Cross-linking by 1-ethyl-3- (3-dimethylaminopropyl)-carbodiimide (EDC) of a collagen/elastin membrane meant to be used as a dermal substitute: effects on physical, biochemical and biological features in vitro.

Next to in vitro-cultured autogeneic keratinocytes for the restoration of epidermis, a suitable dermal matrix is a mandatory component of an artificial skin substitute for the permanent covering of full thickness skin defects. In our model a xenogeneic membrane, consisting of processed native collagen and elastin of porcine origin is meant to serve as a template for the formation of a neo-dermis. In order to improve the resistance of this matrix against enzymatical degradation, we cross-linked it by using the carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) together with N-hydroxysuccinimide. Chemical cross-linking by these agents at two different degrees (shrinkage temperatures 63 degrees C and 81 degrees C) had no relevant effect on mechanical features or water-uptake capacity. The time needed for enzymatic digestion was increased by cross-linking. Concerning growth and spreading of fibroblasts and keratinocytes on and within the structure of this membrane, we did not observe a difference between cross-linked and non-cross-linked material (shrinkage temperature 48 degrees C). We therefore expect that cross-linking by EDC is an effective means to control the degradation of the collagen/elastin membranes in vivo without a significant influence on their biocompatibility.

Extracellular compartments of the blowfly eye: ionic content and topology.

To analyze the elemental composition and topology of the extracellular compartments of the compound eye, the eyes of blowflies Calliphora vicina were rapidly frozen and ultrathin cryosections were freeze dried. Three zones of an ommatidium, peripheral cytosol of visual cells, rhabdomeres, and ommatidial cavities were analyzed by X-ray microprobe analysis. The ommatidial cavity was found to contain sodium and potassium in proportion similar to that in the blowfly hemolymph. Potassium-to-sodium ratio in a cytosol was typical for a cytosol. The rhabdomeres displayed an electrolyte content intermediate between the above compartments. Three topologically connected extracellular compartments were characterized by the experiments with tracers, monastral blue and lanthanum: (1) common intercellular space of ommatidia including peripheral clefts between the visual cells, both tracers entered this compartment; (2) the ommatidial cavity, which is not accessible for monastral blue, however, as revealed by our X-ray microanalysis, it was reachable for lanthanum; (3) rhabdomeric loops, which were accessible for lanthanum entering either via the cavity or from the common intercellular clefts. The above characteristics of the ionic content and topology of ommatidial compartments might suggest higher sodium and lower potassium content in the microvilli as compared with the cytosol. The rhabdomeric and "cavital" plasma membranes are assumed to be permeable for these ions so that a voltage of only 25-30 mV, negative inside, is probably formed across them, much lower than the known resting potential -60 mV across the peripheral plasma membrane of a visual cell.

Comparing properties of brains and computers.

The criterion that pure natural science can only investigate objective phenomena which can be observed by independent observers sets certain limits to pure natural scientific understanding of brain functions. It excludes consciousness and feelings since these are only subjectively accessible and alternatives cannot be decided objectively. The limitations of brain research are discussed by comparing the properties of brains and computers. At least for the time being we do not know of any natural scientific--i.e. physical or chemical--method which allows the objective measurement of consciousness, sensations, and emotions. In addition it is discussed how and in how far our brain understands its surrounding nature.

Kinetics of rhodopsin regeneration in situ and in the excised median eye of Limulus polyphemus measured using electrophysiological methods.

