Stress deprivation from the patellar tendon induces apoptosis of fibroblasts in vivo with activation of mitogen-activated protein kinases

The effect of stress deprivation on the tendon tissue has been an important focus in the field of biomechanics. However, less is known about the in vivo effect of stress deprivation on fibroblast apoptosis as of yet. This study was conducted to test a hypothesis that complete stress deprivation of the patellar tendon induces fibroblast apoptosis in vivo with activation of Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38) within 24 h after treatment. A total of 35 mature rabbits were divided into stress-shielded (n=15), sham-operated (n=15), and control (n=5) groups. To completely shield the patellar tendon from stress, we used an established surgical method. Animals were sacrificed at 24 h, and 2, 4, 7, and 14 days after the treatment. Tendon specimens underwent TUNEL assay and immunohistological examinations of active caspase-3, JNK, and p38. Both the number and the ratio of TUNEL-positive and caspase-3-positive cells were significantly greater (p<0.0001) in the stress-shielded group than in the sham group at 24 h, 2, 4, and 7 days. Concerning JNK and p38, both the number and the ratio were significantly greater (p<0.0001) in the stress-shielded group than in the sham group at 24 h, 2, and 4 days. This study demonstrated that complete stress deprivation induces fibroblast apoptosis in vivo with activation of JNK and p38 within 24 h. This fact suggested that the fibroblast apoptosis caused by stress deprivation is induced via the mitogen-activated protein kinase signaling pathway.     

The human immunodeficiency virus type 1 Vpr protein and its carboxy-terminally truncated form induce apoptosis in tumor cells

The human immunodeficiency virus type 1 (HIV-1) accessory protein Vpr induces apoptosis after cell cycle arrest at the G₂ phase in primate cells. We have reported previously that C81, a carboxy-terminally truncated form of Vpr, interferes with cell proliferation and results in apoptosis without G₂ arrest. Here, we investigated whether this property of Vpr and C81 could be exploited for use as a potential anticancer agent. First, we demonstrated that C81 induced G₁ arrest and apoptosis in all tumor cells tested. In contrast, Vpr resulted in G₂ arrest and apoptosis in HeLa and 293 T cells. Vpr also suppressed the damaged-DNA-specific binding protein 1 (DDB1) in HepG2 cells, thereby inducing apoptosis without G₂ arrest. G₂ arrest was restored when DDB1 was overexpressed in cells that also expressed Vpr. Surprisingly, C81 induced G₂ arrest when DDB1 was overexpressed in HepG2 cells, but not in HeLa or 293 T cells. Thus, the induction of Vpr- and C81-mediated cell cycle arrest appears to depend on the cell type, whereas apoptosis was observed in all tumor cells tested. Overall, Vpr and C81 have potential as novel therapeutic agents for treatment of cancer.     

Mapping of Calf Death in Japanese Black Cattle

Weak calf syndrome (WCS) is a major cause of calf death in Japanese Black cattle. Among IARS disorders, the isoleucyl-tRNA synthetase c.235G>C mutation has been identified as one of the causes of WCS. However, calf deaths differing from those attributed to IARS disorder has been occurring. To identify other genes potentially responsible for these calf deaths, we constructed three populations of three bulls (Bull-1, -2 and -3) that did not carry the IARS mutation, and dead calves (18, 28, and 31 calves) and healthy cattle (18, 15, and 10 cattle) sired by these bulls. The populations were genotyped using the BovineSNP50 BeadChip, but homozygosity mapping did not detect any associated genomic regions with calf death. Linkage analysis performed using each population as a paternal half-sib family of Bull-1, Bull-2, and Bull-3 revealed that, in the Bull-1 population, calf death was mapped to the 8.94?Mb–14.53?Mb and 29.82?Mb–33.77?Mb regions of BTA29. The findings suggested that the incidence of calf death in calves sired by Bull-1 was a hereditary disease exhibiting a dominant, not recessive, inheritance pattern.     

