Biomedical subjects

K Kohler

Publications and source records attributed to K Kohler.

At least 19 recordsLinked to original sources

PubMed2006

Molecular mechanisms of spider silk.

Spiders spin high-performance silks through the expression and assembly of tissue-restricted fibroin proteins. Spider silks are composite protein biopolymers that have complex microstructures. Retrieval of cDNAs and genomic DNAs encoding silk fibroins has revealed an association between the protein sequences and structure-property relationships. However, before spider silks can be subject to genetic engineering for commercial applications, the complete protein sequences and their functions, as well as the details of the spinning mechanism, will require additional progress and collaborative efforts in the areas of biochemistry, molecular biology and material science. Novel approaches to reveal additional molecular constituents embedded in the spider fibers, as well as cloning strategies to manipulate the genes for expression, will continue to be important aspects of spider biology research. Here we summarize the molecular characteristics of the different spider fibroins, the mechanical properties and assembly process of spidroins and the advances in protein expression systems used for recombinant silk production. We also highlight different technical approaches being used to elucidate the molecular constituents of silk fibers.

Amino Acid Motifs↗

PubMed2001

Presence of kynurenic acid and kynurenine aminotransferases in the inner retina.

Kynurenine aminotransferases (KATs I and II) are pivotal to the synthesis of kynurenic acid (KYNA), the only known endogenous glutamate receptor antagonist and neuroprotectant. This study is the first to identify KYNA in the rat retina and to examine immunohistochemically the distribution of KAT isoforms. As determined by HPLC, KYNA concentration in the retina was 99.9 +/- 24.6 pmol/g wet wt. Immunohisto- chemical experiments showed that both KATs were present in the retina. KAT I was preferentially localised on Müller cell endfeet while KAT II was expressed in cells within the ganglion cell layer. In conclusion, KYNA is present and synthesised in the inner retina. This may suggest a modulatory role in glutamate-mediated retinal neurotransmission.

Animals↗

PubMed2001

Identification of purinergic receptors in retinal ganglion cells.

P2X receptors are ligand-gated ion channels activated by adenosine triphosphate and expressed in a broad variety of tissues. The present study demonstrates the expression of various types of purinergic P2X receptors in identified retinal ganglion cells (RGCs) of the adult rat retina. Single-cell reverse transcription polymerase chain reaction (SC-RT-PCR) resulted in a positive amplification signal for all P2X receptor subunit mRNAs examined (P2X(3-5), P2X(7)). Immunohistochemistry with P2X(3,4) receptor subunit-specific antibodies showed a labelling of neurons in the ganglion cell layer and inner nuclear layer. Our data suggest that extracellular ATP acts directly on RGCs via several types of P2X receptors and may provide neuromodulatory influences on information processing in the retina.

Adenosine Triphosphate↗

PubMed2001

Combined HPLC-MS and HPLC-NMR on-line coupling for the separation and determination of lutein and zeaxanthin stereoisomers in spinach and in retina.

The determination and unambiguous identification of carotenoid stereoisomers from biological tissues, avoiding isomerization and oxidation due to the extraction process, is still a major challenge. Particularly, the analysis of lutein and zeaxanthin stereoisomers is of great importance, as these are the main constituents of the macula lutea, the central part of the human retina, and act as possible agents in the prevention and treatment of age-related macular degeneration (AMD). By combining a mild and quick extraction technique such as matrix solid-phase dispersion together with high-performance liquid chromatography (HPLC), the extremely light and oxygen sensitive lutein and zeaxanthin stereoisomers are extracted, enriched, and separated directly from the solid plant or tissue samples, excluding preparation of artifacts. HPLC separations are performed with C30 phases due to their enhanced shape selectivity compared to C18 phases and on-line coupled to mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. By using HPLC-MS with atmospheric pressure chemical ionization, the lutein stereoisomers can be distinguished from the zeaxanthin stereoisomers within one chromatographic run in the upper picogram range, whereas HPLC-NMR coupling allows the unequivocal identification of each stereoisomer with a concentration in the upper nanogram range. This article provides an analytical method for the artifact-free determination of lutein and zeaxanthin stereoisomers directly from the solid biological tissue spinach as a source of carotenoids and retina as the sphere of activity for AMD. In addition, the structures of these stereoisomers were unambiguously elucidated by employing hyphenated analytical techniques.

