Down-regulation of endodermal Shh is required for gland formation in chicken stomach

During the development of the proventriculus (glandular stomach) of the chicken embryo, the endodermal epithelium invades into the surrounding mesenchyme and forms glands. The glandular epithelial cells produce pepsinogen, while the non-glandular (luminal) epithelial cells secrete mucus. Sonic hedgehog is expressed uniformly in the proventricular epithelium before gland formation, but its expression ceases in gland cells. Here we present evidence that down-regulation of Sonic hedgehog is necessary for gland formation in the epithelium using a specific inhibitor of Sonic hedgehog signaling and virus mediated overexpression of Sonic hedgehog. We also show that gland formation is not induced by down-regulation of Sonic hedgehog alone; a mesenchymal influence is also required.     

Adenine excisional repair function of MYH protein on the adenine:8-hydroxyguanine base pair in double-stranded DNA

Adenine paired with 8-hydroxyguanine (oh⁸G), a major component of oxidative DNA damage, is excised by MYH base excision repair protein in human cells. Since repair activity of MYH protein on an A:G mismatch has also been reported, we compared the repair activity of His₆-tagged MYH proteins, expressed in Spodoptera frugiperda Sf21 cells, on A:oh⁸G and A:G mismatches by DNA cleavage assay and gel mobility shift assay. We also compared the repair ability of type 1 mitochondrial protein with type 2 nuclear protein, as well as of polymorphic type 1-Q³²⁴ and 2-Q³¹⁰ proteins with type 1-H³²⁴ and 2-H³¹⁰ proteins by DNA cleavage assay and complementation assay of an Escherichia coli mutM mutY strain. In a reaction buffer with a low salt (0–50 mM) concentration, adenine DNA glycosylase activity of type 2 protein was detected on both A:oh⁸G and A:G substrates. However, in a reaction buffer with a 150 mM salt concentration, similar to physiological conditions, the glycosylase activity on A:G, but not on A:oh⁸G, was extremely reduced and the binding activity of type 2 protein for A:G, but not for A:oh⁸G, was proportionally reduced. The glycosylase activity on A:oh⁸G and the ability to suppress spontaneous mutagenesis were greater for type 2 than type 1 enzyme. There was apparently no difference in the repair activities between the two types of polymorphic MYH proteins. These results indicate that human MYH protein specifically catalyzes the glycosylase reaction on A:oh⁸G under physiological salt concentrations.     

Increased blood spermine levels decrease the cytotoxic activity of lymphokine-activated killer cells: a novel mechanism of cancer evasion

Increased blood polyamine levels, often observed in cancer patients, have negative impacts on patient prognosis and are associated with tumor progression. The purpose of our study was to examine the effects of polyamines on cellular immune function. Peripheral blood mononuclear cells (PBMCs) from healthy volunteers were cultured with the human natural polyamines spermine, spermidine, or putrescine, and the effects on immune cell function were examined. The correlation between post-operative changes in blood polyamine levels and lymphokine-activated killer (LAK) activity was also examined in cancer patients. Spermine decreased the adhesion of non-stimulated PBMCs to tissue culture plastic in a dose- and a time-dependent manner without affecting cell viability or activity. This decrease in adhesion capacity was accompanied by a decrease in the number of CD11a bright-positive and CD56 bright-positive cells. Upon stimulation with interleukin 2 to activate LAK cytotoxicity, PBMCs cultured overnight with 100 or 500 μM spermine showed decreased cytotoxic activity against Daudi cells (91.5 ± 1.7 and 84.9 ± 3.0%, respectively (n = 6) compared to PBMC cultured without polyamines). In a group of 25 cancer patients, changes in blood spermine levels after surgery were negatively correlated with changes in LAK cytotoxicity after surgery (r = −0.510, P = 0.008: n = 25). Increased blood spermine levels may be an important factor in the suppression of anti-tumor immune cell function.     

Biosynthesis of RNA polymerase in Escherichia coli VI. Distribution of RNA polymerase subunits between nucleoid and cytoplasm.

