Immunofluorescence Confocal Laser Scanning Microscopy Research Articles

Up‐regulation of nestin in the infarcted myocardium potentially indicates differentiation of resident cardiac stem cells into various lineages including cardiomyocytes

To identify proteins involved in cardiac regeneration, a proteomics approach was applied. A total of 26 proteins, which displayed aberrant expression in mouse hearts infarcted through ligation of the left anterior descending coronary artery, were identified. These included the intermediate filament protein nestin, which was up-regulated in the infarct border zone. Corresponding changes were observed for its mRNA. Nestin mRNA was also up-regulated in hearts from 17 of 19 patients with end-stage heart failure, including 4 with acute myocardial infarction in comparison with 8 donor hearts. Immunofluorescence confocal laser scanning microscopy revealed that nestin is expressed, on the one hand, in small proportions of cardiomyocytes, endothelial cells, smooth muscle cells, neuronal cells, and fibroblasts. On the other hand, it was found to be coexpressed with the stem cell markers c-kit, Sca-1, Mdr-1, and Abcg2 in small interstitial cells. In infarcted hearts from chimeric mice transplanted with bone marrow from enhanced green fluorescent protein (EGFP) transgenic mice, less than 1% of nestin-positive cells coexpressed EGFP, although EGFP-positive cells were abundant in these. Consequently, enhanced expression of nestin in the injured myocardium might reflect spontaneous regenerative processes supposedly based on the differentiation of resident cardiac stem cells into diverse cardiac cell types.

A neurotropic route for Maize mosaic virus (Rhabdoviridae) in its planthopper vector Peregrinus maidis

To investigate the dissemination route of Maize mosaic virus (MMV, Rhabdoviridae) in its planthopper vector Peregrinus maidis (Delphacidae, Hemiptera), temporal and spatial distribution of MMV was studied by immunofluorescence confocal laser scanning microscopy following 1-week acquisition feeding of planthoppers on infected plants. MMV was detected 1-week post first access to diseased plants (padp) in the midgut and anterior diverticulum, 2-week padp in the esophagus, nerves, nerve ganglia and visceral muscles, and 3-week padp in hemocytes, tracheae, salivary glands and other tissues. MMV is neurotropic in P. maidis; infection was more extensive in the nervous system compared to other tissues. A significantly higher proportion of planthoppers had infected midguts (28.1%) compared to those with infected salivary glands (20.4%) or to those that transmitted MMV (15.7%), suggesting the occurrence of midgut and salivary gland barriers to MMV transmission in P. maidis. In this planthopper, the esophagus and anterior diverticulum are located between the compound ganglionic mass and the salivary glands. We postulate that MMV may overcome transmission barriers in P. maidis by proceeding from the midgut to the anterior diverticulum and esophagus, and from these to the salivary glands via the nervous system: a neurotropic route similar to that of some vertebrate-infecting rhabdoviruses.

Cytoskeleton and motor proteins are required for the transcytosis of Neisseria gonorrhoeae through polarized epithelial cells

Neisseria gonorrhoeae interact with polarized T84 epithelial cells by engaging carcinoembryonic antigen-related cellular adhesion molecule (CEACAM) receptors. Adherent bacteria that are taken up by the cells are able to traverse the epithelial layer from the apical to the basal side. Herein, we demonstrate that the actin cytoskeleton of the cells is not required for the initial adherence of the bacteria, however, it is essential for invasion into and traversal through T84 cells. Furthermore, microtubule inhibitors blocked the traversal, but not the adherence and invasion of the bacteria. Inhibition of the motor activity of myosins reduced invasion and traversal, but not bacterial adherence. Immunofluorescence confocal laser scanning microscopy revealed the colocalization of the microtubule-based kinesin and dynein motors, and the actin-based motor myosin with adherent and intracellular gonococci. Transcytosis was reduced by blocking kinesin and myosin with specific antibodies. This underlines the importance of these motor proteins for the transcytosis of epithelial monolayers by N. gonorrhoeae.