Increase in the activity of fructose-1,6-bisphosphatase in cytosol affects sugar partitioning and increases the lateral shoots in tobacco plants at elevated CO<Subscript>2</Subscript> levels

We generated transgenic tobacco plants with high levels of fructose-1,6-bisphosphatase expressing cyanobacterialfructose-1,6-/sedoheptulose-1,7-bisphosphatase in the cytosol. At ambient CO₂ levels (360 ppm), growth, photosynthetic activity, and fresh weight were unchanged but the sucrose/hexose/starch ratio was slightly altered in the transgenic plants compared with wild-type plants. At elevated CO₂ levels (1200 ppm), lateral shoot, leaf number, and fresh weight were significantly increased in the transgenic plants. Photosynthetic activity was also increased. Hexose accumulated in the upper leaves in the wild-type plants, while sucrose and starch accumulated in the lower leaves and lateral shoots in the transgenic plants. These findings suggest that cytosolic fructose-1,6-bisphosphatase contributes to the efficient conversion of hexose into sucrose, and that the change in carbon partitioning affects photosynthetic capacity and morphogenesis at elevated CO₂ levels.     

Structure and function of the N-terminal nucleolin binding domain of nuclear valosin-containing protein-like 2 (NVL2) harboring a nucleolar localization signal

The N-terminal regions of AAA-ATPases (ATPase associated with various cellular activities) often contain a domain that defines the distinct functions of the enzymes, such as substrate specificity and subcellular localization. As described herein, we have determined the solution structure of an N-terminal unique domain isolated from nuclear valosin-containing protein (VCP)-like protein 2 (NVL2(UD)). NVL2(UD) contains three α helices with an organization resembling that of a winged helix motif, whereas a pair of β-strands is missing. The structure is unique and distinct from those of other known type II AAA-ATPases, such as VCP. Consequently, we identified nucleolin from a HeLa cell extract as a binding partner of this domain. Nucleolin contains a long (～300 amino acids) intrinsically unstructured region, followed by the four tandem RNA recognition motifs and the C-terminal glycine/arginine-rich domain. Binding analyses revealed that NVL2(UD) potentially binds to any of the combinations of two successive RNA binding domains in the presence of RNA. Furthermore, NVL2(UD) has a characteristic loop, in which the key basic residues RRKR are exposed to the solvent at the edge of the molecule. The mutation study showed that these residues are necessary and sufficient for nucleolin-RNA complex binding as well as nucleolar localization. Based on the observations presented above, we propose that NVL2 serves as an unfoldase for the nucleolin-RNA complex. As inferred from its RNA dependence and its ATPase activity, NVL2 might facilitate the dissociation and recycling of nucleolin, thereby promoting efficient ribosome biogenesis.     

β-Glucuronidase from Lactobacillus brevis useful for baicalin hydrolysis belongs to glycoside hydrolase family 30

Baicalin (baicalein 7-O-β-d-glucuronide) is one of the major flavonoid glucuronides found in traditional herbal medicines. Because its aglycone, baicalein, is absorbed more quickly and shows more effective properties than baicalin, the conversion of baicalin into baicalein by β-glucuronidase (GUS) has drawn the attention of researchers. Recently, we have found that Lactobacillus brevis subsp. coagulans can convert baicalin to baicalein. Therefore, we aimed to identify and characterize the converting enzyme from L. brevis subsp. coagulans. First, we purified this enzyme from the cell-free extracts of L. brevis subsp. coagulans and cloned its gene. Surprisingly, this enzyme was found to be a GUS belonging to glycoside hydrolase (GH) family 30 (designated as LcGUS30), and its amino acid sequence has little similarity with any GUS belonging to GH families 1, 2, and 79 that have been reported so far. We then established a high-level expression and simple purification system of the recombinant LcGUS30 in Escherichia coli. The detailed analysis of the substrate specificity revealed that LcGUS30 has strict specificity toward glycon but not toward aglycones. Interestingly, LcGUS30 prefers baicalin rather than estrone 3-(β-d-glucuronide), one of the human endogenous steroid hormones. These results indicated that L. brevis subsp. coagulans and LcGUS30 should serve as powerful tools for the construction of a safe bioconversion system for baicalin. In addition, we propose that this novel type of GUS forms a new group in subfamily 3 of GH family 30.     

