Cell cycle analysis of bovine cultured somatic cells by flow cytometry

This study was undertaken to examine the cell cycle characteristics of bovine fetal and adult somatic cells (fetal fibroblasts, adult skin and muscle cells, and cumulus cells) after culture under a variety of conditions; 1) growth to 60-70% confluency (cycling), 2) serum starvation, 3) culture to confluency. Cell -cycle phases were determined by flow cytometry with propidium iodide staining enabling the calculation of percentages of cells in G0 /G1, S and G2 /M. The majority was in G0/G1 regardless of cell type and treatment. Serum-starved or confluent cultures contained higher percentages of cells in G0/G1 (89.5-95.4%; P < 0.05). Percentages of cells in G0/G1 increased as cell size decreased regardless of the cell type and treatment. In the serum-starved and confluent cultures, about 98% of small cells were in G0/G1 . Serum-starved cultures contained higher percentages of small cells (38.5-66.9%) than cycling and confluent cultures regardless of cell type (P < 0.05) . After trypsinization of fetal fibroblasts and adult skin cells that were serum-starved and cultured to confluency, the percentages of cells in G0/G1 increased (P < 0.05) on incubation for 1.5 (95.7-99.5%) or 3 hr (95.9-98.6%). These results verify that serum starvation and culture to confluency are efficient means of synchronizing bovine somatic cells in G0/G1, and indicate that a more efficient synchronization of the cells in G0/G1 can be established by incubation for a limited time period after trypsinization of serum-starved or confluent cells.     

Matrix-metalloproteinases-2 and -9 production in bovine endometrial cell culture

In vitro cell culture is a convenient tool for studying cellular mechanisms. In the present study, production of matrix-metalloproteinases (MMPs) in bovine endometrial (containing both epithelial and stromal cells) monolayer cells was examined. Blastocysts attached to the endometrial cells in a monolayer culture were examined for their effects on MMP-2 production. Initial attachment of blastocysts to the monolayer inhibited MMP-2 production by endometrial cells. But once trophoblast cells began to migrate into the endometrial cell layer, MMP-2 production increased, and at the same time MMP-9 production also became evident in the medium. In order to understand how blastocysts affected MMP-2 production, we examined the effect of progesterone, estradiol, insulin-like growth factors (IGFs), tumor necrosis factors (TNFs), and interferon-tau (IFN-tau) supplementation. It was IFN-tau that inhibited the production of MMP-2. In addition, progesterone at a lower dose appeared to inhibit MMP-2 production. Both TNF-alpha and TNF-beta strongly stimulated the production of MMP-2 and MMP-9, whereas IGFs had no effect. Based on these findings, it appears that conceptus has the capacity to inhibit MMP activity.     

Electrocorticographic and histological findings in a Shetland sheepdog with intractable epilepsy

A Shetland sheepdog with epilepsy refractory to antiepileptic drugs was brought to the division of Veterinary Radiology at Nippon Veterinary and Animal Science University. Scalp electroencephalography and computed tomography was performed, but no abnormality was detected in either examination. To obtain detailed information, electrodes were implanted on the dura mater, and the electrocorticogram (ECoG) was recorded. In the ECoG, sporadic spikes were detected in the left parietal region, suggesting the presence of the epileptic focus in this region. After the dog's death, abnormalities of gyri were found in the region where spikes were detected in the ECoG. On histopathological examination, laminar malacia of the cingulate gyrus was observed. Furthermore, in the hippocampus, neuronal loss of pyramidal cells was observed.     

Evaluation of intestinal intramucosal pH,arterial and portal venous blood gas values,and intestinal blood flow during small intestinal ischemia and reperfusion in dogs

OBJECTIVES: To determine whether small intestinal ischemia and reperfusion affects intestinal intramucosal pH (pHi), arterial and portal venous blood gas values, and intestinal blood flow (IBF) and to investigate relationships between regional intestinal tissue oxygenation and systemic variables in dogs. ANIMALS: 15 healthy adult Beagles. PROCEDURE: Occlusion of superior mesenteric artery (SMA) for 0, 30, or 60 minutes, followed by reperfusion for 180 minutes, was performed; IBF, pHi, arterial and portal venous blood gas values, arterial pressure, and heart rate were measured at various time points; and intestinal mucosal injury was histologically graded. RESULTS: Occlusion of the SMA induced significant decreases in pHi and IBF. After the release of the occlusion, IBF returned rapidly to baseline values, but improvement in pHi was slow. Arterial and portal venous blood gas analyses were less sensitive than tonometric measurements of pHi, and there was no correlation between results of blood gas analyses and tonometric measurements. Histologic score for intestinal mucosal injury increased significantly, depending on duration of ischemia, and there was a correlation between tonometric results and the histologic score. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that it is difficult to accurately evaluate local oxygenation disorders by monitoring at the systemic level, whereas clinically pHi is the only reliable indicator of inadequate regional intestinal tissue oxygenation in dogs.     

