Expression of Cysteine Proteinases and Their Inhibitor, Cystatin β, in Cultured Rat Mesangial Cells

Matrix expansion in the glomerular mesangial area is observed in diabetic nephropathy. Intracellular breakdown of long-lived proteins was lower in mesangial cells in the high glucose medium than that in the control medium. Enzymatic activity of cathepsin L increased 1.4-fold after 6 h of treatment with the high glucose, and then declined gradually to 72% of control cells after treatment for 36 h. Change in the enzyme activity of cathepsin B showed a similar time course but less magnitude than that of cathepsin L. Immunoblot analysis with anti-cathepsin L antibody showed that change in the enzyme activity of cathepsin L was due to the change in the amount of cathepsin L, and that with anti-cathepsin B antibody showed no change in the amount of cathepsin B in the mesangial cells treated with high glucose. Intracellular cathepsin activities were controlled not only by the amounts but also by the inhibitor cystatin β. Immunoblot analysis with anti-cystatin β antibody showed that intracellar levels of cystatin β increased slightly after 24 h of treatment with high glucose. These changes were derived from changes in mRNA level. These results, therefore, demonstrated that the decrease of intracellular protein breakdown in mesangial cells treated with high glucose medium was due to both suppression of cathepsins and increase of cystatin β.     

Intrahepatic sarcomatous cholangiocarcinoma

A 77-year-old man, diagnosed with a liver tumor, was referred to our hospital. Abdominal ultrasonography demonstrated a low echoic mass in the liver S₂ region, and abdominal CT confirmed the presence of a round low-density mass 7 cm in diameter. Enhanced angio-computed tomography (CT) showed a ring-like form with a pale periphery. In the delayed phase of angio-CT, the inside of the mass was enhanced, showing septal stricture. Abdominal magnetic resonance imaging (MRI) revealed a heterogenous low intensity area in T1-weighted images, with a clear high intensity border becoming apparent in T2-weighted images. Stretching of the hepatic artery was evident on the arterial phase of angiography, while an avascular area was apparent in the lateral segment of the liver in the portal phase. Lateral segmentectomy was performed. The size of the tumor was 6×6×5 cm. On macroscopic cross section, it was white and clearly demarcated from the surrounding tissue. Microscopic observation of H&E-stained specimens did not show any glandular formation. The tumor consisted of an irregular fascicular arrangement of spindle-shaped and round cells with poor intercellular adhesion. While there was no region containing differentiated epithelial components, silver impregnation staining revealed structures resembling regenerating bile ducts. The tumor cells were positive for wide-keratin, and for vimentin staining. Tumor cells were carcinoembryonic antigen (CEA)-positive and alpha-feto protein (AFP)-negative. From the above findings, the tumor was judged to have originated from epithelium rather than from mesenchymal elements. The final diagnosis was intrahepatic cholangiocarcinoma with secondary sarcomatous transformation, rather than hepatocellular carcinoma.     

Apoptotic cells in six patients with IgA nephropathy in repeat biopsies

SUMMARY: Programmed cell death is a selective process of physiological cell deletion and is known as apoptosis. The purpose of the present study was to determine the relationship between the existence of apoptotic cells in glomeruli and the clinical or histopathological findings obtained in repeat renal biopsies of patients with IgA nephropathy. Repeat renal biopsy specimens were obtained from six patients with IgA nephropathy. The nick end labelling method (TUNEL) was used for the detection of apoptotic cells. Clinical laboratory data, i.e. urinary protein excretion, creatinine clearance (Ccr), blood urea nitrogen (BUN) and serum creatinine, (s‐Cr) were obtained from these patients. At the first renal biopsy, apoptotic cells in the glomeruli were observed in three out of six patients using TUNEL. These patients were classified as the severe glomerular damage group. The other three patients without apoptotic cells were in the mild glomerular damage group. Mean levels of urinary protein excretion at the first renal biopsy in the patients with apoptotic cells were slightly higher than those in patients without apoptotic cells. Levels of Ccr in patients with apoptotic cells were lower than those in patients without apoptotic cells. There were no significant differences in the levels of BUN and s‐Cr in patients with or without apoptotic cells. Two patients with apoptotic cells in glomeruli at the first renal biopsy did not show apoptotic cells at the second renal biopsy. These two patients showed improvement not only in clinical laboratory findings but also in histological findings at the second biopsy. Only one patient with apoptotic cells at the first and second biopsies exhibited deterioration at the second biopsy. All three patients without apoptotic cells at the first renal biopsy also showed deterioration of the clinical laboratory and histopathological findings. It is postulated that various factors other than apoptosis might induce progression of renal injuries in such patients. It appears that the clinical laboratory data, i.e. proteinuria, renal function and histopathological findings, might be influenced by apoptosis in patients with IgA nephropathy. It is postulated that apoptosis may induce reduction of excess proliferative glomerular mesangial cells and/or infiltrating cells and tissue repair.     