Chromatography, High Pressure Liquid↗

PubMed2001

Expression of angiotensin-converting enzyme (ACE) in the developing chicken retina.

Angiotensin-converting enzyme (ACE) performs two contrasting enzymatic effects: as part of the renin-angiotensin system it converts angiotensin I into physiologically active angiotensin II, and it inactivates a number of peptides, e.g. substance P. These peptides are well known neurotransmitters in the retina and recently angiotensin II was described in retinal neurons. We therefore investigated a possible involvement of ACE in retinal metabolism by determining the mRNA and protein expression of ACE in the developing and mature chicken retina. ACE-mRNA expression was investigated by RT-PCR in the iris/ciliary body, the choroid, the optic nerve head, pecten, and the retina. Levels of ACE-mRNA were quantified by competitive PCR with heterologous competitor fragments in the retina at different developmental stages. To localize protein expression of ACE in the mature chicken retina an antibody directed against ACE was used. ACE-mRNA was present in all ocular tissues examined. Quantification of ACE-mRNA in avascular retinas of developing chickens revealed small amounts (0.13 attomol microl(-1)) at embryonic day 7 and values of about 0.6 attomol microl(-1)during embryonic days 7-17. ACE-mRNA expression transiently increased ten-fold (7.3 attomol microl(-1)) on postnatal day 1, decreased again to about 1.4 attomol microl(-1)on postnatal day 6, and remained constant thereafter. ACE-immunohistochemistry revealed labeling of photoreceptors, bipolar cells, amacrine cells, and cells in the ganglion cell layer as well as of Müller glia. Our data show that ACE-mRNA is an intrinsic component of the retina and that ACE itself has a widespread but distinct cellular distribution. The transient high expression of ACE-mRNA directly after hatching indicate, that ACE may be involved in fine tuning the neuropeptidergic equipment of the retinal network during the initial phase of visual experience.

Animals↗

PubMed2001

[Subretinal microphotodiode array as replacement for degenerated photoreceptors?].

A survey is given on the status of developments, concerning a subretinal electronic microphotodiode array that aims at replacing degenerated photoreceptors. Various prototypes have been developed, tested, and implanted in various experimental animals up to 18 months. The fact that electrical responses were recorded from the visual cortex of pigs after electrical stimulation by subretinal electrodes and the fact that responses are also recorded in-vitro in degenerated rat retinae, shows the feasibility of this approach. However, there are a number of open questions concerning the biocompatibility, the long-time stability, and the type of transmitted image to be solved before application in patients can be considered.

Animals↗

PubMed2001

[Histological studies of retinal degeneration and biocompatibility of subretinal implants].

Two basic biological premises determine the success of replacement of degenerated photoreceptors by a technical implant. First, the neuronal network in the residual retina of patients selected for implantation must still be capable of processing technically generated signals. Secondly, the implant itself must be biocompatible with tissue, i.e. it may not itself induce further degeneration. Our studies in animal models with advanced retinal degeneration and with donor retinas of retinitis pigmentosa patients have shown that even after complete destruction of the photoreceptors and long periods of blindness, the inner retina in the macular area remains for the most part histologically intact, and that all neurons are demonstrably still present and capable of successfully transmitting and processing signals. Biocompatibility of subretinal implants was studied in pigs. After 14 months of implantation, histological examination of tissue covering the implant showed that the inner retina was completely intact. There were no signs of histopathologic changes.

Animals↗

PubMed2001

Studies on the feasibility of a subretinal visual prosthesis: data from Yucatan micropig and rabbit.