Perlecan-enriched intercellular space of junctional epithelium provides primary infrastructure for leukocyte migration through squamous epithelial cells

The aim of this study was to demonstrate the presence of intraepithelial stroma represented by extracellular matrix (ECM) deposits in the junctional epithelium to clarify its function as a scaffold for leukocyte migration through epithelial cells. Twenty-three biopsy specimens from the gingiva including the junctional epithelium were examined to determine comparative protein and gene level expression profiles for keratin and ECM molecules between the junctional epithelium and the gingival epithelium using immunohistochemistry and in situ hybridization. Intraepithelial leukocyte types and frequencies were also determined and compared between the junctional and gingival epithelia. In the junctional epithelium, which was positive for keratin 19, perlecan was strongly deposited in intercellular space of the whole epithelial layer, while it was faintly positive around the parabasal layer of the gingival epithelium. Perlecan mRNA signals were enhanced to a greater degree in both epithelial and inflammatory cells within the junctional epithelium. In the junctional epithelium, greater numbers of neutrophils and macrophages were found as compared with the gingival epithelium. Our results showed that perlecan is the primary ECM molecule comprising intraepithelial stroma of the junctional epithelium, in which leukocytes may migrate on ECM scaffolds in intercellular space toward the surface of the gingival sulci or pockets.     

Functional and Evolutionary Characterization of the CONSTANS Gene Family in Short-Day Photoperiodic Flowering in Soybean

CONSTANS (CO) plays a central role in photoperiodic flowering control of plants. However, much remains unknown about the function of the CO gene family in soybean and the molecular mechanisms underlying short-day photoperiodic flowering of soybean. We identified 26 CO homologs (GmCOLs) in the soybean genome, many of them previously unreported. Phylogenic analysis classified GmCOLs into three clades conserved among flowering plants. Two homeologous pairs in Clade I, GmCOL1a/GmCOL1b and GmCOL2a/GmCOL2b, showed the highest sequence similarity to Arabidopsis CO. The mRNA abundance of GmCOL1a and GmCOL1b exhibited a strong diurnal rhythm under flowering-inductive short days and peaked at dawn, which coincided with the rise of GmFT5a expression. In contrast, the mRNA abundance of GmCOL2a and GmCOL2b was extremely low. Our transgenic study demonstrated that GmCOL1a, GmCOL1b, GmCOL2a and GmCOL2b fully complemented the late flowering effect of the co-1 mutant in Arabidopsis. Together, these results indicate that GmCOL1a and GmCOL1b are potential inducers of flowering in soybean. Our data also indicate rapid regulatory divergence between GmCOL1a/GmCOL1b and GmCOL2a/GmCOL2b but conservation of their protein function. Dynamic evolution of GmCOL regulatory mechanisms may underlie the evolution of photoperiodic signaling in soybean.     

Diastolic suction in heart failure: Impact of left ventricular geometry,untwist, and flow mechanics

Aims

Vector flow mapping (VFM) can be used to assess intraventricular hemodynamics quantitatively. This study assessed the magnitude of the suction flow kinetic energy with VFM and investigated the relation between left ventricular (LV) function and geometry in patients with an estimated elevated LV filling pressure.

Materials and methods

We studied 24 subjects with an elevated LV filling pressure (EFP group) and 36 normal subjects (normal group). Suction was defined as flow directed toward the apex during the period from soon after systolic ejection to before mitral inflow. The flow kinetic energy index was quantified as the sum of the product of the blood mass and velocity vector and its magnitude to the peak value was measured.

Key findings

Suction flow was observed in 12 (50%) EFP-group patients and 36 (100%) normal-group subjects. The magnitude of the suction kinetic energy index was significantly smaller in EFP versus normal group (2.7 ± 3.8 vs. 5.7 ± 4.4 g/s/cm², P < 0.01). The EFP-group patients with suction had a smaller LV end-systolic volume (ESV) (P < 0.01), greater ellipsoidal geometry (P < 0.05) and untwisting rate (P < 0.01) than the EFP-group patients without suction. A regression analysis indicated a significant linear relation between the suction kinetic energy index and LVEF (r = 0.43, P = 0.04), ESV (r = − 0.40, P = 0.05), eccentricity index (r = 0.44, P = 0.04), and untwisting rate (r = 0.51, P = 0.04).

Significance

The magnitude of the suction flow kinetic energy index derived from VFM may allow the quantitative assessment of the suction flow, which correlates with LV systolic function, geometry, and untwisting mechanics.