Light and electron microscopic analysis of aquaporin 1-like-immunoreactive amacrine cells in the rat retina

Aquaporin 1 (AQP1; also known as CHIP, a channel-forming integral membrane protein of 28 kDa) is the first protein to be shown to function as a water channel and has been recently shown to be present in the rat retina. We previously showed (Kim et al. [1998] Neurosci Lett 244:52-54) that AQP1-like immunoreactivity is present in a certain population of amacrine cells in the rat retina. This study was conducted to characterize these cells in more detail, With immunocytochemistry using specific antisera against AQP1, whole-mount preparations and 50-mum-thick vibratome sections were examined by light and electron microscopy. These cells were a class of amacrine cells, which had symmetric bistratified dendritic trees ramified in stratum 2 and in the border of strata 3 and 4 of the inner plexiform layer (IPL). Their dendritic field diameters ranged from 90 to 230 mum. Double labeling with antisera against AQP1 and gamma-aminobutyric acid or glycine demonstrated that these AQP1-like-immunoreactive amacrine cells were immunoreactive for glycine. Their most frequent synaptic input was from other amacrine cell processes in both sublaminae a and b of the IPL, followed by a few cone bipolar cells. Their primary targets were other amacrine cells and ganglion cells in both sublaminae a and b of the IPL. In addition, synaptic output Onto bipolar cells was rarely observed in sublamina b of the IPL. Thus, the AQP1 antibody labels a class of glycinergic amacrine cells with small to medium-sized dendritic fields in the rat retina. (C) 2002 Wiley-Liss, Inc.

Physiological profiles of restricted endemic plants and their widespread congenors in the North Queensland wet tropics, Australia

Little is known about causes of endemic rarity in plants. This study pioneered an approach that determined environmental variables in the rainforest habitat and generated physiological profiles for light, water, and nutrient relations for three endemically restricted versus widespread congeneric species' pairs. We found no overall consistent differences in the physiological variables between the group of restricted species and the group of widespread species, and congeneric species pairs were therefore examined individually. Availability of soil nutrients did not differ between restricted-widespread species sites suggesting that species grow under comparable nutrient conditions. Under ambient and manipulated higher light conditions, widespread Gardenia ovularis had a greater photosynthetic activity than restricted Gardenia actinocarpa suggesting that the two species differ in their photosynthetic abilities. Differences between Xanthostemon species included lower photosynthetic activity, higher transpiration rate, and a higher foliar manganese concentration in restricted Xanthostemon formosus compared to widespread Xanthostemon chrysanthus. It is suggested that X. formosus is restricted by its high water use to its current rainforest creek edge habitat, while X. chrysanthus grows in a range of environments, although naturally found in riparian rainforest. Restricted Archidendron kanisii had higher electron transport rates, greater dissipative capacity for removal of excess light, and more efficient investment of nitrogen into photosynthetic components, than its widespread relative Archidendron whitei. These observations and previous research suggest that restricted Archidendron kanisii is in the process of expanding its range. Physiological profiles suggest a different cause of rarity for each species. This has implications for the conservation strategies required for each species. (C) 2002 Elsevier Science Ltd. All rights reserved.

Transport and metabolism of xylem cytokinins during lateral bud release in decapitated chickpea (Cicer arietinum) seedlings

Although cytokinins (CKs) are widely thought to have a role in promoting shoot branching, there is little data supporting a causative or even a correlative relationship between endogenous CKs and timing of bud outgrowth. We previously showed that lateral bud CK content increased rapidly following shoot decapitation. However, it is not known whether roots are the source of this CK. Here, we have used shoot decapitation to instantaneously induce lateral bud release in chickpea seedlings. This treatment rapidly alters rate and direction of solvent and solute (including CK) trafficking, which may be a passive signalling mechanism central to initiation of lateral bud release. To evaluate changes in xylem transport, intact and decapitated plants were infiltrated with [H-3]zeatin riboside ([H-3]ZR), a water-soluble blue dye or [H-3]H2O by injection into the hypocotyl. All three tracers were recovered in virtually all parts of the shoot within I h of injection. In intact plants, solute accumulation in the lateral bud at node 1 was significantly less than in the adjacent stipule and nodal tissue. In decapitated plants, accumulation of [H-3]ZR and of blue dye in the same bud position was increased 3- to 10-fold relative to intact plants, whereas content of [H-3]H2O was greatly reduced indicating an increased solvent throughput. The stipule and cut stem, predicted to have high evapotranspiration rates, also showed increased solute content accompanied by enhanced depletion of [H-3]H2O. To assess whether metabolism modifies quantities of active CK reaching the buds, we followed the metabolic fate of [H-3]ZR injected at physiological concentrations. Within 1 h, 80-95% of [H-3]ZR was converted to other active CKs (mainly zeatin riboside-5'phosphate (ZRMP) and zeatin (Z)), other significant, but unconfirmed metabolites some of which may be active (O-acetylZR, O-acetylZRMP and a compound correlated with sites of high CK-concentrations) and inactive catabolites (adenosine, adenine, 5'AMP and water). Despite rapid metabolic degradation, the total active label, which was indicative of CK concentration in buds, increased rapidly following decapitation. It can be inferred that xylem sap CKs represent one source of active CKs appearing in lateral buds after shoot decapitation.

