Liver condition affects bovine oocyte qualities by changing the characteristics of follicular fluid and plasma.

The liver is an important organ that contributes to milk production in dairy cows. The aim of this study was to examine whether liver conditions affect the characteristics of blood plasma and follicular fluid (FF) and whether supplementing in vitro maturation medium with FF from either cows with damaged livers (DL) or those with healthy livers (HL) affects oocyte developmental competence. Biochemical characteristics of FF were significantly correlated with those in plasma. As such, the characteristics of both plasma and FF were similarly affected by liver conditions in that the concentrations of total protein and inorganic phosphorus were higher for the DL cow group than for the HL cow group, whereas the concentrations of albumin, lactate dehydrogenase and calcium were lower for DL cows than for HL cows. In addition, supplementing the medium with DL-FF retarded the progression of the nuclear maturation of oocytes collected from the HL cows. On culturing oocytes in maturation medium containing HL-FF, DL-FF or foetal calf serum, the highest developmental rate to the blastocyst stage was observed in the HL-FF group, while the lowest developmental ratio was observed in the DL-FF group. The growth factor array of the FFs revealed that 10 growth factors were significantly downregulated in the DL-FF compared with those in HL-FF. In conclusion, the characteristics of plasma and FF are affected by liver conditions in a similar way. Concentrations of several growth factors were low in DL-FF, as was the ability of DL-FF to support oocyte maturation compared with that of HL-FF.

Follicle growth and oocyte developmental competence in cows with liver damage.

Follicle growth, oocyte quality or oocyte growing environment (follicular fluid) were evaluated in cows with severe liver damage (haemorrhage, telangiectasis, cholangitis and abscess) that were visually diagnosed at the slaughterhouse. Holstein cows aged 40-90 months with either a healthy liver (HL cow) or damaged liver (DL cow) were selected as donors. Follicle development kinetics was evaluated by counting the follicles at various developmental stages. In addition, the biochemical characteristics of the follicular fluids, developmental competence of preantral follicles cultured for 16 days in vitro and the ability of oocytes to develop to the blastocyst stage 8 days after fertilization were examined. DL cows had fewer secondary follicles than HL cows, and the correlation between the number of secondary follicles and the number of primary follicles differed among DL and HL cows. The follicular fluid of DL cows contained significantly lower levels of albumin and a higher total protein content than that of HL cows. Oocyte nuclear maturation assessed at 5, 16 and 21 h after beginning of culture was slower in DL cows than in HL cows, although the final maturation rates did not differ. The rate of polyspermic fertilization was significantly higher and the proportion of cleavage at 48 h after insemination and blastulation lower in DL cows compared with HL cows. When preantral follicles were cultured in vitro, the rate of follicles with normal morphology was lower in DL cows than in HL cows. These findings suggest that the kinetics of folliculogenesis differ among DL and HL cows and the developmental ability of preantral follicles and oocytes is lower in DL cows than in HL cows.

Culture of stromal cells derived from medullary cavity of human long bone in the presence of 1,25-dihydroxyvitamin D3, recombinant human bone morphogenetic protein-2, or ipriflavone.

We previously showed that stromal cells derived from bone marrow specimens formed at the fracture site of human long bone differentiated during culture to polygonal cells and spindle cells, and polygonal cells, but not spindle cells, produced calcified matrix. To clarify the origin of polygonal and/or spindle cells, and factors necessary for differentiation of marrow stromal cells to osteogenic cells, we cultured stromal cells derived from the normal (unfractured) medullary cavity (SCN) as well as stromal cells from the medullary cavity distant from the fracture site (SCF). After 3 weeks of primary culture and 2 days of secondary culture, the cells were cultured in medium containing 1,25-dihydroxyvitamin D3 (VD), recombinant human bone morphogenetic protein-2 (BMP), or ipriflavone (IF) for 3 weeks. For biochemical analysis, cells reaching confluence after 3 weeks of secondary culture were cultured with one of the factors for 3 days. Some of SCF cultured with VD or IF were transformed to polygonal cells, and showed high alkaline phosphatase (ALPase) activity and high osteocalcin and insoluble calcium production. Cloned polygonal cells from the SCF formed nodules and aggregates consisting of calcium. Other SCF cultured with VD or IF and SCF cultured with BMP were spindle shaped. Some spindle-shaped cells from SCF cultured with BMP or IF revealed high ALPase activity and high osteocalcin production, comparable with the spindle cells from the fracture site. However, spindle-shaped cells from SCF cultured with VD and other spindle-shaped cells from SCF cultured with BMP or IF showed low ALPase activity and low osteocalcin production. The results show that SCF probably contain at least three subpopulations: (a) cells that differentiate to polygonal cells by the influence of VD or IF; (b) cells that differentiate to the spindle cells by the influence of BMP or IF; and (c) cells that are not transformed by the influence of VD, BMP, or IF.

