Candida albicans protein kinase CaHsl1p regulates cell elongation and virulence

Saccharomyces cerevisiae Hsl1p is a Ser/Thr protein kinase that regulates cell morphology. We identified Candida albicans CaHSL1 and analysed its function in C. albicans. Cells lacking CaHsl1p exhibited filamentous growth under yeast growth conditions with the filaments elongating more quickly than did those of the wild type under hyphal growth conditions, suggesting that it plays a role in the suppression of cell elongation. Green fluorescent protein-tagged CaHsl1p colocalized with a septin complex to the bud neck during yeast growth or to a potent septation site during hyphal growth, as expected from the localization in S. cerevisiae. However, the localization of the septin complex did not change in DeltaCahsl1, suggesting that CaHsl1p does not participate in septin organization. CaHsl1p was expressed in a cell cycle-dependent manner and, except for the G1 phase, phosphorylated throughout the cell cycle. In DeltaCahsl1 cells, the phosphorylation of a possible CaHsl1p target CaSwe1p decreased, while that of CaCdc28p at tyrosine18 increased. Either an extra copy of the tyrosine18-mutated CaCdc28p or deletion of CaSWE1 suppressed the cell elongation phenotype caused by CaHSL1 deletion. Furthermore, DeltaCahsl1 exhibited reduced virulence in the mouse systemic candidiasis model. Thus, the CaHsl1p-CaSwe1p-CaCdc28p pathway appears important in the cell elongation of both the yeast and hyphal forms and to the virulence of C. albicans.     

Preparation and immunological features of syngeneic monoclonal anti-idiotypic antibody using complementary determining region (CDR) peptide as the immunogen

A syngeneic monoclonal idiotypic antibody was prepared by immunizing the sequence peptide of complementary determining region-1 (CDRL-1) of 41S-2-L which is an antibody light chain capable of catalytically decomposing the antigen peptide (gp41 peptide:original antigen) as well as the intact gp41 molecule of HIV-1 envelope. The obtained idiotypic antibody, i41SL1-2, showed a high specificity to the CDRL-1 peptide. The intact i41SL1-2 and its heavy and light chains displayed apparent affinity constants to the CDRL-1 peptide of 3.6 × 10⁹, 2.7 × 10⁷, 1.8 × 10⁶/M, respectively. The i41SL1-2 recognized the artificial molecule CA2, which has a more complex steric conformation than the CDRL-1, while the i41SL1-2 showed very low affinity to the original monoclonal antibody 41S-2 and its light chain 41S-2-L. However, a homologous sequence, EGG-D, with the gp41 peptide was expressed in the complementary determining region-3 (CDRH-3) of the heavy chain of i41SL1-2. Furthermore, the consensus sequence EGG was located at the important position of the CDRH-3 loop of i41SL1-2. Although the sequence of CDRL-1 (16 mer) is quite shorter than that of whole light chain (112 mer), the CDRL-1 could induce the rearrangement of CDRH-3 gene of i41SL1-2 so as to express a homologous sequence with the original antigen.     

Mechanism of prolactin release by 5-hydroxytryptophan

Male Wistar rats were intraperitoneally administered 300 mg/kg b.w. of α-methyl-p-tyrosine methyl ester(α-MT). These α-MT pretreated rats were anesthetized with urethane and then 5% glucose or dopamine (1 μg/kg b.w./min) was infused for 45 min. At 1 min before or 15 min after dopamine infusion, 10 or 50 mg/kg of 5-hydroxytryptophan (5-HTP) was injected intraperitoneally, and blood samples were taken from the jugular vein for prolactin determination. In rats treated with α-MT, the administration of 5-HTP increases the serum prolactin level in a dose-related manner. Dopamine infusion caused a marked decrease in serum prolactin level. The concomitant administration of dopamine and 5-HTP prevented the dopamine-induced decrease of serum prolactin in α-MT treated rats. These results indicate that the serotonergic stimulus enhanced prolactin release, not by inhibiting the dopaminergic activity, but by stimulating a prolactin-releasing factor or by activating other neurotransmitter systems.     

