The proximal straight tubule (S3 segment) of the kidney is highly susceptible to ischemia and toxic insults but has a remarkable capacity to repair its structure and function. In response to such injuries, complex processes take place to regenerate the epithelial cells of the S3 segment; however, the precise molecular mechanisms of this regeneration are still being investigated. By applying the "toxin receptor mediated cell knockout" method under the control of the S3 segment-specific promoter/enhancer, Gsl5, which drives core 2 β-1,6-N-acetylglucosaminyltransferase gene expression, we established a transgenic mouse line expressing the human diphtheria toxin (DT) receptor only in the S3 segment. The administration of DT to these transgenic mice caused the selective ablation of S3 segment cells in a dose-dependent manner, and transgenic mice exhibited polyuria containing serum albumin and subsequently developed oliguria. An increase in the concentration of blood urea nitrogen was also observed, and the peak BUN levels occurred 3-7 days after DT administration. Histological analysis revealed that the most severe injury occurred in the S3 segments of the proximal tubule, in which tubular cells were exfoliated into the tubular lumen. In addition, aquaporin 7, which is localized exclusively to the S3 segment, was diminished. These results indicate that this transgenic mouse can suffer acute kidney injury (AKI) caused by S3 segment-specific damage after DT administration. This transgenic line offers an excellent model to uncover the mechanisms of AKI and its rapid recovery.

Cell detachment is essential in culturing adherent cells. Trypsinization is the most popular detachment technique, even though it reduces viability due to the damage to the membrane and extracellular matrix. Avoiding such damage would improve cell culture efficiency. Here we propose an enzyme-free cell detachment method that employs the acoustic pressure, sloshing in serum-free medium from intermittent traveling wave. This method detaches 96.2% of the cells, and increases its transfer yield to 130% of conventional methods for 48 h, compared to the number of cells detached by trypsinization. We show the elimination of trypsinization reduces cell damage, improving the survival of the detached cells. Acoustic pressure applied to the cells and media sloshing from the intermittent traveling wave were identified as the most important factors leading to cell detachment. This proposed method will improve biopharmaceutical production by expediting the amplification of tissue-cultured cells through a more efficient transfer process.

Amyloid deposition, a crucial event of Alzheimer's disease (AD), emerges in distinct brain regions. A key question is what triggers the assembly of the monomeric amyloid ß-protein (Aß) into fibrils in the regions. On the basis of our previous findings that gangliosides facilitate the initiation of Aß assembly at presynaptic neuritic terminals, we investigated how lipids, including gangliosides, cholesterol and sphingomyelin, extracted from synaptic plasma membranes (SPMs) isolated from autopsy brains were involved in the Aß assembly. We focused on two regions of the cerebral cortex; precuneus and calcarine cortex, one of the most vulnerable and one of the most resistant regions to amyloid deposition, respectively. Here, we show that lipids extracted from SPMs isolated from the amyloid-bearing precuneus, but neither the amyloid-free precuneus nor the calcarine cortex, markedly accelerate the Aß assembly in vitro. Through liquid chromatography-mass spectrometry of the lipids, we identified an increase in the ratio of the level of GD1b-ganglioside containing C20:0 fatty acid to that containing C18:0 as a cause of the enhanced Aß assembly in the precuneus. Our results suggest that the local glycolipid environment play a critical role in the initiation of Alzheimer amyloid deposition.

Lipids are key components in the viral life cycle that affect host-pathogen interactions. In this study, we investigated the effect of HCV infection on sphingolipid metabolism, especially on endogenous SM levels, and the relationship between HCV replication and endogenous SM molecular species. We demonstrated that HCV induces the expression of the genes (SGMS1 and 2) encoding human SM synthases 1 and 2. We observed associated increases of both total and individual sphingolipid molecular species, as assessed in human hepatocytes and in the detergent-resistant membrane (DRM) fraction in which HCV replicates. SGMS1 expression had a correlation with HCV replication. Inhibition of sphingolipid biosynthesis with a hepatotropic serine palmitoyltransferase (SPT) inhibitor, NA808, suppressed HCV-RNA production while also interfering with sphingolipid metabolism. Further, we identified the SM molecular species that comprise the DRM fraction and demonstrated that these endogenous SM species interacted with HCV nonstructural 5B polymerase to enhance viral replication. Our results reveal that HCV alters sphingolipid metabolism to promote viral replication, providing new insights into the formation of the HCV replication complex and the involvement of host lipids in the HCV life cycle.

