Characterization of rat follistatin-related gene: effects of estrous cycle stage and pregnancy on its messenger RNA expression in rat reproductive tissues

Follistatin-related gene (FLRG) was first identified as a target of a chromosomal translocation in a human B-cell leukemia. Because FLRG protein binds to activins and bone morphogenetic proteins, FLRG is postulated to be a regulator of these growth factors. However, physiological aspects of FLRG are unclear. To elucidate the physiology of FLRG, we examined expression of FLRG in reproductive tissues of the rat. FLRG mRNA was abundantly expressed in the placenta. FLRG mRNA was also expressed in the ovary, uterus, testis, lung, adrenal gland, pituitary, kidney, small intestine, and heart. During the second half of pregnancy, expression of FLRG in the placenta continuously increased, whereas follistatin mRNA levels decreased from Day 12 to Day 14 and remained low thereafter. FLRG was also expressed in decidua. Levels of decidual FLRG mRNA remained low from Day 12 to Day 16 and then noticeably increased until Day 20. In contrast, follistatin mRNA was highly expressed in the decidua on Day 12, continuously decreased until Day 16, and then remained at relatively low levels thereafter. During the rat estrous cycle, levels of ovarian FLRG mRNA fluctuated diurnally, with highest levels during daytime, and did not change relative to the day of the estrous cycle. The present results suggest that FLRG may play a role in the regulation of reproductive events.     

Synthesis of 5-Alkyl-5-methyl-2-hydroxy-2-cyclopentenones

A hydrogen bacterium strain, N34, and its oxygen-resistant segregant strain, Y38, were subjected to a taxonomical study. Since both strains were capable of N₂-fixation, N₂-fixing facultative hydrogen autotrophs listed in “Bergey’s Manual of Systematic Bacteriology” were used for comparison. Both strains produced a water-insoluble carotenoid pigment, zeaxanthin dirhamnoside, indicating that both should be classified into the genus Xanthobacter. Then, the differential characteristics of the two species of the genus Xanthobacter, X. autotrophicus and X. flavus, were investigated as to both strains. The vitamin requirement, the sensitivity to oxygen under autotrophic conditions, the inducibility of hydrogenase, the substrate range of carbohydrates and N₂-fixing growth characteristics of both strains were almost completely opposite to those of X. flavus. Moreover, both strains coincided exactly with X. autotrophicus in morphological and other physiological characteristics. From these results both strains were identified as Xanthobacter autotrophicus.     

Identification of protein kinase disruptions as suppressors of the calcium sensitivity of S. cerevisiae Δptp2 Δmsg5 protein phosphatase double disruptant

The double disruptant of the S. cerevisiae protein phosphatase (PPase) genes, PTP2 (phosphotyrosine-specific PPase) and MSG5 (phosphotyrosine and phosphothreonine/serine-PPase) causes calcium-sensitive growth (Ca^s). Previous study using Fluorescent-activated cell sorting (FACS) analysis showed that this growth defect with calcium occurs at G1–S transition in the cell cycle. We discovered that six non-essential protein kinase (PKase) disruptions (Δbck1, Δmkk1, Δslt2/Δmpk1, Δmck1, Δssk2 and Δyak1) suppressed the Ca^s-phenotype of the Δptp2 Δmsg5 double disruptant. Bck1p, Mkk1p and Slt2p are components of the mitogen-activated protein kinase (MAPK) cascade of cell wall integrity pathway (Slt2 pathway), and Mck1p is its down regulator. Ssk2p is the MAPK kinase kinase of the high-osmolarity glycerol (HOG) pathway, while Yak1p is a negative regulator for the cAMP-dependent PKA pathway. FACS analysis revealed that only the disruption of Δssk2 and Δyak1 but not Δbck1, Δmkk1, Δslt2 and Δmck1 was able to suppress the delayed G1–S transition, suggesting that suppression of the growth defect is not always accompanied by suppression of the G1–S transition delay. The discovery of these PKases as suppressors revealed that in addition to the previously anticipated Slt2 pathway, HOG, Yak1p and Mck1p regulatory pathways may also be involved in the calcium sensitivity of the Δptp2 Δmsg5 double disruptant.     

Development of an Ultra-Rapid Diagnostic Method Based on Heart-Type Fatty Acid Binding Protein Levels in the CSF of CJD Patients