Culture of marrow stromal cells derived from bone marrow specimens formed at fracture site of human long bone.

To study the process of the internal callus formation, we cultivated marrow stromal cells derived from bone marrow specimens formed at fracture sites of human long bone in alpha-modified Eagle's medium containing 10% fetal calf serum. After 2 weeks of culture, the cells formed two types of colonies; one consisted of spindle cells, and the other comprised of polygonal cells. The two types of colonies were separated and cultured further. The spindle and polygonal cells proliferated to confluence within 3 weeks and after 4 weeks, respectively, after the separation. Both the spindle and polygonal cells showed on the plasma membrane moderate intensity of staining reaction of alkaline phosphatase (ALPase) activity before the period of confluence and strong intensity during the period of confluence. Then, the spindle cells did not produce calcified matrix, but detached from the dish after 6-8 weeks of culture. In the colonies of polygonal cells, however, dense nodules were formed after 9 weeks of culture, which became visible to the naked eye as white aggregates after 11-12 weeks. Electron microscopic studies on the polygonal cells demonstrated matrix vesicles in the intercellular ground substance after 6 weeks of culture, and electron-dense needle-like crystals on the matrix vesicles after 8-10 weeks of culture. On the basis of infrared spectroscopic analysis, the aggregates were composed of hydroxyapatite. Thus, stromal cells derived from bone marrow specimens formed at fracture site of human long bone differentiated to spindle and polygonal cells containing high ALPase activity (a marker for osteogenic capacity) during culture, and the polygonal cells but not spindle cells produced the calcified matrix.

Transcranial magnetic stimulation of the motor cortex in cervical spondylosis and spinal canal stenosis.

STUDY DESIGN: This study investigated the clinical usefulness of motor evoked potentials and a silent period after motor evoked potentials produced by transcranial magnetic stimulation of the brain. OBJECTIVE: The results were correlated with the clinical state of the patients with myelopathy, whereas no abnormality of the conduction time was observed in the patients with spinal canal stenosis. SUMMARY OF BACKGROUND DATA: Magnetic stimulation has been widely used for examination of the descending excitatory motor pathways in the central nervous system, but little attention has been paid to cervical spondylosis and spinal canal stenosis. METHODS: Motor evoked potentials were examined in 35 normal subjects, 67 patients with cervical spondylotic myelopathy, and 24 patients with spinal canal stenosis. Motor evoked potentials were evoked by transcranial brain stimulation during relaxation and during maximum voluntary contraction of the target muscle. RESULTS: The central motor conduction time was found to correlate with the clinical state of the myelopathy patients, whereas no abnormality of the conduction time was observed in the patients with spinal canal stenosis. During maximum voluntary contraction of the target muscle, a silent period was always observed after the motor evoked potentials in the normal subjects, and its duration was markedly shortened in the myelopathy patients. CONCLUSIONS: In cervical myelopathy patients, the central motor conduction time was correlated with clinical evaluation and the silent period was significantly shortened. These findings about duration of the central motor conduction time and the silent period might be a useful parameter of spinal pathology.

[Recurrent episodes of fever of unknown origin as temporal lobe epilepsy].

A 67-year-old woman was admitted to our department for the recurrent fever of unknown origin that occurred once approximately every 1 month for the last 3 years. No clinical and laboratory abnormality were found, except an interictal EEG showing fronto-temporal spike discharges. During hospitalization a characteristic febrile episode was accompanied by automatism, thereby, it became clear that the undetermined periodic febrile episodes were due to temporal lobe epilepsy. In this case, the thermoregulatory center of the hypothalamus might be symptomatic zone of temporal lobe epilepsy.

Interferon-gamma modulates messenger RNA levels of c-sis (PDGF-B chain), PDGF-A chain, and IL-1 beta genes in human vascular endothelial cells.

