Synthesis and Biological Evaluation of 1′-C-Cyano-Pyrimidine Nucleosides

Abstract

2′-Deoxy-, 2′-bromo-, and arabino-1′-C-cyano-pyrimidine nucleosides were synthesized from O² ,2′-cyclouridine. Incorporation of cyano group at the anomeric position was achieved by treatment of 1′,2′-unsaturated uridine with NBS in the presence of pivalic acid followed by TMS-cyanide and stannic chloride. Antineoplastic and antiviral activities of those compounds are also discussed.

     

Effects of Coexistence of β-Casein on Gelation of κ-Carrageenan

The effects of caseins on the rheological properties of κ-carrageenan-calcium gel was investigated by measuring the gel breaking strength. The existence of β-casein in the system promoted the gelation of κ-carrageenan in the presence of calcium ion. Beta-casein increased the strength of calcium gels of κ-carrageenan with increasing NaCl concentration up to 80 mM and strengthened the κ-carrageenan-calcium gel at neutral pH. The values obtained from the slopes of the logarithmic plots of the gel strength versus concentration were 2.15 for κ-carrageenan gel and 2.27 for a β-casein-κcarrageenan mixture gel, suggesting that β-casein may participate in the gelation of κ-carrageenan through the mediation of calcium ions.     

Molecular Characterization of Low-Molecular-Weight Component Protein,Flp, in Actinobacillus actinomycetemcomitans Fimbriae

Fimbriae preparation from Actinobacillus actinomycetemcomitans was found to contain an abundant low-molecular-weight protein (termed Flp) with an apparent molecular mass of approximately 6.5 kDa, in addition to a small amount of 54-kDa protein. Immunogold electron microscopy localized the Flp protein at the bacterial fimbriae but not at the cell surface. The DNA fragment including the flp gene was cloned from A. actinomycetemcomitans 304-a and its nucleotide sequence was determined. An open reading frame of the flp gene was composed of 225 bp encoding a protein of 75 amino acids. Comparison of the translated amino acid sequence with the sequence of native Flp determined by Edman degradation indicated that the N-terminal part of 26 amino acids is leader peptide. The N-terminal sequence of mature Flp exhibited some similarity to type-IV pilin. Furthermore, the processing site of premature Flp is also similar to that of type-IV prepilin, and a gene encoding a protein homologous to type-IV prepilin-like protein leader peptidase was found downstream of the flp gene. These findings indicate that Flp is the major component protein of A. actinomycetemcomitans fimbriae.     

Role of Diffusion Weighted Imaging and Contrast-Enhanced MRI in the Evaluation of Intrapelvic Recurrence of Gynecological Malignant Tumor

Background and Purpose

To investigate the diagnostic performance of diffusion-weighted imaging (DWI) and contrast-enhanced imaging in combination with T2-weighted imaging (T2WI) for magnetic resonance imaging (MRI) evaluation of intrapelvic recurrence of gynecological malignancies.

Materials and Methods

Sixty-two patients with suspected intrapelvic recurrence of gynecological malignancies underwent pelvic MRI including T2WI DWI, and contrast-enhanced imaging. Diagnostic performance for detection of local recurrence, pelvic lymph node and bone metastases, and peritoneal lesions was evaluated by consensus reading of two experienced radiologists using a 5-point scoring system, and compared among T2WI with unenhanced T1-weighted imaging (T1WI) (protocol A), a combination of protocol A and DWI (protocol B), and a combination of protocol B and contrast-enhanced imaging (protocol C). Final diagnoses were obtained by histopathological examinations, radiological imaging and clinical follow-up for at least 6 months. Receiver operating characteristic (ROC) analysis and McNemar test were employed for statistical analysis.

Results

Locally recurrent disease, lymph node recurrence, peritoneal dissemination and bone metastases were present in 48.4%, 29.0%, 16.1%, and 6.5% of the patients, respectively. The patient-based sensitivity, specificity, accuracy, and area under the ROC curve (AUC) for detection of intrapelvic recurrence were 55.0, 81.8, 64.5% and 0.753 for protocol A, 80.0, 77.3, 79.0% and 0.838 for protocol B, and 80.0, 90.9, 83.9% and 0.862 for protocol C, respectively. The sensitivity, accuracy, and AUC were significantly better for protocols B and C than for protocol A (p<0.001). There was no significant difference between protocols B and C.

