Light deprivation slows but does not prevent the loss of photoreceptors in taurine transporter knockout mice.

UNLABELLED: Taurine transporter knockout mice show severe retinal degeneration at an early age. The study was designed to determine whether degeneration also takes place in the absence of light. Mice were maintained up to 6 weeks of age in cyclic lighting or in total darkness. Degeneration took place in both groups, but was more rapid in animals exposed to standard cyclic illumination. At the ultrastructural level the retinas showed features characteristic of apoptosis but not of necrosis. CONCLUSIONS: Cell differentiation is not seriously affected by the lack of a functional taurine transporter but mature photoreceptor cells do not survive without an intact transporter, even in the dark.

Chromaffin cell function and structure is impaired in corticotropin-releasing hormone receptor type 1-null mice.

Corticotropin-releasing hormone (CRH) is both a main regulator of the hypothalamic-pituitary-adrenocortical axis and the autonomic nervous system. CRH receptor type 1 (CRHR1)-deficient mice demonstrate alterations in behavior, impaired stress responses with adrenocortical insufficiency and aberrant neuroendocrine development, but the adrenal medulla has not been analyzed in these animals. Therefore we studied the production of adrenal catecholamines, expression of the enzyme responsible for catecholamine biosynthesis neuropeptides and the ultrastructure of chromaffin cells in CRHR1 null mice. In addition we examined whether treatment of CRHR1 null mice with adrenocorticotropic hormone (ACTH) could restore function of the adrenal medulla. CRHR1 null mice received saline or ACTH, and wild-type or heterozygous mice injected with saline served as controls. Adrenal epinephrine levels in saline-treated CRHR1 null mice were 44% those of controls (P<0.001), and the phenylethanolamine N-methyltransferase (PNMT) mRNA levels in CRHR1 null mice were only 25% of controls (P <0.001). ACTH treatment increased epinephrine and PNMT mRNA level in CRHR1 null mice but failed to restore them to normal levels. Proenkephalin mRNA in both saline- and ACTH-treated CRHR1 null mice were higher than in control animals (215.8% P <0.05, 268.9% P <0.01) whereas expression of neuropeptide Y and chromogranin B did not differ. On the ultrastructural level, chromaffin cells in saline-treated CRHR1 null mice exhibited a marked depletion in epinephrine-storing secretory granules that was not completely normalized by ACTH-treatment. In conclusion, CRHR1 is required for a normal chromaffin cell structure and function and deletion of this gene is associated with a significant impairment of epinephrine biosynthesis.

[Quality control of sonography courses in advanced training of physicians: analysis of present status and potential for improvement]. / Qualitätssicherung von Ultraschallkursen in der ärztlichen Fort- und Weiterbildung: Status-quo-Analyse und Verbesserungspotential.

AIM: Since 1995 sonography represents the diagnostic imaging procedure most commonly applied (52 %) by physicians working in the public health sector. On the account of the high cost of sonography (around 2,5 billion DM in 1998) it seems justified to intensify the quality control of advanced sonography training courses for physicians. This study was set out to evaluate the learning efficiency and teaching quality of sonography training courses for doctors. We also aimed at testing, evaluating and carefully optimising modern concepts for sonography training based on recognised didactic criteria. METHODS: 34 courses in B-mode sonography and colour-Doppler sonography of 5 regional and national institutions were evaluated on the basis of standardised questionnaires by observers taking part in the courses incognito. The control group was formed by 332 physicians taking part in ten sonography courses which were structured in the way of alternating training modules with practical final exams based on OSCE criteria. With the help of modern imaging media it was intended to make these courses as illustrative as possible, including practical exercises in small groups of up to five people. Subsequent evaluation was based on standardised questionnaires. The attendance quota of the participants served as an additional comparative value for the acceptance of different teaching concepts. RESULTS: We observed significant deficits in the didactic concepts of numerous ultrasound courses offered externally: there was a high prevalence of extended frontal teaching with noticeably tiring effects as well as unstructured practical exercises in large groups which resulted in high numbers of absenteeism of the participants. The evaluation of the newly drawn up course concepts, however, demonstrated highly significant and unquestionable advantages of alternating teaching in small groups. DISCUSSION: Background information from the field of pedagogical psychology is supplied and the application of current learning theories to new concepts of ultrasound courses is demonstrated. CONCLUSION: Instructors of sonography courses need to be well trained in modern teaching methods. This study shows a variety of current concepts and provides a list of easily applicable didactic quality criteria for the conception of ultrasound courses.

