Induction of IgA against Haemophilus parainfluenzae antigens in tonsillar mononuclear cells from patients with IgA nephropathy

Much evidence suggests that IgA production in vivo and in vitro is enhanced in patients with IgA nephropathy (IgAN). We have demonstrated glomerular deposition of the outer membranes of Haemophilus parainfluenzae (HP) antigens (OMHP) and the presence of HP-specific IgA in the serum of patients with IgAN. In this study, we investigated the production of IgA and several cytokines by tonsillar mononuclear cells (TMC) from IgAN patients induced by stimulation with OMHP. The spontaneous production of total IgA and TGF-beta by TMC from IgAN patients was higher than that by TMC from patients with chronic tonsillitis (CT) (P < 0.05). Stimulation with OMHP in vitro enhanced the production of HP-specific IgA by TMC from IgAN patients (P < 0.01), but not by TMC from CT patients. OMHP stimulation also enhanced the production of TGF-beta and IL-10 by TMC from IgAN patients (P < 0.001). These results suggest that the infection of HP in the tonsil may be involved in the etiology of IgAN.     

The development of myelinated nociceptors is dependent upon trks in the trigeminal ganglion

Cell size of primary sensory neurons and distribution patterns of neurons that are immunopositive (ip) for VRL-1, a newly cloned capsaicin-receptor homologue, were examined in trigeminal ganglia (TGs) of knockout mice for trkA, trkB or trkC to determine the developmental dependency of myelinated nociceptors on expression of the genes. The number of TG neurons was strongly decreased in the knockout mice as compared to wildtype and heterozygous mice (82%, 39%, and 48% reduction for trkA, trkB and trkC, respectively). The absence of trkA and trkC reduced the number of TG neurons in all cell-size ranges. The number of medium-sized and large TG neurons was decreased in trkB-knockout mice, whereas that of small TG neurons was barely affected by trkB deficiency. TG contained abundant VRL-1-ip neurons in wildtype and heterozygous mice; 9% of TG neurons exhibited immunopositivity. In trkA-knockout mice, VRL-1-ip neurons almost disappeared (1% of TG neurons were VRL-1-ip). However, 13% and 9% of TG neurons in trkB- and trkC-knockout mice, respectively, were immunostained for the ion channel protein. In trkC-knockout mice, the proportion of large VRL-1-ip neurons decreased whereas that of small and medium-sized VRL-1-ip neurons increased. In addition, immunohistochemistry of the protein gene product 9.5 (PGP 9.5) demonstrated that trkA deficiency caused a marked reduction of varicose endings in the epithelium of the palatal mucosa. Loss of trkC diminished the number of PGP 9.5-ip varicose fibers in the deep layer of mucosal connective tissue of the palate. In tooth pulp, PGP 9.5-ip nerve fibers were absent in trkA-knockout mice but abundant in trkB- and trkC-knockout mice. The present study suggests that the development of myelinated nociceptors is dependent on trkA and trkC but not on trkB.     

Anabolic effect of genistein in osteoblastic MC3T3-E1 cells

Genistein is a natural isoflavone found in Leguminosae. The effect of genistein on osteoblastic MC3T3-E1 cells was investigated. Cells were cultured for 48 h in the presence of genistein (10(-7)-10(-5) M). Genistein (10(-6) and 10(-5) M) caused a significant elevation of protein content, alkaline phosphatase activity, and deoxyriboncleic acid (DNA) content in the cells. The effect of genistein (10-5 M) in increasing protein content, alkaline phosphatase activity and DNA content in the cells was completely prevented by the presence of cycloheximide (10(-6) M), an inhibitor of protein synthesis, suggesting that the isoflavone's effect results from a newly synthesized protein component. The effect of genistein (10(-5) M) in elevating cellular protein content and alkaline phosphatase activity was completely inhibited by the presence of trifluo-perazine (10(-5) M), staurosporine (10(-7) M) or vanadate (10(-6) M), various protein kinase inhibitors. Moreover, genistein (10(-5) M)-increased protein content and alkaline phosphatase activity in the cells was clearly abolished by the presence of anti-estrogen tamoxifen (10(-6) M). The effect of 17beta-estradiol (10(-9) M) in elevating protein and alkaline phosphatase activity in the cells was not enhanced by the presence of genistein (10(-5) M). Genistein's effect might be partly involved in estrogen action. The present study demonstrates that genistein has an anabolic effect on osteoblastic MC3T3-E1 cells.     

