A human schwannoma cell line supports the in vitro adhesion of Plasmodium falciparum infected erythrocytes to chondroitin-4-sulfate.

The paucity of human cell lines expressing defined receptors for the cytoadhesion of erythrocytes infected with the human malarial parasite Plasmodium falciparumhas hampered the investigation of this important virulence property. Here, we investigate a permanent cell line derived from a human, malignant schwannoma, termed HMS-97, and show that this cell line expresses chondroitin-4-sulfate as the only surface receptor to which P. falciparum-infected erythrocytes can cytoadhere. Other common receptors for parasite adhesion, including CD36, vascular cellular adhesion molecule-1 (VCAM), intercellular adhesion molecule-1 (ICAM-1), and E-selectin are absent. Thus, HMS-97 cells are a useful tool for the study of P. falciparum adhesion to chondoitin-4-sulfate, the main receptor for parasite sequestration in the placenta. As chondoitin-4-sulfate can be readily cleaved from the cells, HMS-97 cells are also an ideal system for expressing recombinant adhesion receptors and studying their function in binding assays.

A case of neuroendocrine oncogenic osteomalacia associated with a PHEX and fibroblast growth factor-23 expressing sinusidal malignant schwannoma.

Oncogenic osteomalacia is a rare paraneoplastic syndrome that is characterized biochemically by hypophosphatemia and low plasma 1,25-dihydroxyvitamin D3, and clinically by osteomalacia, pseudofractures, bone pain, fatigue, and muscle weakness. We present a patient with a malignant schwannoma as the underlying cause of this disorder. A permanent cell line (HMS-97) derived from this tumor showed evidence of neuroendocrine differentiation by immunohistochemistry and of neurosecretory activity by electron microscopy. The cell line did express PHEX (phosphate-regulating gene with homologies to endopeptidases located on the X-chromosome) and FGF-23 (fibroblast growth factor-23) transcripts on northern hybridization; however, none of the known mutations from the related mendelian disorders of X-linked hypophosphatemic rickets or autosomal-dominant hypophosphatemic rickets could be detected. Tumor cell (HMS-97)-derived conditioned medium did not inhibit phosphate transport in a standard opossum kidney cell assay and in animal experiments. The medium also showed no PTH1- or PTH2-receptor-stimulating bioactivity. HMS-97 cells might be useful for further studies that aim to determine the genetic mechanism that leads to the observed PHEX and FGF-23 expression, both of which might have a direct role in the pathogenesis of oncogenic osteomalacia. In addition, these cells might be a useful tool for the investigation of neuroendocrine Schwann cell function and autoimmune peripheral nerve disease.

Changing serine-485 to alanine in the opossum parathyroid hormone (PTH)/PTH-related peptide receptor enhances PTH stimulation of phospholipase C in a stably transfected human kidney cell line: a useful model for PTH-analog screening?

Using site-directed mutagenesis, we have introduced a serine-485-to-alanine mutation in the opossum parathyroid hormone (PTH) receptor. This amino acid is considered to be phosphorylated by protein kinase A upon ligand binding. Both wild-type (WT) and mutant receptor were stably expressed in 293-EBNA HEK cells. The mutant receptor showed comparable binding characteristics and only a slight increase in cAMP production compared with WT. However, the PTH dose-dependent increase in inositol phosphate production was 24-fold for the mutant receptor vs. 6-fold for the WT receptor. This mutant might prove useful in the sensitive detection of phospholipase C activation through various ligands, as the PTH receptor becomes a target of therapeutic intervention in osteoporosis.

Differential induction of proto-oncogene expression and cell death in ocular tissues following ultraviolet irradiation of the rat eye.

