Cordula R. Malz

Enzymehistochemical demonstration of nitric oxide synthase in the diencephalon of the rainbow trout (Oncorhynchus mickiss)

Nitric oxide, a free radical, has recently been shown to exert major influences on CNS functions in mammals. It is synthesized by NO-synthase. For the first time, this study reveals this enzymes existence in the CNS of a teleost fish and describes its distribution in the diencephalon, where the paraventricular organ displays an extraordinarily high activity. The study contributes to an evolutionary perspective of the biological role played by nitric oxide in the vertebrate CNS, and raises questions regarding the significance of this gas in cerebrospinal fluid-contacting neurons.

Antigenic compartmentation of the primate and tree shrew cerebellum: a common topography of zebrin II in Macaca mulatta and Tupaia belangeri

Despite the apparent uniformity in cellular composition of the adult cerebellar cortex, functional, anatomical, mutational and molecular maps all reveal a complex topography underlying the relatively simple architecture. In particular, zebrin II, a polypeptide antigen identified as aldolase C, is restricted to a subset of Purkinje cells that form a symmetrical and reproducible array of zones and stripes. The vermis of the well‐studied rodent cerebellar cortex is divided into four transverse zones – anterior (∼lobules I–V), central (∼lobules VI and VII), posterior (∼lobule VIII) and nodular (∼lobules IX and X). Each transverse zone is further subdivided mediolaterally into parasagittal stripes. To gain insight into the evolution of cerebellar compartmentation, the pattern of zebrin II expression has been compared between the primate Macaca mulatta and the tree shrew Tupaia belangeri, and the results related to previous findings from other species. Although the somata of most Purkinje cells in the Macaca cerebellum express zebrin II, parasagittal stripes can still be delineated owing to the alternating high and low zebrin II immunoreactivity in the dendrites. In the macaque vermis, a complex set of zebrin II parasagittal compartments is found in all transverse zones. Unlike in rodents, in which uniform expression domains interrupt heterogeneous zones, zebrin II parasagittal stripes in the macaque cerebellum are seen throughout the vermis. In Tupaia, the parasagittal pattern of zebrin II expression also reveals a striking array of stripes in all lobules. The data suggest that cerebellar compartmentation in Tupaia belangeri more closely resembles that of primates than it does rodents or lagomorphs.

Prolyl Hydroxylase Domain (PHD) 2 Affects Cell Migration and F-actin Formation via RhoA/Rho-associated Kinase-dependent Cofilin Phosphorylation

Cells are responding to hypoxia via prolyl-4-hydroxylase domain (PHD) enzymes, which are responsible for oxygen-dependent hydroxylation of the hypoxia-inducible factor (HIF)-1α subunit. To gain further insight into PHD function, we generated knockdown cell models for the PHD2 isoform, which is the main isoform regulating HIF-1α hydroxylation and thus stability in normoxia. Induction of a PHD2 knockdown in tetracycline-inducible HeLa PHD2 knockdown cells resulted in increased F-actin formation as detected by phalloidin staining. A similar effect could be observed in the stably transfected PHD2 knockdown cell clones 1B6 and 3B7. F-actin is at least in part responsible for shaping cell morphology as well as regulating cell migration. Cell migration was impaired significantly as a consequence of PHD2 knockdown in a scratch assay. Mechanistically, PHD2 knockdown resulted in activation of the RhoA (Ras homolog gene family member A)/Rho-associated kinase pathway with subsequent phosphorylation of cofilin. Because cofilin phosphorylation impairs its actin-severing function, this may explain the F-actin phenotype, thereby providing a functional link between PHD2-dependent signaling and cell motility.

