Presenilin-1 differentially facilitates endoproteolysis of the beta-amyloid precursor protein and Notch.

Mutations in the presenilin-1 (PS1) gene are associated with Alzheimer's disease and cause increased secretion of the neurotoxic amyloid-beta peptide (Abeta). Critical intramembraneous aspartates at residues 257 and 385 are required for the function of PS1 protein. Here we investigate the biological function of a naturally occurring PS1 splice variant (PS1 Deltaexon 8), which lacks the critical aspartate 257. Cell lines that stably express PS1 Deltaexon 8 or a PS1 protein in which aspartate residue 257 is mutated secrete significant levels of Abeta, whereas Abeta generation is severely reduced in cells transfected with PS1 containing a mutation of aspartate 385. In contrast, endoproteolytic processing of Notch is almost completely inhibited in cell lines expressing any of the PS1 variants that lack one of the critical aspartates. These data indicate that PS1 may differentially facilitate gamma-secretase-mediated generation of Abeta and endoproteolysis of Notch.

Glycine 384 is required for presenilin-1 function and is conserved in bacterial polytopic aspartyl proteases.

Endoproteolysis of beta-amyloid precursor protein (betaAPP) and Notch requires conserved aspartate residues in presenilins 1 and 2 (PS1 and PS2). Although PS1 and PS2 have therefore been proposed to be aspartyl proteases, no homology to other aspartyl proteases has been found. Here we identify homology between the presenilin active site and polytopic aspartyl proteases of bacterial origin, thus supporting the hypothesis that presenilins are novel aspartyl proteases.

Isolation and characterization of neural crest stem cells from adult human hair follicles.

Neural crest (NC) is a transient embryonic tissue, whose cells are motile and multipotent until they reach their destination and differentiate according to microenvironmental cues into a variety of cell types. However, a subpopulation of these cells remains multipotent. They were found, among other locations, in a bulge of adult murine whisker follicle and were designated epidermal neural crest stem cells (EPI-NCSCs). The aim of this work is to ascertain whether the EPI-NCSCs could be isolated from human hair follicles as well. Due to their exceptional properties, they could represent potential candidates for stem cell therapy. The presented work focuses on the isolation and characterization of EPI-NCSCs from human skin. We obtained a population of cells that expressed markers of NC, NC progeny and general stem cell markers. After prolonged cultivation, the subpopulation of cells spontaneously differentiated into some of NC derivatives, i.e. neurons, smooth muscle cells and Schwann cell progenitors. Targeted differentiation with neuregulin 1 highly increased the number of Schwann cells in the culture. Human EPI-NCSCs could also grow under non-adherent conditions and form 3-dimensional spheres. Microarray analysis was performed and gene profile of human EPI-NCSCs was compared with the list of key genes of murine EPI-NCSCs and the list of genes up-regulated in newly induced NC cells. This revealed 94% and 88% similarity, respectively. All presented results strongly support the NCSC identity and multipotency of isolated human cells. These cells could thus be used in regenerative medicine, especially because of the easy accessibility of donor tissue.

Aeromagnetic and radio echo ice-sounding measurements show much greater area of the dufek intrusion, antarctica.

A combined aeromagnetic and radio echo ice-sounding survey made in 1978 in Antarctica over the Dufek layered mafic intrusion suggests a minimum area of the intrusion of about 50,000 square kilometers, making it comparable in size with the Bushveld Complex of Africa. Comparisons of the magnetic and subglacial topographic profiles illustrate the usefulness of this combination of methods in studying bedrock geology beneath ice-covered areas. Magnetic anomalies range in peak-to-through amplitude from about 50 nanoteslas over the lowermost exposed portion of the section in the Dufek Massif to about 3600 nanoteslas over the uppermost part of the section in the Forrestal Range. Theoretical magnetic anomalies, computed from a model based on the subice topography fitted to the highest amplitude observed magnetic anomalies, required normal and reversed magnetizations ranging from 10(-3) to 10(-2) electromagnetic units per cubic centimeter. This result is interpreted as indicating that the Dufek intrusion cooled through the Curie isotherm during one or more reversals of the earth's magnetic field.

