Restoration of vision after transplantation of photoreceptors.

Cell transplantation is a potential strategy for treating blindness caused by the loss of photoreceptors. Although transplanted rod-precursor cells are able to migrate into the adult retina and differentiate to acquire the specialized morphological features of mature photoreceptor cells, the fundamental question remains whether transplantation of photoreceptor cells can actually improve vision. Here we provide evidence of functional rod-mediated vision after photoreceptor transplantation in adult Gnat1−/− mice, which lack rod function and are a model of congenital stationary night blindness. We show that transplanted rod precursors form classic triad synaptic connections with second-order bipolar and horizontal cells in the recipient retina. The newly integrated photoreceptor cells are light-responsive with dim-flash kinetics similar to adult wild-type photoreceptors. By using intrinsic imaging under scotopic conditions we demonstrate that visual signals generated by transplanted rods are projected to higher visual areas, including V1. Moreover, these cells are capable of driving optokinetic head tracking and visually guided behaviour in the Gnat1−/− mouse under scotopic conditions. Together, these results demonstrate the feasibility of photoreceptor transplantation as a therapeutic strategy for restoring vision after retinal degeneration.

Morphological studies of SnO2 thin films fabricated by using e-beam method.

This paper studies the variations in morphology of SnO2 nanostructures thin films deposited by using e-beam technique with the substrate temperature, oxygen partial pressure and the film thickness. The e-beam conditions were optimized to get crystalline nanosheets of SnO2. The films of 100-700 nm thickness were deposited on quartz substrates at temperatures ranging from room temperature (RT) to 300 degrees C and oxygen partial pressure ranging from 0 to 200 sccm. The nanostructured films have been characterized by means of X-ray diffraction (XRD), field emission scanning electron microscope (FE-SEM) and Energy dispersive spectroscopy (EDS) measurements. XRD results show that the films deposited at RT and 100 degrees C were amorphous, however, for 200 degrees C and 300 degrees C, the films showed crystalline nature with rutile structure. Also, the crystallinity increased with the increase of oxygen partial pressure. FE-SEM images revealed that at RT and 100 degrees C of substrate temperature, the film consist of spherical particles, whereas, the films deposited at 200 degrees C and 300 degrees C consist of sheet like morphology having thickness -40 nm and lateral dimension of 1 microm, respectively. The size of the nanosheets increased with the increase of substrate temperature and oxygen partial pressure due to the enhancement in the crystallinity of the films. A possible growth mechanism of the formation of SnO2 nanosheets is discussed.

The cytoplasmic tail of rhodopsin acts as a novel apical sorting signal in polarized MDCK cells.

All basolateral sorting signals described to date reside in the cytoplasmic domain of proteins, whereas apical targeting motifs have been found to be lumenal. In this report, we demonstrate that wild-type rhodopsin is targeted to the apical plasma membrane via the TGN upon expression in polarized epithelial MDCK cells. Truncated rhodopsin with a deletion of 32 COOH-terminal residues shows a nonpolar steady-state distribution. Addition of the COOH-terminal 39 residues of rhodopsin redirects the basolateral membrane protein CD7 to the apical membrane. Fusion of rhodopsin's cytoplasmic tail to a cytosolic protein glutathione S-transferase (GST) also targets this fusion protein (GST-Rho39Tr) to the apical membrane. The targeting of GST-Rho39Tr requires both the terminal 39 amino acids and the palmitoylation membrane anchor signal provided by the rhodopsin sequence. The apical transport of GST-Rho39Tr can be reversibly blocked at the Golgi complex by low temperature and can be altered by brefeldin A treatment. This indicates that the membrane-associated GST-Rho39Tr protein may be sorted along a yet unidentified pathway that is similar to the secretory pathway in polarized MDCK cells. We conclude that the COOH-terminal tail of rhodopsin contains a novel cytoplasmic apical sorting determinant. This finding further indicates that cytoplasmic sorting machinery may exist in MDCK cells for some apically targeted proteins, analogous to that described for basolaterally targeted proteins.

Cytoplasmic dynein regulation by subunit heterogeneity and its role in apical transport.