The reactivation of rhodopsin after photoregeneration from metarhodopsin in the UV-sensitive cells of the median eye of Limulus was examined by means of extracellular electroretinogram (ERG) measurements. Absorbed photons convert the transducing rhodopsin (Rt) to metarhodopsin, which is thermostable and can be reconverted by another photon to non-transducing rhodopsin (Rn). The amplitude of the ERG is assumed to correlate linearly with the amount of Rt under otherwise constant conditions. The results demonstrate that the reactivation of Rn recorded in vivo in the intact animal is much faster than that in the excised eye [half period: 4 min (in vivo), 24 min (excised eye)]. In the excised eye the ERG amplitudes recover over a sigmoidal time course; however, in vivo the kinetics often appear to be exponential. The in vivo kinetics were measured by several defined molar ratios of Rn to rhodopsin plus metarhodopsin [Rn/(R + M)]. These were adjusted by different durations of pre-illumination with UV light, which is preferentially absorbed by rhodopsin. The in vivo kinetics were fitted by a single exponential function. At very high molar ratios of Rn (> 90%) the kinetics become sigmoidal even in the in vivo experiments. The half-life of the in vivo kinetics depends linearly on the initial molar Rn/(R + M) ratio [half-life: 4-12 min with a rise of 0.1 min/% inactivated metarhodopsin (Mn), 20 degrees C]. The results are consistent with the assumption of multiple-step dephosphorylation being the rate-limiting step in regeneration of rhodopsin in the dark.

Immunological demonstration of Gq-protein in Limulus photoreceptors.

The phototransduction cascade in invertebrates involves the coupling of rhodopsin activation to the action of the enzyme phospholipase C. This step is performed by G-proteins. An antibody against the alpha-subunit of a mouse Gq type G-protein recognized protein bands in Western blots of lateral eye and ventral nerve photoreceptors of Limulus. The protein bands had an apparent molecular mass of about 42 kDa. The antibody also recognized protein bands of a similar molecular mass in immunoblots of brain and intestine tissue. Immunoreactivity was found in lateral eye frozen sections where it was confined to the rhabdom region. When the antibody was applied to ultrathin sections of ventral nerve photoreceptors, the highest density of labeling was found on the rhabdomeral microvilli, but gold particles were also scattered in the cytoplasm. We conclude that a G-protein of the type Gq participates in the phototransduction of Limulus.

The Limulus ventral photoreceptor: light response and the role of calcium in a classic preparation.

The ventral nerve photoreceptor of the horseshoe crab Limulus polyphemus has been used for many years to investigate basic mechanisms of invertebrate phototransduction. The activation of rhodopsin leads in visual cells of invertebrates to an enzyme cascade at the end of which ion channels in the plasma membrane are transiently opened. This allows an influx of cations resulting in a depolarization of the photoreceptor cell. The receptor current of the Limulus ventral photoreceptor consists of three components which differ in several aspects, such as the time course of activation, the time course of recovery from light adaptation, and the reversal potential. Each component is influenced in a different, characteristic way by various pharmacological manipulations. In addition, at least two types of single photon-evoked events (bumps) and three elementary channel conductances are observed in this photoreceptor cell. These findings suggest that the receptor current components are controlled by three different light-activated enzymatic pathways using three different ligands to increase membrane conductance. Probably one of these ligands is cyclic GMP, another one is activated via the IP3-cascade and calcium, the third one might be cyclic AMP. Calcium ions are very important for the excitation and adaptation of visual cells in invertebrates. The extracellular and intracellular calcium concentrations determine the functional state of the visual cell. A rise in the cytosolic calcium concentration appears to be an essential step in the excitatory transduction cascade. Cytosolic calcium is the major intracellular mediator of adaptation. If the cytosolic calcium level exceeds a certain threshold value after exposure to light it causes the desensitization of the visual cell. On the other hand, from a slight rise in cytosolic calcium facilitation results, i.e. increased sensitivity of the photoreceptor.

Interaction of guanosine 5'-triphosphate binding protein Gq from Sepia officinalis with illuminated rhodopsin bound to concanavalin A.