[Formation of the vascular bed: a review of its molecular mechanisms and therapeutic implications]. / Utvárení cévního reciste: prehled molekulárních mechanismu a moznosti terapeutického ovlivnení.

This review provides an update on recent advances in the field of molecular mechanisms of vascular bed development. We introduce the data about growth factors and their receptors and discuss the therapeutic potential of their modulation. The role of tissue hypoxia in vessel development is presented and documented by our own results. We review the role of ephrins and their receptors in differentiation of arterial and venous phenotype of endothelial cells and its loss in vein graft during adaptation to arterial circulation. Role of mutation in Foxc2 associated with valve failure in veins is discussed. Recent findings showing common genetic signals navigating blood vessels and nerves to common pathways are also described. Finally, we summarize current state of knowledge in therapeutic induction and inhibition of angiogenesis.

[Alfred Kohn, professor of histology at German University in Prague]. / Alfred Kohn, profesor histologie na Nemecké Univerzite v Praze.

Prof. Kohn (1867-1959) was the head of the Institute of Histology at the Medical Faculty of German University in Prague for 26 years. In 2007 we commemorated his 140th birthday, and 2009 we will remember the 50th anniversary of his death. He entered the history of medicine by discovery of nature and origin of parathyroid glands and by pioneer research into chromaffin cells and sympathetic paraganglia. Kohn's papers on the pituitary, interstitial cells of testes, and ovaries are also related to endocrinology. All his studies are based on descriptive and comparative histological and embryological observations. Kohn was twice the dean of German Medical Faculty, and a member or honorary member of many important scientific societies. He was repeatedly nominated for Nobel Prize for physiology and medicine. For his Jewish origin he was expelled from Deutsche Gesellschaft der Wissenschaften und Künste für die Tschechoslowakische Republik in 1939 and transported to Terezin ghetto in 1943. After the war he lived in Prague. On the occasion of his 90th birthday he was elected honorary president of Anatomische Gesellschaft and awarded by the Czechoslovak Order of Labour. Alfred Kohn died in 1959. He was one of the outstanding personalities that Prague gave to the world of science.

Retention of a cis Golgi protein requires polar residues on one face of a predicted alpha-helix in the transmembrane domain.

The first membrane-spanning domain (m1) of the model cis Golgi protein M (formerly called E1) from the avian coronavirus infectious bronchitis virus is required for targeting to the Golgi complex. When inserted in place of the membrane-spanning domain of a plasma membrane protein (vesicular stomatitis virus G protein), the chimeric protein ("Gm1") is retained in the Golgi complex of transfected cells. To determine the precise features of the m1 domain responsible for Golgi targeting, we produced single amino acid substitutions in m1 and analyzed their effects on localization of Gm1. Expression at the plasma membrane was used as the criterion for loss of Golgi retention. Rates of oligosaccharide processing were used as a measure of rate and efficiency of transport through the Golgi complex. We identified four uncharged polar residues that are critical for Golgi retention of Gm1 (Asn465, Thr469, Thr476, and Gln480). These residues line one face of a predicted alpha-helix. Interestingly, when the m1 domain of the homologous M protein from mouse hepatitis virus is inserted into the G protein reporter, the chimeric protein is not efficiently retained in the Golgi complex, but transported to the cell surface. Although it possesses three of the four residues we identified as important in the avian m1 sequence, other residues in the membrane-spanning domain from the mouse protein must prevent efficient recognition of the polar face within the lipid bilayer of the cis Golgi.

Oxidative stress and AP-1 activity in tamoxifen-resistant breast tumors in vivo.