BACKGROUND: To estimate the feasibility of the subretinal concept of a visual prosthesis, animal models and prototypes, each representing a certain aspect of the final prosthesis, were utilised to test for requirements for such a medical device: (1) the ability to elicit--by electrical stimulation--event-related central activity in the central visual system, and (2) the long-term biocompatibility and biostability of the implant within the subretinal space. METHODS: (1) In rabbit and Yucatan minipig, cortical evoked potentials were recorded with chronically implanted epidural electrodes during stimulation with light flashes as well as during electrical stimulation in the subretinal space. Voltage pulses ranging from -3 V to +3 V were applied via an acutely implanted electrode array on a wired prototype. (2) For biocompatibility studies a silicon-based micro-photodiode array (MPDA) was used that closely resembled the design and composition of the final prosthesis. Fourteen months after implantation, angiography was performed and the histological findings of the retina in the immediate vicinity of the implant were evaluated. RESULTS: (1) In both rabbit and minipig, subretinal electrical stimulation resulted in evoked cortical potentials that were comparable to visual evoked potentials. The lowest threshold levels for the subretinal stimulation were 0.6 V for rabbits and 2 V for minipigs. (2) Long-term stability of an implanted MPDA and its biocompatibility were proven for a postoperative period of 14 months. CONCLUSIONS: Data from animal experiments with certain prototypes of the final prosthesis suggest the feasibility of the concept of a subretinal visual prosthesis: Both requirements were met: (1) the functioning of the subretinal stimulation and (2) the biocompatibility of the MPDA implant.

Animals↗

PubMed2001

Onset and time course of apoptosis in the developing zebrafish retina.

In mammalian development, apoptosis spreads over the retina in consecutive waves and induces a remarkable amount of cell loss. No evidence for such consecutive waves has been revealed in the fish retina so far. As the zebrafish is of growing importance as a model for retinal development and for degenerative retinal diseases, we examined the onset and time course of apoptosis in the developing zebrafish retina and in adult fish. We found that apoptosis peaked in the ganglion cell layer (GCL) and inner nuclear layer (INL) in early developmental stages (3-4 days post-fertilization; dpf) followed by a second, but clearly smaller wave at 6-7dpf. Apoptosis in the outer nuclear layer (ONL) started at 5dpf and peaked at 7dpf. This late-onset high peak of apoptosis of photoreceptors is different from that of all other species examined to date. With 1.09% of cells in the GCL and 1.10% in the ONL being apoptotic, the rate of apoptosis in the developing zebrafish retina was conspicuously lower than that observed in other vertebrates (up to 50% in GCL). During development (2-21dpf), apoptotic waves were most obvious in the central retina, whereas in the periphery near the marginal zone (MZ), apoptosis was much lower; in adult animals, practically no apoptosis was present in the central retina but it still occurred near the MZ. Our data show that the onset and time course of apoptosis in the GCL and INL of the zebrafish is comparable with other vertebrates; however, the amount of apoptosis is clearly reduced. Thus, apoptosis in the zebrafish retina may serve more as a mechanism for the fine tuning of the retinal neuronal network after mitotic waves during development or in remaining mitotic areas than as a mechanism for eliminating large numbers of excess cells.

Animals↗

PubMed2001

Ocular pulse amplitude is reduced in patients with advanced retinitis pigmentosa.

BACKGROUND/AIMS: The choroid, a low resistance vascular structure carrying 85% of the ocular blood flow, provides nourishment to and removal of potential toxic waste products from the adjacent non-vascularised outer layers of the retina, macula, and optic disc regions. Choroidal perfusion may be reduced in retinitis pigmentosa (RP) and might contribute to retinal pigment epithelium (RPE) degeneration. The aim of this study was to determine whether choroidal perfusion is reduced in RP and whether this is correlated with the stage of disease. METHODS: Ocular pulse amplitude (OPA) evaluated with the ocular blood flow (OBF) system, applanation intraocular pressure (IOP), visual fields, blood pressure (BP), and heart rate (HR) were measured in 75 RP patients having stage RP-I (stage I: visual field size: 7.85-14.67 cm(2); n = 22), stage RP-II (stage II: visual field size: 2.83-7.84 cm(2); n = 29), or stage RP-III (stage III: visual field size: 0.52-2.82 cm(2); n = 24) were compared with matched healthy controls and each other. RESULTS: Neither IOP nor systemic perfusion parameters were significantly (p >0.1) altered, but OPA (mm Hg) in RP patients beginning with stage RP-II (1.6 (0.1), 27.3%, p<0.0001), and RP-III (1.2 (0.1), 45.5%, p<0.0001) was significantly reduced when compared with matched subgroups from a pool of healthy controls (2.2 (0.1), n = 94). CONCLUSIONS: OPA can be used neither for early clinical detection of RP nor to follow the natural course of the disease. However, our data show that in advanced stages of RP not only the retina but also the choroidal circulation is affected.