The heterotrimeric guanosine 5'-triphosphate (GTP)-binding protein Gq was suggested to couple the light receptor rhodopsin with the effector phospholipase C in visual cells of invertebrates. We indirectly linked Gq from Sepia officinalis to a concanavalin A-sepharose column via rhodopsin. Rhodopsin had been solubilized previously with 10 mM n-dodecyl-beta-maltoside from the purified photosensory membrane under illumination. All three subunits of the Gq were released almost pure by elution with 100 microM GTP. The alpha and beta subunits were identified by specific antipeptide antisera. The alpha subunit has a relative molecular mass of 46 kDa, and the beta subunit of 35 kDa. The gamma subunit corresponds to a 9 kDa polypeptide owing to the molecular mass, which is similar to the G gamma subunit of squid. The use of specific antibodies shows that neither actin nor G-protein related to transducin were in the fractions eluted with GTP or alpha-methyl mannoside. We demonstrate that all three subunits of Gq were associated with rhodopsin of invertebrates. Such use of a lectin column might be useful for further investigations of the interaction of rhodopsin and Gq.

The light-stimulated cytosolic calcium transient in Limulus ventral nerve photoreceptors: two components in the rising phase.

1. Light-evoked electrical responses were measured in Limulus ventral nerve photoreceptors simultaneously with changes in the cytosolic free calcium concentration, by means of arsenazo III. 2. It has been shown here for the first time that the rise of the arsenazo signal consists of two phases. Only the slow phase in the rise of the signal depends on the membrane voltage. The reversal potential of the amplitude of this slow rising phase was about +196 mV. After removal of external calcium the reversal potential was about +20 mV. 3. When Na+ in the superfusate was replaced by Li+, the amplitude of the fast rising phase was reduced on the average to 50%, while the slow rising phase was not affected. 4. We conclude that the fast rising phase is caused by release of calcium from internal stores while the slow increase in [Ca2+]i is due to influx across the plasma membrane, possibly through light-activated ion channels.

Ultrastructural changes of the microvillar cytoskeleton in the photoreceptor of the crayfish Orconectes limosus related to different adaptation conditions.

In the retina of crayfish microvilli of seven of the eight photoreceptor cells build highly organized structures, the rhabdoms. Cytoskeletal elements inside the microvilli were investigated in conventional and slightly extracted electron microscopical preparations. In conventional preparations the ultrastructure of these cytoskeletal elements depended on the adaptational state of the animal. They appeared as central filament-like structures inside each microvillus when dark-adapted retinae were prepared and fixed at night in the absence of calcium. Changes of these conditions (light, daytime, or calcium concentration) impaired the detectability of these central filaments; in light-adapted eyes prepared at midday they were rarely seen. Nevertheless, single microvillar filaments were present in light-adapted retinae after mild cell permeabilization with the saponin beta-escin. They appeared as a regular structure in each microvillus, often attached to the membrane. Their fine structure was consistent with the ultrastructure of single actin filaments as indicated by fast-Fourier-analysis and further supported by the presence of anti-actin immunoreactivity in electron microscopical and immunocytochemical preparations. These results indicate that microvillar filaments are not necessarily destroyed by light as previously described; we suggest that their appearance inside the microvillus might be altered by the properties of associated, maybe sidearm-like proteins.

Immunoelectron-microscopic study of G-protein distribution in photoreceptor cells of the cephalopod Sepia officinalis.

Previous studies suggest that more than one single light-stimulated transduction pathway seems to be present in photoreceptor cells of invertebrates. Accordingly, more than one light-dependent G-protein has been detected in squid photoreceptor cells. Two different antibodies were used to locate the distribution of G-proteins in Sepia photoreceptors. One antiserum (anti-G alpha-common) has been raised against the peptide CGAGESGKSTIVKQMK. This sequence is found in most G alpha-protein subunits, it is also present in transducin of vertebrates. This sequence however, is conserved only partially in G alpha q from photosensory membranes of the squid. The second antiserum (anti-G alpha q) has been raised against the peptide QLNLKEYNLV. This sequence is present at the C-terminus of e.g. G alpha q from mouse brain and is identical also in squid. Anti-G alpha q very strongly interacted with the rhabdomeres of the photoreceptor cells from Sepia. In the non-rhabdomeric cytoplasm of the photoreceptor cells gold granules (bound to the second antibody) were almost absent. With anti-G alpha-common, only a small number of gold particles could be found at the rhabdomeres. The non-rhabdomeric cytoplasm was not labelled. Thus, further G-proteins that might be involved in a light dependent phototransduction mechanism besides the phosphoinositide pathway can hardly be expected to belong to the group of 'common' G-proteins.