Cliniconeuropathologic findings of familial frontal lobe epilepsy in Shetland sheepdogs

We examined an epileptic focus by electroencephalography (EEG) by using an international 10-20 electrode system in 11 Shetland sheep dogs affected with familial idiopathic epilepsy. We also performed an evaluation of the amino acids in the cerebrospinal fluid (CSF) and a pathologic examination of the brains of 8 dogs that died from status epilepticus. Continuous electroencephalography demonstrated that an epileptic focus was initially detected in the frontal lobe, particularly the internal area, and that paroxysmal foci developed diffusely in other lobes of affected dogs with recurrent convulsions. The EEG analyses indicated spike and sharp wave complexes, which were considered to be paroxysmal discharges. An increased value for glutamate or aspartate was found in the CSF of some epileptic dogs. Histologically, acute neuronal necrosis and astrocytosis were distributed predominantly in the cingulate cortex and internal area of frontal cortex, less frequently in other areas of the cerebrum. The results of this study suggest that, initially, the dogs have an epileptic focus in the frontal lobe, and that the focus extends gradually to other areas of the cerebrum. Based on the distribution of neuronal necrosis and astrocytosis, acute neuronal damage may be related to the superexcitation of neurons following epilepsy.     

‘Niigata S3’ is a new strawberry cultivar suitable for forcing culture under low temperature and insolation conditions

‘Niigata S3’ is a new strawberry (Fragaria × ananassa Duch.) cultivar that is early flowering and possesses high soluble solid content and good coloration. It was selected from a cross between Kei812 (seed parent) and ‘Asuka-Ruby’ (pollen parent). The first harvest date of ‘Niigata S3’ was December 27, 34 days earlier than ‘Echigohime’ and 9 days earlier than ‘Asuka-Ruby’ (means of 2007 and 2008). The marketable yield of ‘Niigata S3’ was 85% of ‘Echigohime’, 107% of ‘Asuka-Ruby’, while the early yield was 145% of ‘Echigohime’, 85% of ‘Asuka-Ruby’ (based on 2007 and 2008 means). The shape of the fruit is long conical, and its skin color medium-red. The fruit skin hardness of ‘Niigata S3’ was 31.5 g/mm², which was harder than ‘Echigohime’, and its average soluble solid content was 11.4%, which was higher than the values for ‘Echigohime’ and ‘Asuka-Ruby’ (2008). Furthermore, ‘Niigata S3’ did not bear apical overripe fruit. This new cultivar is adaptable to the climatic conditions of Niigata, as well as other regions that experience low winter temperatures and insolation.     

Root phenotyping: important and minimum information required for root modeling in crop plants

As plants cannot relocate, they require effective root systems for water and nutrient uptake. Root development plasticity enables plants to adapt to different environmental conditions. Research on improvements in crop root systems is limited in comparison with that in shoots as the former are difficult to image. Breeding more effective root systems is proposed as the “second green revolution”. There are several recent publications on root system architecture (RSA), but the methods used to analyze the RSA have not been standardized. Here, we introduce traditional and current root-imaging methods and discuss root structure phenotyping. Some important root structures have not been standardized as roots are easily affected by rhizosphere conditions and exhibit greater plasticity than shoots; moreover, root morphology significantly varies even in the same genotype. For these reasons, it is difficult to define the ideal root systems for breeding. In this review, we introduce several types of software to analyze roots and identify important root parameters by modeling to simplify the root system characterization. These parameters can be extracted from photographs captured in the field. This modeling approach is applicable to various legacy root data stored in old or unpublished formats. Standardization of RSA data could help estimate root ideotypes.     

Synthesis and Structure–Activity Relationships of Miticidal 4,5-Dihydropyrazole-5-thiones