The authors have investigated the effects of cytokines and lipopolysaccharide (LPS) on mRNA levels of c-sis (platelet-derived growth factor (PDGF)-B chain), PDGF-A chain, and interleukin 1 beta (IL-1 beta) genes in human vascular endothelial cells (EC). IL-1, tumor necrosis factor (TNF), and LPS not only enhanced the accumulation of c-sis mRNA, but also induced IL-1 beta gene expression. Interferon-gamma (IFN-gamma), in contrast, suppressed the accumulation of c-sis mRNA profoundly and PDGF-A chain mRNA to a lesser extent. The cytokine, in addition, suppressed the release of PDGF-like proteins by EC, while maintaining the growth of EC. IFN-gamma, however, augmented the levels of IL-1 beta mRNA in cultured EC in association with LPS or IL-1, suggesting that the suppression of c-sis expression was not mediated through modulation of IL-1 gene expression by IFN-gamma. These results raise the possibility that IFN-gamma may play a novel regulatory role in the pathogenesis of vascular diseases such as atherosclerosis and vasculitis.

Medical check of competitive canoeists.

We gave a sports injury questionnaire survey to 821 active canoeists, members of the Japan Canoe Association (JCA), and performed a medical check of 63 top competitive JCA canoeists, including physical and laboratory tests and radiographic examinations of the chest, spine, shoulder, elbow, and wrist joints. Completed questionnaires were returned by 417 canoeists, whose reported racing styles were: kayak, 324; Canadian canoe, 71; slalom, 13; and not specified, 9. Of the 417 respondents, 94 canoeists (22. 5%) reported that they experienced lumbago; 20.9% experienced shoulder pain; 3.8%, elbow pain; and 10.8%, wrist pain. On medical examinations, lumbago was found to be mainly of myofascial origin or due to spondylolysis. Impingement syndrome was also observed in 4 canoeists with shoulder problems. The competitive canoeists had low blood pressure, and some had bradycardia. On laboratory examinations, serum hemoglobulin, hematocrit, high-density lipoprotein cholesterol (HDL-CHO), creatine phosphokinase (CK), and creatine (CRTN) in the top competitive canoeists showed high values in comparison with those of an age-matched control group. However, low serum total cholesterol (TP) values were observed in the top competitive canoeists.

Restriction fragment length polymorphism of a lymphocyte surface antigen, Blast-1, in Japanese and Caucasians, and in patients with rheumatoid arthritis.

Analysis of polymorphic systems, demonstrating differences among ethnic groups, provides a valuable tool for biology and medicine. Blast-1 is a member of the immunoglobulin superfamily and an activation-associated glycoprotein expressed on the surface of mononuclear cells. Blast-1 demonstrates DNA polymorphism in healthy controls and patients with rheumatoid arthritis (RA). The sizes of polymorphic restriction endonuclease fragments of genomic DNA encoding Blast-1 were 2.4 and 1.9 kb. In normal controls, the frequency of the homozygote for the 2.4 kb fragment (L-L) was 0.69 and 0.47, and that for the 1.9 kb fragment (S-S) was 0.04 and 0.11 in Caucasians and Japanese, respectively. The frequency of the heterozygote for both fragments (L-S) was 0.27 and 0.42 in Caucasians and Japanese, respectively. The frequencies of the L and S alleles were 0.83 and 0.17 for Caucasians, respectively, and were 0.68 and 0.32 for Japanese, respectively. The difference in the allele frequency between Caucasians and Japanese was significant. In Japanese patients with RA, the frequency of L-L, L-S and S-S types was 0.45, 0.45 and 0.10, respectively. Lung fibrosis in Japanese RA patients was associated with an increase in the L-S and S-S types and a decrease in the L-L type. The present study indicates that the investigation for gene polymorphisms of Blast-1 among distinct ethnic groups is important because Blast-1 appears to be a genetic marker for the manifestation associated with RA.

Characterization of genomic polymorphism of an activation-associated antigen, Blast-1.