BACKGROUND/AIMS: Ultraviolet (UV) irradiation of mammalian cells in culture evokes the transcriptional activation of different proto-oncogenes, among them members of the fos/jun family which are known to play an important role in cell proliferation and differentiation. To investigate in vivo UV induced proto-oncogene expression of irradiated ocular cells, the expression of JunB, JunD, and Egr-1 was analysed in the cornea, lens, and retina. Furthermore, UV radiation is known to induce pleiotrophic events in irradiated cells which include growth arrest, inflammation, and even cell death. In order to determine the type of cell death--for example, apoptosis versus necrosis, sections of UV irradiated rat eyes were further examined for distinct ultrastructural morphology of cell death and DNA fragmentation. METHODS: Eyes of anaesthetised rats were exposed to 1.5 J/cm2 of ultraviolet radiation (280-380 nm). Animals were perfused 6 and 16 hours after irradiation and tissue sections of enucleated bulbi were processed for light and electron microscopy. RESULTS: Under control conditions, Jun B was constitutively expressed in numerous superficial cells but also in scattered basal cells of the corneal epithelium. After UV exposure JunB expression was massively upregulated in many cells of the basal cell layers of the corneal epithelium, although during the entire experiment, both the corneal stroma and endothelium were JunB negative. In contrast, Egr-1 was expressed exclusively in lens epithelium showing only a faint expression pattern under control conditions. However, Egr-1 expression increased after UV exposure, so that many Egr-1 positive cells of the lens epithelium could be found several hours after UV illumination. JunD was expressed in single cells of both the ganglion cell layer and the inner nuclear layer of the retina, a pattern of expression which did not change after UV exposure. Regarding the type of cell death, features of apoptosis were only occasionally present in scattered superficial cells of the corneal epithelium of control eyes. After UV exposure, however, morphological signs of apoptosis and TUNEL positive cells were visible both in the stroma and epithelium of the rat cornea. In contrast, UV irradiated lens epithelial cells exhibited features typical of necrosis. The corneal endothelium and the retina did not show any indications of morphological changes indicative of cell death after UV irradiation. CONCLUSION: Each proto-oncogene encoded protein was found to be expressed in a tissue specific manner and UV irradiation differentially modulates the expression pattern of these transcriptional regulatory proteins. This temporospatial expression pattern of these proteins is accompanied by two morphologically distinct types of cell death in the cornea and lens after UV irradiation.

Expression pattern of candidate cell death effector proteins Bax, Bcl-2, Bcl-X, and c-Jun in sensory and motor neurons following sciatic nerve transection in the rat.

Numerous studies have demonstrated a prolonged expression of c-Jun transcription factor in neurons following axotomy, and it has been hypothesized that c-Jun may be causally involved in neuroregeneration in vivo. By contrast, there is growing evidence from in vitro studies that induction of c-Jun may be necessary for neuronal cell death induced by growth factor starvation. It has been demonstrated that protein levels of cell death repressor Bcl-2 and cell death promotor Bax determine the threshold for neuronal cell death and that their expression is dynamically modulated at the onset of neurodegeneration. In the present study, we investigated by double-immunolabeling methods activation of c-Jun transcription factor and expression of members of the Bcl-2 family of cell death effector proteins in axotomized neurons. Six days after transection of the sciatic nerve in young rats, when axotomized neurons start to degenerate, strong nuclear Jun immunostaining in spinal cord motoneurons was associated with intense cytoplasmic Bax labeling and signs of neuronal atrophy. Bcl-2 and Bcl-X proteins were present only at moderate to low levels. In situ end-labeling by terminal transferase revealed nuclear DNA fragmentation in scattered motoneurons of the ipsilateral ventral horn (1 or 2 labeled nuclei per section). In the L5 dorsal root ganglia (DRG) levels of Bax, Bcl-2, and Bcl-X proteins were highly variable. High levels of Bax immunoreactivity together with intense Jun immunofluorescence were frequently observed in small-diameter sensory neurons. RT-PCR analysis revealed expression of exclusively the anti-apoptotic bcl-xL mRNA isoform in rat DRG which decreased significantly following sciatic nerve transection. These findings indicate that the high susceptibility of central neurons and small-sized DRG neurons to axotomy-induced cell death might be related to their low ratio of cell death repressor Bcl-2 and Bcl-XL to cell death promotor Bax expression. It should be noted, however, that numerous strongly Jun-positive DRG neurons contained low levels of Bax or high levels of Bcl-2 and Bcl-X immunoreactivity. Thus, high levels of c-Jun protein in axotomized neurons do not necessarily suggest a destination to die, and other factors may determine the outcome of axotomy.

Increase in bax expression in substantia nigra following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treatment of mice.

Administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to mammals causes damage to the nigrostriatal pathway similar to that observed in Parkinson's disease. In the present study, we have investigated alterations in cell death effector gene expression induced by the neurotoxin MPTP in mouse substantia nigra. Intraperitoneal MPTP injections in mice resulted in a significant increase in bax mRNA by about two- and three-fold after 3 and 6 days, respectively. The up-regulation of bax mRNA was associated with concomitant increase in Bax immunoreactivity observed mainly in large- and medium-sized neurons in the substantia nigra that are destined to die. Our results indicate a pathophysiological significance of bax, which promotes programmed cell death, in MPTP neurotoxicity.