Despite the existence of multiple subunit isoforms for the microtubule motor cytoplasmic dynein, it has not yet been directly shown that dynein complexes with different compositions exhibit different properties. The 14-kD dynein light chain Tctex-1, but not its homologue RP3, binds directly to rhodopsin's cytoplasmic COOH-terminal tail, which encodes an apical targeting determinant in polarized epithelial Madin-Darby canine kidney (MDCK) cells. We demonstrate that Tctex-1 and RP3 compete for binding to dynein intermediate chain and that overexpressed RP3 displaces endogenous Tctex-1 from dynein complexes in MDCK cells. Furthermore, replacement of Tctex-1 by RP3 selectively disrupts the translocation of rhodopsin to the MDCK apical surface. These results directly show that cytoplasmic dynein function can be regulated by its subunit composition and that cytoplasmic dynein is essential for at least one mode of apical transport in polarized epithelia.

Comparison of antimicrobial resistance patterns between clinical and sewage isolates in a regional hospital in Taiwan.

AIMS: To compare bacterial populations and antimicrobial resistance patterns between clinical and sewage isolates from a regional hospital in northern Taiwan. The dissemination of antibiotic-resistant bacteria from hospital compartments to the hospital sewage treatment plant was examined. METHODS AND RESULTS: A total of 1020 clinical isolates and 435 sewage isolates were collected between July and September 2005. The percentages of Gram-negative bacteria from the clinical and sewage isolates were 87.2% and 91.0%, respectively (P = 0.033). Escherichia coli were the leading bacterial isolates in both groups. Antimicrobial susceptibility testing showed a significant difference (P < 0.001) in resistance to ampicillin (85.6% vs 94.1%), ampicillin/sulbactam (31.7% vs 55.4%), cefazolin (29.2% vs 71.5%) and cefuroxime (20.7% vs 61.9%) between clinical and sewage coliform isolates, respectively. CONCLUSIONS: The sewage isolates had higher antimicrobial resistance rates than the clinical isolates from the same hospital. SIGNIFICANCE AND IMPACT OF THE STUDY: The low efficacy of the hospital sewage treatment may contribute to the dissemination of multidrug resistant bacteria from this hospital compartments to the environment. Practices which limit the disposal of antimicrobial agents into the wastewater system may be the possible measure to prevent the selection of multidrug-resistant bacteria from sewage treatment plants.

Establishment of feline in-house reference intervals for hematologic and biochemical parameters and potential age-related differences.

Reference intervals (RIs) are one of the essential elements in the procedure of disease diagnosis. This is especially true for feline species in which RI is less available than in canine species. RIs are affected by biological, geographical and instrumental factors, yet published RIs with incomplete background are popularly used. Inappropriate interpretations of RIs may affect classification of disease and subsequent treatment. In this study, we demonstrated the step-by-step establishment of feline RIs following the American Society for Veterinary Clinical Pathology (ASVCP) reference interval guideline. A total of 51 parameters were examined, including 20 hematology and 31 biochemistry parameters, and the results were compared to one local RI and two foreign RIs. Overall, about 29% (10/35) of tested parameters were different form local RIs and 60% (30/50) were different from the two foreign RIs, highlighting geographical variations. A higher upper reference limit (URL) in red blood cell count (RBC), hematocrit (Hct), Hemoglobin (Hgb), albumin, creatinine and lower URL in potassium and white blood cell count (WBC) were identified, which may impact the interpretation. In addition, statistical analysis of age and gender were factored separately and indicated that 10 parameters were significantly higher in the adult group. For the impact of gender, percentage of basophil and total iron-binding capacity (TIBC) were lower in female and male cats, respectively. In conclusion, we have demonstrated that it is desirable to establish in-house RIs or RIs of local sources. An age specific RI for the geriatric feline population is advisable for better diagnosis and monitoring the disease.

Expression of class I and class II major histocompatibility antigens on human hepatocellular carcinoma.