BACKGROUND: Most breast cancers, even those that are initially responsive to tamoxifen, ultimately become resistant. The molecular basis for this resistance, which in some patients is thought to involve stimulation of tumor growth by tamoxifen, is unclear. Tamoxifen induces cellular oxidative stress, and because changes in cell redox state can activate signaling pathways leading to the activation of activating protein-1 (AP-1), we investigated whether tamoxifen-resistant growth in vivo is associated with oxidative stress and/or activation of AP-1 in a xenograft model system where resistance is caused by tamoxifen-stimulated growth. METHODS: Control estrogen-treated, tamoxifen-sensitive, and tamoxifen-resistant MCF-7 xenograft tumors were assessed for oxidative stress by measuring levels of antioxidant enzyme (e.g., superoxide dismutase [SOD], glutathione S-transferase [GST], and hexose monophosphate shunt [HMS]) activity, glutathione, and lipid peroxidation. AP-1 protein levels, phosphorylated c-jun levels, and phosphorylated Jun NH(2)-terminal kinase (JNK) levels were examined by western blot analyses, and AP-1 DNA-binding and transcriptional activities were assessed by electrophoretic mobility shift assays and a reporter gene system. All statistical tests are two-sided. RESULTS: Compared with control estrogen-treated tumors, tamoxifen resistant tumors had statistically significantly increased SOD (more than threefold; P=.004) and GST (twofold; P=.004) activity and statistically significantly reduced glutathione levels (greater than twofold; P<.001) and HMS activity (10-fold; P<.001). Lipid peroxides were not significantly different between control and tamoxifen-resistant tumors. We observed no differences in AP-1 protein components or DNA-binding activity. However, AP-1-dependent transcription (P=.04) and phosphorylated c-Jun and JNK levels (P<.001) were statistically significantly increased in the tamoxifen-resistant tumors. CONCLUSION: Our results suggest that the conversion of breast tumors to a tamoxifen-resistant phenotype is associated with oxidative stress and the subsequent antioxidant response and with increased phosphorylated JNK and c-Jun levels and AP-1 activity, which together could contribute to tumor growth.

In vitro differences of neonatal and later postnatal keratinocytes and dermal fibroblasts.

Skin healing process is postnatally always associated with scarring of various extent. Based on the clinical experience of plastic surgeons, the healing after lip cleft reconstruction is surprisingly almost scar-less when it is carried out within a few first days after birth. This phenomenon is not seen in delayed cases. In order to decipher causative mechanism, we have isolated and studied principal cell populations, keratinocytes and fibroblast, from residual tissue samples after reconstructive operation (N=39) performed at various age (0-9 years). These cells play the pivotal role in the healing and that is why we focused on description of their phenotype and also functionality with respect to age. We have identified a population of remarkably small cells in explants from newborns (day 0-10). These small cells were strongly positive for markers of low differentiated keratinocytes, keratin-8 and -19, and moreover also for vimentin. In the explants cultures from older babies this population was missing. Fibroblasts from newborns and older patients differed namely in terms of nestin expression and also in the production of extracellular matrix components. We conclude that in vitro described properties of keratinocytes and fibroblasts in newborns could participate on the almost scar-less wound healing in earliest neonatal period.

Effect of a novel series of macrocyclic hypolipidemic agents on plasma lipid and lipoprotein levels of four non-primate species.

The ansamycins are structurally novel hypolipidemic agents derived from rifampicin, but lacking antibacterial activity. Oral or intravenous administration resulted in rapid lowering of plasma cholesterol in rats, hamsters, guinea pigs and dogs. In the chow-fed rat, three related compounds (CGP 43371, CGS 23810 and CGS 24565) exhibited ED50 values of 13.7, 3.1 and 0.18 mg/kg, respectively. A feature common to the lipid lowering documented in these four species was the concomitant reduction of low density lipoprotein (LDL) and high density lipoprotein (HDL) cholesterol. In the chow-fed rat, however, apolipoprotein AI (apo AI) levels were much less affected than were those of HDL cholesterol. CGP 43371 at 3 and 10 mg/kg, lowered HDL cholesterol by 20% and 39%, respectively, whereas plasma apo AI was reduced by only 1% and 12%. Similarly, in lipoprotein fractions separated by ultracentrifugation, apo AI was unchanged in the d = 1.019-1.21 g/ml fraction after treatment with 3 or 10 mg/kg of CGP 43371, but HDL cholesterol was reduced 12% and 26% in this fraction at the two dose levels. Plasma and lipoprotein apo B levels, on the other hand, were reduced to a level equivalent to that of the reduction in cholesterol. The ansamycins thus represent a new structural series which may possess a novel mechanism of action as well, involving differential effects on HDL cholesterol and protein.