Interferon (IFN)-gamma and interleukin (IL)-4 regulate many types of immune responses. Here we report that acidic glycosphingolipids (AGLs) of Hypsizigus marmoreus and Pleurotus eryngii induced secretion of IFN- gamma and IL-4 from T cells in a CD11c-positive cell-dependent manner similar to that of alpha-galactosylceramide (alpha-GalCer) and isoglobotriaosylceramide (iGb3), although activated T cells by AGLs showed less secretion of cytokine than those activated by alpha-GalCer. In addition, stimulation of these mushroom AGLs induced proliferation of NK1.1 alpha/beta TCR-double positive cells in splenocytes. Administration of a mixture of alpha-GalCer and AGLs affected the stimulation of alpha-GalCer and generally induced a subtle Th1 bias for splenocytes but induced an extreme Th2 bias for thymocytes. These results suggested that edible mushroom AGLs contribute to immunomodulation.

Hantavirus pulmonary syndrome (HPS) is an increasing health problem in Brazil because of encroachment of sprawling urban, agricultural, and cattle-raising areas into habitats of subfamily Sigmodontinae rodents, which serve as hantavirus reservoirs. From 1993 through June 2007, a total of 884 cases of HPS were reported in Brazil (case-fatality rate 39%). To better understand this emerging disease, we collected 89 human serum samples and 68 rodent lung samples containing antibodies to hantavirus from a 2,500-km-wide area in Brazil. RNA was isolated from human samples and rodent tissues and subjected to reverse transcription-PCR. Partial sequences of nucleocapsid protein and glycoprotein genes from 22 human and 16 rodent sources indicated only Araraquara virus and Juquitiba virus lineages. The case-fatality rate of HPS was higher in the area with Araraquara virus. This virus, which may be the most virulent hantavirus in Brazil, was associated with areas that have had greater anthropogenic changes.

Megalin is a 600-kDa single-spanning transmembrane glycoprotein and functions as an endocytic receptor, distributed not only in the kidney but also in other tissues. Structurally and functionally distinct ligands for megalin have been identified. Megalin has 30 potential N-glycosylation sites in its extracellular domain. We found that megalin interacts with its ligands in a glycoform-dependent manner.

The assembly and deposition of amyloid β protein (Aβ) is a fundamental event during the early stages of Alzheimer's disease (AD) and cerebral amyloid angiopathy. A growing body of evidence indicates that gangliosides form a pathological platform for the generation of ganglioside-bound Aβ, which facilitates the assembly of soluble Aβs; however, the molecular mechanisms underlying the binding of Aβ to gangliosides in the brain remain unclear due to the lack of an in vivo system that may address this issue. In insects, including the fruit fly Drosophila melanogaster, gangliosides are not intrinsically present at a detectable level. We herein demonstrate that ganglioside expression is inducible in Drosophila via the expression of transgenes of ganglioside synthesis enzymes and the feeding of exogenous sialic acid, and also that the induction of ganglioside synthesis significantly accelerates Aβ assembly in vivo. Our results support the hypothesis that gangliosides are responsible for Aβ assembly in vivo and also provide an opportunity to develop a valuable model for basic research as well as a therapeutic strategy for AD.

Proteinases that digest the extracellular matrix are usually used to harvest cells from culture vessels in a general culture process, which lowers the initial adhesion rate in regenerative medicine. Cell sheet engineering is one of the most important technologies in this field, especially for transplantation, because fabricated cell sheets have rich extracellular matrixes providing strong initial adhesion. Current cell sheet fabrication relies on temperature-responsive polymer-coated dishes. Cells are cultured on such specialized dishes and subjected to low temperature. Thus, we developed a simple but versatile cell sheet fabrication method using ubiquitous culture dishes/flasks without any coating or temperature modulation. Confluent mouse myoblasts (C2C12 cell line) were exposed to ultrasonic vibration from underneath and detached as cell sheets from entire culture surfaces. Because of the absence of low temperature, cell metabolism was statically increased compared with the conventional method. Furthermore, viability, morphology, protein expression, and mRNA expression were normal. These analyses indicated no side effects of ultrasonic vibration exposure. Therefore, this novel method may become the standard for cell sheet fabrication. Our method can be easily conducted following a general culture procedure with a typical dish/flask, making cell sheets more accessible to medical experts.