Photosynthetic light and temperature responses of Eucalyptus cloeziana and Eucalyptus argophloia

Acclimation of gas exchange to temperature and light was determined in 18-month-old plants of humid coastal (Gympie) and dry inland ( Hungry Hills) provenances of Eucalyptus cloeziana F. Muell., and in those of a dry inland provenance of Eucalyptus argophloia Blakely. Plants were acclimated at day/night temperatures of 18/13, 23/18, 28/23 and 33/ 28 degreesC in controlled-temperature glasshouses for 4 months. Light and temperature response curves were measured at the beginning and end of the acclimation period. There were no significant differences in the shape and quantum-yield parameters among provenances at 23, 28 and 33 degreesC day temperatures. Quantum yield [mumol CO2 mumol(- 1) photosynthetic photon flux density (PPFD)] ranged from 0.04 to 0.06 and the light response shape parameter ranged from 0.53 to 0.78. Similarly, no consistent trends in the rate of dark respiration for plants of each provenance were identified at the four growth temperatures. Average values of dark respiration for the plants of the three provenances ranged from 0.61 to 1.86 mumol m(-2) s(-1). The optimum temperatures for net photosynthesis increased from 23 to 32 degreesC for the humid- and from 25 to 33 degreesC for the dry-provenance E. cloeziana and from 21 to 33 degreesC for E. argophloia as daytime temperature of the growth environment increased from 18 to 33 degreesC. These results have implications in predicting survival and productivity of E. cloeziana and E. argophloia in areas outside their natural distribution.

Caveolin interacts with the angiotensin II type 1 receptor during exocytic transport but not at the plasma membrane

The mechanisms involved in angiotensin II type 1 receptor (AT(1)-R) trafficking and membrane localization are largely unknown. In this study, we examined the role of caveolin in these processes. Electron microscopy of plasma membrane sheets shows that the AT(1)-R is not concentrated in caveolae but is clustered in cholesterol-independent microdomains; upon activation, it partially redistributes to lipid rafts. Despite the lack of AT(1)-R in caveolae, AT(1)-R. caveolin complexes are readily detectable in cells co-expressing both proteins. This interaction requires an intact caveolin scaffolding domain because mutant caveolins that lack a functional caveolin scaffolding domain do not interact with AT(1)-R. Expression of an N-terminally truncated caveolin-3, CavDGV, that localizes to lipid bodies, or a point mutant, Cav3-P104L, that accumulates in the Golgi mislocalizes AT(1)-R to lipid bodies and Golgi, respectively. Mislocalization results in aberrant maturation and surface expression of AT(1)-R, effects that are not reversed by supplementing cells with cholesterol. Similarly mutation of aromatic residues in the caveolin-binding site abrogates AT(1)-R cell surface expression. In cells lacking caveolin-1 or caveolin-3, AT(1)-R does not traffic to the cell surface unless caveolin is ectopically expressed. This observation is recapitulated in caveolin-1 null mice that have a 55% reduction in renal AT(1)-R levels compared with controls. Taken together our results indicate that a direct interaction with caveolin is required to traffic the AT(1)-R through the exocytic pathway, but this does not result in AT(1)-R sequestration in caveolae. Caveolin therefore acts as a molecular chaperone rather than a plasma membrane scaffold for AT(1)-R.

Synaptic vesicle transport and synaptic membrane transporter sites in excitatory amino acid nerve terminals in Alzheimer disease

The apparent L-[H-3]glutamate uptake rate (v') was measured in synaptic vesicles isolated from cerebral cortex synaptosomes prepared from autopsied Alzheimer and non-Alzheimer dementia cases, and age-matched controls. The initial synaptosome preparations exhibited similar densities of D-[H-3]aspartate membrane binding sites (B-MAX values) in the three groups. In control brain the temporal cortex D-[H-3]aspartate B-MAX was 132% of that in motor cortex, parallel with the L- [H-3]glutamate v' values (temporal = 139% of motor; NS). Unlike D- [H-3]aspartate B-MAX values, L- [H-3]glutamate v' values were markedly and selectively lower in Alzheimer brain preparations than in controls, particularly in temporal cortex. The difference could not be attributed to differential effects of autopsy interval or age at death. Non-Alzheimer dementia cases resembled controls. The selective loss of vesicular glutamate transport is consistent with a dysfunction in the recycling of transmitter glutamate.

