Quantitative and noninvasive assessment of prenatal X-ray-induced CNS abnormalities using magnetic resonance imaging

Our purpose was to noninvasively assess formation of the microvasculature, blood-brain barrier (BBB) and blood-CSF barrier formation of prenatal X-ray-induced CNS abnormalities using quantitative MRI. Eight pregnant female Sprague-Dawley rats were divided into two groups consisting of control and X-irradiated animals. After birth, 20 neonatal male rats were divided into four groups of five rats. To evaluate the development of the BBB, changes in T(1) induced by Gd-DTPA were compared quantitatively in normal and prenatally irradiated animals in the formative period 1 to 2 weeks after birth. To assess the abnormalities of the microvasculature, quantitative perfusion MRI and MR angiography were also used. Histology was also performed to evaluate the BBB (albumin) and vascular endothelial cells (laminin). Decreased cerebral blood flow (CBF) and angioarchitectonic abnormalities were observed in the prenatally irradiated rats. However, abnormalities of the BBB and blood-CSF barrier were not observed using Gd-enhanced MRI and albumin staining. Quantitative perfusion MRI, MR angiography and Gd-enhanced T(1) mapping are useful for assessing CNS disturbance after prenatal exposure to radiation. These techniques provide important diagnostic information for assessing the condition of patients during the early stages of life after accidental or unavoidable prenatal exposure to radiation.     

Bacterial symbionts of the giant jewel stinkbug Eucorysses grandis (Hemiptera: Scutelleridae)

Microbiological characterization of gut symbiotic bacteria in a limited number of stinkbugs of the families Acanthosomatidae, Plataspidae, Pentatomidae, Scutelleridae, Parastrachiidae, Alydidae and Pyrrhocoridae has shown symbiotic association with midgut bacteria to be common in phytophagous taxa of these heteropteran insects. Here we investigated the midgut bacterial symbiont of Eucorysses grandis, a stinkbug of the family Scutelleridae. A specific gammaproteobacterium was consistently identified in insects from five different geographic origins. The bacterium was detected in 64 of 64 insects sampled from three host populations. Phylogenetic analyses revealed that the bacterium constitutes a distinct lineage in the Gammaproteobacteria, neither closely related to the gut symbiont of another scutellerid stinkbug, Cantao ocellatus, nor to gut symbionts of other stinkbugs. Diagnostic PCR, in situ hybridization and electron microscopy demonstrated that the bacterium is located extracelluarly, in the midgut fourth section, which possesses crypts. These results indicate that the primary gut symbionts have multiple evolutionary origins in the Scutelleridae. A Sodalis-allied facultative symbiont was also identified in some insects from natural populations. Biological aspects of the primary gut symbiont and the secondary Sodalis-allied symbiont are discussed.     

Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 associates with insulin receptor substrate-1 and enhances insulin actions and adipogenesis

Peptidyl-prolyl cis/trans isomerase NIMA-interacting 1 (Pin1) is a unique enzyme that associates with the pSer/Thr-Pro motif and catalyzes cis-trans isomerization. We identified Pin1 in the immunoprecipitates of overexpressed IRS-1 with myc and FLAG tags in mouse livers and confirmed the association between IRS-1 and Pin1 by not only overexpression experiments but also endogenously in the mouse liver. The analysis using deletion- and point-mutated Pin1 and IRS-1 constructs revealed the WW domain located in the N terminus of Pin1 and Ser-434 in the SAIN (Shc and IRS-1 NPXY binding) domain of IRS-1 to be involved in their association. Subsequently, we investigated the role of Pin1 in IRS-1 mediation of insulin signaling. The overexpression of Pin1 in HepG2 cells markedly enhanced insulin-induced IRS-1 phosphorylation and its downstream events: phosphatidylinositol 3-kinase binding with IRS-1 and Akt phosphorylation. In contrast, the treatment of HepG2 cells with Pin1 siRNA or the Pin1 inhibitor Juglone suppressed these events. In good agreement with these in vitro data, Pin1 knock-out mice exhibited impaired insulin signaling with glucose intolerance, whereas adenoviral gene transfer of Pin1 into the ob/ob mouse liver mostly normalized insulin signaling and restored glucose tolerance. In addition, it was also demonstrated that Pin1 plays a critical role in adipose differentiation, making Pin1 knock-out mice resistant to diet-induced obesity. Importantly, Pin1 expression was shown to be up-regulated in accordance with nutrient conditions such as food intake or a high-fat diet. Taken together, these observations indicate that Pin1 binds to IRS-1 and thereby markedly enhances insulin action, essential for adipogenesis.     