Alkaline phosphatase and dipeptidylpeptidase IV staining of tissue components of skeletal muscle: a comparative study.

A combined alkaline phosphatase (AP) and dipeptidlypeptidase IV (DPP IV) staining reaction has demonstrated enzymatic heterogeneity of the arterial and venous segments of capillaries in rat skeletal muscle. This study compared the staining reactions of skeletal muscles in many commonly used laboratory animals, including the axolotl, chick, quail, Monodelphys, rat, mouse, hamster, guinea pig, rabbit, dog, monkey, and human. DPP IV activity was found in the venous ends of the capillaries and in the endothelium of some larger veins in many of the species but was never demonstrated in the arterial side of the circulation. AP was found in the arterial ends of capillaries in all species except the axolotl, and it was also found in the endothelium of larger arteries of most species. AP activity was absent in venous endothelium of all species except for birds and Monodelphys. DPP IV activity was found in the perineurium of intramuscular nerves of most species, and AP activity was commonly seen in tendons and intramuscular connective tissue. The interspecies variability found in this study shows that care must be taken in comparing experimental data involving this technique from one species to another, but within a species the technique allows a fine level of discrimination between functionally distinct compounds of skeletal muscle tissue.

Localization of dipeptidylpeptidase IV and alkaline phosphatase in developing spinal cord meninges and peripheral nerve coverings of the rat.

Localization of dipeptidylpeptidase IV was studied in the spinal cord meninges and peripheral nerve coverings of fetal and postnatal rats. In the same sections, the localization of alkaline phosphatase was monitored. In the prenatal period, dipeptidylpeptidase (DPP) IV activity in the differentiating meninges appeared at the time of cerebrospinal fluid spaces formation (on day 16 in the cervical region and on day 18 in the lumbar region). In adult animals DPP IV was found in cells of those meningeal lamellae which delineated the cerebrospinal fluid spaces (the outer, intermediate and inner lamellae), in the perineurium, in Schwann cells and in some fibroblasts of the bulk of dura mater. It is suggested that DPP IV plays a role in the metabolism of neuropeptides by their interaction with cerebrospinal fluid. Alkaline phosphatase activity was detectable earlier than DPP IV activity. Positivity was first observed in some cells of the meninx primitiva and, later on, in the ectomeninx and also in the differentiating endomeninx where it disappeared postnatally. The developing ectomeninx exhibited activities of both enzymes. Alkaline phosphatase occupied its external layers, while DPP IV was localized in its inner layers. This enzymatic heterogeneity of the ectomeningeal layers suggests that the ectomeninx gives rise not only to dura mater (which in adult animals exhibits alkaline phosphatase activity) but also to the outer arachnoid layer (positive for DPP IV in adult rats).

Morphogenesis of the human gluteus maximus muscle arising from two muscle primordia.

In human embryos and fetuses a supernumerary muscle was found situated on the distal margin of the gluteus maximus muscle and supplied by the most distal main branch of the inferior gluteus nerve. According to its origin and insertion it is being named the coccygeofemoralis muscle. In embryos and fetuses of up to 40 mm in CR length the coccygeofemoralis muscle is separated by loose connective tissue from neighbouring fetal muscles. Later on, close contact between the coccygeofemoralis and the distal margin of the fetal gluteus maximus muscle develops, and during the prenatal period both fetal muscles gradually fuse. Postnatally, the coccygeofemoralis muscle is incorporated into the gluteus maximus muscle of which the pars sacroiliaca corresponds to the fetal gluteus maximus itself and the pars coccygea represents the fetal coccygeofemoralis muscle. With respect to the general process of muscle morphogenesis, the developmental pattern described for the gluteus maximus muscle demonstrates that adult muscles may be formed by a fusion of several fetal muscles.

Sensory nerve endings in the beak skin of Japanese quail.