3-Hydroxy fatty acids found in capsules of Cryptococcus neoformans

Using immunofluorescence confocal laser scanning microscopy, immunogold transmission electron microscopy and gas chromatography--mass spectrometry, we demonstrated the presence of 3-hydroxy fatty acids in Cryptococcus neoformans. Our results suggest that these oxylipins accumulate in capsules where they are released as hydrophobic droplets through tubular protuberances into the surrounding medium.

Identification of Endosomal Epidermal Growth Factor Receptor Signaling Targets by Functional Organelle Proteomics

Epidermal growth factor (EGF) receptor (EGFR) signal transduction is organized by scaffold and adaptor proteins, which have specific subcellular distribution. On a way from the plasma membrane to the lysosome EGFRs are still in their active state and can signal from distinct subcellular locations. To identify organelle-specific targets of EGF receptor signaling on endosomes a combination of subcellular fractionation, two-dimensional DIGE, fluorescence labeling of phosphoproteins, and MALDI-TOF/TOF mass spectrometry was applied. All together 23 EGF-regulated (phospho)proteins were identified as being differentially associated with endosomal fractions by functional organelle proteomics; among them were proteins known to be involved in endosomal trafficking and cytoskeleton rearrangement (Alix, myosin-9, myosin regulatory light chain, Trap1, moesin, cytokeratin 8, septins 2 and 11, and CapZbeta). Interestingly R-Ras, a small GTPase of the Ras family that regulates cell survival and integrin activity, was associated with endosomes in a ligand-dependent manner. EGF-dependent association of R-Ras with late endosomes was confirmed by confocal laser scanning immunofluorescence microscopy and Western blotting of endosomal fractions. EGFR tyrosine kinase inhibitor gefitinib was used to confirm EGF-dependent regulation of all identified proteins. EGF-dependent association of signaling molecules, such as R-Ras, with late endosomes suggests signaling specification through intracellular organelles.

Hypoxic preconditioning protects human brain endothelium from ischemic apoptosis by Akt-dependent survivin activation

Preconditioning-induced ischemic tolerance is well documented in the brain, but cell-specific responses and mechanisms require further elucidation. The aim of this study was to develop an in vitro model of ischemic tolerance in human brain microvascular endothelial cells (HBMECs) and to examine the roles of phosphatidylinositol 3-kinase (PI3-kinase)/Akt and the inhibitor-of- apoptosis protein, survivin, in the ability of hypoxic preconditioning (HP) to protect endothelium from apoptotic cell death. Cultured HBMECs were subjected to HP, followed 16 h later by complete oxygen and glucose deprivation (OGD) for 8 h; cell viability was quantified at 20 h of reoxygenation (RO) by the 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazolium bromide assay. HBMECs were examined at various times after HP or OGD/RO using immunoblotting and confocal laser scanning immunofluorescence microscopy for appearance of apoptotic markers and expression of phosphorylated (p)-Akt and p-survivin. Causal evidence for the participation of the PI3-kinase/Akt pathway in HP-induced protection and p-survivin upregulation was assessed by the PI3-kinase inhibitor LY-294002. HP significantly reduced OGD/RO-induced injury by 50% and also significantly reduced the OGD-induced translocation of apoptosis-inducing factor (AIF) from mitochondria to nucleus and the concomitant cleavage of poly(ADP-ribose) polymerase-1 (PARP-1). PI3-kinase inhibition blocked HP-induced increases in Akt phosphorylation, reversed the effects of HP on OGD-induced AIF translocation and PARP-1 cleavage, blocked HP-induced survivin phosphorylation, and ultimately attenuated HP-induced protection of HBMECs from OGD. Thus HP promotes an antiapoptotic phenotype in HBMECs, in part by activating survivin via the PI3-kinase/Akt pathway. Survivin and other phosphorylation products of p-Akt may be therapeutic targets to protect cerebrovascular endothelium from apoptotic injury following cerebral ischemia.