Vitamin K2 Biosynthetic Enzyme,UBIAD1 Is Essential for Embryonic Development of Mice

UbiA prenyltransferase domain containing 1 (UBIAD1) is a novel vitamin K₂ biosynthetic enzyme screened and identified from the human genome database. UBIAD1 has recently been shown to catalyse the biosynthesis of Coenzyme Q10 (CoQ10) in zebrafish and human cells. To investigate the function of UBIAD1 in vivo, we attempted to generate mice lacking Ubiad1, a homolog of human UBIAD1, by gene targeting. Ubiad1-deficient (Ubiad1 ^−/−) mouse embryos failed to survive beyond embryonic day 7.5, exhibiting small-sized body and gastrulation arrest. Ubiad1 ^−/− embryonic stem (ES) cells failed to synthesize vitamin K₂ but were able to synthesize CoQ9, similar to wild-type ES cells. Ubiad1 ^+/− mice developed normally, exhibiting normal growth and fertility. Vitamin K₂ tissue levels and synthesis activity were approximately half of those in the wild-type, whereas CoQ9 tissue levels and synthesis activity were similar to those in the wild-type. Similarly, UBIAD1 expression and vitamin K₂ synthesis activity of mouse embryonic fibroblasts prepared from Ubiad1 ^+/− E15.5 embryos were approximately half of those in the wild-type, whereas CoQ9 levels and synthesis activity were similar to those in the wild-type. Ubiad1 ^−/− mouse embryos failed to be rescued, but their embryonic lifespans were extended to term by oral administration of MK-4 or CoQ10 to pregnant Ubiad1 ^+/− mice. These results suggest that UBIAD1 is responsible for vitamin K₂ synthesis but may not be responsible for CoQ9 synthesis in mice. We propose that UBIAD1 plays a pivotal role in embryonic development by synthesizing vitamin K₂, but may have additional functions beyond the biosynthesis of vitamin K₂.     

Ribosomes in a Stacked Array: ELUCIDATION OF THE STEP IN TRANSLATION ELONGATION AT WHICH THEY ARE STALLED DURING S-ADENOSYL-l-METHIONINE-INDUCED TRANSLATION ARREST OF CGS1 mRNA*

Expression of CGS1, which codes for an enzyme of methionine biosynthesis, is feedback-regulated by mRNA degradation in response to S-adenosyl-l-methionine (AdoMet). In vitro studies revealed that AdoMet induces translation arrest at Ser-94, upon which several ribosomes stack behind the arrested one, and mRNA degradation occurs at multiple sites that presumably correspond to individual ribosomes in a stacked array. Despite the significant contribution of stacked ribosomes to inducing mRNA degradation, little is known about the ribosomes in the stacked array. Here, we assigned the peptidyl-tRNA species of the stacked second and third ribosomes to their respective codons and showed that they are arranged at nine-codon intervals behind the Ser-94 codon, indicating tight stacking. Puromycin reacts with peptidyl-tRNA in the P-site, releasing the nascent peptide as peptidyl-puromycin. This reaction is used to monitor the activity of the peptidyltransferase center (PTC) in arrested ribosomes. Puromycin reaction of peptidyl-tRNA on the AdoMet-arrested ribosome, which is stalled at the pre-translocation step, was slow. This limited reactivity can be attributed to the peptidyl-tRNA occupying the A-site at this step rather than to suppression of PTC activity. In contrast, puromycin reactions of peptidyl-tRNA with the stacked second and third ribosomes were slow but were not as slow as pre-translocation step ribosomes. We propose that the anticodon end of peptidyl-tRNA resides in the A-site of the stacked ribosomes and that the stacked ribosomes are stalled at an early step of translocation, possibly at the P/E hybrid state.     