Systemic application of pyrethroid insecticides evokes differential expression of c-Fos and c-Jun proteins in rat brain.

Expression of the c-Fos and c-Jun transcription factor was investigated by immunocytochemistry in the thalamus, hypothalamus, hippocampus and cortex of adult rats following intraperitoneal application of proconvulsant doses of the pyrethroid insecticides, cypermethrin and permethrin. Pyrethroid insecticides are used world-wide and their uptake, e.g., by nutrition and inhalation evokes severe neurological symptoms in animals and humans, but their effects on neuronal gene expression has not been elucidated. Cypermethrin induced a strong expression of c-Fos and c-Jun in all the thalamic nuclei, except the ventro-posterior complex and substantia nigra, and in all the hypothalamic nuclei. In general, the immunoreactivities (IR) persisted for 8 h on their maximal levels, and were still above control levels after 24 h in several thalamic and hypothalamic areas. c-Fos-IR was strongly increased in all cortical layers with a predominance in the superficial layers II-IV, whereas c-Jun-IR was only slightly increased. In the hippocampus, cypermethrin induced a weak expression of c-Fos, but not of c-Jun, in the dentate gyrus and CA-3 area. Permethrin that has a lower pharmacological potency, evoked a similar pattern of c-Fos and c-Jun expression, however, intensity and persistence of the neuronal labeling were less pronounced. Our results demonstrate that the neurotoxic effects of pyrethroid insecticides comprise molecular genetic alterations in the brain such as early and lasting induction of Fos and Jun transcription factor proteins. These changes in the neuronal program are prominent in the hypothalamus and thalamus that are involved in the regulation of the autonomic and visceral nervous systems.

Differential regulation of bcl-2, bax, c-fos, junB, and krox-24 expression in the cerebellum of Purkinje cell degeneration mutant mice.

Purkinje cell degeneration (pcd) is an autosomal recessive mutation in the mouse characterized by an almost complete loss of cerebellar Purkinje neurons between postnatal days 22 and 28. The pcd gene has not been identified, however, a relationship between activation of specific genes and cell death has been suggested in other models of neuronal cell death. In the present study we analyzed the expression of several candidate cell death effector genes (bax, c-fos, junB, krox-24) and a cell death repressor gene (bcl-2) in the cerebellum of pcd homozygotes and wild-type mice. At postnatal day 22, when Purkinje cells start to degenerate, levels of c-fos, junB, and krox-24 mRNA increased about 5-fold in mutants. To the contrary, the amount of bcl-2 mRNA declined and bax transcripts remained unchanged compared to wild-type animals. Immunoreactivity for c-Fos and Jun could be detected exclusively in cerebellar Purkinje neurons of pcd mice but not in wild-types, whereas the number of Bcl-2 immunopositive Purkinje cells decreased significantly in mutants. Both double labeling experiments and immunostaining of consecutive sections revealed lack of colocalization of Jun with Bcl-2. These results demonstrate an induction of members of the fos and jun family and a downregulation of antiapoptotic bcl-2 in cerebellar Purkinje neurons that are destined to die. Fos and Jun transcription factor proteins may be implicated in the regulation of bcl-2 expression and in the signal cascade leading to Purkinje cell death.

Up-regulation of bax and down-regulation of bcl-2 is associated with kainate-induced apoptosis in mouse brain.

Systemic administration of kainate induces cell death in vulnerable regions of the rodent brain. Neuronal degeneration is associated with internucleosomal DNA fragmentation and induction of presumptive cell death effector genes (e.g. p53, c-fos) suggesting that kainate activates an apoptotic pathway. In the present study, kainate-induced DNA damage has been demonstrated at the cellular level by in situ nick translation in the mouse hippocampus and neocortex at 24 h and 48 h after intraperitoneal injections. In the same regions, the intensity of Bcl-2 immunoreactivity decreased by about 45% as measured by digital image analysis. Most important, kainate treatment evoked a nearly 3-fold increase in bax mRNA levels within the mouse brain. The down-regulation of bcl-2, which promotes cell survival, and the up-regulation of bax, which promotes programmed cell death, may have functional significance in kainate-mediated excitotoxicity and in the selective vulnerability of specific brain regions.

Differential expression of bcl-2 and bax mRNA in axotomized dorsal root ganglia of young and adult rats.