Conclusion

MRI using a combination of DWI and T2WI gives comparatively acceptable results for assessment of intrapelvic recurrence of gynecological malignancies.     

The First Nationwide Survey and Genetic Analyses of Bardet-Biedl Syndrome in Japan

Bardet-Biedl syndrome (BBS) is an autosomal recessive disorder characterized by central obesity, mental impairment, rod-cone dystrophy, polydactyly, hypogonadism in males, and renal abnormalities. The causative genes have been identified as BBS1-19. In Western countries, this disease is often reported, but remains undiagnosed in many patients until later in life, while only a few patients with no mutations identified have been reported in Japan. We thus conducted the first nationwide survey of BBS in Japan by sending questionnaires to 2,166 clinical departments with board-certified specialists and found 7 patients with clinically definite BBS. We performed exome analyses combined with analyses of mRNA and protein in these patients. We identified 2 novel mutations in the BBS5 gene (p.R89X and IVS7-27 T>G) in 2 sibling patients. The latter mutation that resided far from the authentic splicing site was associated with skipping of exon 8. We also found 3 previously reported mutations in the BBS2 (p.R413X and p.R480X) and BBS7 (p.C243Y) genes in 2 patients. To our knowledge, a nationwide survey of BBS has not been reported in any other country. In addition, this is the first study to identify genetic alterations in Japanese patients with BBS. Our results indicate that BBS in Japan is genetically heterogeneous and at least partly shares genetic features with BBS in other countries.     

Alterations of Regional Cerebral Blood Flow in Tinnitus Patients as Assessed Using Single-Photon Emission Computed Tomography

Tinnitus is the perception of phantom sound without an external auditory stimulus. Using neuroimaging techniques, such as positron emission tomography, electroencephalography, magnetoencephalography, and functional magnetic resonance imaging (fMRI), many studies have demonstrated that abnormal functions of the central nervous system are closely associated with tinnitus. In our previous research, we reported using resting-state fMRI that several brain regions, including the rectus gyrus, cingulate gyrus, thalamus, hippocampus, caudate, inferior temporal gyrus, cerebellar hemisphere, and medial superior frontal gyrus, were associated with tinnitus distress and loudness. To reconfirm these results and probe target regions for repetitive transcranial magnetic stimulation (rTMS), we investigated the regional cerebral blood flow (rCBF) between younger tinnitus patients (<60 years old) and the age-matched controls using single-photon emission computed tomography and easy Z-score imaging system. Compared with that of controls, the rCBF of tinnitus patients was significantly lower in the bilateral medial superior frontal gyri, left middle occipital gyrus and significantly higher in the bilateral cerebellar hemispheres and vermis, bilateral middle temporal gyri, right fusiform gyrus. No clear differences were observed between tinnitus patients with normal and impaired hearing. Regardless of the assessment modality, similar brain regions were identified as characteristic in tinnitus patients. These regions are potentially involved in the pathophysiology of chronic subjective tinnitus.     

Potential Biomarkers of Fatigue Identified by Plasma Metabolome Analysis in Rats

In the present study, prior to the establishment of a method for the clinical diagnosis of chronic fatigue in humans, we validated the utility of plasma metabolomic analysis in a rat model of fatigue using capillary electrophoresis-mass spectrometry (CE-MS). In order to obtain a fatigued animal group, rats were placed in a cage filled with water to a height of 2.2 cm for 5 days. A food-restricted group, in which rats were limited to 10 g/d of food (around 50% of the control group), was also assessed. The food-restricted group exhibited weight reduction similar to that of the fatigued group. CE-MS measurements were performed to evaluate the profile of food intake-dependent metabolic changes, as well as the profile in fatigue loading, resulting in the identification of 48 metabolites in plasma. Multivariate analyses using hierarchical clustering and principal component analysis revealed that the plasma metabolome in the fatigued group showed clear differences from those in the control and food-restricted groups. In the fatigued group, we found distinctive changes in metabolites related to branched-chain amino acid metabolism, urea cycle, and proline metabolism. Specifically, the fatigued group exhibited significant increases in valine, leucine, isoleucine, and 2-oxoisopentanoate, and significant decreases in citrulline and hydroxyproline compared with the control and food-restricted groups. Plasma levels of total nitric oxide were increased in the fatigued group, indicating systemic oxidative stress. Further, plasma metabolites involved in the citrate cycle, such as cis-aconitate and isocitrate, were reduced in the fatigued group. The levels of ATP were significantly decreased in the liver and skeletal muscle, indicative of a deterioration in energy metabolism in these organs. Thus, this comprehensive metabolic analysis furthered our understanding of the pathophysiology of fatigue, and identified potential diagnostic biomarkers based on fatigue pathophysiology.     

