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BACKGROUND & AIMS: Acute diarrheal diseases are the second most common cause of infant mortality in developing countries. This is contributed to by lack of effective drug therapy that shortens the duration or lessens the volume of diarrhea. The epithelial brush border sodium (Na+)/hydrogen (H+) exchanger 3 (NHE3) accounts for a major component of intestinal Na+ absorption and is inhibited in most diarrheas. Because increased intestinal Na+ absorption can rehydrate patients with diarrhea, NHE3 has been suggested as a potential druggable target for drug therapy for diarrhea. METHODS: A peptide (sodium-hydrogen exchanger 3 stimulatory peptide [N3SP]) was synthesized to mimic the part of the NHE3 C-terminus that forms a multiprotein complex that inhibits NHE3 activity. The effect of N3SP on NHE3 activity was evaluated in NHE3-transfected fibroblasts null for other plasma membrane NHEs, a human colon cancer cell line that models intestinal absorptive enterocytes (Caco-2/BBe), human enteroids, and mouse intestine in vitro and in vivo. N3SP was delivered into cells via a hydrophobic fluorescent maleimide or nanoparticles. RESULTS: N3SP uptake stimulated NHE3 activity at nmol/L concentrations under basal conditions and partially reversed the reduced NHE3 activity caused by elevated adenosine 3,5-cyclic monophosphate, guanosine 3,5-cyclic monophosphate, and Ca2+ in cell lines and in in vitro mouse intestine. N3SP also stimulated intestinal fluid absorption in the mouse small intestine in vivo and prevented cholera toxin-, Escherichia coli heat-stable enterotoxin-, and cluster of differentiation 3 inflammation-induced fluid secretion in a live mouse intestinal loop model. CONCLUSIONS: These findings suggest pharmacologic stimulation of NHE3 activity as an efficacious approach for the treatment of moderate/severe diarrheal diseases.

Photoreceptor cells (PRCs) across the animal kingdom are characterized by a stacking of apical membranes to accommodate the high abundance of photopigment. In arthropods and many other invertebrate phyla PRC membrane stacks adopt the shape of densely packed microvilli that form a structure called rhabdomere. PRCs and surrounding accessory cells, including pigment cells and lens-forming cells, are grouped in stereotyped units, the ommatidia. In larvae of holometabolan insects, eyes (called stemmata) are reduced in terms of number and composition of ommatidia. The stemma of Drosophila (Bolwig organ) is reduced to a bilateral cluster of subepidermal PRCs, lacking all other cell types. In the present paper we have analyzed the development and fine structure of the Drosophila larval PRCs. Shortly after their appearance in the embryonic head ectoderm, PRC precursors delaminate and lose expression of apical markers of epithelial cells, including Crumbs and several centrosome-associated proteins. In the early first instar larva, PRCs show an expanded, irregularly shaped apical surface that is folded into multiple horizontal microvillar-like processes (MLPs). Apical PRC membranes and MLPs are covered with a layer of extracellular matrix. MLPs are predominantly aligned along an axis that extends ventro-anteriorly to dorso-posteriorly, but vary in length, diameter, and spacing. Individual MLPs present a "beaded" shape, with thick segments (0.2-0.3 μm diameter) alternating with thin segments (>0.1 μm). We show that loss of the glycoprotein Chaoptin, which is absolutely essential for rhabdomere formation in the adult PRCs, does not lead to severe abnormalities in larval PRCs.

During immune surveillance, T cells survey the surface of antigen-presenting cells. In searching for peptide-loaded major histocompatibility complexes (pMHCs), they must solve a classic trade-off between speed and sensitivity. It has long been supposed that microvilli on T cells act as sensory organs to enable search, but their strategy has been unknown. We used lattice light-sheet and quantum dot-enabled synaptic contact mapping microscopy to show that anomalous diffusion and fractal organization of microvilli survey the majority of opposing surfaces within 1 minute. Individual dwell times were long enough to discriminate pMHC half-lives and T cell receptor (TCR) accumulation selectively stabilized microvilli. Stabilization was independent of tyrosine kinase signaling and the actin cytoskeleton, suggesting selection for avid TCR microclusters. This work defines the efficient cellular search process against which ligand detection takes place.