Creutzfeldt-Jakob disease (CJD) is a transmissible, fatal, neurodegenerative disease in humans. Recently, various drugs have been reported to be useful in the treatment of CJD; however, for such treatments to be useful it is essential to rapidly and accurately diagnose CJD. 124 CJD patients and 87 with other diseases causing rapid progressive dementia were examined. Cerebral spinal fluid (CSF) from CJD patients was analyzed by 2D-PAGE and the protein expression pattern was compared with that from healthy subjects. One of three CJD-specific spots was found to be fatty acid binding protein (FABP), and heart-type FABP (H-FABP) was analyzed as a new biochemical marker for CJD. H-FABP ELISA results were compared between CJD patients and patients with other diseases (n = 211). Visual readout accuracy of the Rapicheck^® H-FABP test panel for CSF was analyzed using an independent measure of CSF H-FABP concentration. The distribution of H-FABP in the brains of CJD patients was examined by immunohistochemistry. ELISA sensitivity and specificity were 90.3% and 92.9%, respectively, and Rapicheck^® H-FABP sensitivity and specificity were 87.9% and 96.0%, respectively. ELISA and Rapicheck^® H-FABP assays provided comparable results for 14-3-3 protein and total tau protein. Elevated H-FABP levels were associated with an accumulation of abnormal prion protein, astrocytic gliosis, and neuronal loss in the cerebral cortices of CJD patients. In conclusion, Rapicheck^® H-FABP of CSF specimens enabled quick and frequent diagnosis of CJD. H-FABP represents a new biomarker for CJD distinct from 14-3-3 protein and total tau protein.     

Assessment of the number of local cytotoxic T lymphocytes required for degradation of micrometer-size tumor spheroids

Adoptive immunotherapy with human cytotoxic T lymphocytes (CTL) is a promising cancer treatment. Previously we showed that human CTLs against various types of tumors can be efficiently produced by coculturing peripheral blood cells with target cells. The aims of this study were to simulate the interaction of CTLs and micrometer-size tumor tissues in vitro and to assess the required number of CTLs at local tumor sites for degradation of a tumor. Allogeneic CTLs against a human transitional cell carcinoma cell line and autologous CTLs against a renal cell carcinoma cell derived from a surgical specimen were generated. The cytotoxic activities of CTLs against tumor cells in monolayer culture and tumor spheroids formed in U-bottom 96-well culture plates were assessed. Both allogeneic and autologous CTLs showed greater destructive activity than lymphokine activated killer (LAK) cells against target tumor spheroids. CTLs inoculated at E/T ratios of 0.1 to 1 coexisted with the tumor spheroid for 5 to 6 days and then increased in number with apparently lethal activity against the tumor spheroid. In contrast to CTLs, the increase in LAK cell numbers was scarcely observed, and the proliferated LAK cells did not show cytotoxicity against the tumor spheroid. These observations suggest that, when a small number of CTLs reach a local tumor site, they can destroy micrometer-size tumors after considerable local proliferation. This revised version was published online in August 2006 with corrections to the Cover Date.     

Expression of human immunoglobulin E epsilon chain cDNA in E. coli.

Using the cDNA of human epsilon chain, three expression plasmids that code directly the constant portion of the epsilon chain (C epsilon 1-C epsilon 4, C epsilon 2-C epsilon 4 and C epsilon 3-C epsilon 4 domains) were constructed. These epsilon chain peptides were synthesized in E. coli under the control of the trp promoter-operator. The bacterially produced peptides have the antigenicity of human epsilon chain and gave the molecular weights equal to the values calculated from the amino acid sequence of the constructed plasmids.     

Nicotine suppresses tunicamycin-induced, but not thapsigargin-induced, expression of GRP78 during ER stress-mediated apoptosis in PC12 cells

We previously reported that nicotine protected against tunicamycin (Tm)-induced ER stress-mediated apoptosis, but not thapsigargin (Tg)-induced apoptosis in PC12 cells. In the present study, we report that the expression of glucose-regulated protein 78 (GRP78) was suppressed by nicotine in Tm-treated PC12 cells. Interestingly, the GRP78 expression was not changed by nicotine in Tg-treated cells. Moreover, nicotine reduced the activation of caspase-12 in Tm-treated cells, but not in Tg-treated cells. These results suggest that nicotine prevented Tm-induced ER stress-mediated apoptosis by attenuating an early stage of Tm-induced ER stress. It was possible that the suppression of GRP78 expression by nicotine was achieved through the suppression of the Ire1-XBP1 and/or ATF6 pathways. We observed that nicotine suppressed the Tm-induced, but not Tg-induced, splicing of XBP1 mRNA, and also suppressed the Tm-induced, but not Tg-induced, production of cleaved ATF6 in PC12 cells. These results indicate that the suppression of Ire1-XBP1 and ATF6 pathways contributes to the suppression of GRP78 expression by nicotine in Tm-treated PC12 cells, suggesting that nicotine suppresses a common step upstream of both the Ire1-XBP1 and ATF6 pathways which are required for the expression of GRP78 during Tm-induced ER stress.     

A crucial role for antigen-presenting cells and alloantigen expression in graft-versus-leukemia responses

Graft-versus-leukemia (GVL) response after allogeneic bone marrow transplantation (BMT) represents one of the most potent forms of immunotherapy against malignant diseases. Antigen-presenting cells (APCs) are crucial for the induction of graft-versus-host disease (GVHD), the most serious complication of allogeneic BMT, but their role in GVL responses is unclear. Using a series of clinically relevant mouse GVL tumor models, we found that APCs and alloantigen expression on tumors are crucial for GVL. Moreover, APCs of host origin predominated in GVL responses although donor APCs contributed as the acuity of tumor burden decreased.