[Innovative course concept for small group teaching in clinical methods. Results of a longitudinal, 2-cohort study in the setting of the medical didactic pilot project in Dusseldorf]. / Innovative Kurskonzepte für Kleingruppenpraktika in bildgebenden Verfahren. Ergebnisse einer Längsschnitt-2-Kohorten-Studie im Rahmen des medizindidaktischen Pilotprojektes Düsseldorf.

BACKGROUND AND OBJECTIVE: Medical students in Germany often criticize the anonymous and not practice-orientated way they are taught. It was the aim of this study to develop, try out and evaluate cost-effectiveness of practice-oriented interdisciplinary teaching in small groups. PATIENTS AND METHODS: The study was based on ca. 300 medical students/year, a practicable number in relation the average annual total intake by German medical schools. Between 1995 and 1998, 300 medical students annually, chosen by lot, were offered voluntary additional teaching of imaging methods (computed tomography, endoscopy and sonography), from a total of 2485 students (1276 females, 1209 males; mean age 25.8 years), the reminder serving as controls. After lecture-demonstration to all, the selected small groups were separately taught. They performed sonography on each other in small cohorts of 5 or they practised endoscopies on phantom models under supervision of a student tutor. In addition, lecturers circulated among the groups to answer additional questions and provide further help. All participants evaluated the course concept and the instructors on a questionnaire provided by the interdisciplinary centre for tertiary education. The effects of teaching on the participants were tested by a practical test in the form of an objective structured clinical examination (OSCE). RESULTS: Ca. 80% of course participants reached the intended aim and passed the OSCE examination. They gave the instructions in the pilot project highest marks, in all its criteria nearly one mark higher than those of other evaluated teaching sessions. Pre-clinical students judged the programme even more favourably than those in their clinical years. The additional cost incurred was moderately low at ca. DM 67 per year per student. CONCLUSION: Modern practice-oriented small group teaching will provide high quality results even with large numbers of students. The concept has already been taken over by other medical faculties.

Regulation of thylakoid protein phosphorylation at the substrate level: reversible light-induced conformational changes expose the phosphorylation site of the light-harvesting complex II.

Light-dependent activation of thylakoid protein phosphorylation regulates the energy distribution between photosystems I and II of oxygen-evolving photosynthetic eukaryotes as well as the turnover of photosystem II proteins. So far the only known effect of light on the phosphorylation process is the redox-dependent regulation of the membrane-bound protein kinase(s) activity via plastoquinol bound to the cytochrome bf complex and the redox state of thylakoid dithiols. By using a partially purified thylakoid protein kinase and isolated native chlorophyll (chl) a/b light-harvesting complex II (LHCII), as well as recombinant LHCII, we find that illumination of the chl-protein substrate exposes the phosphorylation site to the kinase. Light does not activate the phosphorylation of the LHCII apoprotein nor the recombinant pigment-reconstituted complex lacking the N-terminal domain that contains the phosphothreonine site. The suggested light-induced conformational change exposing the N-terminal domain of LHCII to the kinase is evidenced also by an increase in its accessibility to tryptic cleavage after light exposure. Light activates preferentially the trimeric form of LHCII, and the process is paralleled by chl fluorescence quenching. Both phenomena are slowly reversible in darkness. Light-induced exposure of the LHCII N-terminal domain to the endogenous protein kinase(s) and tryptic cleavage occurs also in thylakoid membranes. These results demonstrate that light may regulate thylakoid protein phosphorylation not only via the signal transduction chain connecting redox reactions to the protein kinase activation, but also by affecting the conformation of the chl-protein substrate.

Inhibition of collagen IV deposition promotes regeneration of injured CNS axons.

Scarring impedes axon regrowth across the lesion site and is one major extrinsic constraint to effective regeneration in the adult mammalian central nervous system. In the present study we determined whether specific biochemical or immunochemical modulation of one major component of the scar, the basal membrane (BM), would provide a means to stimulate axon regeneration in the mechanically transected postcommissural fornix of the adult rat. Basal membrane developed within the first 2 weeks after transection in spatiotemporal coincidence with the abrupt growth arrest of spontaneously regrowing axons. Local injection of anticollagen IV antibodies or alpha, alpha'-dipyridyl, an inhibitor of collagen triple helix formation and synthesis, significantly reduced lesion-induced BM deposition. This treatment allowed massive axon elongation across the lesion site. Anterograde tracing provided unequivocal evidence that regenerating axons follow their original pathway, reinnervate the appropriate target, the mammillary body, and become remyelinated with compact myelin. Presynaptic electrophysiological recordings of regenerated fibre tracts showed recovery to nearly normal conduction properties. Our results indicate that lesion-induced BM is an impediment for successful axonal regeneration and its reduction is a prerequisite and sufficient condition for regrowing axons to cross the lesion site.