Cholesterol is a crucial lipid within the plasma membrane of mammalian cells. Recent biochemical studies showed that one pool of cholesterol in the plasma membrane is "accessible" to binding by a modified version of the cytolysin perfringolysin O (PFO*), whereas another pool is sequestered by sphingomyelin and cannot be bound by PFO* unless the sphingomyelin is destroyed with sphingomyelinase (SMase). Thus far, it has been unclear whether PFO* and related cholesterol-binding proteins bind uniformly to the plasma membrane or bind preferentially to specific domains or morphologic features on the plasma membrane. Here, we used nanoscale secondary ion mass spectrometry (NanoSIMS) imaging, in combination with 15N-labeled cholesterol-binding proteins (PFO* and ALO-D4, a modified anthrolysin O), to generate high-resolution images of cholesterol distribution in the plasma membrane of Chinese hamster ovary (CHO) cells. The NanoSIMS images revealed preferential binding of PFO* and ALO-D4 to microvilli on the plasma membrane; lower amounts of binding were detectable in regions of the plasma membrane lacking microvilli. The binding of ALO-D4 to the plasma membrane was virtually eliminated when cholesterol stores were depleted with methyl-β-cyclodextrin. When cells were treated with SMase, the binding of ALO-D4 to cells increased, largely due to increased binding to microvilli. Remarkably, lysenin (a sphingomyelin-binding protein) also bound preferentially to microvilli. Thus, high-resolution images of lipid-binding proteins on CHO cells can be acquired with NanoSIMS imaging. These images demonstrate that accessible cholesterol, as judged by PFO* or ALO-D4 binding, is not evenly distributed over the entire plasma membrane but instead is highly enriched on microvilli.

Intestinal M (microfold) cells are specialized epithelial cells overlying lymphoid tissues in the small intestine. Unlike common enterocytes, M cells lack an organized apical brush border, and are able to transcytose microparticles across the mucosal barrier to underlying antigen-presenting cells. We found that in both the dextran sodium sulfate and Citrobacter rodentium models of colitis, significantly increased numbers of Peyer's patch (PP) phenotype M cells were induced at the peak of inflammation in colonic epithelium, often accompanied by loosely organized lamina propria infiltrates. PP type M cells are thought to be dependent on cytokines, including tumor necrosis factor (TNF)-α and receptor activator of nuclear factor kappa-B ligand; these cytokines were also found to be induced in the inflamed tissues. The induction of M cells was abrogated by anti-TNF-α blockade, suggesting that anti-TNF-α therapies may have similar effects in clinical settings, although the functional consequences are not clear. Our results suggest that inflammatory cytokine-induced PP type M cells may be a useful correlate of chronic intestinal inflammation.

Giardia lamblia is a protistan parasite that infects and colonizes the small intestine of mammals. It is widespread and particularly endemic in the developing world. Here we present a detailed structural study by 3-D negative staining and cryo-electron tomography of a unique Giardia organelle, the ventral disc. The disc is composed of a regular array of microtubules and associated sheets, called microribbons that form a large spiral, held together by a myriad of mostly unknown associated proteins. In a previous study we analyzed by cryo-electron tomography the central microtubule portion (here called disc body) of the ventral disc and found a large portion of microtubule associated inner (MIPs) and outer proteins (MAPs) that render these microtubules hyper-stable. With this follow-up study we expanded our 3-D analysis to different parts of the disc such as the ventral and dorsal areas of the overlap zone, as well as the outer disc margin. There are intrinsic location-specific characteristics in the composition of microtubule-associated proteins between these regions, as well as large differences between the overall architecture of microtubules and microribbons. The lateral packing of microtubule-microribbon complexes varies substantially, and closer packing often comes with contracted lateral tethers that seem to hold the disc together. It appears that the marginal microtubule-microribbon complexes function as outer, laterally contractible lids that may help the cell to clamp onto the intestinal microvilli. Furthermore, we analyzed length, quantity, curvature and distribution between different zones of the disc, which we found to differ from previous publications.

