Analysis of circulating microRNA during early gestation in Japanese black cattle.

Circulating microRNAs (miRNAs) have been used as biomarkers for various diseases and physiological conditions in humans and mice; studies in domestic animals, particularly cattle, are limited. The importance of early pregnancy diagnosis (especially within the 21-d cow estrous cycle) in the livestock industry is extremely high. This study compared the circulating miRNAs in bred non-pregnant and pregnant Japanese Black cows, explored miRNAs as biomarkers for early pregnancy diagnosis, and established a measurement system that included selecting an appropriate reference miRNA and determining the effect of hemolysis on miRNA quantification in plasma. miRNA was extracted from the plasma of Japanese Black cows on day 21 after artificial insemination and subjected to a customized bovine oligonucleotide microarray for expression analysis. Differentially expressed miRNAs and reference miRNA candidates were selected and validated using reverse transcription-quantitative PCR (RT-qPCR). An appropriate endogenous reference miRNA for normalization was selected using NormFinder software. To evaluate the effect of hemolysis on miRNA quantification, hemolyzed samples were prepared using plasma from four cows in the estrous cycle and subjected to RT-qPCR. A total of 124 miRNAs were detected in bovine plasma by microarray analysis in bred non-pregnant and pregnant cows. The levels of five circulating miRNAs were significantly higher in pregnant cows than in bred non-pregnant cows, and 24 miRNAs were detected only in the pregnant group. NormFinder analysis and RT-qPCR validation showed that miR-2455 was an appropriate reference miRNA in the plasma of bred non-pregnant and pregnant Japanese Black cows, and miR-19b, miR-25, miR-29a, and miR-148a were significantly higher in the pregnant group. These four circulating miRNAs did not change during the estrous cycle and were less affected by hemolysis. In the current study, we found four miRNAs, miR-19b, miR-25, miR-29a, and miR-148a, which were present at high levels in the plasma of pregnant Japanese Black cows. Since these miRNAs are less affected by hemolysis, they may potentially be used as biomarkers for early pregnancy diagnosis in cattle.

Mutations in the gene encoding c-Abl-binding protein SH3BP2 cause cherubism.

Cherubism (MIM 118400) is an autosomal dominant inherited syndrome characterized by excessive bone degradation of the upper and lower jaws followed by development of fibrous tissue masses, which causes a characteristic facial swelling. Here we describe seven mutations in the SH3-binding protein SH3BP2 (MIM 602104) on chromosome 4p16.3 that cause cherubism.

Characterization of expressed genes in the SLL2 region of self-compatible Arabidopsis thaliana.

Self-incompatibility in Brassica species is regulated by a set of S-locus genes: SLG, SRK, and SP11/SCR. In the vicinity of the S-locus genes, several expressed genes, SLL2 and SP2/ClpP, etc., were identified in B. campestris. Arabidopsis thaliana is a self-compatible Brassica relative, and its complete genome has been sequenced. From comparison of the genomic sequences between B. campestris and A. thaliana, microsynteny between gene clusters of Arabidopsis and Brassica SLL2 regions was observed, though the S-locus genes, SLG, SRK, and SP11/SCR were not found in the region of Arabidopsis. Almost all genes predicted in this region of Arabidopsis were expressed in both vegetative and reproductive organs, suggesting that the genes in the SLL2 region might not be related to self-incompatibility. Considering the recent speculation that the S-locus genes were translocated as a single unit between Arabidopsis and Brassica, the translocation might have occurred in the region between the SLL2 and SP7 genes.

Highly divergent sequences of the pollen self-incompatibility (S) gene in class-I S haplotypes of Brassica campestris (syn. rapa) L.

Self-incompatibility (SI) enables flowering plants to discriminate between self- and non-self-pollen. In Brassica, SI is controlled by the highly polymorphic S locus. The recently identified male determinant, termed SP11 or SCR, is thought to be the ligand of S receptor kinase, the female determinant. To examine functional and evolutionary properties of SP11, we cloned 14 alleles from class-I S haplotypes of Brassica campestris and carried out sequence analyses. The sequences of mature SP11 proteins are highly divergent, except for the presence of conserved cysteines. The phylogenetic trees suggest possible co-evolution of the genes encoding the male and female determinants.

IL-4 and transforming growth factor-beta suppress human immunoglobulin secretion in vitro by surface IgD- B cells.