Adult↗

PubMed2001

Evaluation of the rhodopsin knockout mouse as a model of pure cone function.

PURPOSE: To determine a time window in the rhodopsin knockout (Rho(-/-)) mouse during which retinal function is already sufficiently developed but cone degeneration is not yet substantial, thus representing an all-cone retina. METHODS: Electroretinograms (ERGs) were obtained from 14 homozygous Rho(-/-) mice and eight C57Bl/6 control mice. The same individuals were tested every 7 days, beginning as early as postnatal day (P)14. The ERG protocols included flash and flicker stimuli, both under photopic and scotopic conditions. Retinal and choroidal morphology was observed in animals of comparable age. RESULTS: Functionally, the developmental phase lasted until postnatal week (PW)3 in both the Rho(-/-) mice and the control animals. During PW4 to 6, the Rho(-/-) mice showed a plateau in ERG parameters with normal or even supernormal cone responses and complete absence of rod contributions. At PW7, there was a marked onset of degeneration, which progressed so that no ERG signals were left at PW13, when the control eyes still had normal ERG responses. Microscopically, cone degeneration paralleled the functional changes, beginning at approximately PW6 and almost complete at PW13, whereas retinal pigment epithelium (RPE) and choroid did not show any abnormalities. CONCLUSIONS: From PW4 to 6, Rho(-/-) mice appear to have normal cone and no rod function. Despite the missing rod outer segment (OS), the structure of retina, RPE, and choroid remained unchanged. Therefore, the Rho(-/-) mice can serve during this age period as a model for pure cone function. Such a model is particularly useful to evaluate rod-cone interaction and to dissect rod- from cone-mediated signaling pathways in vivo.

Animals↗

PubMed2000

Self-induced shapiro effect in semiconductor superlattices

We observe that the oscillatory motion of photoinjected electron-hole pairs in a biased semiconductor superlattice (Bloch oscillation) is accompanied by a coherent quasi-dc current that is generated by the interaction of the carriers with the self-induced oscillating field. It is shown that this novel macroscopic quantum effect, which is a coherent analog of the Shapiro effect observed in Josephson junctions, can be controlled by changing the spectral position of the exciting laser pulse, which in turn determines the amplitude and phase of the wave packet oscillations. It is thereby possible to coherently drive the electrons either downwards or upwards in the potential of the static field.

Journal Article↗

PubMed2000

The distribution and developmental regulation of NMDA receptor subunit proteins in the outer and inner retina of the rat.

In order to investigate whether N-methyl-D-aspartate (NMDA) receptors with distinct pharmacological properties are differentially distributed within the retinal layers, the spatial distribution and temporal regulation of all NMDA receptor subunits was analyzed in parallel on the protein level in the rat retina during development. Immunohistochemistry was performed on retinal sections at different developmental ages between embryonic (E) days 20/21 and the adult stage using specific antibodies against NMDA subunits (NR1, NR2A-D). All NMDA subunits were expressed in the rat retina postnatally but showed different spatial patterns. In particular, and in contrast to previous in situ hybridization studies, labeling of NR2 subunits was observed in horizontal cell bodies and in the outer plexiform layer, indicating that functional NMDA receptors are expressed in this retinal cell type in the rat. Expression of NR2D was restricted to the inner retina and seemed to be involved in neurotransmission within the rod pathway. In the inner plexiform layer (IPL), distinct patterns of labeling were observed for different NMDA subunits. NR1 was found in two bands which can be related to the off- and on-signal pathways, whereas NR2A and NR2B were located in two bands within the off-sublaminae of the IPL. The antibody against NR2C was distributed throughout the whole IPL, and NR2D was expressed exclusively in the innermost part of the IPL where rod bipolar cell terminals terminate. Distinct bands of immunoreactivity in the IPL were observed only from P14 on. In conclusion, there are clear differences in the spatial distribution and temporal expression of NMDA receptor subtypes in the rodent retina. This indicates that specific retinal cells selectively express glutamate receptors composed of different subunit combinations and thus display different pharmacological and kinetic properties.