Blast-1 is a human activation-associated glycoprotein expressed on the surface of mononuclear cells, and a possible genetic marker for the manifestation of rheumatoid arthritis. In the present study, genomic polymorphism of the Blast-1 gene was analyzed using 100 healthy subjects. Restriction fragment length polymorphism (RFLP) of the Blast-1 gene was recognized only by Bam HI digestion among 46 restriction enzymes tested. The sizes of polymorphic fragments were 2.4 kilobase (kb) on the L band, and 1.9 kb on the S band. A family study demonstrated that the two alleles of the Blast-1 gene were inherited in a co-dominant Mendelian fashion. The genotype frequencies of homozygosity for the L and S bands were 47% and 42%, respectively, while the frequency of heterozygosity was 11%. The allele frequencies of the L and S bands were 0.68 and 0.32, respectively. The distribution of the Blast-1 genotypes in the present study was concordant with Hardy-Weinberg equilibrium (p greater than 0.7), which indicates that the frequency of the Blast-1 gene in the population is derived from random mating in preceding generations. The results of the present study may provide useful information in disease associations with the Blast-1 gene.

Differential expression of transforming growth factor-beta and its receptors in hepatocytes and nonparenchymal cells of rat liver after CCl4 administration.

BACKGROUND/AIMS: Transforming growth factor-beta (TGF-beta) is a family of multifunctional proteins that regulate hepatocyte proliferation, and biosynthesis of the extracellular matrix. In this study we examined whether modulation of TGF-beta receptor expression contributes to the liver diseases. METHODS: The mRNA expression of TGF-beta1, TGF-beta type I receptor (TGFbetaRI), TGF-beta type II receptor (TGFbetaRII) and TGF-beta type III receptor (TGFbetaRIII) in rat livers injured by CCl4 administration was studied by Northern blotting. The mRNA expression patterns were confirmed by in situ hybridization. RESULT: The peak of TGF-beta1 mRNA expression was observed 48 h after acute intoxication with CCl4 in nonparenchymal cells. However, the levels of TGFbetaRI and TGFbetaRII mRNA expression decreased from 24 h to 48 h and from 12 h to 48 h, respectively, and returned to the normal level by 72 h. TGFbetaRII mRNA expression was depressed more and for longer than that of TGFbetaRI mRNA. Analysis in separated hepatocytes and nonparenchymal cells from the injured livers indicated that the mRNA changes occurred in hepatocytes. Nonparenchymal cells expressed TGFbetaRI and TGFbetaRII mRNAs at constant levels during liver regeneration. TGFbetaRIII mRNA, which also decreased after 12 h, was not apparent in hepatocytes but only in nonparenchymal cells. CONCLUSIONS: These observations suggest that: (i) whenever TGF-beta1 is increased in CCl4-treated livers, it may induce liver fibrogenesis via nonparenchymal cells; (ii) the mitoinhibitory effect of TGF-beta1 on hepatocytes is transiently relieved by down-regulation of TGF-beta receptors for 72 h post-damage; and (iii) the resistance to TGF-beta growth inhibition between 24 to 48 h may be predominantly due to down-regulation of the expression of TGFbetaRII.

Down-regulation of TGF-beta receptors in human colorectal cancer: implications for cancer development.

Many colorectal cancer cells are resistant to the anti-proliferative effects of transforming growth factor-beta (TGF-beta). TGF-beta also acts as paracrine factor from cancer cells on their mesenchymal cells. The aim of this study was to examine the expression of TGF-beta and its receptors in human colorectal cancer tissue and determine any relationship with cancer growth. In situ hybridization and Northern blot hybridization detection of TGF-beta1, type I and type II receptor mRNA and immunohistochemical staining of TGF-beta1 were performed using 11 human colorectal adenomas, 22 colorectal cancers and ten normal colorectal mucosas as control. TGF-beta receptor mRNAs were expressed mainly by normal colorectal epithelial cells and adenoma. However, mRNAs for TGF-beta receptors were only faintly, if at all, expressed in eight of 22 human colorectal cancers. In addition, intense signals of TGF-beta1 mRNA and the protein were detected in all colorectal cancers. TGF-beta receptor mRNAs and TGF-beta1 protein were also distributed in fibroblasts and endothelial cells in the interstitium. Moreover, Smad 4 protein was translocated to nucleus in primarily cultured adenoma cells, but not in cancer cells after TGF-beta stimulation. The escape of human colon cancer from TGF-beta-mediated growth inhibition by down-regulation of TGF-beta receptors as well as the effects of TGF-beta on stroma formation and angiogenesis indicate a possible role for TGF-beta in the progression of colon cancer in an intact host.