The conformational study of two carbocyclic nucleosides: why carbocyclic nucleic acids (CarNAs) form more stable duplexes with RNA than DNA does

Replacement of the furanose moiety of DNA with a cyclopentane ring produces a modified sugar analogue: Carbocyclic nucleic acid (CarNA). UV melting-temperature experiments demonstrate that the incorporation of 2'-deoxycarbaguanosine ((c)G) and 2'-deoxyaristeromycin ((c)A) of carbocyclic nucleosides into a DNA strand increases the stability of the CarNA/RNA hybrid. Circular Dichroism (CD) study indicates that the CarNA/RNA hybrid adopts an A-like conformation. To elucidate the molecular basis of the increased stability of the CarNA/RNA, the conformation of (c)G and (c)A were examined by (1)H NMR conformational analysis of (3)J(HH) coupling constants and ab initio molecular orbital (MO) calculations. These results show that the populations of N-type of (c)G and (c)A are higher than those of dG and dA, respectively, at different temperatures [For example, 37% (N%) of (c)G vs. 28%of dG, 36% (N%) of (c)A vs. 25% of dA at 278 K], which suggest that the cyclopentane rings of (c)G and (c)A prefer the N-type conformation in two-state N-S pseudorotional equilibrium in comparison with the furanose rings of dG and dA. The DeltaH degrees of (c)G (DeltaH degrees = - 0.43 kcal mol(-1)) and (c)A (DeltaH degrees = - 0.41kcal mol(-1)) are lower than that of dG (dG = - 1.8 kcal mol(-1)) and dA (dA = - 1.0 kcal mol(-1)), respectively, which suggest that the gauche effect in the (c)A and (c)G driving N-S pseudorotional equilibrium to S-type is reduced by replacement of the 4'-oxygen by a CH(2) group. These results suggest that the preferred N-type of the (c)G and (c)A leads to the A-like conformation, which contributes to the stability of CarNA/RNA hybrid.     

Injectable cell scaffold restores impaired cell-based therapeutic angiogenesis in diabetic mice with hindlimb ischemia

The clinical success of cell-based therapeutic angiogenesis has been limited in diabetic patients with critical limb ischemia. We previously reported that an injectable cell scaffold (ICS), which is a nano-scaled hydroxyapatite (HAp)-coated polymer microsphere, enhances therapeutic angiogenesis. Subsequently, we developed a modified ICS for clinical use, measuring 50 μm in diameter using poly(l-lactide-co-ε-caprolactone) as a biodegradable polymer, which achieved appropriately accelerated absorption in vivo. The aim of the present study was to evaluate the effectiveness of this practical ICS in diabetic hindlimb ischemia.     

Motor Function of the Upper-Extremity after Transection of the Second Thoracic Nerve Root during Total En Bloc Spondylectomy

Background

In total en bloc spondylectomy (TES) of upper thoracic spine including the second thoracic (T2) vertebra, T2 nerve roots are usually transected. In this study, we examined the association between transection of the T2 nerve roots and upper-extremity motor function in patients with upper thoracic TES.

Methods

We assessed 16 patients who underwent upper thoracic TES with bilateral transection of the T2 nerve roots. Patients were divided into three groups: 3 patients without any processing of T1 and upper nerve roots (T2 group), 7 with extensive dissection of T1 nerve roots (T1–2 group), and 6 with extensive dissection of T1 and upper nerve roots (C–T2 group). Postoperative upper-extremity motor function was compared between the groups.

Results

Postoperative deterioration of upper-extremity motor function was observed in 9 of the 16 patients (56.3%). Three of the 7 patients in the T1–2 group and all 6 patients in the C–T2 group showed deterioration of upper-extremity motor function, but there was no deterioration in the T2 group. In the T1–2 group, 3 patients showed mild deterioration that did not affect their activities of daily living and they achieved complete recovery at the latest follow-up examination. In contrast, severe dysfunction occurred frequently in the C–T2 group, without recovery at the latest follow-up.

Conclusions

The transection of the T2 nerve roots alone did not result in upper-extremity motor dysfunction; rather, the dysfunction is caused by the extensive dissection of the T1 and upper nerve roots. Therefore, transection of the T2 nerve roots in upper thoracic TES seems to be an acceptable procedure with satisfactory outcomes.     