A series of novel 4,5-dihydropyrazole-5-thiones (DHPs) was synthesised by treating the corresponding dihydropyrazolones with ‘Lawesson’s reagent and evaluated for miticidal activity against two-spotted spider mites (Tetranychus urticae Koch). Of these, 3-(4-chlorophenyl)-4,4-dimethyl-1-phenyl-4,5-dihydropyrazole-5-thione, 3-(4-chlorophenyl)-4-ethyl-4-methyl-1-phenyl-4,5-dihydropyrazole-5-thione, 3-(4-chlorophenyl)-1-phenyl-4,5-dihydropyrazole-5-thione-4-spirocyclopentane and 4,4-dimethyl-1-phenyl-3-(4-trifluoromethyl-phenyl)-4,5-dihydropyrazole-5-thione were highly active (pEC₅₀>4·0) and were more effective than the miticide dicofol (pEC₅₀=3·879), which has traditionally been used for the control of phytophagous mites. Structure–activity relationship (SAR) studies were performed on each position of the pyrazole ring of DHPs. The results indicated that the unsubstituted phenyl, 4-substituted phenyl and thioxo groups on the 1-, 3- and 5-positions of DHPs respectively were required for activity. Quantitative SAR studies using physicochemical parameters of substituents and the capacity factor k′ as a hydrophobicity index suggested that: (a) the activities of all types of DHPs examined were mainly dominated by hydrophobicity, (b) the bulkiness of 4-substituents of the 3-phenyl ring favoured the activity and (c) the log k′ optimum for all DHPs was 1·675, equivalent to a log P_ow value of c. 5·0.     

MytiLec,a Mussel R-Type Lectin,Interacts with Surface Glycan Gb3 on Burkitt’s Lymphoma Cells to Trigger Apoptosis through Multiple Pathways

MytiLec; a novel lectin isolated from the Mediterranean mussel (Mytilus galloprovincialis); shows strong binding affinity to globotriose (Gb3: Galα1-4Galβ1-4Glc). MytiLec revealed β-trefoil folding as also found in the ricin B-subunit type (R-type) lectin family, although the amino acid sequences were quite different. Classification of R-type lectin family members therefore needs to be based on conformation as well as on primary structure. MytiLec specifically killed Burkitt''s lymphoma Ramos cells, which express Gb3. Fluorescein-labeling assay revealed that MytiLec was incorporated inside the cells. MytiLec treatment of Ramos cells resulted in activation of both classical MAPK/ extracellular signal-regulated kinase and extracellular signal-regulated kinase (MEK-ERK) and stress-activated (p38 kinase and JNK) Mitogen-activated protein kinases (MAPK) pathways. In the cells, MytiLec treatment triggered expression of tumor necrosis factor (TNF)-α (a ligand of death receptor-dependent apoptosis) and activation of mitochondria-controlling caspase-9 (initiator caspase) and caspase-3 (activator caspase). Experiments using the specific MEK inhibitor U0126 showed that MytiLec-induced phosphorylation of the MEK-ERK pathway up-regulated expression of the cyclin-dependent kinase inhibitor p21, leading to cell cycle arrest and TNF-α production. Activation of caspase-3 by MytiLec appeared to be regulated by multiple different pathways. Our findings, taken together, indicate that the novel R-type lectin MytiLec initiates programmed cell death of Burkitt’s lymphoma cells through multiple pathways (MAPK cascade, death receptor signaling; caspase activation) based on interaction of the lectin with Gb3-containing glycosphingolipid-enriched microdomains on the cell surface.     

Effects of management, environment and landscape conditions on establishment of hardwood seedlings and saplings in central Japanese coniferous plantations

There is an increasing need to restore natural hardwood forests in landscapes dominated by monocultural conifer plantations. A convenient restoration approach is to exploit natural regeneration processes. Natural regeneration, however, is affected by diverse interacting factors, for which better understanding is required, in order to optimize restoration programs. To identify optimal management practices for improving natural regeneration of hardwood trees in coniferous plantations, we examined the effects of multiple factors on the abundance of seedlings, small saplings and large saplings (height <0.3, 0.3-1.3 and ?1.3 m, respectively) of hardwood tree and shrub species in both line thinned (LT) and unthinned (UT) plantations of sugi (Cryptomeria japonica) and hardwood forests (HF) in central Japan. The effects of management practices (number of the times of weeding and cleaning, thinning method, years after thinning and forest age), environment (slope position, slope angle and canopy openness), and landscape conditions (distance from nearest hardwood forest, altitude and landuse before planting) on the number of hardwood individuals were examined by using the data obtained from the LT plantations. We also compared hardwood density between LT and UT plantations to examine the effect of line thinning. Finally, we examined species composition of LT plantations and HF to identify hardwood forest components in the thinned plantations. The effects on hardwood regeneration of environmental conditions, landscape factors and management practices applied in the plantations varied, depending on the size class and life form of the regenerating species. The abundance of large saplings of tall tree species was affected by several management factors, especially number of the times of weeding. Landscape conditions (distance from the nearest hardwood forest and altitude) affected the abundance of small saplings and seedlings of tall tree species, but not the other classes. Seedlings and small saplings of many tall tree species that contribute to hardwood forest canopies were less abundant in the LT plantations. The results show that numerous factors affect the establishment and abundance of naturally regenerating hardwood tree species, and suggest that successful establishment during early plantation stages can have long-lasting effects on natural regeneration of tall tree species.