Abnormal extracellular matrix protein transport associated with increased apoptosis of vascular smooth muscle cells in Marfan syndrome and bicuspid aortic valve thoracic aortic aneurysm

Background - Marfan syndrome (MS) is a genetic disorder caused by a mutation in the fibrillin gene FBN1. Bicuspid aortic valve (BAV) is a congenital heart malformation of unknown cause. Both conditions are associated with ascending aortic aneurysm and premature death. This study examined the relationship among the secretion of extracellular matrix proteins fibrillin, fibronectin, tenascin, and vascular smooth muscle cell (VSMC) apoptosis. The role of matrix metalloproteinase (MMP)- 2 in VSMC apoptosis was studied in MS aneurysm. Methods and Results - Aneurysm tissue was obtained from patients undergoing surgery ( MS: 4 M, 1 F, age 27 - 45 years; BAV: 3 M, 2 F, age 28 - 65 years). Normal aorta from subjects with nonaneurysm disease was also collected ( 4 M, 1 F, age 23 - 93 years). MS and BAV aneurysm histology showed areas of cystic medial necrosis (CMN) without inflammatory infiltrate. Immunohistochemical study of cultured MS and BAV VSMC showed intracellular accumulation and reduction of extracellular distribution of fibrillin, fibronectin, and tenascin. Western blot showed no increase in expression of fibrillin, fibronectin, or tenascin in MS or BAV VSMC and increased expression of MMP-2 in MS VSMCs. There was 4-fold increase in loss of cultured VSMC incubated in serum-free medium for 24 hours in both MS ( 27 +/- 8%) and BAV ( 32 +/- 14%) compared with control ( 7 +/- 5%). Conclusions - In MS and BAV there is alteration in both the amount and quality of secreted proteins and an increased degree of VSMC apoptosis. Up-regulation of MMP-2 might play a role in VSMC apoptosis in MS VSMC. The findings suggest the presence of a fundamental cellular abnormality in BAV thoracic aorta, possibly of genetic origin.

A unified model of flames as gasdynamic discontinuities

Viewed on a hydrodynamic scale, flames in experiments are often thin so that they may be described as gasdynamic discontinuities separating the dense cold fresh mixture from the light hot burned products. The original model of a flame as a gasdynamic discontinuity was due to Darrieus and to Landau. In addition to the fluid dynamical equations, the model consists of a flame speed relation describing the evolution of the discontinuity surface, and jump conditions across the surface which relate the fluid variables on the two sides of the surface. The Darrieus-Landau model predicts, in contrast to observations, that a uniformly propagating planar flame is absolutely unstable and that the strength of the instability grows with increasing perturbation wavenumber so that there is no high-wavenumber cutoff of the instability. The model was modified by Markstein to exhibit a high-wavenumber cutoff if a phenomenological constant in the model has an appropriate sign. Both models are postulated, rather than derived from first principles, and both ignore the flame structure, which depends on chemical kinetics and transport processes within the flame. At present, there are two models which have been derived, rather than postulated, and which are valid in two non-overlapping regions of parameter space. Sivashinsky derived a generalization of the Darrieus-Landau model which is valid for Lewis numbers (ratio of thermal diffusivity to mass diffusivity of the deficient reaction component) bounded away from unity. Matalon & Matkowsky derived a model valid for Lewis numbers close to unity. Each model has its own advantages and disadvantages. Under appropriate conditions the Matalon-Matkowsky model exhibits a high-wavenumber cutoff of the Darrieus-Landau instability. However, since the Lewis numbers considered lie too close to unity, the Matalon-Matkowsky model does not capture the pulsating instability. The Sivashinsky model does capture the pulsating instability, but does not exhibit its high-wavenumber cutoff. In this paper, we derive a model consisting of a new flame speed relation and new jump conditions, which is valid for arbitrary Lewis numbers. It captures the pulsating instability and exhibits the high-wavenumber cutoff of all instabilities. The flame speed relation includes the effect of short wavelengths, not previously considered, which leads to stabilizing transverse surface diffusion terms.