Ultraviolet-induced suppressor T cells and factor(s) in murine contact photosensitivity. II. Igh-V restriction of T-cell-suppressor factor

In murine contact photosensitivity (CPS) to 3,3',4',5-tetrachlorosalicylanilide, we have reported that antigen-specific suppressor T cells and factor(s) (TsF) are induced by preexposure of the photosensitizing site to low doses of ultraviolet B. The TsF is a single-chain factor bearing both antigen-binding site(s) and I-J determinants. In this report, we examined the genetic restriction of the factor in terms of both H-2 and Igh-associated genes. The CPS responses of BALB/c (H-2d, Igh-VaCa) and BALB.B (H-2b, Igh-VaCa) but not DBA/2 (H-2d, Igh-VcCc) were suppressed by the injection of the BALB/c TsF, and reciprocally, the response of BALB/c was suppressed by the BALB.B TsF. This demonstrated that H-2 identity was not a requirement for TsF function. Furthermore, the BALB/c TsF significantly suppressed the CPS reaction in BAB-14 (H-2d, Igh-VaCb) but not in either C.B-20 (H-2d, Igh-VbCb) or C.AL-20 (H-2d, Igh-VdCd). In addition, the BAB-14 TsF, but not the C.B-20 factor, induced suppression in BALB/c mice. These results indicated that identity at the Igh-V locus of the strain producing the factor and the recipient was required for suppression. Because of the single-chain nature of the factor, it seems that the I-J+ molecule present in our TsF is closely related to not only recognition but also Igh-V restriction functions.     

Role of intracellular Mg2+ in the activation of muscarinic K+ channel in cardiac atrial cell membrane

Effects of intracellular Mg²⁺ in the activation of a muscarinic K⁺ channel were examined in single atrial cells, using patch-recording techniques. In cell-attached patch recordings, acetylcholine (ACh) or adenosine (Ado), present in the pipette, activated a specific population of K⁺ channels. In inside-out patches, openings of the K⁺ channel by ACh or Ado diminished and did not resume until Mg²⁺ was added to the perfusate which contained GTP or GTP-S, a non-hydrolyzable GTP analogue. Channel openings caused by GTP faded by removing Mg²⁺, while GTP-S-induced openings persisted steadily even when both Mg²⁺ and GTP-S were removed. In contrast to the case of GTP-induced channel openings, the GTP-S-induced openings were not inhibited by the A protomer of pertussi toxin with NAD. From these observations, we concluded: 1) Intracellular Mg²⁺ is essential for GTP to activate the GTP-binding protein. 2) Deactivation of the N protein may be caused by hydrolysis of GTP to GDP. This process may not require Mg²⁺. 3) During the activation by GTP analogues, the N protein may be dissociated into its subunits.     

A morphological study on glutamate-induced swelling of cultured astrocytes: involvement of calcium and chloride ion mechanisms.

Morphological changes in cultured astrocytes exposed to L-glutamate (Glu) were examined light and electron microscopically. The treatment with 0.1 mM Glu for 60 min caused marked swelling of the cells, which was characterized by reduction in staining of cytoplasm with Toluidine blue, disappearance of the cytoplasmic granular ground substances, swollen mitochondrion and nucleus, and dispersed chromatin. The above changes were prevented by the removal of Na+, Ca2+ or Cl- from the incubation medium for Glu treatment. However, the Glu treatment in a Cl(-)-free medium caused conspicuous aggregation of 10 nm filaments.     

Overexpression of mRNAs of TGFbeta-1 and related genes in fibroblasts of Werner syndrome patients

We analyzed mRNAs that were up- or down-regulated in fibroblasts from Werner syndrome (WS) patients compared with those from normal individuals. The mRNAs from normal and WS cells were first screened by differential display, and those mRNAs that were apparently up- or down-regulated were selected except for mRNAs related to extra-cellular matrix (ECM) proteins that are already known to be up-regulated in WS fibroblasts. Then, the expression levels of these mRNAs were semiquantified by northern blot analysis, and six up-regulated and two down-regulated mRNAs were identified in WS cell lines. Among the six up-regulated mRNAs were three mRNAs that coded TGFbeta-1 and two proteins, their expressions of which were increased by TGFbeta-1. These results together with the fact that TGFbeta-1 up-regulates the expression of ECM proteins strongly suggest that TGFbeta-1 has a key role in accelerated cellular senescence of fibroblasts of WS patients.     

Construction of promoter-probe vectors for Candida maltosa,a n-alkane-assimilating yeast,using the LEU2 gene of Saccharomyces cerevisiae

For the purpose of isolation of promoter regions which are regulated by a carbon source in the medium in an n-alkane-assimilating yeast, Candida maltosa, two promoter-probe vectors were constructed. Each of them consists of the LEU2 gene of Saccharomyces cerevisiae whose 5′-non-coding region was trimmed with BAL31, an autonomously replicating sequence isolated from C. maltosa genome (the TRA region) which we have previously isolated, and the pBR322 sequence. One of them, pPLC2, having the TATA box, lacks the regulatory sequence (“sequence L”) of the LEU2 gene, and the other, pPLC1, lacks both the TATA box and sequence L. Using pPLC1 as a shot-gun cloning vector in C. maltosa, many promoter regions which were active when glucose was present in the medium as a carbon source were obtained from the genome of C. maltosa. The sizes of the inserted fragments of two of them were determined. (In this paper, a promoter region refers to a promoter which includes a TATA box, plus a regulatory sequence such as an UAS (upstream activating sequence)-like sequence).