Innate immune signaling via TLR4 plays critical roles in pathogenesis of metabolic disorders, but the contribution of different lipid species to metabolic disorders and inflammatory diseases is less clear. GM3 ganglioside in human serum is composed of a variety of fatty acids, including long-chain (LCFA) and very-long-chain (VLCFA). Analysis of circulating levels of human serum GM3 species from patients at different stages of insulin resistance and chronic inflammation reveals that levels of VLCFA-GM3 increase significantly in metabolic disorders, while LCFA-GM3 serum levels decrease. Specific GM3 species also correlates with disease symptoms. VLCFA-GM3 levels increase in the adipose tissue of obese mice, and this is blocked in TLR4-mutant mice. In cultured monocytes, GM3 by itself has no effect on TLR4 activation; however, VLCFA-GM3 synergistically and selectively enhances TLR4 activation by LPS/HMGB1, while LCFA-GM3 and unsaturated VLCFA-GM3 suppresses TLR4 activation. GM3 interacts with the extracellular region of TLR4/MD2 complex to modulate dimerization/oligomerization. Ligand-molecular docking analysis supports that VLCFA-GM3 and LCFA-GM3 act as agonist and antagonist of TLR4 activity, respectively, by differentially binding to the hydrophobic pocket of MD2. Our findings suggest that VLCFA-GM3 is a risk factor for TLR4-mediated disease progression.

We describe the genetic analysis of samples from hantavirus pulmonary syndrome (HPS) patients from southern and southeastern states of Brazil and rodents captured at the presumed site of infection of these patients. A total of 65 samples that were antibody-positive for Sin Nombre or Laguna Negra virus by enzyme-linked immunosorbent assay were processed by nested reverse transcription-polymerase chain reaction (RT-PCR) by using several primer combinations in the M and S genome segments. PCR products were amplified and sequenced from samples from 11 HPS patient and 7 rodent samples. Phylogenetic analysis of nucleotide sequence differences showed the cocirculation of Araraquara and Juquitiba-like viruses, previously characterized from humans. Our genetic data indicate that Araraquara virus is associated with Bolomys lasiurus (hairy-tailed Bolo mouse) and the Juquitiba-like virus is associated with Oligoryzomys nigripes (black-footed pigmy rice rat).

Glycan biosynthesis occurs though a multi-step process that requires a variety of enzymes ranging from glycosyltransferases to those involved in cytosolic sugar metabolism. In many cases, glycan biosynthesis follows a glycan-specific, linear pathway. As glycosyltransferases are generally regulated at the level of transcription, assessing the overall transcriptional profile for glycan biosynthesis genes seems warranted. However, a systematic approach for assessing the correlation between glycan expression and glycan-related gene expression has not been reported previously.

We demonstrate the preferential orders of molecular chaperones glucose-regulated protein 94 (GRP94), binding immunoglobulin protein (BiP), and calreticulin (CRT) in an endoplasmic reticulum (ER) fraction from rat liver using columns conjugated with denatured myoglobin, RNase A, or β-lactoglobulin as client proteins in the presence or absence of ATP. The results showed that BiP, CRT, and GRP94 preferentially contributed myoglobin, RNase A, and β-lactoglobulin, respectively, in the presence of ATP. In the absence of ATP, GRP94 and CRT preferentially recognized misfolded myoglobin (α-helix-rich protein), whereas BiP preferentially recognized misfolded RNase A (α-helix/β-sheet mixed protein) and β-lactoglobulin (β-sheet-rich protein). The preferential order of ER chaperones may be dynamically regulated by ER conditions and the higher-order structure of client proteins.

Glycosphingolipids (GSLs) are composed of a polar glycan chain and a hydrophobic tail known as ceramide. Together with variation in the glycan chain, ceramides exhibit tissue-specific structural variation in the long-chain base (LCB) and N-acyl chain moieties in terms of carbon chain length, degree of desaturation, and hydroxylation. Here, we report the structural variation in GSLs in the urinary bladders of mice and humans. Using TLC, we showed that the major GSLs are hexosylceramide, lactosylceramide, globotriaosylceramide, globotetraosylceramide, Neu5Ac-Gal-Glc-Ceramide, and Neu5Ac-Neu5Ac-Gal-Glc-Ceramide. Our LC-MS analysis indicated that phytoceramide structures with a 20-carbon LCB (4-hydroxyeicosasphinganine) and 2-hydroxy fatty acids are abundant in hexosylceramide and Neu5Ac-Gal-Glc-Ceramide in mice and humans. In addition, quantitative PCR demonstrated that DES2 and FA2H, which are responsible for the generation of 4-hydroxysphinganine and 2-hydroxy fatty acid, respectively, and SPTLC3 and SPTSSB, which are responsible for the generation of 20-carbon LCBs, showed significant expressions in the epithelial layer than in the subepithelial layer. Immunohistochemically, dihydroceramide:sphinganine C4-hydroxylase (DES2) was expressed exclusively in urothelial cells of the urinary bladder. Our findings suggest that these ceramide structures have an impact on membrane properties of the stretching and shrinking in transitional urothelial cells.