Early Staphylococcal Biofilm Formation on Solid Orthopaedic Implant Materials: In Vitro Study

Biofilms forming on the surface of biomaterials can cause intractable implant-related infections. Bacterial adherence and early biofilm formation are influenced by the type of biomaterial used and the physical characteristics of implant surface. In this in vitro research, we evaluated the ability of Staphylococcus epidermidis, the main pathogen in implant-related infections, to form biofilms on the surface of the solid orthopaedic biomaterials, oxidized zirconium-niobium alloy, cobalt-chromium-molybdenum alloy (Co-Cr-Mo), titanium alloy (Ti-6Al-4V), commercially pure titanium (cp-Ti) and stainless steel. A bacterial suspension of Staphylococcus epidermidis strain RP62A (ATCC35984) was added to the surface of specimens and incubated. The stained biofilms were imaged with a digital optical microscope and the biofilm coverage rate (BCR) was calculated. The total amount of biofilm was determined with the crystal violet assay and the number of viable cells in the biofilm was counted using the plate count method. The BCR of all the biomaterials rose in proportion to culture duration. After culturing for 2–4 hours, the BCR was similar for all materials. However, after culturing for 6 hours, the BCR for Co-Cr-Mo alloy was significantly lower than for Ti-6Al-4V, cp-Ti and stainless steel (P<0.05). The absorbance value determined in the crystal violet assay and the number of viable cells on Co-Cr-Mo were not significantly lower than for the other materials (P>0.05). These results suggest that surface properties, such as hydrophobicity or the low surface free energy of Co-Cr-Mo, may have some influence in inhibiting or delaying the two-dimensional expansion of biofilm on surfaces with a similar degree of smoothness.     

WFS1 protein modulates the free Ca(2+) concentration in the endoplasmic reticulum

The WFS1 gene, encoding an endoplasmic reticulum (ER) membrane glycoprotein, is mutated in Wolfram syndrome characterized by diabetes mellitus and optic atrophy. Herein, Ca(2+) dynamics were examined in WFS1-knockdown and -overexpressing HEK293 cells. Studies using ER-targeted Ca(2+)-sensitive photoprotein aequorin demonstrated WFS1 protein to positively modulate ER Ca(2+) levels by increasing the rate of Ca(2+) uptake. Furthermore, Ca(2+) imaging with Fura-2 showed the magnitude of the store-operated Ca(2+) entry to parallel WFS1 expression levels. These data indicate that WFS1 protein participates in the regulation of cellular Ca(2+) homeostasis, at least partly, by modulating the filling state of the ER Ca(2+) store.     

A sensitive method for quantitation of steroid hydroxylase activities of individual P-450 in tissue homogenates

Assay methods which allow measurements of the level of individual P-450's in a homogenate of steroidogenic tissues have been developed. The assay is based simply on the determination of the specific products of steroid monooxygenase reactions under conditions in which sufficient amounts of the purified electron-donating components as well as lipids and detergents are supplemented so that the membrane-associated P-450 is able to exhibit its maximum activity. Under these conditions, an at least 10-fold increase in the NADPH-dependent monooxygenase activity of P-450 occurred as compared to that measured in the unenriched system. In addition, the present assay used ascorbate as an O2- -scavenger which effectively prevented possible inhibition caused by initiation of O2- -formation. Under the present assay conditions, nearly quantitative recovery of activities was accomplished of each purified P-450 that had been added to the homogenates as an internal standard. Furthermore, the high sensitivity of this assay allows the measurement of P-450 in small specimens (at least 100 mg of tissues), and could be used for measurements in autopsied cases or the glands of small animals.