The distribution of DNA-dependent RNA polymerase in Escherichia coli was analysed by measuring enzyme subunits in nucleoid (folded chromosome) and cytoplasm. Two independent methods, two-dimensional polyacrylamide gel electrophoresis of total proteins and sodium dodecyl sulphate/polyacrylamide gel electrophoresis of antibody precipitates, gave essentially the same results; with wild-type cells growing at a doubling time of 70 minutes, about two-thirds of the core enzyme but little σ subunit are present in the nucleoid. Pulse-chase experiments indicated that the distribution of the pulse-labelled proteins was at equilibrium within 1·5 minutes for β′, 5 minutes for β, and 15 minutes for α subunit. This order of appearance of the newly synthesized core subunits into the nueleoid is in good agreement with that into complete enzyme structure. This finding, together with the known sequence of subunit assembly (2α → α₂ → α₂β → α₂ββ′ → E), indicates that the assembly of RNA polymerase takes place in the cytoplasm. In concert with the conclusion, the amounts of pulse-labelled subunits in the cytoplasm of temperature-sensitive assembly defective mutants coincide well with those of intermediate subassemblies accumulated in the mutant cells. However, it is not known if the premature core is activated in cytoplasm prior to binding to the nucleoid or shortly after association with the nucleoid.     

Isolation and characterization of two entomopathogenic fungi attackingDiaphorina citri (Homoptera, Psylloidea) in Indonesia

In an attempt to suppress the propagation of citrus greening disease in Indonesia, we explored pathogens ofDiaphorina citri which vectors the disease. At two orange orchards, manyD. citri adults were found to be dead and covered with fungal mycelia. Two fungi,Paecilomyces fumosoroseus andHirsutella citriformis, were consistently isolated from the infected insects. Molecular phylogeny of their 18S rDNA sequences showed that they belong to the ascomycetous clade of the Clavicipitales/Hypocreales, which embraces many entomopathogenic fungi. When healthy adults ofD. citri were inoculated with conidia of theP. fumosoroseus, the insects died within 6 d.     

Structural basis for regulation of poly‐SUMO chain by a SUMO‐like domain of Nip45

Post‐translational modification by small ubiquitin‐like modifier (SUMO) provides an important regulatory mechanism in diverse cellular processes. Modification of SUMO has been shown to target proteins involved in systems ranging from DNA repair pathways to the ubiquitin‐proteasome degradation system by the action of SUMO‐targeted ubiquitin ligases (STUbLs). STUbLs recognize target proteins modified with a poly‐SUMO chain through their SUMO‐interacting motifs (SIMs). STUbLs are also associated with RENi family proteins, which commonly have two SUMO‐like domains (SLD1 and SLD2) at their C terminus. We have determined the crystal structures of SLD2 of mouse RENi protein, Nip45, in a free form and in complex with a mouse E2 sumoylation enzyme, Ubc9. While Nip45 SLD2 shares a β‐grasp fold with SUMO, the SIM interaction surface conserved in SUMO paralogues does not exist in SLD2. Biochemical data indicates that neither tandem SLDs or SLD2 of Nip45 bind to either tandem SIMs from either mouse STUbL, RNF4 or to those from SUMO‐binding proteins, whose interactions with SUMO have been well characterized. On the other hand, Nip45 SLD2 binds to Ubc9 in an almost identical manner to that of SUMO and thereby inhibits elongation of poly‐SUMO chains. This finding highlights a possible role of the RENi proteins in the modulation of Ubc9‐mediated poly‐SUMO formation. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

K-ras Gene Mutation Related to Histological Atypias in Human Colorectal Adenomas

To investigate the relationship of oncogene analysis to morphology, we analyzed K-ras gene mutations by dot-blot hybridization with and without consideration of histological atypias in individual colorectal adenomas. Each of 54 colon polyps were divided into two parts after fixation. One part was used as a mass to assess point mutations; the remaining portion of each polyp was paraffin-embedded, stained with hematoxylin and eosin, and examined for point mutations related to histological atypias. In the first part of our study, K-ras gene mutations at codon 12 were detected in 13 cases (24%). In the second part of our study, 12 cases had distinctly different histological atypias. From each of these 12 cases, two areas, one with higher or one with lower grade atypia in the same polyp were excised to analyze for K-ras gene mutation. Two of these 12 cases (17%) had the mutation in different areas of the same tumor. These two cases contained the mutation only in the areas with higher grade atypia, and only one case added information regarding ras mutation upon microdissection when compared to the entire biopsy. These results suggest that oligonucleotide hybridization can identify the majority of cases containing ras mutations despite regional morphologic variation. Individual cases, however, may contain clonal subpopulations within adenomas with different ras sequences from other regions within the same adenoma.     