Cellular and subcellular distributions of β1- and β2-Adrenoceptors in the CA1 and CA3 regions of the rat hippocampus

β-Adrenoceptors (ARs) in the hippocampus play an important role in regulating synaptic plasticity and memory consolidation. However, little is known about the distributions of β-ARs in the hippocampus, especially in the cornu ammonis (CA)1 and CA3 regions of Sprague-Dawley rats. Here, we report that β1- and β2-ARs in the CA1 and CA3 regions have differential subcellular distributions. Using double immunofluorescence labeling and confocal laser scanning microscopy, we found that almost all of the neuronal nuclei positive cells express β1- and β2-ARs, while few glial fibrillary acidic protein positive cells express them. Interestingly, β1-ARs are predominantly distributed in the cell membrane and cytoplasm, whereas β2-ARs are predominantly distributed not only in the membrane and cytoplasm, but also in the nucleus. The differential subcellular distribution of β1- and β2-ARs may have functional significance.

The Brefeldin A-inhibited Guanine Nucleotide-exchange Protein, BIG2, Regulates the Constitutive Release of TNFR1 Exosome-like Vesicles

The type I, 55-kDa tumor necrosis factor receptor (TNFR1) is released from cells to the extracellular space where it can bind and modulate TNF bioactivity. Extracellular TNFR1 release occurs by two distinct pathways: the inducible proteolytic cleavage of TNFR1 ectodomains and the constitutive release of full-length TNFR1 in exosome-like vesicles. Regulation of both TNFR1 release pathways appears to involve the trafficking of cytoplasmic TNFR1 vesicles. Vesicular trafficking is controlled by ADP-ribosylation factors (ARFs), which are active in the GTP-bound state and inactive when bound to GDP. ARF activation is enhanced by guanine nucleotide-exchange factors that catalyze replacement of GDP by GTP. We investigated whether the brefeldin A (BFA)-inhibited guanine nucleotide-exchange proteins, BIG1 and/or BIG2, are required for TNFR1 release from human umbilical vein endothelial cells. Effects of specific RNA interference (RNAi) showed that BIG2, but not BIG1, regulated the release of TNFR1 exosome-like vesicles, whereas neither BIG2 nor BIG1 was required for the IL-1beta-induced proteolytic cleavage of TNFR1 ectodomains. BIG2 co-localized with TNFR1 in diffusely distributed cytoplasmic vesicles, and the association between BIG2 and TNFR1 was disrupted by BFA. Consistent with the preferential activation of class I ARFs by BIG2, ARF1 and ARF3 participated in the extracellular release of TNFR1 exosome-like vesicles in a nonredundant and additive fashion. We conclude that the association between BIG2 and TNFR1 selectively regulates the extracellular release of TNFR1 exosome-like vesicles from human vascular endothelial cells via an ARF1- and ARF3-dependent mechanism.

OLIG2 is a useful immunohistochemical marker in differential diagnosis of clear cell primary CNS neoplasms

To analyse OLIG2 expression in clear cell primary central nervous system (CNS) tumours to clarify the diagnostic usefulness of OLIG2 immunohistochemistry in this subset of brain tumours. We analysed OLIG2 expression in 60 oligodendroglial neoplasms (57 with and three without chromosome 1p aberration), 10 central neurocytomas, 10 clear cell ependymomas, nine dysembryoplastic neuroepithelial tumours (DNTs) and two clear cell meningiomas using immunohistochemistry. Additionally, we analysed oligodendroglial neoplasms with numerous gliofibrillary and minigemistocytic oligodendrocytes for OLIG2/glial fibrillary acidic protein (GFAP) coexpression and central neurocytoma for coexpression of neurone-specific nuclear protein (NeuN) and OLIG2 using double immunofluorescent labelling and confocal laser scanning microscopy. All oligodendroglial neoplasms and DNTs showed widespread OLIG2 expression. Eight of 10 central neurocytomas, all clear cell meningiomas and 8/10 clear cell ependymomas were negative for OLIG2. Two of 10 central neurocytomas and 2/10 clear cell ependymomas showed focal OLIG2 expression. We found prominent coexpression of GFAP and OLIG2 in gliofibrillary and minigemistocytic oligodendrocytes. Further, we found coexpression of NeuN and OLIG2 in single cells in central neurocytoma. Widespread OLIG2 expression discriminates oligodendroglial neoplasms or DNTs from other clear cell primary brain tumour types. In clear cell primary brain tumours lacking OLIG2 expression, differential diagnosis may require additional immunohistochemical markers.

