Structural studies of [2',6'-dimethyl-L-tyrosine1]endomorphin-2 analogues: enhanced activity and cis orientation of the Dmt-Pro amide bond

Analogues of endomorphin-2 (EM-2: Tyr-Pro-Phe-Phe-NH(2)) (1) were designed to examine the importance of each residue on mu-opioid receptor interaction. Replacement of Tyr(1) by 2',6'-dimethyl-L-tyrosine (Dmt) (9-12) exerted profound effects: [Dmt(1)]EM-2 (9) elevated mu-opioid affinity 4.6-fold (K(i mu=0.15 nM) yet selectivity fell 330-fold as delta-affinity rose (K(i)delta=28.2 nM). This simultaneous increased mu- and delta-receptor bioactivities resulted in dual agonism (IC(50)=0.07 and 1.87 nM, respectively). While substitution of Phe(4) by a phenethyl group (4) decreased mu affinity (K(i)mu=13.3 nM), the same derivative containing Dmt (12) was comparable to EM-2 but also acquired weak delta antagonism (pA(2)=7.05). 1H NMR spectroscopy revealed a trans configuration (1:2 to 1:3, cis/trans) in the Tyr-Pro amide bond, but a cis configuration (5:3 to 13:7, cis/trans) with Dmt-Pro analogues.     

Urocystis tranzscheliana,a newly recorded smut fungus onPrimula sieboldii from Japan

A smut fungus onPrimula sieboldii was newly found in Japan and identified asUrocystis tranzscheliana by comparative morphology. This species causes systemic infection ofP. sieboldii and produces sori in its ovaries.Contribution No. 121, Laboratories of Plant Pathology and Mycology, Institute of Agriculture and Forestry, University of Tsukuba.     

Lattice population dynamics for plants with dispersing seeds and Vegetative propagation

The population dynamics of plants in a lattice structured habitat are studied theoretically. Plants are assumed to propagate both by producing seeds that scatter over the population and by vegetative reproduction by extending runners, rhizomes, or roots, to neighboring vacant sites. In addtion, the seed production rate may be dependent on the local density in the neighborhood, indicating beneficial or harmful crowding effects. Two sets of population dynamical equation(s) are derived: one based onmean-field approximation and the other based onpair approximation (tracing both global and local densities simultaneously). We examine the accuracy of these approximate dynamics by comparing them with direct computer simulation of the stochastic lattice model. Pair approximation is much more accurate than mean-field approximation. Mean-field approximation overestimates the parameter range for persistence if crowding effects on seed production are harmful or weakly beneficial, but underestimates it if crowding effects are highly beneficial. Dynamics may show bistability (both population persistence and extinction) if the effect of crowding is strongly beneficial. If there is a linear trade-off between seed production and vegetative reproduction, the equilibrium abundance of the population may be maximised by a mixture of seed production and vegetative reproduction, rather than by pure seed production or by pure vegetative reproduction. This result is correctly predicted by pair approximation but not by mean-field approximation.     

ATP-induced shrinkage of DNA with MukB protein and the MukBEF complex of Escherichia coli

Fluorescence microscopic observation of individual T4 DNA molecules revealed that the MukBEF complex (bacterial condensin) and its subunit, the MukB (a member of the SMC [structural maintenance of chromosomes] superfamily) homodimer, of Escherichia coli markedly shrunk large DNA molecules in the presence of hydrolyzable ATP. In contrast, in the presence of ADP or ATP-gammaS, the conformation of DNA was almost not changed. This suggests that the ATPase activity of subunit MukB is essential for shrinking large DNA molecules. Stretching experiments on the shrunken DNA molecules in the presence of ATP and MukBEF indicated a cross-bridging interaction between DNA molecules.     

Oral lipase activities and fat-taste receptors for fat-taste sensing in chickens

It has been reported that a functional fat-taste receptor, GPR120, is present in chicken oral tissues, and that chickens can detect fat taste in a behavioral test. However, although triglycerides need to be digested to free fatty acids to be recognized by fat-taste receptors such as GPR120, it remains unknown whether lipase activities exist in chicken oral tissues. To examine this question, we first cloned another fat-taste receptor candidate gene, CD36, from the chicken palate. Then, using RT-PCR, we determined that GPR120 and CD36 were broadly expressed in chicken oral and gastrointestinal tissues. Also by RT-PCR, we confirmed that several lipase genes were expressed in both oral and gastrointestinal tissues. Finally, we analyzed the lipase activities of oral tissues by using a fluorogenic triglyceride analog as a lipase substrate. We found there are functional lipases in oral tissues as well as in the stomach and pancreas. These results suggested that chickens have a basic fat-taste reception system that incorporates a triglycerides/oral-lipases/free fatty acids/GPR120 axis and CD36 axis.     

DNA Polymerase δ Is Required for Early Mammalian Embryogenesis

Background

In eukaryotic cells, DNA polymerase δ (Polδ), whose catalytic subunit p125 is encoded in the Pold1 gene, plays a central role in chromosomal DNA replication, repair, and recombination. However, the physiological role of the Polδ in mammalian development has not been thoroughly investigated.