Driving forces behind the fluctuating growth of the number of successful nests in an inland population of white-tailed eagles in Hokkaido,Japan

This study aimed to describe the change in the number of successful nests of the white-tailed eagle, Haliaeetus albicilla, for 25 years (1997–2021) along the Teshio River (100 km), Japan, which is a new habitat for this endangered species and identify factors driving the number of nests. The number of nests grew from two to nine. The logistic function fitted in well with the growth, and the capacity of the study area sustaining the successful nests was estimated at 6.5. The precipitation in January and April explained the deviation of the observed values from the model prediction. In particular, heavy rain in April was associated with low numbers. Forty-six nest remains were collected from 17 nest locations. Twelve genera of birds, six genera of mammals, and four genera of fishes were identified. Fish and bird items occupied 93.6% of prey individuals. The fish proportion was similar between high-performance years when the observed number of successful nests was higher than the model prediction and low-performance years with a lower number than the prediction (55.2% and 51.0%). However, it was higher in the nests with two fledglings (63.0%) than those with a single fledgling (41.5%). The nearest neighbor distance (NND) of the successful nests declined with the increase in the number of nests. Based on territory size (the mean NND = 7.8 km), the study area can hold 13 nests. The process and mechanism of the dynamics of the number of nests were discussed, focusing on territoriality and weather effects.     

Agonist-promoted heteromeric oligomerization between adenosine A(1) and P2Y(1) receptors in living cells

We have explored the process of oligomerization of G protein-coupled purinergic receptors, adenosine A(1) receptor (A(1)R) and P2Y(1) receptor (P2Y(1)R), in intact HEK293T cells by means of modified bioluminescence resonance energy transfer technology (BRET(2)) that offers greatly improved separation of the emission spectra of the donor and acceptor moieties compared to traditional BRET. This approach identified both constitutive and agonist-promoted heteromeric oligomerization between Myc-tagged P2Y(1)R fused to a donor, Renilla luciferase (Myc-P2Y(1)R-Rluc) and HA-tagged A(1)R fused to an acceptor, a different form of green fluorescent protein (HA-A(1)R-GFP(2)). The BRET(2) signal increased in a time-dependent manner in the cells expressing HA-A(1)R-GFP(2)/Myc-P2Y(1)R-Rluc upon addition of agonists for both receptors, which could be inhibited by pretreatment with the P2Y(1)R antagonist MRS2179. A high degree of HA-A(1)R-GFP(2) and Myc-P2Y(1)R-Rluc co-localization in the co-transfected HEK293T cells was also observed by confocal laser microscopy. These results indicate that A(1)R and P2Y(1)R can form constitutive hetero-oligomers in living cells and this process is promoted by the simultaneous activation of both receptors.     

Lifetime reproductive success in reproductively suppressed female voles

A population of the grey red-backed vole, Clethrionomys rufocanus bedfordiae, was investigated on a 1 ha control grid and a 1 ha grid on which the voles were fed within a 2.1 ha outdoor enclosure in Hokkaido, Japan by live trapping from 1984 to 1986, for testing the Reproductive Suppression Model of Wasser and Barash (1983)-females can optimize their lifetime reproductive success by suppressing reproduction when future conditions for the survival of offspring are likely to be sufficiently better than present ones as to exceed the costs of the suppression itself. Age at the first pregnancy more varied in a higher density population on the experimental grid and females could be classified into the early and the late reproductive type in two generations (A: females born from February to June 1985; B: females born from September to November 1985). Lifetime reproductive success (the number of pregnancies, the number of successful litters, and the number of offspring) was not different between the early and the late reproducing females. The late reproducing females lived for longer periods than the early reproducing females, so that the loss by delayed start of reproduction was compensated for by a longer life span. Life span was not different between offspring of the early and the late reproducing females. These facts supported the Reproductive Suppression Model.