The Transcriptional Co-Repressor C-Terminal Binding Protein (CtBP) Associates with Centrosomes During Mitosis

CtBP is a co-repressor of transcription that acts by inhibiting co-activators and recruiting to promoter control elements (via interactions with DNA-binding transcription factors) a complex of proteins that modify histones, repress transcription and silence gene expression. There are two highly homologous genes, CtBP1 and CtBP2 that encode CtBP. In addition, a CtBP1-related protein has been described that has 11 fewer amino acids at its N-terminus than CtBP1. This variant of CtBP1 – known as CtBP3 or BARS [Brefeldin A (BFA) ribosylated substrate] – plays a critical role in the fragmentation of the Golgi complex at the onset of mitosis. Although there are some reports of CtBP with a cytoplasmic distribution after transfection, it is unclear how in normal cells a nuclear co-repressor regulates the behaviour of the Golgi complex at the onset of mitosis. Using polyclonal rabbit antibodies that were raised against a human CtBP1-GST fusion-protein, here we show by immunofluorescence laser-scanning confocal microscopy that in mitotic cells a species of CtBP becomes associated with the centrosomes at the onset of prophase and then throughout mitosis. The association can be seen in both cycling mitotic cells and nocodazole-arrested cells. The interaction was confirmed by co-immunoprecipitation and centrosome isolation. Since centrosomes are considered to be the organising centre for Golgi morphogenesis, the interaction demonstrated here may explain how a nuclear co-repressor of transcription can exert a regulatory effect on the Golgi complex at a specific stage of the cell cycle.

Early colonization of barley roots by Pseudomonas fluorescens studied by immunofluorescence technique and confocal laser scanning microscopy

Highly specific polyclonal antibodies against two Pseudomonas fluorescens strains (DF57 and Ag1), which differed by approximately 10% of their utilizable substrates as tested in Biolog GN plates, were used for in situ labelling of bacterial cells colonizing barley roots grown in sterile soil. By using a confocal laser scanning microscope single bacteria of both strains could be detected on the roots. Seed-inoculated bacteria rapidly colonized the root surface (rhizoplane) by active migration; after 1 day the anterior part of the root was densely covered by bacteria occupying the crevices between epidermal root cells. As the roots became longer, this bacterial population in the rhizoplane formed long strings of closely associated cells. After 7 days, however, the rhizoplane population of string-forming cells was partially detached developing a patchy distribution along the root; a separate population of cells localized in the slime matrix (mucigel) surrounding the root was well developed at this time. Using two different fluorochromes attached to the antibodies, the two strains could be detected simultaneously in co-inoculation experiments. The recordings, however, gave no indications of competition between the two strains during root colonization.

Oxylipin covered ascospores of Eremothecium coryli

Eremothecium coryli is known to produce intriguing spindle-shaped ascospores with long and thin whip-like appendages. Here, ultra structural studies using scanning electron microscopy, indicate that these appendages serve to coil around themselves and around ascospores causing spore aggregation. Furthermore, using immunofluorescence confocal laser scanning microscopy it was found that hydrophobic 3-hydroxy oxylipins cover the surfaces of these ascospores. Using gas chromatography-mass spectrometry, only the oxylipin 3-hydroxy 9:1 (a monounsaturated fatty acid containing a hydroxyl group on carbon 3) could be identified. Sequential digital imaging suggests that oxylipin-coated spindle-shaped ascospores are released from enclosed asci probably by protruding through an already disintegrating ascus wall.