This study is concerned with the distribution and ultrastructure of sensory nerve endings in the beak skin of adult Japanese quail (Coturnix coturnix japonica). The following nerve endings were found: free nerve endings, clusters of dermal Merkel nerve endings, Herbst corpuscles and Ruffini corpuscles. The latter were found only in the dermis of the tip of the upper beak. The remaining endings were present in the skin of all areas of upper and lower beak. Free nerve endings were supplied by either thin myelinated axons or unmyelinated C-fibers and were localized in the dermis close to the basal layer of the epidermis. Merkel cells formed clusters (up to 50) localized below and between the epidermal cones of the beak skin. Disc-shaped thickenings of nerve endings were squeezed between individual Merkel cells. Small Herbst corpuscles were found in the dermis close to the epidermal cones of the beak skin. Large Herbst corpuscles occurred in deep layers of the dermis. The Ruffini corpuscles were cylindrical in shape (80 microns x 400 microns) and arranged in groups of up to ten corpuscles. Each corpuscle was surrounded by an incomplete fibrous capsule.

Muscle morphogenesis in the absence of myogenic cells.

Experimental evidence indicates, that the myogenic cells themselves are not responsible for the muscle pattern formation. We report on a chance observation that reveals that muscle pattern formation can occur even in the absence of myogenic cells. Epiblastic cells from a quail embryo in the primitive streak stage were implanted into the wing bud of a chick embryo. The grafted quail cells developed into mononucleate, fibroblast-like cells that formed the muscle belly of the extensor medius longus muscle. This showed essentially normal form and topography as revealed by computer-aided 3D-reconstruction. This finding shows, that the formation of muscles does not depend on the presence of myogenic cells.

Developmental origin of avian Merkel cells.

We have investigated the developmental origin and ultrastructure of avian Merkel cells by electron microscopy and chick/quail transplantation experiments. On embryonic day 3, chick leg primordia were homotopically grafted onto Japanese quail host embryo. Fourteen days later, quail cells that had migrated into grafted chick legs were identified according to the masses of heterochromatin associated with the nucleolus that are characteristic for quail. Both in chick and quail, Merkel cells are usually located in the dermis just below the epidermis. They are placed between nerve terminals either individually or in small groups wrapped in sheaths that are formed by glial cell processes. Occasionally, some Merkel cells appear in nerve fascicles and within Herbst corpuscles. Merkel cells, as well as glial cells, in grafted chicken legs were of quail origin. This finding provides evidence against the epidermal origin of avian Merkel cells and indicates that Merkel cells are derived from neural crest cells that colonise, together with glial cells and melanocytes, the developing limb primordium.

Schwann cells are not required for guidance of motor nerves in the hindlimb in Splotch mutant mouse embryos.

The topogenesis of the hindlimb nerves of Splotch homozygous mutant mouse embryos was studied using light and electron microscopy. Homozygous mutants show multiple defects of neural crest-derived tissues. The defects increase along a rostro-caudal gradient. The cervical and upper thoracic segments have small spinal ganglia, and Schwann cells are associated with the spinal nerves. In the lumbo-sacral region neurulation is not complete, and the derivatives of the neural crest are missing. The lumbo-sacral nerve trunks are formed by ventral roots only. They are occasionally associated with presumptive glial cells that have migrated from the spinal cord for a short distance. Beyond the vertebral primordia, the spinal nerves are not accompanied by Schwann cells. No compartmentalization of the axons within the lumbo-sacral nerves was visible, whereas Schwann cells did segment the nerve into the fascicles in brachial nerves. The lumbo-sacral plexus develops, and its branches grow into the hindlimb despite the absence of Schwann cells. On day 13.5 of gestation, the lumbo-sacral nerve trunks extend well into the distal calf. They are topographically correctly positioned. Their branches enter the muscle primordia and form contacts with their mesenchymal cells though the cutaneous branches are missing. Generally, the outgrowth of lumbo-sacral nerves is slower than in phenotypically normal littermates, whose nerves reach the foot plate at corresponding stages of development. These results demonstrate that the lumbo-sacral plexus and the topographically correct position of lumbo-sacral nerve trunks develop despite the absence of Schwann cells. Therefore Schwann cells are not necessary for the outgrowth and guidance of axons within the limb.