Previous reports indicate that human hepatocytes do not express class I and class II MHC antigens. Our analyses on 10 human hepatocellular carcinoma (HCC) cell lines by immunofluorescence tests and RIA, demonstrate that all the human HCC cell lines tested express class I MHC antigens and among them, three poorly differentiated human HCC cell lines also express class II MHC antigens. Results of immunoprecipitation and/or Western blotting experiments indicate similarity in the chemical nature of both the class I and class II MHC antigens expressed by the human HCC cell lines and by a human B lymphoblastoid cell line Raji. Furthermore, a new variant form of class I antigen was detected in some of these HCC cell lines. Immunohistochemical studies of HCC tissues using the peroxidase-antiperoxidase staining method indicated that class I and class II antigens were detectable in 7 out of 11 and 3 out of 11 HCC tissues from patients, respectively. The availability of MHC class I antigen-positive cultured HCC cell lines, including the poorly differentiated lines that also express MHC class II antigen, has provided us with interesting models to study the relationship between expression of MHC antigen and transformation and differentiation of human hepatocytes. These studies will also allow us some insight into the role of MHC class I and class II antigen in the immunosensitivity and immunogenicity of HCC cells to the host-immune response.

Subunit heterogeneity of cytoplasmic dynein: Differential expression of 14 kDa dynein light chains in rat hippocampus.

Cytoplasmic dynein is a multi-subunit protein complex in which each subunit is encoded by a few genes. How these subunit isoforms are assembled and regulated to mediate the diverse functions of cytoplasmic dynein is unknown. We previously have shown that two highly conserved 14 kDa dynein light chains, Tctex-1 and RP3, have different cargo-binding abilities. In this report, coimmunoprecipitation revealed that Tctex-1 and RP3 were present in mutually exclusive dynein complexes of brain. Two specific antibodies were used to examine the localization of these two dynein light chains in adult rat hippocampal formation and cerebral cortex. By light microscopy, Tctex-1 and RP3 immunoreactivities exhibited distinct and almost complementary distribution patterns in both brain regions. In hippocampal formation, Tctex-1 immunoreactivity was most enriched in somata of newly generated granule cells and scant in the mature granule and pyramidal cell somata. In contrast, RP3 immunoreactivity was abundant in pyramidal and granule cell somata. Ultrastructural analysis of the dentate gyrus revealed both dynein light chains were associated with various membranous organelles that often were affiliated with microtubules. In addition, Tctex-1 and RP3 immunoreactivities were preferentially and highly enriched on membranous organelles and/or vesicles of axon terminals and dendritic spines, respectively. These results suggest that dynein complexes with different subunit composition, and possibly function, are expressed differentially in a spatially and temporally regulated manner. Furthermore, Tctex-1 and RP3 may play important roles in synaptic functions.

Rhodopsin trafficking and its role in retinal dystrophies.

We review the sorting/targeting steps involved in the delivery of rhodopsin to the outer segment compartment of highly polarized photoreceptor cells. The transport of rhodopsin includes (1) the sorting/budding of rhodopsin-containing vesicles at the trans-Golgi network, (2) the directional translocation of rhodopsin-bearing vesicles through the inner segment, and (3) the delivery of rhodopsin across the connecting cilium to the outer segment. Several independent lines of evidence suggest that the carboxyl-terminal, cytoplasmic tail of rhodopsin is involved in the post-Golgi trafficking of rhodopsin. Inappropriate subcellular targeting of naturally occurring rhodopsin mutants in vivo leads to photoreceptor cell death. Thus, the genes encoding mutations in the cellular components involved in photoreceptor protein transport are likely candidate genes for retinal dystrophies.

Phenotypes of stop codon and splice site rhodopsin mutations causing retinitis pigmentosa.