Effects of perinatal diazepam exposure on the liver of rats.

Diazepam is routinely used in obstetrics in the treatment of maternal eclampsia or as anesthetic induction during deliveries or cesarean sections. We investigated here the effects of perinatal diazepam treatment on the liver of Sprague Dawley rats. From 15th day of gestation to birth pregnant rats were treated daily in the first experimental group (DZ1) with 2.5 mg DZ/kg b.w., s.c., the second (DZ2) with 5 and the third (DZ3) with 10 mg DZ/kg b.w. After birth respective treatment was continued to the pups till 5th day postnatal. One half of pups from DZ2 and control was sacrificed at 12th day, the other were raised to 120th day and then sacrificed. After fixation in formaldehyde serial paraffin sections were processed with proliferating cell nuclear antigen (PCNA), autofluorescence of lipofuscin and Elastica v. Gieson staining. Livers from 12 day old rats showed increased levels of PCNA marked hepatocytes and lipofuscin containing cells. Livers from 120 day old animals exhibited increased levels of PCNA cells especially with weakly stained nuclei, the number of lipofuscin containing cells were decreased in DZ1 and DZ2 but reached in DZ3 the similar number as control. Statistical analysis showed that quantification of Kupffer cells lacked significance. To conclude livers of 12 day old animals demonstrated a stimulated DNA synthesis and an accumulation of nondegradable residues. Livers from 120 day old rats demonstrated an increase in number of potentially proliferating hepatocytes by increased levels of weakly stained PCNA nuclei. At lower dosage DZ seems to inhibit lipofuscinogenesis, at highest dosage DZ accumulates non-degradable residues. Elastica v. Gieson staining was not suitable to quantify Kupffer cells with certainty.

Topographical distribution of lactate dehydrogenase activity in human clear eye lenses and in lenses with different types of senile cataract: a histochemical investigation.

BACKGROUND: Homogenates of human clear lenses show an age-dependent reduction of enzyme activities. Topographical patterns of enzymes in clear and cataractous lenses can be visualized by histochemistry. MATERIAL AND METHODS: Human lenses were characterized by slit-lamp investigations as bearing different types of senile cataracts. Subsequently, lenses were removed by intracapsular extraction. Clear human lenses served as controls. Bovine lenses served to standardize freeze-cutting and incubation for lactate dehydrogenase histochemistry. RESULTS: Bovine lenses show a sharp demarcation between the enzyme reaction of cortical fibers bearing cell nuclei and the non-reacting deeper fibers not exhibiting cell nuclei. Clear human lenses, lenses with deep supranuclear cortical cataracts, and lenses with nuclear cataracts exhibit the same borderline. However, in lenses with a subcapsular cortical cataract only the epithelium and a very thin layer of the most superficially located fibers show positive enzyme reactions. CONCLUSION: In growing clear human and bovine lenses, independent of age, the more peripherally located cortical fibers bearing cell nuclei exhibit strong enzyme-histochemical reactions. More centrally located lens areas lacking cell nuclei increase in volume in an age-dependent manner. These lens regions do not exhibit enzyme activities detectable by our histochemical technique. Therefore the lens areas free of histochemical reaction product become larger with increasing age, whereas the peripherally located lens fibers apparently do not change their enzyme activities with age. Thus, homogenates of total lenses show age-dependent reductions of enzyme activities, although enzyme activities remain at a physiological level in cortical lens fibers with recognizable cell nuclei. In lenses with immature supranuclear cortical and (particularly) in lenses with black nuclear cataracts, cortical fibers still can exhibit high enzyme activities. Unexpectedly, also ruptured and broken fibers in immature deep supranuclear cortical cataracts show strong enzyme activities. In contrast, in lenses with (incipient) subcapsular cortical cataracts only the most superficially located lens fibers exhibit some enzyme activity.