Methodology/Principal Findings

To examine this role, we used a gene targeting strategy to generate two kinds of Pold1 mutant mice: Polδ-null (Pold1 ^−/−) mice and D400A exchanged Polδ (Pold1 ^exo/exo) mice. The D400A exchange caused deficient 3′–5′ exonuclease activity in the Polδ protein. In Polδ-null mice, heterozygous mice developed normally despite a reduction in Pold1 protein quantity. In contrast, homozygous Pold1 ^−/− mice suffered from peri-implantation lethality. Although Pold1 ^−/− blastocysts appeared normal, their in vitro culture showed defects in outgrowth proliferation and DNA synthesis and frequent spontaneous apoptosis, indicating Polδ participates in DNA replication during mouse embryogenesis. In Pold1 ^exo/exo mice, although heterozygous Pold1 ^exo/+ mice were normal and healthy, Pold1 ^exo/exo and Pold1 ^exo/− mice suffered from tumorigenesis.

Conclusions

These results clearly demonstrate that DNA polymerase δ is essential for mammalian early embryogenesis and that the 3′–5′ exonuclease activity of DNA polymerase δ is dispensable for normal development but necessary to suppress tumorigenesis.     

Demonstration and Characterization of the Heterodimerization of ZnT5 and ZnT6 in the Early Secretory Pathway

The majority of CDF/ZnT zinc transporters form homo-oligomers. However, ZnT5, ZnT6, and their orthologues form hetero-oligomers in the early secretory pathway where they load zinc onto zinc-requiring enzymes and maintain secretory pathway functions. The details of this hetero-oligomerization remain to be elucidated, and much more is known about homo-oligomerization that occurs in other CDF/ZnT family proteins. Here, we addressed this issue using co-immunoprecipitation experiments, mutagenesis, and chimera studies of hZnT5 and hZnT6 in chicken DT40 cells deficient in ZnT5, ZnT6, and ZnT7 proteins. We found that hZnT5 and hZnT6 combine to form heterodimers but do not form complexes larger than heterodimers. Mutagenesis of hZnT6 indicated that the sites present in transmembrane domains II and V in which many CDF/ZnT proteins have conserved hydrophilic amino acid residues are not involved in zinc binding of hZnT6, although they are required for zinc transport in other CDF/ZnT family homo-oligomers. We also found that the long N-terminal half of hZnT5 is not necessary for its functional interaction with hZnT6, whereas the cytosolic C-terminal tail of hZnT5 is important in determining hZnT6 as a partner molecule for heterodimer formation. In DT40 cells, cZnT5 variant lacking the N-terminal half was endogenously induced during periods of endoplasmic reticulum stress and so seemed to function to supply zinc to zinc-requiring enzymes under these conditions. The results outlined here provide new information about the mechanism of action through heterodimerization of CDF/ZnT proteins that function in the early secretory pathway.     

Secreted Nucleobindin-2 Inhibits 3T3-L1 Adipocyte Differentiation

Nucleobindin-2 is a 420 amino acid EF-hand Ca2+ binding protein that can be further processed to generate an 82 amino terminal peptide termed Nesfatin-1. To examine the function of secreted Nucleobindin-2 in adipocyte differentiation, cultured 3T3-L1 cells were incubated with either 0 or 100 nM of GST, GST-Nucleobindin-2, prior to and during the initiation of adipocyte differentiation. Nucleobindin-2 treatment decreased neutral lipid accumulation (Oil-Red O staining) and expression of several marker genes for adipocyte differentiation (PPARγ, aP2, and adipsin). When Nucleobindin- 2 was constitutively secreted into cultured medium, cAMP content and insulin stimulated CREB phosphorylation were significantly reduced. On the other hand, intracellularly overexpressed Nucleobindin-2 failed to affect cAMP content and CREB phosphorylation. Taken together, these data indicate that secreted Nucleobindin-2 is a suppressor of adipocyte differentiation through inhibition of cAMP production and insulin signal.     

The transition of adult patients with childhood-onset chronic diseases from pediatric to adult healthcare systems: a survey of the perceptions of Japanese pediatricians and child health nurses

Background

Advances in medical science have enabled many children with chronic diseases to survive to adulthood. The transition of adult patients with childhood-onset chronic diseases from pediatric to adult healthcare systems has received attention in Europe and the United States. We conducted a questionnaire survey among 41 pediatricians at pediatric hospitals and 24 nurses specializing in adolescent care to compare the perception of transition of care from pediatric to adult healthcare services for such patients.

Findings

Three-fourths of the pediatricians and all of the nurses reported that transition programs were necessary. A higher proportion of the nurses realized the necessity of transition and had already developed such programs. Both pediatricians and nurses reported that a network covering the transition from pediatric to adult healthcare services has not been established to date.

Conclusions

It has been suggested that spreading the importance of a transition program among pediatricians and developing a pediatric-adult healthcare network would contribute to the biopsychosocial well-being of adult patients with childhood-onset chronic disease.