Treatment of Mid‐Pregnant Mice with KRN633, an Inhibitor of Vascular Endothelial Growth Factor Receptor Tyrosine Kinase,Induces Abnormal Retinal Vascular Patterning in Their Newborn Pups

We previously reported that treatment of mid‐pregnant mice with KRN633, a vascular endothelial growth factor receptor tyrosine kinase inhibitor, caused fetal growth restriction resulting from diminished vascularization in the placenta and fetal organs. In this study, we examined how the treatment of mid‐pregnant mice with KRN633 affects the development and morphology of vascular components (endothelial cells, pericytes, and basement membrane) in the retinas of their newborn pups. Pregnant mice were treated with KRN633 (5 mg/kg) once daily from embryonic day 13.5 until the day of delivery. Vascular components were examined using immunohistochemistry with specific markers for each component. Radial vascular growth in the retina was slightly delayed until postnatal day 4 (P4) in the newborn pups of KRN633‐treated mothers. On P8, compared with the pups of control mothers, the pups of KRN633‐treated mothers exhibited decreased numbers of central arteries and veins and abnormal branching of the central arteries. No apparent differences in pericytes or basement membrane were observed between the pups of control and KRN633‐treated mothers. These results suggest that a critical period for determining retinal vascular patterning is present at the earliest stages of retinal vascular development, and that the impaired vascular endothelial growth factor signaling during this period induces abnormal architecture in the retinal vascular network     

Antiobesity and emetic effects of a short-length peptide YY analog and its PEGylated and alkylated derivatives

Neuropeptide Y2 receptor (Y2R) agonism is an important anorectic signal and a target of antiobesity drug discovery. Recently, we synthesized a short-length Y2R agonist, PYY-1119 (4-imidazolecarbonyl-[d-Hyp²⁴,Iva²⁵,Pya(4)²⁶,Cha^27,36,γMeLeu²⁸,Lys³⁰,Aib³¹]PYY(23–36), 1) as an antiobesity drug candidate. Compound 1 induced marked body weight loss in diet-induced obese (DIO) mice; however, 1 also induced severe vomiting in dogs at a lower dose than the minimum effective dose administered to DIO mice. The rapid absorption of 1 after subcutaneous administration caused the severe vomiting. Polyethylene glycol (PEG)- and alkyl-modified derivatives of 1 were synthesized to develop Y2R agonists with improved pharmacokinetic profiles, i.e., lower maximum plasma concentration (C_max) and longer time at maximum concentration (T_max). Compounds 5 and 10, modified with 20?kDa PEG at the N-terminus and eicosanedioic acid at the Lys³⁰ side chain of 1, respectively, showed high Y2R binding affinity and induced significant body weight reduction upon once-daily administration to DIO mice. Compounds 5 and 10, with their relatively low C_max and long T_max, partially attenuated emesis in dogs compared with 1. These results indicate that optimization of pharmacokinetic properties of Y2R agonists is an effective strategy to alleviate emesis induced by Y2R agonism.     

Characterization of Glial Populations in the Aging and Remyelinating Mouse Corpus Callosum

Cells in the white matter of the adult brain have a characteristic distribution pattern in which several cells are contiguously connected to each other, making a linear array (LA) resembling pearls-on-a-string parallel to the axon axis. We have been interested in how this pattern of cell distribution changes during aging and remyelination after demyelination. In the present study, with a multiplex staining method, semi-quantitative analysis of the localization of oligodendrocyte lineage cells (oligodendrocyte progenitors, premyelinating oligodendrocytes, and mature oligodendrocytes), astrocytes, and microglia in 8-week-old (young adult) and 32-week-old (aged) corpus callosum showed that young adult cells still include immature oligodendrocytes and that LAs contain a higher proportion of microglia than isolated cells. In aged mice, premyelinating oligodendrocytes were decreased, but microglia continued to be present in the LAs. These results suggest that the presence of microglia is important for the characteristic cell localization pattern of LAs. In a cuprizone-induced demyelination model, we observed re-formation of LAs after completion of cuprizone treatment, concurrent with remyelination. These re-formed LAs again contained more microglia than the isolated cells. This finding supports the hypothesis that microglia contribute to the formation and maintenance of LAs. In addition, regardless of the distribution of cells (LAs or isolated cells), astrocytes were found to be more abundant than in the normal corpus callosum at 24 weeks after cuprizone treatment when remyelination is completed. This suggests that astrocytes are involved in maintaining the functions of remyelinated white matter.

     

Dielectric relaxation of DNA in aqueous solutions.

Using a four-electrode cell and a new electronic system for direct detection of the frequency differences specturm of solution impedance, the complex dielectric constant of calf thymus DNA (M_r = 4 × 10⁶) in aqueous NaCl at 10°C is measured at frequencies ranging from 0.2 Hz to 30 kHz. The DNA concentrations are C_p = 0.01% and 0.05%, and the NaCl concentrations are varied from C_s = 10^?4 M to 10^?3 M. A single relaxation regions is found in this frequency range, the relaxation frequency being 10 Hz at C_p = 0.01% and C_s = 10^?3 M. At C_p = 0.05% it is evidenced that the DNA chains have appreciable intermolecular interactions. The dielectric relaxaton time τ_d at C_p = 0.01% agrees well with the rotational relaxation time estimated from the reduced visocisty on the assumption that the DNA is not representable as a rigid rod but a coiled chain. It is concluded that the dielectric relaxiatioinis ascribed to the rotation of the molecule. Observed values of dielectric increment and other experimental findings are reasonably explained by assuming that the dipole moment of DNA results from the slow counterion fluctuation which has a longer relaxation time than τ_d.     