The effect of IL-4 and transforming growth factor-beta (TGF-beta) on immunoglobulin secretion in vitro by peripheral blood mononuclear cells (PBMC) or purified B cells activated with murine EL4 thymoma cells and phorbol myristate acetate (PMA) was investigated. As previously reported, IL-4 induced IgE and IgG4 secretion by B cells in PBMC preparations and B cells activated with EL4 cells and PMA. However, when B cells, either in PBMC preparations or purified and activated with EL4 cells and PMA, spontaneously secreted large quantities of immunoglobulin, IL-4 suppressed the immunoglobulin secretion of all isotypes. IL-4 also suppressed the IgE secretion by B cells from an atopic dermatitis patient. This suppressive effect was not reversed by adding IL-2 or interferon-gamma (IFN-gamma) to the cultures. We also showed that TGF-beta suppressed the immunoglobulin secretion by purified B cells activated by EL4 cells and PMA. To investigate whether IL-4 or TGF-beta suppressed immunoglobulin secretion by in vivo 'switched' and isotype-committed B cells, sIgD- B cells were isolated, activated with EL4 cells and PMA and cultured with IL-4 or TGF-beta. Such activated B cells secreted large quantities of IgG1, IgG2, IgG3, IgA1, IgA2 and IgM, and IL-4 and TGF-beta suppressed all these isotypes by greater than 80%. The data demonstrated that IL-4 and TGF-beta suppress immunoglobulin secretion in vitro by in vivo isotype-committed sIgD- B cells, suggesting that these lymphokines may play a down-regulatory role on differentiated isotype-committed B cells in an isotype-unrestricted manner. The data also showed that IL-4 and TGF-beta acted directly on isolated B cells.

Distribution and clinical significance of lupus anticoagulant and anticardiolipin antibody in 349 patients with systemic lupus erythematosus.

Samples from 349 patients with systemic lupus erythematosus (SLE) were tested simultaneously for lupus anticoagulant (LAC) and anticardiolipin antibodies (ACL). LAC was detected in 27.2% of 349 SLE patients by a modified mixing kaolin clotting time. ACL was detected in 34.7% by enzyme-linked immunosorbent assay. Only half of the patients who had LAC or ACL were positive for both of them. In addition, isotypes of ACL in these patients were studied. The IgG isotype was detected in 81.8% of 121 patients, and more than half had only the IgG isotype. When clinical features of patients with LAC or ACL were studied, the incidence of thrombosis, fetal loss, and thrombocytopenia were significantly higher in both groups compared with patients without LAC or ACL. In particular, the patients with both LAC and ACL showed the highest risk of fetal loss (89%) during pregnancy. These results indicate that LAC and ACL are detected in partly different groups of SLE patients, but both of these groups are clinically similar.

Orthostatic tremor associated with voice tremor.

We report a 67-year-old female with orthostatic and voice tremor. Her orthostatic tremor mainly affected her lower extremities, alternating between antagonist muscle groups at a frequency of 4.4-4.8 Hz. The voice tremor ranged between 4.8 and 8.8 Hz. In this case, the frequency of voice tremor was same as that of orthostatic tremor, suggesting a common origin from a tremor-generating mechanism. These tremors were diagnosed as 'forme fruste' of the essential tremor, not the incipient stage of Parkinson's disease. Medications including clonazepam, perphenazine, Dopa and trihexyphenidyl hydrochloride had no effect on both the orthostatic and voice tremors, but propranolol was somewhat beneficial on voice tremor.

Action mechanism of amphipathic peptides gramicidin S and melittin on erythrocyte membrane.

Amphipathic peptides gramicidin S and melittin caused a characteristic colloid-osmotic hemolysis on human erythrocytes; that is, the peptides produced initially a small membrane lesion in erythrocyte membrane, followed by the release of hemoglobin. The size of membrane lesion increased with an increase in the concentration of peptide. Under the conditions causing membrane lesion, we observed the release of membrane fragments containing phospholipids. The present results show that both the peptides have the ability to stimulate the release of membrane fragments out of the cells and this brings about the perforation of molecules of small size, leading to a colloid-osmotic hemolysis.

Increased frequency of non-insulin-dependent diabetes mellitus among narcoleptic patients.

Forty-eight narcoleptic patients were randomly selected from a group of 197 narcoleptic outpatients, and a 50-g oral glucose tolerance test (OGTT) was performed. Blood glucose levels were determined by oximetry before and 30, 60, and 120 min after the glucose administration. Serum insulin concentrations (IRI) were determined by enzyme immunoassay, and an insulinogenic index (II) was calculated. (II = the ratio of IRI increment to blood glucose increment in 30 min following glucose load). From the results of the OGTT, six patients with definite diabetes mellitus (DM) were identified (12.5%, 4 men and 2 women) according to World Health Organization criteria (1980). The II of the DM patients was significantly low, with an average of 0.13 +/- 0.03. From recent data in the literature on the prevalence of DM in the general Japanese adult population (1.75-5.5%), it was concluded that the frequency of DM among our randomly selected narcoleptic patients was significantly increased. The positive results for diabetes in our six narcoleptic patients could not be attributed to obesity, because there was no difference between the obesity indices of DM and nonDM narcoleptic patients, nor was there a difference between the frequency of DM in obese and nonobese patients.