The metabolic syndrome including obesity and diabetes mellitus is known to be a major health problem worldwide. A recent study reported that obesity causes endoplasmic reticulum (ER) stress and subsequently leads to insulin resistance and type 2 diabetes. However, little is known about the alterations in the components of the calnexin/calreticulin (CNX/CRT) cycle, which promote glycoprotein folding in obese and diabetic conditions. To understand the operating status of the lectin-like chaperones related to the CNX/CRT cycle in the metabolic syndrome, we analyzed the chaperones for the activity, protein expression, and mRNA expression levels using Zucker fatty (ZF) and Zucker diabetic fatty (ZDF) rat models for obesity and diabetes, respectively. We demonstrated that misfolded proteins were gradually increased with progression of the syndrome, obesity to diabetes. The individual chaperone activities of CNX and CRT were both decreased in the ZF rat ER and, in contrast, were increased in the ZDF rat ER. The protein quantities and mRNA expressions of CNX and CRT were decreased in the ZF rats, but increased in the ZDF rats compared with those of the healthy model. Therefore, these results indicate that obesity down-regulates CNX and CRT expressions and their activities and diabetes up-regulates the expressions and activities of CNX and CRT. Our findings clearly suggest that metabolic syndrome affects the lectin-like chaperones in the CNX/CRT cycle at both the activity and expression levels.

Homer proteins, which regulate the signaling pathway of metabotropic glutamate receptors, may contribute to the glutamatergic modulation of dopamine neurons in the basal ganglia. This study examined whether the induction of Homer 1 genes is or not associated with the methamphetamine-induced dopaminergic neurotoxicity in the discrete brain regions of rats. Basal levels of Homer 1a and 1c mRNAs in the forebrain regions were higher than those in the substantia nigra, whereas Homer 1b mRNA levels were higher in the substantia nigra than those in the forebrain regions examined. A neurotoxic dose (40 mg/kg, i.p.) of methamphetamine increased the mRNA and protein levels of Homer 1a in the striatum and nucleus accumbens, but not in the medial prefrontal cortex or the substantia nigra. Both Homer 1b and 1c mRNAs were not affected in any brain regions examined. These results suggest that the induction of Homer 1a gene may be involved at least in part in the methamphetamine-induced dopaminergic neurotoxicity, possibly through the glutamate-dopaminergic interaction.

Megalin, a receptor-like transporter glycoprotein, is expressed on kidney proximal tubular cells and reabsorbs small-molecular-weight proteins from the glomerular filtrate. Here, we report that mouse megalins differently modified with core 2 beta6GlcNAc transferase had different kinetic properties to a fluorescence-labeled ligand, retinol-binding protein (RBP). BALB/c mice, a wild-type strain in terms of the expression of kidney-specific core 2 beta6GlcNAc transferase, express megalin carrying the core 2 extended Le(x) epitope, while DBA/2 mice, a mutant-strain of the core 2 beta6GlcNAc transferase, express megalin lacking the epitope. We purified these two types of megalin using lentil lectin chromatography and measured the ligand-binding activities of the megalins using Cy5-labeled RBP by applying gel permeation chromatography (GPC) and fluorescence correlation spectroscopy (FCS). The analysis by GPC indicated that the apparent V(max) of the interaction between Cy5-labeled RBP and the megalins of BALB/c and DBA/2 mice was 60 microM and 30 microM, respectively, and the apparent K(m) was 11 microM and 17 microM, respectively. Scatchard analysis demonstrated the presence of two binding sites. Linear regression analysis resulted in a two-binding-site model characterized by a high-affinity site (K(dBALB)=12.0 microM; K(dDBA)=20.9 microM) and a low-affinity site (K(dBALB)=36.2 microM; K(dDBA)=58.8 microM). FCS analysis exhibited quite different K(m) and V(max) values from those obtained by GPC, but similar K(m) values for the two types of megalin, and a lower V(max) value for DBA/2 megalin than BALB/c megalin. These results suggest that the core 2 GlcNAc extended glycan chains on megalin can change the ligand-binding affinity and capacity.