Loss of HB-EGF in smooth muscle or endothelial cell lineages causes heart malformation

Epidermal growth factor (EGF) and ErbB family molecules play a role in heart development and function. To investigate the role of EGF family member, heparin-binding EGF-like growth factor (HB-EGF) in heart development, smooth muscle and endothelial cell lineage-specific HB-EGF knockout mice were generated using the Cre/loxP system in combination with the SM22alpha or TIE2 promoter. HB-EGF knockout mice displayed enlarged heart valves, and over half of these mice died during the first postnatal week, while survivors showed cardiac hypertrophy. These results suggest that expression of HB-EGF in smooth muscle and/or endothelial cell lineages is essential for proper heart development and function in mice.     

Is the ClC-2 chloride channel involved in the Cl- secretory mechanism of gastric parietal cells?

It has been controversial whether the ClC-2 chloride channel is involved in hydrochloric acid secretion of gastric parietal cells. Here, we investigated whether ClC-2 is the apical Cl- channel associated with gastric acid secretion. Two anti-ClC-2 antibodies used in this study reacted with cloned ClC-2 protein expressed in HEK293 cells. In isolated rabbit gastric glands, significant expression of ClC-2 mRNA was observed, but the presence of ClC-2 protein was not clear. Furthermore, no expression of ClC-2 protein was observed in isolated rat and human gastric mucosa. Immunohistochemistry on the rat gastric mucosa showed no significant expression of ClC-2 protein in the parietal cells which showed abundant expression of H+,K+-ATPase. These results indicate that ClC-2 may not be a Cl- -transporting protein for gastric acid secretion in parietal cells.     

Mutations affecting liver development and function in Medaka, Oryzias latipes, screened by multiple criteria

We report here mutations affecting various aspects of liver development and function identified by multiple assays in a systematic mutagenesis screen in Medaka. The 22 identified recessive mutations assigned to 19 complementation groups fell into five phenotypic groups. Group 1, showing defective liver morphogenesis, comprises mutations in four genes, which may be involved in the regulation of growth or patterning of the gut endoderm. Group 2 comprises mutations in three genes that affect the laterality of the liver; in kendama mutants of this group, the laterality of the heart and liver is uncoupled and randomized. Group 3 includes mutations in three genes altering bile color, indicative of defects in hemoglobin-bilirubin metabolism and globin synthesis. Group 4 consists of mutations in three genes, characterized by a decrease in the accumulation of fluorescent metabolite of a phospholipase A(2) substrate, PED6, in the gall bladder. Lipid metabolism or the transport of lipid metabolites may be affected by these mutations. Mutations in Groups 3 and 4 may provide animal models for relevant human diseases. Group 5 mutations in six genes affect the formation of endoderm, endodermal rods and hepatic bud from which the liver develops. These Medaka mutations, identified by morphological and metabolite marker screens, should provide clues to understanding molecular mechanisms underlying formation of a functional liver.     

Identification of high excision capacity for 5-hydroxymethyluracil mispaired with guanine in DNA of Escherichia coli MutM,Nei and Nth DNA glycosylases

The oxidation and deamination of 5-methylcytosine (5mC) in DNA generates a base-pair between 5-hydroxymethyluracil (5hmU) and guanine. 5hmU normally forms a base-pair with adenine. Therefore, the conversion of 5mC to 5hmU is a potential pathway for the generation of 5mC to T transitions. Mammalian cells have high levels of activity of 5hmU-DNA glycosylase, which excises 5hmU from DNA. However, glycosylases that similarly excise 5hmU have not been observed in yeast or Escherichia coli. Recently, we found that E.coli MutM, Nei and Nth have DNA glycosylase activity for 5-formyluracil, which is another type of oxidation product of the thymine methyl group. In this study, we examined whether or not E.coli MutM, Nei and Nth have also DNA glycosylase activity that acts on 5hmU in vitro. When incubated with synthetic duplex oligonucleotides containing 5hmU:G or 5hmU:A, purified MutM, Nei and Nth cleaved the 5hmU:G oligonucleotide 58, 5 and 37 times, respectively, more efficiently than the 5hmU:A oligonucleotide. In E.coli, the 5hmU-DNA glycosylase activities of MutM, Nei and Nth may play critical roles in the repair of 5hmU:G mispairs to avoid 5mC to T transitions.