Light and electron microscopy of S-antigen- and opsin-immunoreactive photoreceptors in the retina of turtle, chicken, and hedgehog.

S-antigen and opsin-immunoreactive sites were studied in the retina of turtle (Pseudemys scripta elegans), chicken (Gallus domesticus), and hedgehog, (Erinaceus roumanicus) by light-microscopic avidin-biotin-peroxidase and electron-microscopic protein A-gold and immunoglobulin-gold (immunogold) techniques. In general, S-antigen and opsin immunoreactions were exclusively localized to certain photoreceptors of the retinae studied. In turtle and chicken, strong S-antigen immunoreactivity was found in the outer and inner segments, perikarya and synaptic bases of rod photoreceptors. Some--apparently green--single cones also exhibited immunoreactive outer and inner segments. In hedgehog, the inner segments, perikarya and pedicles of rods displayed immunoreaction of varying intensities. In all three species there was an accumulation of immunogold particles on the distal portion of the inner segment, the connecting piece and the proximal portion of the outer segment. Opsin immunoreactivity was found in the outer segments of rod photoreceptors, and in those of--apparently green--single cones of turtle and chicken. The localization and significance of the S-antigen immunoreaction is discussed in connection with the occurrence of different photopigments in the retinae studied. The correlation of the presence of S-antigen and of either rhodopsin or porphyropsin in rod and--apparently green--single cone photoreceptors was emphasized.

Retinal S-antigen: immunocytochemical and immunochemical studies on distribution in animal photoreceptors and pineal organs.

Antiserum against bovine retinal S-antigen, a soluble protein (MW = 50 kDa) thought to be involved in phototransduction, was used in an immunohistochemical and immunochemical study of vertebrate eyes and pineal systems and invertebrate photoreceptor organs. Positive reactions, not seen with antiserum preabsorbed with highly purified S-antigen, were observed in planarian and starfish ocelli; scallop eyes; polychaete eye; crayfish compound eye; lamprey, salmon, frog, turtle, quail and hamster eyes. A specific reaction was also seen in the pineal organ of all the vertebrates examined, albeit weak in turtle and quail. In addition, several structures associated with photoreceptor organs, including the reduced frontal eyes of crayfish, the organ of Bellonci in crayfish eyestalk, and bipolar cells resembling those giving rise to Landolt's clubs in quail and golden hamster retinae, were immunopositive. Immunochemical studies revealed the presence of a single immunopositive band of protein which was similar but not identical in size in all vertebrate eyes and pineal organs (except that of chicken pineal) and invertebrate tissue examined. The wide distribution of positive reaction in photoreceptive tissue indicates that the retinal S-antigen determinant has been highly conserved during evolution.

Cyclic renewal of whole pineal photoreceptor outer segments.

In the light-sensitive epiphysis cerebri of adult frogs (Rana esculenta) adapted in summer to an artificial long day (L:D = 17:7; L = 90 lx at 2,300 degrees K; controlled temperature of 8 degrees C) outer segments of cone-like pineal photoreceptor cells exhibit a cyclic and complete renewal. Shortly after 'lights off' inner segments protruding into the pineal lumen bear exclusively bulbous cilia of the sensory type. Considering these results, concepts dealing with different types of sensory pinealocytes have to be re-evaluated.

Progress in microfluorometric identification of monoamine fluorophores.

Techniques for the microfluorometric identification of monoamine fluorophores have been put forward along two different lines: (1) extension of the excitation range into the far UV and (2) computer-assisted on-line scanning recordings in combination with an automatic microfluorometric identification of fluorophores. By an extension of the excitation range to 250 nm it is possible to distinguish fluorophores of DOPA from those of dopamine, fluorophores of noradrenaline from those of adrenaline, and fluorophores of 5-OH-tryptophan from those of 5-OH-tryptamine. In a computer-assisted on-line scanning procedure using a high-aperture inverted illuminating microscope equipped with UV transmitting optics the specific formaldehyde-induced catecholamine fluorescence is characterized microfluorometrically by corrected ratios of excitation maxima (370:320 nm and 320:275 nm). Excitation data are plotted out with corresponding coordinates in the x- and in the y-axis. Furthermore the digitized values of fluorescence intensities obtained with the scanning procedure are processed according to principles of automatic image-analysis procedures.

Cytometric investigations in histochemically identified neurons--changes of fluorescence intensities and nuclear diameters in dopamine neurons of the arcuate nucleus in the cycling rat.