Polymorphism of the kinase domain of the S-locus receptor kinase gene (SRK) in Brassica oleracea L.

 DNA polymorphism of the S-locus receptor kinase gene (SRK) participating in self-incompatibility in Brassica was analyzed by PCR-RFLP and nucleotide sequencing. In the screening of primers for specific amplification of polymorphic DNA fragments of SRK, the best combination was that of a forward primer (PK1) having the nucleotide sequence of the second exon of S6 SRK and a reverse primer (PK4) having the complementary nucleotide sequence of the fifth exon of S6 SRK. PCR using this primer pair amplified DNA fragments of 0.9–1.0 kb from 36 S haplotypes out of 42 tested. These DNA fragments showed high polymorphism in polyacrylamide-gel electrophoresis after digestion with restriction endonuclease(s): 25 types were found in a double digestion with MboI and AfaI. Nucleotide sequencing of the DNA fragments amplified from five S haplotypes showed that the third, fourth, and fifth exons of SRK are highly conserved, and that there are high variations of the second and third introns of SRK, which produced polymorphism of the band pattern in PCR-RFLPs. Another forward primer (PK5) having the nucleotide sequence of the second exon, which is derived from S2 SRK, amplified DNA fragments of almost the same region of SRK from 27 S haplotypes in combination with PK4. Although SRK alleles of the class-II S haplotypes were not amplified, all of the class-I S-haplotypes were amplified with a primer mixture of PK1, PK4 and PK5. The DNA fragments of both SRK alleles in S heterozygotes, or a 1 : 1 mixture of the genomic DNA of different S homozygotes, were amplified without exception, suggesting the usefulness of these primers for the identification of S heterozygotes. The DNA fragment sizes obtained by digestion with restriction endonucleases served as markers for the identification of S haplotypes. Received: 15 December 1996 / Accepted: 14 February 1997     

Immunohistochemical, in situ hybridization and biochemical studies on endogenous cellulase of Corbicula japonica

We previously reported on the endogenous cellulase gene of Corbicula japonica, CjCel9A. In this study, the tissue localization of the mRNA and translated products of CjCel9A was investigated in order to understand how this gene is physiologically involved in cellulose decomposition by C. japonica. Antiserum against recombinant CjCel9A protein was prepared. Multiple bands were observed mainly on western blot analysis of the crystalline style, and the band sizes partially corresponded to the active bands detected using zymographic analysis. In situ hybridization and immunohistochemical analyses clarified the exclusive production and secretion of this cellulase by the secretory cells localized in the epithelium of the digestive tubules in the digestive gland. These data strongly support our previous assumption that the endogenous cellulase of C. japonica is produced in the digestive gland and transported to the crystalline style to act as a component of its cellulolytic activity.     

Yeast plasmids resembling 2 micron DNA: regional similarities and diversities at the molecular level.

The nucleotide sequence of two Zygosaccharomyces plasmids, pSB2 (5,415 base pairs), isolated from Zygosaccharomyces bailii, and pSM1 (5,416 base pairs), isolated from Zygosaccharomyces fermentati Naganishi, was determined. The predicted amino acid sequences of open reading frames among six yeast plasmids that resemble 2 microns DNA indicated regional sequence similarities among FLP proteins. Greater similarities were seen among Zygosaccharomyces plasmids (pSB2, pSB3, pSR1, and pSM1) than other combinations. A putative recognition site for the FLP enzyme of a Zygosaccharomyces plasmid also showed some conservation, especially in the 4 nucleotides flanking the central spacer region. From comparative studies of the sequences of putative genes of each plasmid, we propose an apparent phylogenetic relationship among yeast plasmids resembling 2 micron DNA. Among the Zygosaccharomyces plasmids, pSB2 and pSR1 are most closely related, since not only were the FLP enzymes of these two plasmids most closely related, but also the amino acid sequence of the putative P gene of pSR1 showed clear homology with that of open reading frame B of pSB2.     

Association of meteorological and day-of-the-week factors with emergency hospital admissions in Fukuoka, Japan

We carried out a statistical study of the influence of meteorological and day-of-the-week factors on the intrinsic emergency patients transported to hospitals by ambulance. Multiple piecewise linear regression analysis was performed on data from 6,081 emergency admissions for 1 year between April 1997 and March 1998 in Fukuoka, Japan. The response variable was the daily number of emergency patients admitted with three types of disease: cerebrovascular, respiratory and digestive diseases. The results showed that the number of emergency patients admitted daily with cerebrovascular disease was significantly associated with temperature on the day of admission and whether the day was Sunday. As it became colder than 12 degrees C, emergency admissions of patients with cerebrovascular disease increased drastically, reaching a plateau at 4 degrees C. On the 3rd and 7th days after the temperature fell below 10 degrees C, the daily admission of patients with respiratory disease significantly increased. We also observed a weak association between emergency admissions of patients suffering from digestive disease and rising barometric pressure on the day of admission.