Occlusable corneas in toadfishes: light transmission, movement and ultrastruture of pigment during light- and dark-adaptation

The toadfishes Tetractenos hamiltoni and Torquigener pleurogramma (Tetraodontidae) possess occlusable yellow corneas. We examine the light transmission and location of the yellow/orange pigment throughout the cornea, the temporal properties of pigment migration and the ultrastructure of the pigmented processes during light- and dark-adaptation. Each species was dark-adapted during the day and light-adapted during the night and then exposed to either sun illumination or darkness for different lengths of time (0-70 min). Movement of corneal pigment could be induced in both species regardless of time of day or night. The pigment was able to migrate in a dorsal or ventral direction and changed from minimal to maximal pigmentation within 60 min. Three types of transmission curves were found with varying degrees of transmission in the 400-500 nm waveband, indicating that the pigment distribution is not uniform across the cornea; some areas of the cornea transmit near UV light, while others absorb blue light. The gradual change of the transmission characteristics in different areas of the cornea indicates the presence of different concentrations of a single type of pigment. Ultrastructural examination of the corneas showed that the layer containing the pigment is situated within the scleral cornea either surrounding (T. pleurogramma) or abutting (T. hamiltoni) an iridescent layer. Long sheet-like processes or chromatophores extending centrally from dorsal and ventral reservoirs are filled with pigment during the light-adapted state but empty in the dark-adapted state.

Rab23, a negative regulator of hedgehog signaling, localizes to the plasma membrane and the endocytic pathway

The regulation of hedgehog signaling by vesicular trafficking was exemplified by the finding that Rab23, a Rab-GTPase vesicular transport protein, is mutated in open brain mice. In this study, the localization of Rab23 was analyzed by light and immunoelectron microscopy after expression of wild-type (Rab23-GFP), constitutively active Rab23 (Rab23Q68L-GFP), and inactive Rab23 (Rab23S23N-GFP) in a range of mammalian cell types. Rab23-GFP and Rab23Q68L-GFP were predominantly localized to the plasma membrane but were also associated with intracellular vesicular structures, whereas Rab23S23N-GFP was predominantly cytosolic. Vesicular Rab23-GFP colocalized with Rab5Q79L and internalized transferrin-biotin, but not with a marker of the late endosome or the Golgi complex. To investigate Rab23 with respect to members of the hedgehog signaling pathway, Rab23-GFP was coexpressed with either patched or smoothened. Patched colocalized with intracellular Rab23-GFP but smoothened did not. Analysis of patched distribution by light and immunoelectron microscopy revealed it is primarily localized to endosomal elements, including transferrin receptor-positive early endosomes and putative endosome carrier vesicles and, to a lesser extent, with LBPA-positive late endosomes, but was excluded from the plasma membrane. Neither patched or smoothened distribution was altered in the presence of wild-type nor mutant Rab23-GFP, suggesting that despite the endosomal colocalization of Rab23 and patched, it is likely that Rab23 acts more distally in regulating hedgehog signaling.

Intracellular sorting and transport of proteins

The secretory and endocytic pathways of eukaryotic organelles consist of multiple compartments, each with a unique set of proteins and lipids. Specific transport mechanisms are required to direct molecules to defined locations and to ensure that the identity, and hence function, of individual compartments are maintained. The localisation of proteins to specific membranes is complex and involves multiple interactions. The recent dramatic advances in understanding the molecular mechanisms of membrane transport has been due to the application of a multi-disciplinary approach, intergrating membrane biology, genetics, imaging, protein and lipid biochemistry and structural biology. The aim of this review is to summarise the general principles of protein sorting in the secretory and endocytic pathways and to highlight the dynamic nature of these processes. The molecular mechanisms involved in this transport along the secretory and endocytic pathways are discussed along with the signals responsible for targeting proteins to different intracellular locations. (C) 2003 Elsevier Science Ltd. All rights reserved.

Using the flexibility index to compare batch and continuous activated sludge processes

This paper considers the question of which is better: the batch or the continuous activated sludge processes? It is an important question because dissension still exists in the wastewater industry as to the relative merits of each of the processes. A review of perceived differences in the processes from the point of view of two related disciplines, process engineering and biotechnology, is presented together with the results of previous comparative studies. These reviews highlight possible areas where more understanding is required. This is provided in the paper by application of the flexibility index to two case studies. The flexibility index is a useful process design tool that measures the ability of the process to cope with long term changes in operation.

Retinal specializations in the blue marlin: eyes designed for sensitivity to low light levels

The large eyes and well-developed visual system of billfishes suggest that vision is an important sense for the detection and interception of prey and lures. Investigations of visual abilities in these large pelagic fishes are difficult, however anatomical studies of billfish eyes and retinas allow prediction of a number of visual capabilities. From the density of ganglion cells in the blue marlin (Makaira nigricans) retina, visual acuities of less than 10 cycles per degree were derived, a surprisingly low visual resolution given the absolute size of the marlin eye. Cone photoreceptors, on the other hand, were present in high densities, resulting in a presumed summation of cones to ganglion cells at a ratio of 40:1, even in the area of best vision. The optical sensitivity of the marlin eye was high owing to the large dimensions of the cone photoreceptors. These results indicate that the marlin eye is specifically adapted to cope with the low light levels encountered during diving. Since the marlin is likely to use its vision at depth, it is suggested that this line of research could help estimate the limits of vertical distribution based on light level.