Limits of natural science: brain research and computers.

The criterion that pure natural science can only investigate objective phenomena which can be observed by independent observers sets certain limits to our scientific understanding of brain functions. The methods and the present state of brain research and of computer development are described. The limitations of brain research are discussed by comparing the properties of brains and computers. At least for the time being we do not know of any natural scientific--i.e. physical or chemical--method which allows the objective measurement of consciousness, sensations, and emotions.

On the Ca(2+)-dependence of inositol-phospholipid-specific phospholipase C of microvillar photoreceptors from Sepia officinalis.

The breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) by the action of a phosphoinositide-specific phospholipase C is a key event in the signal transduction mechanism of microvillar photoreceptors. The enzymatic activity of phospholipase C from photoreceptor cell membranes of the cuttlefish Sepia officinalis was measured at different concentrations of free calcium. A bell-shaped curve with a maximum of enzyme activity at about 0.2 to 0.7 microM of free calcium was obtained. In the range of 10 to 90 microM free Ca2+, the activity of the phospholipase C decreased to about 30%. The activity of the phospholipase was also modulated by strontium ions. The presence of 10 microM Sr2+ strongly reduced the dependence of the phospholipase from a given calcium concentration. Similarly to 10 mM EGTA, the presence of 10 microM La3+ reduced the enzymatic activity more than ten-fold. A comparison of these values with the changes of the calcium concentration measured in ventral photoreceptor cells of Limulus polyphemus in the dark (approximately 0.5 microM Ca2+) and under illumination (up to 30-100 microM Ca2+) indicates that in microvillar photoreceptor cells an elevation of the calcium concentration in the physiological range reduces the activity of the phospholipase C. The IP3 produced by the action of phospholipase C is known to elevate the intracellular Ca2+ concentration transiently, which in turn will be able to reduce the further IP3 formation. A negative feedback in this early part of the enzyme cascade of vision in invertebrates may substantially contribute to the process of adaptation.

A newly discovered pathway of melanin formation in cultured retinal pigment epithelium of cattle.

According to a recent hypothesis the melanin granules in the retinal pigment epithelium of mammals originate from photosensory membrane degradation. To test this hypothesis the retinal pigment epithelium of cattle was kept in tissue culture and exposed to gold-labelled rod outer segments. Gold granules were later detected inside phagosomes, melanosomes and mature melanin granules. Tyrosinase, the key enzyme in melanogenesis, was additionally localized inside phagosomes. These results indicate that in cultured retinal pigment epithelium the matrix of the melanosome can originate from phagosomes. Therefore, the melanosome is a specialized lysosome.

Cardioinhibitory peptides from Limulus polyphemus: modulation of the neurogenic heart.

Four neuropeptides have been isolated and sequenced from acetone extracts of brains of the horseshoe crab Limulus polyphemus. They belong to a newly discovered peptide family in invertebrates. A possible role of the four peptides from Limulus as cardioregulatory neurotransmitters has been tested on the isolated Limulus heart. Three of the peptides (DEGHKMLYFamide, GHSLLHFamide, and PDHHMMYFamide) produce dose-dependent decreases in both amplitude and rate of the heart contractions, whereas DHGNMLYFamide reduces only the amplitude of the heartbeat. All four peptides differ in threshold, potency and duration of their effects.

Cyclic 3',5'-nucleotide phosphodiesterase: cytochemical localization in photoreceptor cells of the fly Calliphora erythrocephala.