Hunner-Type (Classic) Interstitial Cystitis: A Distinct Inflammatory Disorder Characterized by Pancystitis,with Frequent Expansion of Clonal B-Cells and Epithelial Denudation

Interstitial cystitis (IC) is a chronic bladder disease with urinary frequency, bladder discomfort or bladder pain of unknown etiology. Based on cystoscopic findings, patients with IC are classified as either Hunner-type/classic IC (HIC), presenting with a specific Hunner lesion, or non-Hunner-type IC (NHIC), presenting with no Hunner lesion, but post-hydrodistension mucosal bleeding. Inflammatory cell infiltration, composed predominantly of lymphocytes, plasma cells and epithelial denudation, has in the past been documented as a major pathological IC finding. However, the significance of the pathological evaluation of IC, especially with regard to the difference between HIC and NHIC, has been downplayed in recent years. In this study, we performed immunohistochemical quantification of infiltrating T-lymphocytes, B-lymphocytes and plasma cells, and measured the amount of residual epithelium in urinary bladder biopsy specimens taken from patients with HIC and NHIC, and those with no IC, using image analysis software. In addition, in situ hybridization of the light chains was performed to examine clonal B-cell expansion. Lymphoplasmacytic infiltration was significantly more severe in HIC specimens than in NHIC specimens (P <0.0001). Substantial lymphoplasmacytic inflammation (≥200 cells/mm²) was observed in 93% of HIC specimens, whereas only 8% of NHIC specimens were inflamed. Plasmacytic infiltration was more prominent in HIC specimens compared with NHIC and non-IC cystitis specimens (P <0.005). Furthermore, expansion of light-chain-restricted B-cells was observed in 31% of cases of HIC. The amount of residual epithelium was decreased in HIC specimens compared with NHIC specimens and non-IC cystitis specimens (P <0.0001). These results suggest that NHIC and HIC are distinct pathological entities, with the latter characterized by pancystitis, frequent clonal B-cell expansion and epithelial denudation. An abnormality in the B-cell population may be involved in the pathogenesis of HIC.     

Spheroid formation and expression of liver-specific functions of human hepatocellular carcinoma-derived FLC-4 cells cultured in lactose-silk fibroin conjugate sponges

This study presents a hepatic tissue engineering application of three-dimensional (3D) porous sponges composed of lactose-silk fibroin (SF) conjugates (Lac-CY-SF) bearing β-galactose residues, hepatocyte-specific ligands. Lac-CY-SF sponges were prepared by freeze-drying, followed by immersion in a series of methanol aqueous solutions. Lac-CY-SF sponges showed heterogeneous pore structure with round pores about 100 μm in diameter and elongated pores 250-450 μm in length and 100-150 μm in breadth. To employ a 3D Lac-CY-SF culture system, human hepatocellular carcinoma-derived FLC-4 cells were seeded in Lac-CY-SF sponges and cultured up to 3 weeks. FLC-4 cell culture in collagen and SF sponges was also performed for comparison with the cell response to Lac-CY-SF sponges. Within 5 days of culture, FLC-4 cells cultured in Lac-CY-SF sponges, as well as the cells cultured in collagen sponges, formed multicellular spheroids with diameters from 30 to 100 μm more efficiently than did the cells cultured in SF sponges. After 3 weeks of culture, WST-1 viability assay revealed that shrinkage suppression of Lac-CY-SF sponges enabled the maintenance of viable FLC-4 cells for a long time, while the shrinkage and disintegration of collagen sponges prevented the maintenance of the cells. FLC-4 cells cultured in Lac-CY-SF sponges exhibited greater elevation of albumin secretion and sustained a higher albumin level compared with the cells cultured in collagen and SF sponges during the 3 week cultivation period. FLC-4 cells cultured in Lac-CY-SF sponges for 3 weeks expressed genes related to liver-specific functions such as transferrin and HNF-4α. On the other hand, the cells cultured in collagen and SF sponges for 3 weeks did not express these genes. These results indicated the very promising properties of Lac-CY-SF sponges as a scaffold for long-term culture of functional FLC-4 cells to study drug toxicity and hepatocyte metabolism in humans and develop a bioartificial liver model.     