The relative formaldehyde-induced fluorescence intensities and the diameter of cell nuclei of fluorescent perikarya of the arcuate nucleus were recorded in serial cross-sectioned hypothalamic arcuate nuclei of regularly cycling rats (5 estrous and 4 proestrous animals). Stress-induced changes of cytometric parameters were avoided by preadaptation of animals to handling procedures. Dopamine neurons in a 75 micrometer thick periventricular layer of the arcuate nucleus exhibited significantly smaller nerve cell nuclei and significantly reduced relative fluorescence intensities in proestrous rats. Both of these cytometric parameters indicate a decrease in the activity of periventricular dopamine neurons of the arcuate nucleus. The reported findings might support the hypothesis that dopamine inhibits the release of LH-RH.

Aminergic systems in pulmonate gastropod molluscs. III. Microspectrofluorometric characterization of the monoamines in the reproductive system.

Histochemical fluorescence (Falck-Hillarp) and microspectrofluorometric (MSF) methods were used to characterize different types of catecholamine-containing cellular elements located in the reproductive system of fresh-water snails (Bulinus truncatus, Planorbarius corneus) and land snails [Archachatina marginata, Helix (Cryptomphalus) aspersa]. Transverse sections through the genital tract display a common structural pattern of tubular differentiations: (1) an internal epithelium bordering the lumen and containing variable numbers of monoaminergic cells; (2) an enveloping sheath of connective and muscular tissue containing fine nerve fibers in the form of a network that exhibits a variable degree of density. MSF determinations showed that the formaldehyde-induced fluorophores of the intraepithelial aminergic cells belong to the following classes: (1) the DOPA/dopamine group in the duct of the albumen gland of B. truncatus and the carrefour of A. marginata; and (2) the norepinephrine/epinephrine group in the duct of the albumen gland and in the oviduct sac of P. corneus. In the reproductive systems of B. truncatus and P. corneus (duct of the albumen gland, oviduct sac, vagina), A. marginata and H. aspersa (duct of the fertilization pocket, origin of the receptaculum seminis, carrefour), the MSF analysis revealed norepinephrine/epinephrine-containing intramural nerve fibers. On the other hand, the small neurons in the vagina of B. truncatus belong to the DOPA/dopamine group.

Indoleamines in the pineal complex of Lampetra planeri (Petromyzontidae). A fluorescence microscopic and microspectrofluorimetric study.

Using the technique of Falck and Hillarp, the pineal and parapineal organs of Lampetra planeri were found to exhibit a labile yellow fluorescence characteristic of indoleamines. Microspectrofluorimetric analysis of the yellow fluorophores yielded emission and excitation spectra very similar to those of the melatonin precursors serotonin (5-HT) or 5-hydroxytryptophan (5-HTP). 5-HT/5-HTP fluorophores are concentrated essentially in the region of the atrium, along the pineal tract and in the rostral part of the parapineal organ. The fluorescence is strong in early autumn, decreases in winter and is weak or absent in spring, implying a seasonal variation in indoleamine metabolism. Fluorescence intensity increases after injections of nialamide or nialamide and 5-HTP and is lowered by injection of reserpine. Indoleamine fluorescence is localized in the recently identified sensory-type cells: the photoneuroendocrine cells (Meiniel, 1980). The ependymal-like cells, the sensory neurons and the classical photoreceptor cells (Collin, 1969 a) do not appear to be involved in indole metabolism. The absence of catecholamine fibres in the perivascular spaces suggests that indole biosynthesis is regulated via the direct photosensitivity of the pineal complex.

Distribution patterns of formaldehyde-induced fluorescence of hypothalamic monoamines.

In a circumscribed area of the preoptic periventricular nucleus of a male rat, formaldehyde-induced monoamine fluorophores modified by treatment with HCl vapors were investigated microfluorometrically (measurement of excitation peak ratio 370 : 320 nm) in all fluorescent terminals and preterminals. Microfluorometric recordings of an individual fluorescent structure were performed without UV irradiation of neighboring fluorophores. Recorded data were sampled and corrected by a microcomputer (Wang PCS II). 19 neuronal processes (axons) contained noradrenaline fluorophores; 11 contained dopamine fluorophores; 6 exhibited uncharacteristic excitation peak ratios; and in 19 recordings technical problems did not allow identification of the fluorophore content.