Pattern of calretinin immunoreactivity in the main olfactory system and the vomeronasal system of the tree shrew, Tupaia belangeri

The distribution of the calcium‐binding protein calretinin was studied in peripheral and central parts of the main olfactory system (MOS) and the vomeronasal system ( VNS ) of adult tree shrew Tupaia belangeri. The calretinin immunoreaction was carried out with a peroxidase‐coupled polyclonal antibody. In the VNS, complete labeling of all receptor cells and vomeronasal nerve fibers was observed, whereas only a subset of the somata and dendrites of receptor cells and of the olfactory nerve fibers of the MOS was immunoreactive. From the immunoreactive dendritic clubs of vomeronasal receptor cells, calretinin‐labeled structures, presumably clumps of microvilli, arose that terminated within immunopositive portions of the mucus. In the main olfactory bulb, the neuropil of some of the glomeruli was immunoreactive. All periglomerular and many mitral cells were labeled. The external plexiform layer was subdivided into a faintly immunoreactive superficial half and a strongly immunoreactive deep half. Immunoreactive basal dendrites of mitral cells could be followed into either the deep half or the superficial half. In the laminated internal granular layer, a subset of immunopositive granule cells extended dendrites into the external plexiform layer. Mitral cells and granule cells with dendrites ascending to different levels of the external plexiform layer may represent functional subclasses. In the accessory olfactory bulb, all vomeronasal nerve fibers, glomeruli, and mitral/tufted cells were labeled, whereas immunoreactive periglomerular cells and internal granule cells were only scattered. In Tupaia, calretinin immunoreactivity is a more general property of the primary projecting neurons of the VNS than of the MOS and possibly indicates the involvement of calretinin in the perception of certain of the olfactory qualities. J. Comp. Neurol. 420:428–436, 2000.

Nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase activity in the brain of a cichlid fish, with remarkable findings in the entopeduncular nucleus: a histochemical study.

In the entire brain of the African cichlid fish, Haplochromis burtoni, nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) activity was visualized histochemically in fewer nuclei compared to other teleost fish. Intensively labeled perikarya were found in the ventral hypothalamic area, the nucleus of the medial longitudinal fascicle, the nucleus of the midbrain tegmentum, the nucleus of the lateral longitudinal fascicle, the trigeminal motor nucleus and the octavolateral area. Compared to other NADPH-d labeled nuclei in the brain, we saw an unusual localization of NADPH-d activity in the rostral, dorsal, ventral and caudal part of the entopeduncular nucleus. Additionally, some isolated perikarya of different morphological appearance were found at the levels of the preglomerular nucleus, the diffuse nucleus of the lateral torus and the lateral longitudinal fascicle. A widespread distribution of labeled fibers was identified throughout the brain. The remarkable NADPH-d activity, particularly in the entopeduncular nucleus, differs significantly from the existing data on other teleosts. Taking into account the sensory functions of the entopeduncular nucleus described in other vertebrates, the massive NADPH-d activity in this nucleus may indicate an important role of NADPH-d in the modulation of sensory functions.

Localization of calcium-binding protein (calretinin, 29 kD) in the brain and pituitary gland of teleost fish: an immunohistochemical study.

Immunocytochemical techniques were used to investigate the distribution of calretinin in the brain and pituitary gland of the hardhead catfish Arius felis. Calretinin immunoreactive neurons were found in the telencephalon (lateral nucleus of ventral telencephalic area), diencephalon (around the medial forebrain bundle, lateral tuberal nucleus, central pretectal nucleus, posterior periventricular hypothalamic nucleus, medial preglomerular nucleus, diffuse nucleus of the inferior lobe), mesencephalon (nucleus of the medial longitudinal fascicle, ventral nucleus of the semicircular torus), cerebellum (valvula cerebelli, eurydendroid cells) and rhombencephalon (secondary gustatory nucleus, isthmic nucleus, trigeminal motor nucleus, medial auditory nucleus of the medulla, medial and inferior reticular formation, anterior, descending, posterior and tangential octaval nuclei). Calretinin-labeled fibers were observed in the optic nerve and at the levels of the central pretectal nucleus, the nucleus of the medial longitudinal fascicle, the ventral nucleus of the semicircular torus, the secondary gustatory nucleus, the trigeminal motor nucleus, the eurydendroid cells, the medial auditory nucleus of the medulla and the octaval nucleus. For the first time, we are reporting on calretinin-positive cells in the rostral and proximal pars distalis of the adenohypophysis. Although, it seems speculatory, calretinin-expressing cells in the pituitary gland may be involved in hormonal regulation and hence, calretinin might play a significant role in governing hypophysial functions in fishes. Our results suggest that calretinin shows species-specific variations also among the teleost fish, similar to mammals.