Regulation of the Contribution of Integrin to Cell Attachment on Poly(2-Methoxyethyl Acrylate) (PMEA) Analogous Polymers for Attachment-Based Cell Enrichment

Cell enrichment is currently in high demand in medical engineering. We have reported that non-blood cells can attach to a blood-compatible poly(2-methoxyethyl acrylate) (PMEA) substrate through integrin-dependent and integrin-independent mechanisms because the PMEA substrate suppresses protein adsorption. Therefore, we assumed that PMEA analogous polymers can change the contribution of integrin to cell attachment through the regulation of protein adsorption. In the present study, we investigated protein adsorption, cell attachment profiles, and attachment mechanisms on PMEA analogous polymer substrates. Additionally, we demonstrated the possibility of attachment-based cell enrichment on PMEA analogous polymer substrates. HT-1080 and MDA-MB-231 cells started to attach to poly(butyl acrylate) (PBA) and poly(tetrahydrofurfuryl acrylate) (PTHFA), on which proteins could adsorb well, within 1 h. HepG2 cells started to attach after 1 h. HT-1080, MDA-MB-231, and HepG2 cells started to attach within 30 min to PMEA, poly(2-(2-methoxyethoxy) ethyl acrylate-co-butyl acrylate) (30:70 mol%, PMe2A) and poly(2-(2-methoxyethoxy) ethoxy ethyl acrylate-co-butyl acrylate) (30:70 mol%, PMe3A), which suppress protein adsorption. Moreover, the ratio of attached cells from a cell mixture can be changed on PMEA analogous polymers. These findings suggested that PMEA analogous polymers can be used for attachment-based cell enrichment.     

Minimally Invasive Electro-Magnetic Navigational Bronchoscopy-Integrated Near-Infrared-Guided Sentinel Lymph Node Mapping in the Porcine Lung

BackgroundThe use of near-infrared (NIR) fluorescence imaging with indocyanine green (ICG) for sentinel lymph node (SN) mapping has been investigated in lung cancer; however, this has not been fully adapted for minimally invasive surgery (MIS). The aim of our study was to develop a minimally invasive SN mapping integrating pre-operative electro-magnetic navigational bronchoscopy (ENB)-guided transbronchial ICG injection and intraoperative NIR thoracoscopic imaging.MethodsA NIR thoracoscope was used to visualize ICG fluorescence. ICG solutions in a 96-well plate and ex vivo porcine lungs were examined to optimize ICG concentrations and injection volumes. Transbronchial ICG injection (n=4) was assessed in comparison to a traditional transpleural approach (n=3), where after thoracotomy an ICG solution (100μL at 100μg/mL) was injected into the porcine right upper lobe for SN identification. For further translation into clinical use, transbronchial ICG injection prior to thoracotomy followed by NIR thoracoscopic imaging was validated (n=3). ENB was used for accurate targeting in two pigs with a pseudo-tumor.ResultsThe ICG fluorescence at 10 μg/mL was the brightest among various concentrations, unchanged by the distance between the thoracoscope and ICG solutions. Injected ICG of no more than 500μL showed a localized fluorescence area. All 7 pigs showed a bright paratracheal lymph node within 15 minutes post-injection, with persistent fluorescence for 60 minutes. The antecedent transbronchial ICG injection succeeded in SN identification in all 3 cases at the first thoracoscopic inspection within 20 minutes post-injection. The ENB system allowed accurate ICG injection surrounding the pseudo-tumors.ConclusionsENB-guided ICG injection followed by NIR thoracoscopy was technically feasible for SN mapping in the porcine lung. This promising platform may be translated into human clinical trials and is suited for MIS.