Developmental changes in the biochemical and immunological characters of the carbohydrate moiety of neuroglycan C, a brain-specific chondroitin sulfate proteoglycan

Neuroglycan C (NGC), a brain-specific transmembrane proteoglycan, is thought to bear not only chondroitin sulfate but also N- and O-linked oligosaccharides on its core protein. In this study, we isolated and purified NGC from rat brains at various developmental stages by immunoaffinity column chromatography or by immunoprecipitation, and examined the structural characters of its carbohydrate moiety. The chondroitin sulfate disaccharide composition of NGC at postnatal day 10 was significantly different from those of two secreted chondroitin sulfate proteoglycans, neurocan and phosphacan, purified from the brain at the same developmental stage; higher levels of 4-sulfate unit and E unit, a disulfated disaccharide unit, and a lower level of 6-sulfate unit. The levels of both 6-sulfate and E units decreased with a compensatory increase of 4-sulfate unit with postnatal development of the brain. Lectin-blot analysis of the NGC core glycoprotein prepared by chondroitinase digestion confirmed that NGC actually bore both N- and O-linked carbohydrates, and also revealed that lectin-species reactive with NGC did not always recognize other brain-specific proteoglycans, neurocan and phosphacan, and vice versa, even though they were isolated from the brain at the same stage. The reactivity of NGC with lectins and with the HNK-1 antibody markedly changed as the brain matured. These findings indicate that the structure of the carbohydrate moiety of NGC is developmentally regulated, and differs from those of neurocan and phosphacan. The developmentally-regulated structural change of the carbohydrates on NGC may be partly implicated in the modulation of neuronal cell recognition during brain development.     

Distinct narcolepsy syndromes in Orexin receptor-2 and Orexin null mice: molecular genetic dissection of Non-REM and REM sleep regulatory processes

Narcolepsy-cataplexy, a neurological disorder associated with the absence of hypothalamic orexin (hypocretin) neuropeptides, consists of two underlying problems: inability to maintain wakefulness and intrusion of rapid eye movement (REM) sleep into wakefulness. Here we document, using behavioral, electrophysiological, and pharmacological criteria, two distinct classes of behavioral arrests exhibited by mice deficient in orexin-mediated signaling. Both OX2R(-/-) and orexin(-/-) mice are similarly affected with behaviorally abnormal attacks of non-REM sleep ("sleep attacks") and show similar degrees of disrupted wakefulness. In contrast, OX2R(-/-) mice are only mildly affected with cataplexy-like attacks of REM sleep, whereas orexin(-/-) mice are severely affected. Absence of OX2Rs eliminates orexin-evoked excitation of histaminergic neurons in the hypothalamus, which gate non-REM sleep onset. While normal regulation of wake/non-REM sleep transitions depends critically upon OX2R activation, the profound dysregulation of REM sleep control unique to the narcolepsy-cataplexy syndrome emerges from loss of signaling through both OX2R-dependent and OX2R-independent pathways.     

Topology of cytochrome b558 in neutrophil membrane analyzed by anti-peptide antibodies and proteolysis.

Cytochrome b558 is an important constituent of the superoxide-generating system in neutrophils and B lymphocytes. In this paper, the topology of the cytochrome in human neutrophil membrane was studied using antibodies raised in rabbits against synthetic peptides corresponding to various regions in the large and small subunits of the cytochrome. The antibodies recognized the cytochrome subunits in immunoblots and the cytochrome in situ. An antibody raised against residues 150-172 in the large subunit (anti-L123) bound to intact neutrophils, indicating that this region is exposed to the outside of the cells. In contrast, antibodies raised against any of the carboxyl-terminal regions of the large and small subunits (anti-LC and anti-SC, respectively) or the amino-terminal region of the small subunit (anti-SN), bound to neutrophils only after the cells were made permeable by freezing and thawing. The region close to the carboxyl terminus of the large subunit was digested by extracellularly added papain and, as a result, an 18-kDa carboxyl-terminal fragment was detected. Thus the carboxyl-terminal region of the large subunit is cytoplasmic and/or buried in the membrane, and the region around residues 369-398 is exposed on the cell surface. In contrast to the large subunits, the small subunit in neutrophils was resistant to any of the proteinases tested, although the subunit in membrane or Triton-solubilized preparation was digestible with papain. These results indicate that the large subunit of cytochrome b558 is a transmembrane protein with at least two regions exposed on the cell surface and that the carboxyl terminus of this subunit and both termini of the small subunit are exposed to the cytoplasmic side.     