A diagnostic caution in screening for depressed college students

The Beck Depression Inventory (BDI) is the most frequently used research measure for selecting samples of depressed college students, and a cutoff score of 10 is most often used. Although previous studies have examined the diagnostic characteristics of subjects selected in this manner, these studies have been limited to examination of depressive diagnostic features and have focused on samples selected on the basis of a less commonly used cutoff score of 16. This study extends previous research by assessing a broad range of diagnostic characteristics of depressed college students identified using single (1-point) and dual (2-point) administrations of the BDI. Sixty-three college students meeting the BDI criterion for at least mild depression participated in structured interviews based on DSM-III. Results showed a high degree of diagnostic heterogeneity for both methods, although the 2-point criterion increased homogeneity marginally. The BDI screenings occasionally resulted in the selection of individuals with substance abuse disorders, precluding the diagnosis of an affective disorder. A second group of selected individuals contained students with past histories of psychiatric disorders, but no clinical symptoms at present. It is suggested that researchers carefully control for heterogeneity of student samples by following 2-point screening sessions with a brief diagnostic interview.This research was conducted as part of the first author's M.S. thesis. We express our appreciation to the following without whose help this study could not have been conducted: Madeline Barnes, Allan F. Chino, Louis Damis, Michael Kippes, Christine Childers Michael, and Sue Slade.     

Glomerular changes in the KK-Ay/Ta mouse: a possible model for human type 2 diabetic nephropathy

BACKGROUND: In type 2 diabetic nephropathy, there is no animal model which has been completely matched with humans. Advanced glycation end products (AGE) and transforming growth factor-beta (TGF-beta) are closely related to hyperglycaemia and their pathobiochemistry could explain diabetic nephropathy. The objective of the present study was to evaluate the KK-A(y)/Ta mouse as a suitable model for type 2 diabetic nephropathy including pathological changes and immunohistochemical analyses of AGE and TGF-beta, compared with the non-diabetic BALB/cA mouse. METHODS: The urinary albumin/creatinine ratio (ACR), body weight (BW), fasting and casual blood glucose, blood haemoglobin A(1c) (HbA(1c)), creatinine clearance (Ccr) and blood pressure were measured for phenotypic characterisation. The pathological changes of glomeruli were evaluated by light microscopy, immunofluorescence and electron microscopy. AGE and TGF-beta accumulation were evaluated by immunoperoxidase staining. RESULTS: The mean levels of ACR, casual blood glucose, blood HbA(1c) and Ccr in KK-A(y)/Ta mice were higher than those in age-matched non-diabetic BALB/cA mice after 12 weeks of age. There were no significant changes in the levels of systemic blood pressure among all groups. The pathological changes of glomeruli in KK-A(y)/Ta mice were consistent with those in the early stage of human diabetic nephropathy. AGE and TGF-beta protein appeared to be localised in the glomerular mesangial matrices. CONCLUSION: It appears that KK-A(y)/Ta mice, especially in terms of histopathological findings, are a suitable animal model for the early stage of type 2 diabetic nephropathy.     

The behavior of vascular smooth muscle cells and platelets onto epigallocatechin gallate-releasing poly(l-lactide-co-epsilon-caprolactone) as stent-coating materials

Localized drug delivery from drug-eluting stents has been accepted as one of the most promising treatment methods for preventing restenosis after stenting. However, thrombosis, inflammation, and restenosis are still major problems for the utility of cardiovascular prostheses such as vascular grafts and stents. Epigallocatechin-3-O-gallate (EGCG), a major polyphenolic constituent of green tea, has been shown to have anti-thrombotic, anti-inflammatory and anti-proliferative activities. It was hypothesized that controlled release of EGCG from biodegradable poly(lactide-co-epsilon-caprolactone, PLCL) stent coatings would suppress migration and invasion of vascular smooth muscle cells (VSMCs) as well as platelet-mediated thrombosis. EGCG-releasing PLCL (E-PLCL) was prepared by blending PLCL with 5% EGCG. The surface morphology, roughness and melting temperature of PLCL were not changed despite EGCG addition. EGCG did, however, EGCG appreciably increase the hydrophilicity of PLCL. EGCG was found to be uniformly dispersed throughout E-PLCL without direct chemical interactions with PLCL. E-PLCL displayed diffusion controlled release of EGCG release for periods up to 34 days. E-PLCL significantly suppressed the migration and invasion of VSMCs as well as the adhesion and activation of platelets. E-PLCL coatings were able to smooth the surface of bare stents with neither cracks nor webbings after balloon expansion. The structural integrity of coatings was sufficient to resist delamination or destruction during 90% dilatation. These results suggest that EGCG-releasing polymers can be effectively applied for fabricating an EGCG-eluting vascular stent to prevent in-stent restenosis and thrombosis.     