Use of Immunofluorescence Confocal Laser Scanning Microscopy to Study Distribution of the Bacterium Corn Stunt Spiroplasma in Vector Leafhoppers (Hemiptera: Cicadellidae) and in Host Plants

The distribution of corn stunt spiroplasma (Spiroplasma kunkelii, class Mollicutes, family Spiroplasmataceae, CSS) in its leafhopper vector Dalbulus maidis (DeLong & Wolcott) was studied by immunofluorescence confocal laser scanning microscopy (iCLSM), by using three preparation methods: dissected whole-mount leafhopper organs, thick sections of paraffin-embedded leafhopper parts, and smears of leafhopper hemocytes. These methods combined revealed the localization of CSS in the midgut, filter chamber, Malpighian tubules, hindgut, fat tissues, hemocytes, muscles, tracheae, and in several lobes of the salivary glands. However, CSS was not detected in nerve cells of the brain or other nerve ganglia of CSS-infected D. maidis. The percentage of leafhoppers with detected CSS in various organs/tissues 2 to 3 wk post-exposure to CSS-infected plants for 1 wk was up to 95%, and 75% of these leafhoppers inoculated CSS into maize, Zea mays L., test seedlings before iCLSM processing. Using thick sections from CSS-infected maize leaves, iCLSM was also used to localize CSS in phloem tissues of maize plants. Although transmission electron microscopy (TEM) provides higher resolution for the localization of mollicutes or other pathogens in host plants and insect vectors/hosts, iCLSM has the following advantages compared with TEM: 1) processing and examination of specimens is considerably faster; 2) much larger and more numerous samples can be processed; and 3) the distribution of pathogens can be studied in whole organs or even the entire insect (through thick sectioning). Additionally, compared with epifluorescence microscopy, iCLSM provides three-dimensional images of the studied organs, indicating their spatial relationships, which can be valuable for studying the routes of pathogens in their vectors.

Estrogen receptors α and β (ERα and ERβ) and androgen receptor (AR) in human sperm: localization of ERβ and AR in mitochondria of the midpiece

The central role of estrogens and androgens in the male reproductive system has focused attention on the presence and distribution of their cognate receptors [estrogen receptor (ER) alpha, ERbeta and androgen receptor (AR)] in male reproductive tissues and cells. Since the presence of steroid hormone receptors in mitochondria of mammalian cells has been well documented, we investigated the possibility of mitochondrial localization of sex steroid hormone receptors in sperm. Applying immunofluorescence labelling and confocal laser scanning microscopy we show that the estrogen receptor beta and the AR of human sperm are specifically enriched in the midpiece, at the site of the mitochondria, which were visualized by labelling with the vital dye CMX. Nuclear and mitochondrial localization of AR was also detected in LnCap human prostate cancer cells. Differentially, most of the ERalpha immunostaining is in the form of a compact zone at a region corresponding to the equatorial segment of the upper post-acrosomal region of the sperm head. Immunoblotting experiments using sperm extracts revealed the presence of a 66 and a 45 kDa protein reacting with the ERalpha antibody, one 64 kDa protein reacting with the ERbeta antibody and a 110 and a 90 kDa protein reacting with the antibody against AR. Our findings suggest that the differential localization of AR and ER isoforms in human sperm reveals distinct roles of these receptors in the physiology of sperm cells and, perhaps, also in the process of fertilization.