PURPOSE: To understand the pathophysiology of retinitis pigmentosa caused by mutations in the rhodopsin gene that lead to truncation of the protein. METHODS: Heterozygotes with the glutamine-64-to-ter (Q64ter), the intron 4 splice site, and the glutamine-344-to-ter (Q344ter) mutations in the rhodopsin gene, representing families with at least three generations of affected members, were studied with clinical examinations and measurements of rod and cone sensitivity across the visual field, rod- and cone-isolated electroretinograms (ERGs), rod dark adaptation, and rhodopsin levels. RESULTS: There was a range of severity of disease expression in each family, some heterozygotes having moderate or severe retinal degeneration and others with a mild phenotype. The mildly affected heterozygotes had normal results on ocular examination but decreased rod sensitivities at most loci across the visual field, abnormalities in rod-isolated ERG a- and b-waves, and reduced rhodopsin levels. Rod dark adaptation followed an approximately normal time course of recovery in patients with the Q64ter mutation. Patients with the splice site or Q344ter mutations both had prolonged recovery of sensitivity, but the time course was different in the two genotypes. CONCLUSIONS: There is allele specificity for the pattern of retinal dysfunction in the Q64ter, intron 4 splice site, and Q344ter rhodopsin mutations. The pattern of dysfunction in all three mutations suggests the mutant opsins interfere with normal rod cell function, and there is subsequent rod and cone cell death.

Protection of mouse photoreceptors by survival factors in retinal degenerations.

PURPOSE: To examine the protective effect of a number of survival factors on degenerating photoreceptors in mutant mice with naturally occurring inherited retinal degenerations, including retinal degeneration (rd/rd), retinal degeneration slow (rds/rds), nervous (nr/nr), and Purkinje cell degeneration (pcd/pcd), in three different forms of mutant rhodopsin transgenic mice and in light damage in albino mice. METHODS: Various survival factors were injected intravitreally into one eye of mice at or soon after the beginning of photoreceptor degeneration, with the opposite eye serving as the control, and the eyes were examined histologically at later ages. The survival factors included brain-derived neurotrophic factor (BDNF), neurotrophin-3, neurotrophin-4, ciliary neurotrophic factor (CNTF), Axokine (a mutein of CNTF), leukemia inhibitory factor, basic fibroblast growth factor, and nerve growth factor and insulin-like growth factor II, either alone or in various combinations. RESULTS: Photoreceptor degeneration was slowed in rd/rd and nr/nr mutant mice and in Q344ter mutant rhodopsin mice by certain forms of CNTF; the degeneration in Q344ter mice was slowed by Axokine and by leukemia inhibitory factor; and the degeneration in a few nr/nr mice was slowed by BDNF. The other agents were ineffective in these mice, and none of the agents were effective in the other mutants and other mutant rhodopsin transgenic mice. However, light damage experiments that compared agent effectiveness in albino mice versus rats suggested a significant delivery problem with the very small mouse eye, thereby making the interpretation of negative findings equivocal in mutant mice. Basic fibroblast growth factor failed to protect the mouse retina from the damaging effects of constant light, whereas it showed a strong protective effect in the rat, indicating an important species difference. CONCLUSIONS: The slowing of degeneration in the rd/rd and Q344ter mutant mice demonstrated that intraocularly injected survival factors can protect photoreceptors from degenerating in animal models with the same or similar genetic defects as those in human inherited retinal degenerations.

Retinal function and rhodopsin levels in autosomal dominant retinitis pigmentosa with rhodopsin mutations.

We studied rod and cone function in 20 patients from six families with autosomal dominant retinitis pigmentosa, who represented five different point mutations in the gene encoding rhodopsin. In a family with a stop codon mutation at the carboxyl end of the molecule (glutamine-344), young members with the mutation were asymptomatic and clinically unaffected but showed about 1 log unit of rod sensitivity loss across the visual field and decreased rhodopsin levels; at this stage, cone function was essentially normal. In three families with mutations at the border of a transmembrane segment (arginine-135-leucine and arginine-135-tryptophan), there was neither detectable rod function nor measurable rhodopsin; cone function was variably impaired. Two families carrying different mutations (threonine-17-methionine and threonine-58-arginine) had altitudinal visual field defects with less impaired rod and cone function in the inferior than in the superior field. Rod adaptation was abnormal in both families, but the time course of adaptation differed between patients with the two mutations. Differences in the pattern of retinal dysfunction were therefore discernible in patients with different rhodopsin mutations.