Bacillus thuringiensis subsp. israelensis (Bti) is widely used for the biological control of mosquito populations. However, the mechanism of Bti toxins is still not fully understood. To further elucidate the mechanism of Bti toxins, we developed an Aedes aegypti resistant strain that shows high-level resistance to Cry11Aa toxin. After 27 selections with Cry11Aa toxin, the larvae showed a 124-fold resistance ratio for Cry11Aa (strain G30). G30 larvae showed cross-resistance to Cry4Aa (66-fold resistance), less to Cry4Ba (13-fold), but not to Cry11Ba (2-fold). Midguts from these resistant larvae did not show detectable difference in the processing of the Cry11Aa toxin compared to that in susceptible larvae (WT). Brush border membrane vesicles (BBMV) from resistant larvae bound slightly less Cry11Aa compared to WT BBMV. To identify potential proteins associated with Cry11A resistance, not only transcript changes in the larval midgut were analyzed using Illumina sequencing and qPCR, but alterations of previously identified receptor proteins were investigated using immunoblots. The transcripts of 375 genes were significantly increased and those of 208 genes were down regulated in the resistant larvae midgut compared to the WT. None of the transcripts for previously identified receptors of Cry11Aa (Aedes cadherin, ALP1, APN1, and APN2) were altered in these analyses. The genes for the identified functional receptors in resistant larvae midgut did not contain any mutation in their sequences nor was there any change in their transcript expression levels compared to WT. However, ALP proteins were expressed at reduced levels (∼ 40%) in the resistant strain BBMV. APN proteins and their activity were also slightly reduced in resistance strain. The transcript levels of ALPs (AAEL013330 and AAEL015070) and APNs (AAEL008158, AAEL008162) were significantly reduced. These results strongly suggest that ALPs and APNs could be associated with Cry11Aa resistance in Ae. aegypti.

Cry proteins from Bacillus thuringiensis are insecticidal PFTs (pore-forming toxins). In the present study, we show that two distinct functional pre-pores of Cry1Ab are formed after binding of the protoxin or the protease-activated toxin to the cadherin receptor, but before membrane insertion. Both pre-pores actively induce pore formation, although with different characteristics, and contribute to the insecticidal activity. We also analysed the oligomerization of the mutant Cry1AbMod protein. This mutant kills different insect populations that are resistant to Cry toxins, but lost potency against susceptible insects. We found that the Cry1AbMod-protoxin efficiently induces oligomerization, but not the activated Cry1AbMod-toxin, explaining the loss of potency of Cry1AbMod against susceptible insects. These data are relevant for the future control of insects resistant to Cry proteins. Our data support the pore-formation model involving sequential interaction with different midgut proteins, leading to pore formation in the target membrane. We propose that not only different insect targets could have different receptors, but also different midgut proteases that would influence the rate of protoxin/toxin activation. It is possible that the two pre-pore structures could have been selected for in evolution, since they have differential roles in toxicity against selected targets, increasing their range of action. These data assign a functional role for the protoxin fragment of Cry PFTs that was not understood previously. Most PFTs produced by other bacteria are secreted as protoxins that require activation before oligomerization, to finally form a pore. Thus different pre-pores could be also part of the general mechanism of action of other PFTs.

Bacillus thuringiensis Cyt proteins are pore-forming toxins that have insecticidal activity mainly against dipteran insects. However, certain Cyt proteins have toxicity to some insect orders, but not toxicity of Cyt1Aa against lepidopteran larvae has been found. Insect specificity has been proposed to rely in specific binding to certain lipids on the brush border membrane of midgut cells since no protein receptors have been described so far. To determine the molecular basis of Cyt1Aa insect specificity we compared different steps of Cyt1Aa mode of action in a susceptible insect as the dipteran Aedes aegypti and also in the non-susceptible lepidopteran Manduca sexta. Our data shows that the lack toxicity of Cyt1Aa to M. sexta larvae does not rely on protoxin processing, membrane binding interaction, and oligomerization of Cyt1Aa since these steps were similar in the two insect species analyzed.

Filopodia are fine actin-based cellular projections used for both environmental sensing and cell motility, and they are essential organelles for metazoan cells. In this study, we reconstruct the origin of metazoan filopodia and microvilli. We first report on the evolutionary assembly of the filopodial molecular toolkit and show that homologs of many metazoan filopodial components, including fascin and myosin X, were already present in the unicellular or colonial progenitors of metazoans. Furthermore, we find that the actin crosslinking protein fascin localizes to filopodia-like structures and microvilli in the choanoflagellate Salpingoeca rosetta. In addition, homologs of filopodial genes in the holozoan Capsaspora owczarzaki are upregulated in filopodia-bearing cells relative to those that lack them. Therefore, our findings suggest that proteins essential for metazoan filopodia and microvilli are functionally conserved in unicellular and colonial holozoans and that the last common ancestor of metazoans bore a complex and specific filopodial machinery.