Bcl-2 and Bax have recently been identified as putative repressor and effector proteins respectively, in the cell death program of growth factor-deprived haematopoietic cell lines. Overexpression of bcl-2 in neuronal cell culture prevents apoptosis induced by removal of neurotrophic factors. In the present in vivo study the expression of bcl-2 and bax mRNA has been investigated in dorsal root ganglia of young and adult rats using polymerase chain reaction. A high constitutive expression was observed for both genes in control ganglia. Unilateral transection of the sciatic nerve led to a dramatic decrease in bcl-2 mRNA levels in ganglia of young animals within 5 days following nerve lesion and a partial recovery thereafter. In contrast, the decline in bcl-2 mRNA was much less pronounced in axotomized ganglia of adults. The amount of bax transcripts did not change significantly in ganglia of both young and adult rats up to 20 days after nerve injury. The decrease in bcl-2 expression in dorsal root ganglia may be part of the molecular mechanism leading to neuronal cell death after axotomy-induced deprivation of neurotrophic factors. The age-dependent decline in the ratio of bcl-2 to bax gene products may explain the greater susceptibility of immature neurons to apoptosis.

[In vivo autofluorescence. Measurements of human crystalline lenses with cataract and normal findings after excitation with monochromatic light]. / In-vivo-Eigenfluoreszenz. Messungen von menschlichen Augenlinsen mit Katarakt und Normalbefund bei Anregung mit monochromatischem Licht.

In 25 eyes with nuclear cataract, 18 eyes with posterior subcapsular cataract, 25 eyes with cortical cataract, and 23 eyes without any pathological lens changes, the maximal fluorescence intensity was determined after excitation with monochromatic light at 365 nm, 405 nm, 436 nm, and 485 nm. The coefficient of variation was smaller than 5%. All eyes with cataract underwent cataract surgery a few days after the fluorescence measurements. The fluorescence spectrometer, especially constructed for in vivo measurements, consists of a modified slit lamp (Zeiss 75 SL) and an optical multichannel analyser (OMA) for gauging the data. The clinical trial was undertaken to determine whether, considering the influence of age, there is a difference between the fluorescence intensities in eyes with the above named cataracts and noncataractous eyes. The data were analyzed to determine the effect of age upon fluorescence intensity for all excitation wavelengths in both cataractous and noncataractous eyes. Age had an influence on the fluorescence intensities for all four excitation wavelengths. Assuming that the influence of age was not dependent on the state of the lens, it was quantified for all measurements and an "age-corrected" fluorescence intensity was calculated. The statistical analyses of these "age-corrected" fluorescence intensities revealed a significant difference (P < 0.001) for all of the types of cataracts examined and for normal eyes. The cataract types examined and the normal eyes showed differences in their fluorescence feature. To assess the fluorescence intensities obtained after excitation with the wavelengths mentioned above, one must take into consideration the influence of age on the measurements.

[Absorption and fluorescence spectrometry measurement of intraocular lenses]. / Absorptions- und fluoreszenzspektrometrische Messungen an Intraokularlinsen.

Light absorption and fluorescence measurements can be used to detect accumulations of small molecules in intraocular lenses (IOLs). It was shown that HEMA IOLs can store and to some extent release a variety of drugs and fluorescein. PMMA and silicone IOLs, however, do not have this characteristic. UV-blocking IOLs absorb UV light completely. The absorption limit varies between 390 and 410 nm, depending on the make. Neither absorption nor fluorescence response were affected by exposure to UV light or heat.

[Natural fluorescence of nuclear cataract lenses and melanomas of the eye]. / Natürliche Fluoreszenz von Kern-Katarakt-Linsen und von Melanomen am Auge.

For various reasons, it is desirable to have an objective cataract classification system that does not depend on either the opinion of the physician or the patient. Since both highly molecular protein aggregates and chromophores are formed in cataractous lenses, which fluoresce under suitable stimulation, we have developed a fluorescence apparatus that illuminates the lens with five monochromatic wavelengths between 350 and 500 nm in situ. The fluorescence spectrum is recorded up to 100 ms with an optical multi-channel analyzer. This fluorescence method is non-invasive and does not harm the eye; it also uses the native fluorophore. The set of fluorescence spectra obtained by this method describes the condition of the cataractous lens both quantitatively and qualitatively. Because of the sensitivity of the method, it can also be used to assess the disease objectively in the early stages and to test and determine the effectiveness of anti-cataract drugs. Another use was discovered by using the fluorescence measurements for the diagnosis of malignant melanomas. With this equipment, melanomas in the eye and the surrounding area can be diagnosed both rapidly and precisely.