Abnormal rod dark adaptation in autosomal dominant retinitis pigmentosa with proline-23-histidine rhodopsin mutation.

We studied rod and cone function in 13 patients from four families with autosomal dominant retinitis pigmentosa and the proline-23-histidine rhodopsin mutation. In patients with early stages of this disease, rod sensitivity was mildly abnormal throughout the retina and cone sensitivity was normal. In more severely affected patients, sensitivity loss varied with retinal region, some regions showing mild rod loss only and other regions having pronounced rod and cone dysfunction. Rhodopsin levels were decreased below normal by amounts that indicated the rod sensitivity loss was determined by the reduced ability to absorb light. The most characteristic abnormality of this genotype was a slowed rod branch of dark adaptation, which was present regardless of the extent or severity of disease. The time required for recovery of rod sensitivity was more than twice the normal time. These findings with dark-adapted perimetry, fundus reflectometry, and dark adaptometry showed intrafamilial and interfamilial consistency.

Localization of Tctex-1, a cytoplasmic dynein light chain, to the Golgi apparatus and evidence for dynein complex heterogeneity.

To date, much attention has been focused on the heavy and intermediate chains of the multisubunit cytoplasmic dynein complex; however, little is known about the localization or function of dynein light chains. In this study, we find that Tctex-1, a light chain of cytoplasmic dynein, localizes predominantly to the Golgi apparatus in interphase fibroblasts. Immunofluorescent staining reveals striking juxtanuclear staining characteristic of the Golgi apparatus as well as nuclear envelope and punctate cytoplasmic staining that often decorates microtubules. Tctex-1 colocalization with Golgi compartment markers, its distribution upon treatment with various pharmacological agents, and the cofractionation of Tctex-1-associated membranes with Golgi membranes are all consistent with a Golgi localization. The distribution of Tctex-1 in interphase cells only partially overlaps with the dynein intermediate chain and p150(Glued) upon immunofluorescence, but most of Tctex-1 is redistributed onto mitotic spindles along with other dynein/dynactin subunits. Using sequential immunoprecipitations, we demonstrate that there is a subset of Tctex-1 not associated with the intermediate chain at steady state; the converse also appears to be true. Distinct populations of dynein complexes are likely to exist, and such diversity may occur in part at the level of their light chain compositions.

Rhodopsin mutations responsible for autosomal dominant retinitis pigmentosa. Clustering of functional classes along the polypeptide chain.

Over 40 mutations in the rhodopsin gene have been identified in patients with autosomal dominant retinitis pigmentosa. Twenty-one of these mutations have been introduced into a human rhodopsin cDNA by site-directed mutagenesis, and the encoded proteins have been produced by transfection of a human embryonic kidney cell line (293S). Three of the mutant proteins (G51V, V345M, and P347S) resemble the wild type in yield, regenerability with 11-cis-retinal, and accumulation in the plasma membrane (class I). The remaining 18 mutant proteins are produced at lower levels, regenerate variably or not at all with 11-cis-retinal, and accumulate partially or predominantly in the endoplasmic reticulum (class II). Together with an earlier analysis of 13 mutant rhodopsins (Sung, C.-H., Schneider, B., Agarwal, N., Papermaster, D.S., and Nathans, J. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 8840-8844), these experiments define distinct classes of biochemical defects in human rhodopsin and further show that amino acid substitutions in class II reside within the transmembrane and extracellular domains, whereas class I mutants cluster in the first transmembrane domain and at the extreme carboxyl terminus.

A rhodopsin gene mutation responsible for autosomal dominant retinitis pigmentosa results in a protein that is defective in localization to the photoreceptor outer segment.