P(i) uptake in the small intestine occurs predominantly through the NaPi-2b (SLC34a2) co-transporter. NaPi-2b is regulated by changes in dietary P(i) but the mechanisms underlying this regulation are largely undetermined. Sequence analyses show NaPi-2b has a PDZ binding motif at its C terminus. Immunofluorescence imaging shows NaPi-2b and two PDZ domain containing proteins, NHERF1 and PDZK1, are expressed in the apical microvillar domain of rat small intestine enterocytes. Co-immunoprecipitation studies in rat enterocytes show that NHERF1 associates with NaPi-2b but not PDZK1. In HEK co-expression studies, GFP-NaPi-2b co-precipitates with FLAG-NHERF1. This interaction is markedly diminished when the C-terminal four amino acids are truncated from NaPi-2b. FLIM-FRET analyses using tagged proteins in CACO-2(BBE) cells show a distinct phasor shift between NaPi-2b and NHERF1 but not between NaPi-2b and the PDZK1 pair. This shift demonstrates that NaPi-2b and NHERF1 reside within 10 nm of each other. NHERF1(-/-) mice, but not PDZK1(-/-) mice, had a diminished adaptation of NaPi-2b expression in response to a low P(i) diet. Together these studies demonstrate that NHERF1 associates with NaPi-2b in enterocytes and regulates NaPi-2b adaptation.

The kidney is a key regulator of phosphate homeostasis. There are two predominant renal sodium phosphate cotransporters, NaPi2a and NaPi2c. Both are regulated by parathyroid hormone (PTH), which decreases the abundance of the NaPi cotransporters in the apical membrane of renal proximal tubule cells. The time course of PTH-induced removal of the two cotransporters from the apical membrane, however, is markedly different for NaPi2a compared with NaPi2c. In animals and in cell culture, PTH treatment results in almost complete removal of NaPi2a from the brush border (BB) within 1 h whereas for NaPi2c this process in not complete until 4 to 8 h after PTH treatment. The reason for this is poorly understood. We have previously shown that the unconventional myosin motor myosin VI is required for PTH-induced removal of NaPi2a from the proximal tubule BB. Here we demonstrate that myosin VI is also necessary for PTH-induced removal of NaPi2c from the apical membrane. In addition, we show that, while at baseline the two cotransporters have similar diffusion coefficients within the membrane, after PTH addition the diffusion coefficient for NaPi2a initially exceeds that for NaPi2c. Thus NaPi2c appears to remain "tethered" in the apical membrane for longer periods of time after PTH treatment, accounting, at least in part, for the difference in response times to PTH of NaPi2a versus NaPi2c.

We developed an optical imaging method based on a feedback principle in which the specific scan pattern is adapted according to the shape of the sample. The feedback approach produces nanometer-resolved 3D images of very small and moving features in live cells in seconds. We show images of microvilli in live cultured opossum kidney cells expressing NaPi co-transporter proteins with different GFP constructs and images of cell protrusions in a collagen matrix with a resolution of about 20 nm. We found that in the microvilli the NaPi proteins can be found clustered. Along cell protrusions in 3D we identified cellular adhesions to the extracellular matrix. Our approach to super-resolution and to 3D nanoimaging is different than other proposed methods that break the diffraction limit using non-linear effects or are based on single molecule localization.

Gliomas are invasive cancers that resist all forms of attempted therapy. Immunotherapy using Ag-pulsed dendritic cells has improved survival in some patients. We present evidence that another level of complexity may also contribute to lack of responses by the lymphocytes toward gliomas. Atomic force microscopy of four different glioma types-human U251 and rat T9 and F98 glioma cells, including freshly isolated human glioblastoma multiforme neurosphere cultures (containing "stem cell-like cells")-revealed a complex surface topography with numerous microvilli and filopodia. These structures were not found on other cell types. Electron microscopy and immunofluorescence microscopy of glioma cells confirmed that microvilli are present. U251 cells with microvilli resisted the cytolytic actions of different human effector cells, (lymphokine-activated killer cells, γδ T cells, conventional CTLs, and chimeric Ag-receptor-redirected T cells) better than their nonmicrovilli-expressing counterparts. Killer lymphocytes released perforin, which was detected within the glioma's microvilli/filopodia, indicating these structures can receive the cytolytic effector molecules, but cytotoxicity is suboptimal. Air-dried gliomas revealed nodes within the microvilli/filopodia. The microvilli that penetrated 0.4-μm transwell chamber's pores resisted the actions of CTLs and physical damage. Those nodelike structures may represent a compartmentalization that resists physical damage. These microvilli may play multiple roles in glioma biology, such as invasion and resistance to lymphocyte-mediated killing.