Origin of spinal cord meninges, sheaths of peripheral nerves, and cutaneous receptors including Merkel cells. An experimental and ultrastructural study with avian chimeras.

The origin of cells covering the nervous system and the cutaneous receptors was studied using the quail-chick marking technique and light and electron microscopy. In the first experimental series the brachial neural tube of the quail was grafted in place of a corresponding neural tube segment of the chick embryo at HH-stages 10 to 14. In the second series the leg bud of quail embryos at HH-stages 18-20 was grafted in place of the leg bud of the chick embryos of the same stages and vice versa. It was found that all meningeal layers of the spinal cord, the perineurium and the endoneurium of peripheral nerves, as well as the capsular and inner space cells of Herbst sensory corpuscles, develop from the local mesenchymal cells. Schwann cells and cells of the inner core of sensory corpuscles are of neural crest origin. The precursors of Merkel cells migrate similarly to the Schwann cells into the limb bud where they later differentiate. This means that in addition to the Schwann cells and the melanocytes a further neural crest-derived subpopulation of cells enters the limb.

Crural Herbst corpuscles in chicken and quail: numbers and structure.

Herbst corpuscles were studied in the crural region of perinatal and adult chicken and quail in order to find out their number and dimensions and to learn more about their structure, especially in relation to size. Crural corpuscles are arrayed in an encapsulated string between tibia and fibula. They are closely packed together; a small number of corpuscles is found apart from the string, often attached to the periost. The strings of corpuscles are approximately 40 mm long in adult chicken and 20 mm long in the quail. The crural region of the chicken contains 382.8 +/- 90.9 (mean +/- SD) corpuscles, the numbers ranging from 301 to 582; in the quail, the mean number is 119.2 +/- 27.9, with a range from 83 to 167 corpuscles. In the chicken, one axon supplies an average of 1.60 corpuscles; in the quail, the relation of axons to corpuscles is approximately 0.92. In both species, final numbers of crural corpuscles are already attained before hatching and no difference is found in the mean number and range of corpuscles between perinatal and adult birds. In both chicken and quail, individual strings contain corpuscles of various sizes, from large to very small. The chicken corpuscles are generally twice as large in diameter and often longer than those of the quail. The corpuscles are composed of an axon terminal that projects two rows of axonal spines into the clefts of the inner core and ends with an ultraterminal bulb; the terminal is surrounded with a bilaterally symmetrical inner core, amorphous inner space containing collagen fibrils of various thickness, and a capsule. Large chicken corpuscles contain inner cores composed of up to 100 lamellae, while quail inner cores have half that number at the most. The capsules are usually composed of 8 to 10 lamellar layers in both species, but they are thicker in the chicken than in the quail. The possible functional significance of individual structural components of Herbst corpuscles is discussed.

The Splotch mutation interferes with muscle development in the limbs.

Homozygosity for the Splotch mutation causes neural tube and neural crest defects in mice. It has been demonstrated that Splotch mutant mice carry mutations in the homeodomain of the Pax-3 gene. Pax-3 is expressed in the neural tube, some neural crest derivatives, the mesenchyme of the limb bud and the somites. We have examined the development of the somite-derived skeletal muscles in homozygotes carrying the Splotch (Sp1H) mutation. Our results suggest that the Splotch mutation affects the development of skeletal muscles in a region-specific way: 1. The expression of the CMZ transgene in homozygotes reveals a disorganisation of the dermomyotome in whole stained embryos. 2. The axial musculature is reduced in size along a rostro-caudal gradient. 3. The muscle anlagen in the limbs develop much more slowly. Muscles of the head and the ventral body wall are normally developed in the mutant on day 13.5 of gestation. Recently, it has been shown that the myogenic precursors of the limbs are derived from the lateral half of the somite. The specific disturbance of muscle development in the limbs of Splotch mutants thus suggests a role for Pax-3 in the organisation of the somite, the production of trophic factors in the limb mesenchyme or an alteration of myogenic and mesenchymal cells.