Role of PKA signaling in D2 receptor-expressing neurons in the core of the nucleus accumbens in aversive learning

The nucleus accumbens (NAc) serves as a key neural substrate for aversive learning and consists of two distinct subpopulations of medium-sized spiny neurons (MSNs). The MSNs of the direct pathway (dMSNs) and the indirect pathway (iMSNs) predominantly express dopamine (DA) D1 and D2 receptors, respectively, and are positively and negatively modulated by DA transmitters via Gs- and Gi-coupled cAMP-dependent protein kinase A (PKA) signaling cascades, respectively. In this investigation, we addressed how intracellular PKA signaling is involved in aversive learning in a cell type-specific manner. When the transmission of either dMSNs or iMSNs was unilaterally blocked by pathway-specific expression of transmission-blocking tetanus toxin, infusion of PKA inhibitors into the intact side of the NAc core abolished passive avoidance learning toward an electric shock in the indirect pathway-blocked mice, but not in the direct pathway-blocked mice. We then examined temporal changes in PKA activity in dMSNs and iMSNs in behaving mice by monitoring Förster resonance energy transfer responses of the PKA biosensor with the aid of microendoscopy. PKA activity was increased in iMSNs and decreased in dMSNs in both aversive memory formation and retrieval. Importantly, the increased PKA activity in iMSNs disappeared when aversive memory was prevented by keeping mice in the conditioning apparatus. Furthermore, the increase in PKA activity in iMSNs by aversive stimuli reflected facilitation of aversive memory retention. These results indicate that PKA signaling in iMSNs plays a critical role in both aversive memory formation and retention.Aversive stimuli induce not only rapid avoidance behavior, but also memory formation to escape from uncomfortable environments, and thus strongly influence animal behavior (1–3). The mesolimbic dopaminergic (DA) system plays a critical role in both rapid aversive reaction and memory formation (3–5). The nucleus accumbens (NAc) receives DA inputs from the ventral tegmental area (VTA) and serves as a key neural substrate for the control of aversive learning (6–8). The NAc consists of two subpopulations of medium-sized spiny neurons (MSNs) (9–11). The MSNs of the direct pathway (dMSNs) send their axons to the substantia nigra pars reticulata (SNr) and VTA, and selectively express dopamine D1 receptors, whereas the MSNs of the indirect pathway (iMSNs) indirectly project to the SNr and VTA via the ventral pallidum (VP) and predominantly express D2 receptors (12, 13). D1 receptors stimulate the cAMP-dependent protein kinase A (PKA) signaling cascade via Gs and exhibit a low affinity for DA (14–16). Conversely, D2 receptors inhibit the cAMP-PKA cascade via Gi and show a high affinity for DA (14–16). Thus, these two distinct types of MSNs, constituting two parallel pathways, contribute to the dynamic modulation of neuronal cell excitability and synaptic plasticity in the NAc circuitry (14–16).Although accumulated evidence indicates that DA modulation of the NAc is critical for both reward-based and aversive reactions (3, 5, 6, 17), the response of DA neurons in the VTA to aversive stimuli is not uniform; that is, some DA neurons are stimulated in response to aversive stimuli, whereas most others react by transiently suppressing their firing (18–22). Recent optogenetic studies have revealed that not only activation of iMSNs, but also inactivation of the VTA neurons, which down-regulates DA levels in the NAc, evoke an aversive reaction and learning (23–26); however, how intracellular cAMP-PKA signaling is involved in the induction and retention of aversive memory in a cell type-dependent manner in the NAc circuit remains largely elusive.In the present investigation, we addressed this issue using two approaches. We first used asymmetric reversible neurotransmission blocking (aRNB) techniques (27, 28), in which either the direct or indirect pathway at one side of the NAc was selectively blocked by the pathway-specific expression of transmission-blocking tetanus toxin and the other intact side was manipulated by injection of PKA inhibitors. In the second approach, we examined temporal changes in PKA activities of these two pathways in the formation of aversive memory by monitoring Förster resonance energy transfer (FRET) responses of PKA selective for either dMSNs or iMSNs with the aid of in vivo microendoscopic analysis (29, 30). These two different approaches explicitly demonstrated that the activation of PKA in iMSNs plays a key role in both the formation and the retention of aversive memory.