In dietary potassium deficiency there is a decrease in the transport activity of the type IIa sodium/phosphate cotransporter protein (NaPi) despite an increase in its apical membrane abundance. This novel posttranslational regulation of NaPi activity is mediated by the increased glycosphingolipid content of the potassium-deficient apical membrane. However, the mechanisms by which these lipids modulate NaPi activity have not been determined. We determined if in potassium deficiency NaPi is increasingly partitioned in cholesterol-, sphingomyelin-, and glycosphingolipid-enriched microdomains of the apical membrane and if the increased presence of NaPi in these microdomains modulates its activity. By using a detergent-free density gradient flotation technique, we found that 80% of the apical membrane NaPi partitions into the low density cholesterol-, sphingomyelin-, and GM1-enriched fractions characterized as "lipid raft" fractions. In potassium deficiency, a higher proportion of NaPi was localized in the lipid raft fractions. By combining fluorescence correlation spectroscopy and photon counting histogram methods for control and potassium-deficient apical membranes reconstituted into giant unilamellar vesicles, we showed a 2-fold decrease in lateral diffusion of NaPi protein and a greater than 2-fold increase in size of protein aggregates/clusters in potassium deficiency. Our results indicate that NaPi protein is localized in membrane microdomains, that in potassium deficiency a larger proportion of NaPi protein is present in these microdomains, and that NaPi lateral diffusion is slowed down and NaPi aggregation/clustering is increased in potassium deficiency, both of which could be associated with the decreased Na/Pi cotransport activity in potassium deficiency.

One key tenet of the raft hypothesis is that the formation of glycosphingolipid- and cholesterol-rich lipid domains can be driven solely by characteristic lipid-lipid interactions, suggesting that rafts ought to form in model membranes composed of appropriate lipids. In fact, domains with raft-like properties were found to coexist with fluid lipid regions in both planar supported lipid layers and in giant unilamellar vesicles (GUVs) formed from 1) equimolar mixtures of phospholipid-cholesterol-sphingomyelin or 2) natural lipids extracted from brush border membranes that are rich in sphingomyelin and cholesterol. Employing headgroup-labeled fluorescent phospholipid analogs in planar supported lipid layers, domains typically several microns in diameter were observed by fluorescence microscopy at room temperature (24 degrees C) whereas non-raft mixtures (PC-cholesterol) appeared homogeneous. Both raft and non-raft domains were fluid-like, although diffusion was slower in raft domains, and the probe could exchange between the two phases. Consistent with the raft hypothesis, GM1, a glycosphingolipid (GSL), was highly enriched in the more ordered domains and resistant to detergent extraction, which disrupted the GSL-depleted phase. To exclude the possibility that the domain structure was an artifact caused by the lipid layer support, GUVs were formed from the synthetic and natural lipid mixtures, in which the probe, LAURDAN, was incorporated. The emission spectrum of LAURDAN was examined by two-photon fluorescence microscopy, which allowed identification of regions with high or low order of lipid acyl chain alignment. In GUVs formed from the raft lipid mixture or from brush border membrane lipids an array of more ordered and less ordered domains that were in register in both monolayers could reversibly be formed and disrupted upon cooling and heating. Overall, the notion that in biomembranes selected lipids could laterally aggregate to form more ordered, detergent-resistant lipid rafts into which glycosphingolipids partition is strongly supported by this study.

Microscopy images of the generalized polarization (GP) of the lipid probe 2-dimethylamino-6-lauroylnapthalene (LAURDAN) in phospholipid vesicles and in natural membranes were obtained by using two-photon excitation. The images show different distribution of the GP value depending on membrane composition. The use of linearly polarized excitation allowed the attribution of the GP heterogeneity to coexisting membrane domains of different dynamic properties. Based on the photoselection operated by the excitation polarization, a model is proposed to explain the results and to estimate the domains' dimension.