The distribution of cyclic 3',5'-nucleotide phosphodiesterase activity was determined in photoreceptor cells of the fly Calliphora erythrocephala. With cAMP as substrate, staining was most intense within the phototransducing region of these cells, the rhabdomeral microvilli and also in the extracellular space surrounding the microvilli and in the mitochondria. With cGMP as substrate, the intensity within the rhabdomeres was less marked, while their extracellular surroundings were stained heavily. Thus, compared to cGMP, cAMP is the better substrate for the phosphodiesterase in the rhabdomeres of the fly. For comparison, the same cytochemical method was used to localize the well-known phosphodiesterase activity in retinal tissue of the mouse. Under the same conditions as used for fly photoreceptors, a very intense reaction product was obtained in rod outer segments. With regard to the conflicting reports concerning the light-stimulated changes of cyclic nucleotides in invertebrate photoreceptor cells, the results presented here further argue for an important role of a cyclic nucleotide in the process of phototransduction of invertebrates.

Disturbing GTP-binding protein function through microinjection into the visual cell of Limulus.

We have tested the action of three agents microinjected into the ventral nerve photoreceptor of Limulus on the electrical response to dim light. 1. A monoclonal antibody (mAb 4A) against the G alpha subunit of frog transducin reduces the size of the receptor current to 60%, suggesting an interaction with G alpha in the Limulus photoreceptor. 2. Injection of Clostridium botulinum ADP-ribosyltransferase C3 reduces the size to 46%; latency is not affected. The results imply that small GTP-binding proteins play a functional role in photoreception of invertebrates. 3. Injection of GDP-beta-S reduces dose-dependently the size of the receptor current to 15% and prolongs the latency to 200%, presumably by reducing number and rate of G-protein activations.

Isolation of the rhabdomeral microvillar cytoskeleton of the crayfish (Orconectes limosus) photoreceptor by a crosslinking reagent.

1. Rhabdomeral microvilli of photoreceptor cells of invertebrates contain a labile central cytoskeleton. For stabilization of the rhabdomeral cytoskeleton of the crayfish Orconectes limosus the crosslinking reagent suberic acid bis (N-hydroxysuccinimide ester) was used. 2. It was found that this crosslinking reagent can be successfully used to stabilize and isolate the microvillar cytoskeleton of crayfish photoreceptors. 3. After detergent treatment cytosolic proteins and the cell membranes were removed. 4. By the combined use of crosslinker and detergent the accessibility of antibodies or other markers to the microvillar cytoskeleton is possible. 5. This method may be useful, because at present little is known about the proteins associated with the central filament of invertebrate photoreceptors.

The light-induced rise in cytosolic calcium starts later than the receptor current of the Limulus ventral photoreceptor.

The intracellular arsenazo signal indicating the transient light-evoked change in cytosolic Ca2+ (or Sr2+) concentration was measured in Limulus ventral photoreceptors simultaneously with the receptor current under voltage clamp conditions at 15 degrees C. The latency of the light-evoked arsenazo response was consistently more than 25 msec longer than the latency of the electrical light response (receptor current or -potential). Replacing calcium by strontium in the superfusate caused, within 30-40 min, reversible changes: an enlargement of the arsenazo response and a considerable prolongation of both latencies, that of the electrical and that of the arsenazo response; the difference between the two latencies, however, stayed essentially constant.

The effect of neuropeptides on the ERG of the crayfish Orconectes limosus.

CCAP (Crustacean Cardioactive Peptide), Proctolin, FMRFamide, Met- and Leu-enkephalin, Substance P, RPCH (red pigment concentrating hormone) and PDH (pigment dispersing hormone) were applied to the isolated retina of the crayfish Orconectes limosus. Changes in light sensitivity, measured as changes of the amplitude of the electroretinogram (ERG) were observed after application of RPCH, PDH and CCAP. RPCH caused an increase of the ERG amplitude to 133% of its reference value whereas PDH and CCAP decreased the amplitude to 78% and 30% respectively. A dose-response curve showed that 10(-9) mol/l CCAP produce a half-maximal effect.