Centrifugal Phe-Met-Arg-Phe-NH2-like immunoreactive innervation of the retina in a non-teleost bony fish, Lepisosteus osseus

In all teleosts studied, Phe-Met-Arg-Phe-NH2- (FMRFamide-) like immunoreactive fibers originating from structures related to the olfactory system project to the retina. A complete report on this olfacto-retinalis projection in fish that are phylogenetically older than teleosts is still missing. We have visualized FMRFamide-like immunoreactive fibers in the optic nerve, the optic chiasm and in the retina of the longnose gar, Lepisosteus osseus. They terminate on amacrine or horizontal cells in the internal nuclear layer. Additionally, some of them appear to contact retinal ganglion cells.

Interspecific variation of isthmo-optic projections in poikilothermic vertebrates

Retinopetal projections from isthmic and caudal mesencephalic regions were studied in a number of poikilothermic vertebrates. The tested animals were elasmobranchs, bony fishes, amphibians, and reptiles. Isthmo-optic projections were absent in elasmobranchs, sturgeons, most teleosts, all anuran and urodel amphibians, and in anolis lizards. Tegmental projections to the retina were found in a few fishes and in most reptiles. Interspecific variations regarding three morphological parameters were as follows: (i) isthmic projections to the contralateral eye originate from one or from two distinct nuclei; (ii) major interspecific differences exist regarding numbers and aggregation densities of cells in the nuclei of origin; (iii) the percentage of retinopetal fibres that innervate the ipsilateral retina varies significantly between species.

Knockdown of prolyl-4-hydroxylase domain 2 inhibits tumor growth of human breast cancer MDA-MB-231 cells by affecting TGF-β1 processing

The prolyl‐4‐hydroxylase domain 1–3 (PHD1–3) enzymes are regulating the protein stability of the α‐subunit of the hypoxia‐inducible factor‐1 (HIF‐1), which mediates oxygen‐dependent gene expression. PHD2 is the main isoform regulating HIF‐1α hydroxylation and thus stability in normoxia. In human cancers, HIF‐1α is overexpressed as a result of intratumoral hypoxia which in turn promotes tumor progression. The role of PHD2 for tumor progression is in contrast far from being thoroughly understood. Therefore, we established PHD2 knockdown clones of MDA‐MB‐231 breast cancer cells and analyzed their tumor‐forming potential in a SCID mouse model. Tumor progression was significantly impaired in the PHD2 knockdown MDA‐MB‐231 cells, which could be partially rescued by re‐establishing PHD2 expression. In a RNA profile screen, we identified the secreted phosphoprotein 1 (SPP1) as one target, which is differentially regulated as a consequence of the PHD2 knockdown. Knockdown of PHD2 drastically reduced the SPP1 expression in MDA‐MB‐231 cells. A correlation of SPP1 and PHD2 expression was additionally verified in 294 invasive breast cancer biopsies. In subsequent analyses, we identified that PHD2 alters the processing of transforming growth factor (TGF)‐β1, which is highly involved in SPP1 expression. The altered processing capacity was associated with a dislocation of the pro‐protein convertase furin. Thus, our data demonstrate that in MDA‐MB‐231 cells PHD2 might affect tumor‐relevant TGF‐β1 target gene expression by altering the TGF‐β1 processing capacity.

Localization of the neuronal form of nitric oxide synthase (bNOS) in the diencephalon and pituitary gland of the catfish, Synodontis multipunctatus: an immunocytochemical study

The distribution of the neuronal form of nitric oxide synthase (bNOS) was investigated in the brain and pituitary gland of the catfish, Synodontis multipunctatus. Immunoreactive neurons were found mainly in the nucleus praeopticus periventricularis, the parvocellular and supraoptic subdivisions of the nucleus praeopticus, the nucleus recessus lateralis and the nucleus recessus posterioris. In addition, some scattered bNOS labeled somata were noted in the dorsal hypothalamic area. A few positive cells in the adenohypophysis and some reactive fibers in the pituitary stalk were also seen. Our results are compatible with the notion that the cells expressing bNOS in the diencephalon and hypophysis are involved in the control of hormone regulation. Moreover, the presence of bNOS positive cells in the rostral pars distalis of the pituitary gland supports a role of nitric oxide in osmoregulation.