation activity of the D540R variant arises from effects on the ligase adenylylation step and on substrate DNA binding in particular.