Complementary Stimulation of Hepatobiliary Transport and Detoxification Systems by Rifampicin and Ursodeoxycholic Acid in Humans

Rifampicin (RIFA) and ursodeoxycholic acid (UDCA) improve symptoms and biochemical markers of liver injury in cholestatic liver diseases by largely unknown mechanisms. We aimed to study the molecular mechanisms of action of these drugs in humans. Thirty otherwise healthy gallstone patients scheduled for cholestectomy were randomized to RIFA (600 mg/day for 1 week) or UDCA (1 g/day for 3 weeks) or no medication before surgery. Routine biochemistry, lipids, and surrogate markers for P450 activity (4beta-hydroxy cholesterol, 4beta-OH-C) and bile acid synthesis (7alpha-hydroxy-4-cholesten-3-one, C-4) were measured in serum. Bile acids were analyzed in serum, urine, and bile. A wedge liver biopsy specimen was taken to study expression of hepatobiliary ABC transporters as well as detoxification enzymes and regulatory transcription factors. RIFA enhanced bile acid detoxification as well as bilirubin conjugation and excretion as reflected by enhanced expression of CYP3A4, UGT1A1, and MRP2. These molecular effects were paralleled by decreased bilirubin and deoxycholic acid concentrations in serum and decreased lithocholic and deoxycholic acid concentrations in bile. UDCA on the other hand stimulated the expression of BSEP, MDR3, and MRP4. UDCA became the predominant bile acid after UDCA treatment and lowered the biliary cholesterol saturation index. RIFA enhances bile acid detoxification as well as bilirubin conjugation and export systems, whereas UDCA stimulates the expression of transporters for canalicular and basolateral bile acid export as well as the canalicular phospholipid flippase. These independent but complementary effects may justify a combination of both agents for the treatment of cholestatic liver diseases.

Changes of occludin expression in intestinal mucosa after burn in rats

In severely burned rats, hyperemia, edema and other pathological injuries occur in the intestinal mucosa. Ultramicroscopically, the microvilli, tight junction and organelles are disrupted. Occludin is a functional component of tight junctions. The purpose of the present study is to investigate changes of occludin expression, and to further elucidate the relationship between occludin expression and ultrastructure damage. The fluorescence intensity of occludin was detected in intestinal wall by the method of immunofluorescence histochemistry and confocal laser scanning microscopy (CLSM). Expression of occludin and its mRNA were determined by western blotting and RT-PCR, respectively. Changes of intestinal mucosa ultrastructure were observed by TEM. The results showed that fluorescence intensity of occludin at 3PBH was enhanced, higher than that of the control group, being 80.77 ± 8.38 and 72.86 ± 4.74, respectively, and reached a peak at 12PBH (116.14 ± 6.89). The expression levels of occludin at 3PBH and 6PBH were 1.21 ± 0.02 and 1.53 ± 0.14 times that of the control group, respectively, and there were significant differences ( P < 0.01) between 3PBH group and 6PBH group and control group. The levels of occludin mRNA were also enhanced. At 12PBH, the level reached a peak ( P < 0.01), being 2.00 ± 0.24 times that of the control group. Coincidently, the structure of the tight junction between epithelial cells was disrupted on a large scale under TEM. We speculate that up-regulation of epithelial occludin may play a role in enhancing paracellular permeability and be related to the damage to the tight junction.

Differential subcellular distribution of estrogen receptor isoforms: Localization of ERα in the nucleoli and ERβ in the mitochondria of human osteosarcoma SaOS-2 and hepatocarcinoma HepG2 cell lines

The localization of estrogen receptors alpha (ERα) and beta (ERβ) in osteosarcoma SaOS-2 and hepatocarcinoma HepG2 cells was studied by immunofluorescence labelling and confocal laser scanning microscopy, as well as by subcellular fractionation and immunoblotting of the proteins of the fractions with respective antibodies. In both cell types, ERα was localized mainly in the nucleus, particularly concentrated on nuclear structures, which on the basis of their staining with pyronin and with antibodies against the nucleoli-specific Ki67 antigen and C23-nucleolin, were characterized as nucleoli. A faint, diffuse ERα staining was also observed in the cytoplasm. ERβ was specifically enriched at the site of the mitochondria, visualized by labelling with the vital dye CMX and antibody against the mitochondrial-specific cytochrome oxidase subunit I. Immunoblotting experiments corroborated the immunofluorescence labelling distribution of ERα and ERβ. These findings support the concept of a direct action of steroid/thyroid hormones on mitochondrial functions by way of their cognate receptors and also suggest a direct involvement of ERα in nucleolar-related processes.