Feline infectious peritonitis viruses (FIPVs) are classified into type I and type II serogroups. Here, we report that feline aminopeptidase N (APN), a cell-surface metalloprotease on the intestinal, lung and kidney epithelial cells, is a receptor for type II FIPV but not for type I FIPV. A monoclonal antibody (MAb) R-G-4, which blocks infection of Felis catus whole fetus (fcwf-4) cells by type II FIPV, was obtained by immunizing mice with fcwf-4 cells which are highly susceptible to FIPV. This MAb also blocked infection of fcwf-4 cells by type II feline enteric coronavirus (FECV), canine coronavirus (CCV), and transmissible gastroenteritis virus (TGEV). On the other hand, it did not block infection by type I FIPVs. MAb R-G-4 recognized a polypeptide of relative molecular mass 120-130 kDa in feline intestinal brush-border membrane (BBM) proteins. The polypeptide possessed aminopeptidase activity, and the first 15 N-terminal amino acid sequence was identical to that of the feline APN. Feline intestinal BBM proteins and the polypeptide reacted with MAb R-G-4 (feline APN) inhibited the infectivity of type II FIPV, type II FECV, CCV and TGEV to fcwf-4 cells, but did not inhibit the infectivity of type I FIPVs.

A model system is described for defining the physiologic functions of mammalian cadherins in vivo. 129/Sv embryonic stem (ES) cells, stably transfected with a dominant negative N-cadherin mutant (NCAD delta) under the control of a promoter that only functions in postmitotic enterocytes during their rapid, orderly, and continuous migration up small intestinal villi, were introduced into normal C57B1/6 (B6) blastocysts. In adult B6129/Sv chimeric mice, each villus receives the cellular output of several surrounding monoclonal crypts. A polyclonal villus located at the boundary of 129/Sv- and B6-derived intestinal epithelium contains vertical coherent bands of NCAD delta-producing enterocytes plus adjacent bands of normal B6-derived enterocytes. A comparison of the biological properties of these cell populations established that NCAD delta disrupts cell-cell and cell-matrix contacts, increases the rate of migration of enterocytes along the crypt-villus axis, results in a loss of their differentiated polarized phenotype, and produces precocious entry into a death program. These data indicate that enterocytic cadherins are critical cell survival factors that actively maintain intestinal epithelial function in vivo.

A polymerase chain reaction strategy was devised to identify new members of the mammalian myosin I family of actin-based motors. Using cellular RNA from mouse granular neurons and PC12 cells, we have cloned and sequenced three 1.2-kb polymerase chain reaction products that correspond to novel mammalian myosin I genes designated MMI alpha, MMI beta, MMI gamma. The pattern of expression for each of the myosin I's is unique: messages are detected in diverse tissues including the brain, lung, kidney, liver, intestine, and adrenal gland. Overlapping clones representing full-length cDNAs for MMI alpha were obtained from mouse brain. These encode a 1,079 amino acid protein containing a myosin head, a domain with five calmodulin binding sites, and a positively charged COOH-terminal tail. In situ hybridization reveals that MMI alpha is highly expressed in virtually all neurons (but not glia) in the postnatal and adult mouse brain and in neuroblasts of the cerebellar external granular layer. Expression varies in different brain regions and undergoes developmental regulation. Myosin I's are present in diverse organisms from protozoa to vertebrates. This and the expression of three novel members of this family in brain and other mammalian tissues suggests that they may participate in critical and fundamental cellular processes.

Steady-state fluorescence properties of 6-dodecanoyl-2-dimethylaminonapththalene (Laurdan) have been used to determine the coexistence of separate lipid-phase domains in apical brush border membrane vesicles isolated from the rat renal cortex. The temperature dependence of generalized polarization has been utilized to quantitate the lipid phases. Finally, the effect of cholesterol enrichment on these parameters has been studied. The results indicate the coexistence of lipid-phase domains in brush border membranes based on the values of the generalized polarization and assuming that the membrane state can be described by coexistence of gel and liquid crystalline state. At 37 degrees C, approximately 19-32% of the brush border membrane lipids are in the gel phase, and cholesterol enrichment causes a further concentration-dependent increase in the gel phase of brush border membrane lipids.