Animals↗

PubMed2000

Coupled bloch-phonon oscillations in semiconductor superlattices

We investigate coherent Bloch oscillations in GaAs/AlxGa1-xAs superlattices with electronic miniband widths larger than the optical phonon energy. In these superlattices the Bloch frequency can be tuned into resonance with the optical phonon. Close to resonance a direct coupling of Bloch oscillations to LO phonons is observed which gives rise to the coherent excitation of LO phonons. The density necessary for driving coherent LO phonons via Bloch oscillations is about 2 orders of magnitude smaller than the density necessary to drive coherent LO phonons in bulk GaAs. The experimental observations are confirmed by the theoretical description of this phenomenon [A.W. Ghosh et al., Phys. Rev. Lett. 85, 1084 (2000)].

Journal Article↗

PubMed2000

Expression of purinergic receptors in bipolar cells of the rat retina.

P2X receptors are ligand-gated ion channels which are activated by excitatory neurotransmitter ATP. Despite considerable evidence of signaling by extracellular nucleotides in other sensory systems, P2X receptors in the visual system have only rarely been studied, and almost nothing is known about their functional significance in the retina. To determine whether ATP plays a role in the modulation of vertical retinal signal pathways, we examined the expression of P2X receptor mRNA in freshly isolated bipolar cells of the rat retina (Brown Norway, P25) using the single-cell RT-PCR technique. Positive amplification signals were found in about 33% of the bipolar cells for P2X(3), P2X(4) and P2X(5) but not for P2X(7) mRNA. We conclude that at least a subpopulation of bipolar cells in the rat retina expresses ionotropic P2 receptors of the P2X type and that these possibly exert a neuromodulatory influence on information processing in the retina.

Animals↗

PubMed2000

Direct observation of depolarization shift of the intersubband resonance

We have studied the intersubband resonance of GaAs/AlGaAs multi-quantum-well systems by comparing photon drag and absorption spectra obtained by in-plane photocurrent and photoconduction measurements. The peak absorption at room temperature is found to be blueshifted from the photon drag resonance by as much as 33 cm(-1). We argue that this difference gives directly the depolarization shift, since the resonant photon drag current is driven by the Doppler effect, which is a k-vector dependent single particle process.

Journal Article↗

PubMed2000

[Evaluating the Pro Ton applanation tonometer in rabbits].

PURPOSE: To assess use of the tonometer ProTon for measuring intraocular pressure in rabbits. MATERIALS AND METHODS: Eleven chinchilla-bastard rabbits were measured under standardized conditions with the applanation tonometer ProTon for 62 +/- 15 days. One rabbit was excluded because of intensively defensive reactions. RESULTS: Intraocular pressure in the ten rabbits was 12.15 +/- 3.63 mmHg in the right eye and 12.11 +/- 3.52 mmHg in the left eye. CONCLUSION: Intraocular pressure can be measured reliably over a long period with the applanation tonometer ProTon. It is therefore suitable for determining intraocular pressure in experimentally induced glaucoma in rabbits.

Animals↗

PubMed2000

Cell differentiation, synaptogenesis, and influence of the retinal pigment epithelium in a rat neonatal organotypic retina culture.

This study was focused on the analysis of cell type differentiation and synaptogenesis as well as outer segment formation in an organotypic culture of the neonatal rat retina during a 6-14 day period of in vitro development. Moreover, the effects of the retinal pigment epithelium (RPE) on these processes were investigated. The in vitro development resulted in a retinal architecture and lamination comparable to that of in vivo retinas. The RPE influences the proper alignment of photoreceptors as well as the formation of the outer limiting membrane (OLM), but not processes of cell differentiation, synaptogenesis and inner retinal lamination.

Animals↗