Over 45 mutations in the rhodopsin gene have been identified in patients with autosomal dominant retinitis pigmentosa, including a cluster near the extreme carboxy-terminus, a region of the protein for which no function has yet been assigned. To elucidate the biochemical defect(s) in this group of mutants, we have studied a naturally occurring stop codon mutation that removes the last five amino acids of rhodopsin (Q344ter). When produced in transfected tissue culture cells, the mutant protein is indistinguishable from the wild type in light-dependent activation of the photoreceptor G-protein (transducin), and in serving as a light-dependent substrate for rhodopskin kinase. Mice that express a Q344ter transgene in rod photoreceptors show nearly normal light responses as determined by suction electrode recordings of the membrane current from single rod outer segments; the main difference between transgenic and nontransgenic responses is a 15% longer time-to-peak in the response of transgenic rods. In the Q344ter transgenic retina, direct immunofluorescent staining with antibodies specific for either wild-type or Q344ter rhodopsin shows abnormal accumulation of the Q344ter, but not the endogenous rhodopsin, in the plasma membrane of the photoreceptor cell body. These data indicate that rhodopsin's carboxy-terminus is required for efficient transportation to or retention in the outer segment.

Apoptotic photoreceptor cell death in mouse models of retinitis pigmentosa.

Retinitis pigmentosa (RP) is a group of inherited human diseases in which photoreceptor degeneration leads to visual loss and eventually to blindness. Although mutations in the rhodopsin, peripherin, and cGMP phosphodiesterase genes have been identified in some forms of RP, it remains to be determined whether these mutations lead to photoreceptor cell death through necrotic or apoptotic mechanisms. In this paper, we report a test of the hypothesis that photoreceptor cell death occurs by an apoptotic mechanism in three mouse models of RP: retinal degeneration slow (rds) caused by a peripherin mutation, retinal degeneration (rd) caused by a defect in cGMP phosphodiesterase, and transgenic mice carrying a rhodopsin Q344ter mutation responsible for autosomal dominant RP. Two complementary techniques were used to detect apoptosis-specific internucleosomal DNA fragmentation: agarose gel electrophoresis and in situ labeling of apoptotic cells by terminal dUTP nick end labeling. Both methods showed extensive apoptosis of photoreceptors in all three mouse models of retinal degeneration. We also show that apoptotic death occurs in the retina during normal development, suggesting that different mechanisms can cause photoreceptor death by activating an intrinsic death program in these cells. These findings raise the possibility that retinal degenerations may be slowed by interfering with the apoptotic mechanism itself.

Functional heterogeneity of mutant rhodopsins responsible for autosomal dominant retinitis pigmentosa.

Thirteen mutant rhodopsins responsible for autosomal dominant retinitis pigmentosa (ADRP) have been produced by transfection of cloned cDNA into tissue culture cells. Three mutants [class I: Phe-45----Leu, Gln-344----termination (deletion of C-terminal positions 344-348), and Pro-347----Leu] resemble wild-type rhodopsin in yield, regenerability with 11-cis-retinal, and plasma membrane localization. Ten mutants [class II: Thr-17----Met, Pro-23----His, Thr-58----Arg, Val-87----Asp, Gly-89----Asp, Gly-106----Trp, Arg-135----Leu, Arg-135----Trp, Tyr-178----Cys, and Asp-190----Gly] accumulate to significantly lower levels, regenerate with 11-cis-retinal variably or not at all, and are transported inefficiently to the plasma membrane, remaining primarily in the endoplasmic reticulum. These data suggest that there are at least two distinct biochemical defects associated with different rhodopsin mutants in ADRP.

Rhodopsin's carboxy-terminal cytoplasmic tail acts as a membrane receptor for cytoplasmic dynein by binding to the dynein light chain Tctex-1.

The interaction of cytoplasmic dynein with its cargoes is thought to be indirectly mediated by dynactin, a complex that binds to the dynein intermediate chain. However, the roles of other dynein subunits in cargo binding have been unknown. Here we demonstrate that dynein translocates rhodopsin-bearing vesicles along microtubules. This interaction occurs directly between the C-terminal cytoplasmic tail of rhodopsin and Tctex-1, a dynein light chain. C-terminal rhodopsin mutations responsible for retinitis pigmentosa inhibit this interaction. Our results point to an alternative docking mechanism for cytoplasmic dynein, provide novel insights into the role of motor proteins in the polarized transport of post-Golgi vesicles, and shed light on the molecular basis of retinitis pigmentosa.