Glucose is reabsorbed from the glomerular filtrate in the proximal segment of the renal tubule in two stages. The first stage is uphill transport across the brush border membrane by Na(+)-glucose cotransport and the second stage is downhill transport across the basolateral membrane by facilitated diffusion. Genes for both a renal Na(+)-glucose cotransporter (SGLT1) and a renal facilitated glucose transporter (GLUT2) have been cloned and sequenced. To examine whether SGLT1 and GLUT2 colocalize to the same tubular epithelial cells in rat kidney, double-immunoperoxidase studies with dual chromogens and paraformaldehyde perfusion-fixed frozen sections of rat kidney were performed. Antipeptide antisera were prepared against rat GLUT2 (amino acids 510-522) and rabbit SGLT1 (amino acids 402-420). Proximal tubules were identified immunocytochemically with an antiserum raised against a synthetic peptide corresponding to the 21 amino acids at the COOH-terminal of the heavy chain of rat gamma-glutamyl transpeptidase, which is a proximal tubule-specific enzyme. The anti-GLUT2 antiserum strongly stained the basolateral membrane of 46% of cortical tubules, whereas the SGLT1 antiserum stained the brush border of 56% of the cortical tubules. The gamma-glutamyl transpeptidase antiserum also stained the brush border of 51% of the cortical tubules. GLUT2 and SGLT1 colocalized to 40% of cortical epithelium, but 16% of cortical epithelial cells were immunopositive for brush border SGLT1 and immunonegative for basolateral GLUT2. These gamma-glutamyl transpeptidase staining results suggest that at least 50% of the tubules in the cortex are proximal tubules and that SGLT1 and GLUT2 colocalize to most proximal tubules. The fact that SGLT1 antiserum immunoreacted with tubules unreactive to the GLUT2 antiserum suggests that either the SGLT1 epitope is conserved on a related brush border protein or that there is another GLUT transporter responsible for the exit of sugar from these proximal tubule cells.

Glucose is reabsorbed from the glomerular filtrate in the proximal segment of the renal tubule in two stages. The first stage is uphill transport across the brush border membrane by Na(+)-glucose cotransport and the second stage is downhill transport across the basolateral membrane by facilitated diffusion. Genes for both a renal Na(+)-glucose cotransporter (SGLT1) and a renal facilitated glucose transporter (GLUT2) have been cloned and sequenced. To examine whether SGLT1 and GLUT2 colocalize to the same tubular epithelial cells in rat kidney, double-immunoperoxidase studies with dual chromogens and paraformaldehyde perfusion-fixed frozen sections of rat kidney were performed. Antipeptide antisera were prepared against rat GLUT2 (amino acids 510-522) and rabbit SGLT1 (amino acids 402-420). Proximal tubules were identified immunocytochemically with an antiserum raised against a synthetic peptide corresponding to the 21 amino acids at the COOH-terminal of the heavy chain of rat gamma-glutamyl transpeptidase, which is a proximal tubule-specific enzyme. The anti-GLUT2 antiserum strongly stained the basolateral membrane of 46% of cortical tubules, whereas the SGLT1 antiserum stained the brush border of 56% of the cortical tubules. The gamma-glutamyl transpeptidase antiserum also stained the brush border of 51% of the cortical tubules. GLUT2 and SGLT1 colocalized to 40% of cortical epithelium, but 16% of cortical epithelial cells were immunopositive for brush border SGLT1 and immunonegative for basolateral GLUT2. These gamma-glutamyl transpeptidase staining results suggest that at least 50% of the tubules in the cortex are proximal tubules and that SGLT1 and GLUT2 colocalize to most proximal tubules. The fact that SGLT1 antiserum immunoreacted with tubules unreactive to the GLUT2 antiserum suggests that either the SGLT1 epitope is conserved on a related brush border protein or that there is another GLUT transporter responsible for the exit of sugar from these proximal tubule cells.

Blastocrithidia papi is a unique trypanosomatid in that its life cycle is synchronized with that of its host, and includes an obligate stage of development in Malpighian tubules (MTs). This occurs in firebugs, which exited the winter diapause. In the short period, preceding the mating of overwintered insects, the flagellates penetrate MTs of the host, multiply attached to the epithelial surface with their flagella, and start forming cyst-like amastigotes (CLAs) in large agglomerates. By the moment of oviposition, a large number of CLAs are already available in the rectum. They are discharged on the eggs' surface with feces, used for transmission of bugs' symbiotic bacteria, which are compulsorily engulfed by the newly hatched nymphs along with the CLAs. The obligate development of B. papi in MTs is definitely linked to the life cycle synchronization. The absence of peristalsis allow the trypanosomatids to accumulate and form dense CLA-forming subpopulations, whereas the lack of peritrophic structures facilitates the extensive discharge of CLAs directly into the hindgut lumen. The massive release of CLAs associated with oviposition is indispensable for maximization of the infection efficiency at the most favorable time point. [ABSTRACT FROM AUTHOR]