Evolution of craniofacial novelty in parrots through developmental modularity and heterochrony

Parrots (order Psittaciformes) have developed novel cranial morphology. At the same time, they show considerable morphological diversity in the cranial musculoskeletal system, which includes two novel structures: the suborbital arch and the musculus (M.) pseudomasseter. To understand comprehensively the evolutionary pattern and process of novel cranial morphology in parrots, phylogenetic and developmental studies were conducted. Firstly, we undertook phylogenetic analyses based on mitochondrial ribosomal RNA gene sequences to obtain a robust phylogeny among parrots, and secondly we surveyed the cranial morphology of parrots extensively to add new information on the character states. Character mapping onto molecular phylogenies indicated strongly the repeated evolution of both the suborbital arch and the well-developed M. pseudomasseter within parrots. These results also suggested that the direction of evolutionary change is not always identical in the two characters, implying that these characters are relatively independent or decoupled structures behaving as separate modules. Finally, we compared the developmental pattern of jaw muscles among bird species and found a difference in the timing of M. pseudomasseter differentiation between the cockatiel Nymphicus hollandicus (representative of a well-developed condition) and the peach-faced lovebird Agapornis roseicollis (representative of an underdeveloped condition). On the basis of this study, we suggest that in the development of novel traits, modularity and heterochrony facilitate the diversification of parrot cranial morphology.     

Pheophytinization of bacteriochlorophyll c and energy transfer in cells of Chlorobium tepidum

Bacteriochlorophyll (BChl) c in whole cells of Chlorobium tepidum grown at 46 °C changed into bacteriopheophytin (BPhe) c within 10 days after reaching full growth. When a small amount of C. tepidum cells in which BChl c had been completely pheophytinized were transferred to a new culture medium, normal growth was observed after a short lag phase, and the absorption spectrum of the growing cells showed the presence of a normal amount of BChl c. During the growth of C. tepidum in the new culture, the BChl c concentration was nearly proportional to the cell density measured by turbidity (OD₆₄₀). These results indicate that C. tepidum can survive even when BChl c has been completely pheophytinized and that BChl c is newly synthesized in such cells when transferred to a new culture medium. In partly pheophytinized cells, upon excitation of BPhe c at 550 nm the fluorescence emission spectrum showed maxima at 775 and 810 nm, which correspond to emissions from BChl c and BChl a, respectively. This indicates energy transfer from BPhe c to BChl c and BChl a. In cells in which BChl c was completely pheophytinized, fluorescence measurements were indicative of direct energy transfer from BPhe c to baseplate BChl a. These findings suggest that when BChl c in C. tepidum cells is pheophytinized, the product (BPhe c) remains in the chlorosomes and continues to work as a light-harvesting pigment. Received: 2 October 1998 / Accepted: 22 April 1999     

Molecular and cellular changes associated with the evolution of novel jaw muscles in parrots

Vertebrates have achieved great evolutionary success due in large part to the anatomical diversification of their jaw complex, which allows them to inhabit almost every ecological niche. While many studies have focused on mechanisms that pattern the jaw skeleton, much remains to be understood about the origins of novelty and diversity in the closely associated musculature. To address this issue, we focused on parrots, which have acquired two anatomically unique jaw muscles: the ethmomandibular and the pseudomasseter. In parrot embryos, we observe distinct and highly derived expression patterns for Scx, Bmp4, Tgfβ2 and Six2 in neural crest-derived mesenchyme destined to form jaw muscle connective tissues. Furthermore, immunohistochemical analysis reveals that cell proliferation is more active in the cells within the jaw muscle than in surrounding connective tissue cells. This biased and differentially regulated mode of cell proliferation in cranial musculoskeletal tissues may allow these unusual jaw muscles to extend towards their new attachment sites. We conclude that the alteration of neural crest-derived connective tissue distribution during development may underlie the spatial changes in jaw musculoskeletal architecture found only in parrots. Thus, parrots provide valuable insights into molecular and cellular mechanisms that may generate evolutionary novelties with functionally adaptive significance.     

Development of the pseudothumb in frogs

Frogs have highly conserved hand and foot morphology, possessing four fingers and five toes. As an exception, two Japanese ranid frog species, the Otton frog Babina subaspera and the dagger frog Babina holsti, possess a unique thumb-like structure (the pseudothumb) in the forelimb, giving an appearance of a total of five fingers on the hand. To obtain insights into the developmental mechanisms that generate this novel character, we investigated the hand morphogenesis of the Otton frog. The unique morphological pattern of the pseudothumb was already established in juveniles. Surprisingly, the bud-like structure, which is similar to the area of inductive activity (e.g. feather buds in birds and the carapacial ridge in turtles), was detected over the site where the future prepollex develops in larvae. By contrast, this bud-like structure was not found in larvae of other ranid species. We discuss possible scenarios that would favour the evolution of this very unusual trait in frogs.