Cytoplasmic Location of α1A Voltage-Gated Calcium Channel C-Terminal Fragment (Cav2.1-CTF) Aggregate Is Sufficient to Cause Cell Death

The human α_1A voltage-dependent calcium channel (Ca_v2.1) is a pore-forming essential subunit embedded in the plasma membrane. Its cytoplasmic carboxyl(C)-tail contains a small poly-glutamine (Q) tract, whose length is normally 4∼19 Q, but when expanded up to 20∼33Q, the tract causes an autosomal-dominant neurodegenerative disorder, spinocerebellar ataxia type 6 (SCA6). A recent study has shown that a 75-kDa C-terminal fragment (CTF) containing the polyQ tract remains soluble in normal brains, but becomes insoluble mainly in the cytoplasm with additional localization to the nuclei of human SCA6 Purkinje cells. However, the mechanism by which the CTF aggregation leads to neurodegeneration is completely elusive, particularly whether the CTF exerts more toxicity in the nucleus or in the cytoplasm. We tagged recombinant (r)CTF with either nuclear-localization or nuclear-export signal, created doxycyclin-inducible rat pheochromocytoma (PC12) cell lines, and found that the CTF is more toxic in the cytoplasm than in the nucleus, the observations being more obvious with Q28 (disease range) than with Q13 (normal-length). Surprisingly, the CTF aggregates co-localized both with cAMP response element-binding protein (CREB) and phosphorylated-CREB (p-CREB) in the cytoplasm, and Western blot analysis showed that the quantity of CREB and p-CREB were both decreased in the nucleus when the rCTF formed aggregates in the cytoplasm. In human brains, polyQ aggregates also co-localized with CREB in the cytoplasm of SCA6 Purkinje cells, but not in other conditions. Collectively, the cytoplasmic Ca_v2.1-CTF aggregates are sufficient to cause cell death, and one of the pathogenic mechanisms may be abnormal CREB trafficking in the cytoplasm and reduced CREB and p-CREB levels in the nuclei.     

Association of Eosinophilic Inflammation with FKBP51 Expression in Sputum Cells in Asthma

Background

Airway eosinophilia is a predictor of steroid responsiveness in steroid-naïve asthma. However, the relationship between airway eosinophilia and the expression of FK506-binding protein 51 (FKBP51), a glucocorticoid receptor co-chaperone that plays a role in steroid insensitivity in asthma, remains unknown.

Objective

To evaluate the relationship between eosinophilic inflammation and FKBP51 expression in sputum cells in asthma.

Methods

The FKBP51 mRNA levels in sputum cells from steroid-naïve patients with asthma (n = 31) and stable asthmatic patients on inhaled corticosteroid (ICS) (n = 28) were cross-sectionally examined using real-time PCR. Associations between FKBP51 levels and clinical indices were analyzed.

Results

In steroid-naïve patients, the FKBP51 levels were negatively correlated with eosinophil proportions in blood (r = −0.52) and sputum (r = −0.57), and exhaled nitric oxide levels (r = −0.42) (all p<0.05). No such associations were observed in patients on ICS. In steroid-naïve patients, improvement in forced expiratory volume in one second after ICS initiation was correlated with baseline eosinophil proportions in blood (r = 0.74) and sputum (r = 0.76) and negatively correlated with FKBP51 levels (r = −0.73) (all p<0.0001) (n = 20). Lastly, the FKBP51 levels were the lowest in steroid-naïve asthmatic patients, followed by mild to moderate persistent asthmatic patients on ICS, and the highest in severe persistent asthmatic patients on ICS (p<0.0001).

Conclusions

Lower FKBP51 expression in sputum cells may reflect eosinophilic inflammation and glucocorticoid responsiveness in steroid-naïve asthmatic patients.