The morphology and ultrastructure of the midgut of Gampsocleis gratiosa (Orthoptera: Tettigoniidae) was examined by light and electron microscopy. The midgut consists of two bulbous gastric caeca and a tubular ventriculus. The general organization of the gastric caeca is similar to that of the ventriculus. They are composed of a peritrophic membrane, an epithelium, a basal lamina and muscle layer from the inside to outside. Three types of cells were identified: regenerative, principal, and endocrine. Regenerative cells occur in groups (called nidi) at the base of principal cells. Principal cells grow from regenerative cells. Rare endocrine cells are scattered throughout the epithelium. Principal cells exhibit intense secretory activity, and regional differences in their ultrastructure were observed along the entire midgut. The microvilli are longer than those in any other region in the posterior region of the midgut. Lysosomes, multivesicular bodies (MVBs), autophagosomes, abundant Golgi apparatuses and lipid droplets primarily occur in the gastric caeca. Three pathways of secretion (merocrine, apocrine and holocrine) occur within the midgut epithelium, and a distinctive type of apocrine bleb was found in the gastric caeca. Therefore, these gastric caeca may be evolving toward a special type of gland. [ABSTRACT FROM AUTHOR]

Noise is a prevalent and sometimes even dominant aspect of many biological processes. While many natural systems have adapted to attenuate or even usefully integrate noise, the variability it introduces often still delimits the achievable precision across biological functions. This is particularly so for visual phototransduction, the process responsible for converting photons of light into usable electrical signals (quantum bumps). Here, randomness of both the photon inputs (regarded as extrinsic noise) and the conversion process (intrinsic noise) are seen as two distinct, independent and significant limitations on visual reliability. Past research has attempted to quantify the relative effects of these noise sources by using approximate methods that do not fully account for the discrete, point process and time ordered nature of the problem. As a result the conclusions drawn from these different approaches have led to inconsistent expositions of phototransduction noise performance. This paper provides a fresh and complete analysis of the relative impact of intrinsic and extrinsic noise in invertebrate phototransduction using minimum mean squared error reconstruction techniques based on Bayesian point process (Snyder) filters. An integrate-fire based algorithm is developed to reliably estimate photon times from quantum bumps and Snyder filters are then used to causally estimate random light intensities both at the front and back end of the phototransduction cascade. Comparison of these estimates reveals that the dominant noise source transitions from extrinsic to intrinsic as light intensity increases. By extending the filtering techniques to account for delays, it is further found that among the intrinsic noise components, which include bump latency (mean delay and jitter) and shape (amplitude and width) variance, it is the mean delay that is critical to noise performance. As the timeliness of visual information is important for real-time action, this delay could potentially limit the speed at which invertebrates can respond to stimuli. Consequently, if one wants to increase visual fidelity, reducing the photoconversion lag is much more important than improving the regularity of the electrical signal. [ABSTRACT FROM AUTHOR]

Purpose: This study aimed to determine if rebamipide eyedrops can improve ocular surface damage caused by the use of glaucoma eyedrops. Methods: Female Kbl:Dutch rabbits were used to evaluate glaucoma eyedrop-induced ocular surface damage; one eye of each rabbit was untreated and the other was administered glaucoma eyedrops for 30 days. To evaluate the effects of rebamipide on ocular surface damage, one eye of each rabbit was administered vehicle-treated glaucoma eyedrops and the other was administered rebamipide-treated glaucoma eyedrops for 30 days. Corneal and conjunctival epithelial damage was evaluated using fluorescein and rose bengal staining, respectively. Conjunctival inflammation was observed by light microscopy with hematoxylin-eosin staining. Dark cells (in which the corneal microvilli were damaged) were analyzed by scanning electron microscopy. Results: There were no significant differences in fluorescein staining between the untreated and glaucoma eyedrop-treated groups; however, rose bengal staining and the number of inflammatory cells in the conjunctiva significantly increased after glaucoma eyedrop treatment. There was a four-fold increase in the number of dark cells in the glaucoma eyedrop-treated group compared to untreated. In contrast, in the conjunctiva of the rebamipide-treated glaucoma eyedrop group, rose bengal staining scores, the number of inflammatory cells, and the number of dark cells were decreased compared to the vehicle-treated glaucoma eyedrop group. Conclusions: Results from our in vivo rabbit study demonstrated that short-term use of glaucoma eyedrops induces corneal epithelium disorders at the cellular level, but that simultaneous use of rebamipide has the potential to protect and repair the ocular surface. [ABSTRACT FROM AUTHOR]