Calbindin D28k-containing neurons in the paratrigeminal nucleus receive convergent nociceptive information and project to nucleus of the solitary tract in rat

The paratrigeminal nucleus (PTN) receives orofacial somatic and visceral afferent fibers and contains many calbindin-D28k neurons (CB-containing neurons) that project to nucleus of the solitary tract (NTS). In the present study, retrograde and transganglionic tracing methods combined with immunofluorescence histochemistry and confocal laser scanning microscopy were used. After Fluoro-gold (FG) injection into the unilateral NTS, 74.4% FG-labeled neurons of ipsilateral PTN were double-labeled with CB. Furthermore, 41.0% and 32.5% FG/CB double-labeled neurons co-existed with Fos induced by nociceptive stimulation of the lips and the upper alimentary tract, respectively. In the PTN unilateral to FG injection site, 26.6% CB-LI neurons were double-labeled with PAG, 61.5% and 79.0% CB/PAG double-labeled neurons were triple-labeled with FG and Fos, and 22.9% FG/CB double-labeled neurons were triple-labeled with PAG, 84.3% FG/PAG double-labeled neurons expressed Fos induced by the upper alimentary tract stimulation. In the intact animals, 62.8% CB-LI neurons and 88.3% PAG-LI neurons co-existed with GABA BR, respectively. In addition, some terminals from the inferior alveolar nerve (IAN) were closely apposed to CB/Fos double-labeled or CB single-labeled neurons. These results suggested that CB-containing neurons in the PTN receive the nociceptive information converge from the orofacial area and visceral organs, and comprising the glutamatergic excitatory transmission pathway from the PTN to the NTS. This pathway might be modulated by GABA via the GABA B receptor.

GLAST expression on bullfrog Müller cells is regulated by dark/light

Using immunofluorescence double-labeling, Western blotting and confocal laser scanning microscopy, we investigated if and how the expression of glial high-affinity glutamate/aspartate transporter (GLAST) in bullfrog Müller cells may be regulated by dark/light. Compared with light-adapted retinas, the expression of GLAST in Müller cells was overall up-regulated in retinas dark-adapted for 30 min but declined in retinas dark-adapted for longer (>30 min) periods. The declined expression level of GLAST during prolonged dark adaptation was raised by immersion with 1 mM glutamate. These results suggest that glutamate uptake mediated by GLAST could be regulated dynamically and efficiently in accord with dark/light-induced changes in glutamate release of retinal neurons.

Contribution of Microscopy to a Better Knowledge of the Biology ofGiardia lamblia

Giardia lamblia is a flagellated protozoan of great medical and biological importance. It is the causative agent of giardiasis, one of the most prevalent diarrheal disease both in developed and third-world countries. Morphological studies have shown that G. lamblia does not present structures such as peroxisomes, mitochondria, and a well-elaborated Golgi complex. In this review, special emphasis is given to the contribution made by various microscopic techniques to a better knowledge of the biology of the protozoan. The application of video microscopy, immunofluorescence confocal laser scanning microscopy, and several techniques associated with transmission electron microscopy (thin section, enzyme cytochemistry, freeze-fracture, deep-etching, fracture-flip) to the study of the cell surface, peripheral vesicles, endoplasmic reticulum-Golgi complex system, and of the encystation vesicles found in trophozoites and during the process of trophozoite-cyst transformation are discussed.