Atsuko Uyeda

CLONING AND SEQUENCING OF HEN MAGNUM CDNAS ENCODING VITELLINE MEMBRANE OUTER LAYER PROTEIN I (VMO-I)

Two cDNAs encoding hen vitelline membrane outer layer protein I (VMO-I), which is classified as a new type of multi-beta-sheet assembly, were cloned and sequenced. Northern blot analysis using vmo-I cDNA as a probe showed the presence of three mRNA species. Strikingly, expression of these mRNAs was restricted to a specific region of the hen oviduct, the area joining the infundibulum to the magnum.

Neurocrescin: a novel neurite-outgrowth factor secreted by muscle after denervation.

REGENERATION of injured axons at neuromuscular junctions has been assumed to be regulated by extracellular factors that promote neurite outgrowth. We report here the cloning of a novel neurite outgrowth factor, designated neurocrescin, from chick denervated skeletal muscle. A recombinant neurocrescin promoted neurite outgrowth from cultured neurons of spinal cord and telencephalon of chick embryo. It was expressed predominantly in neural tissue and muscle, and was secreted extracellularly after intramolecular cleavage. This truncated form was detected in denervated muscle but not in innervated muscle. Thus, neurocrescin appears to be a novel neurite outgrowth factor that is secreted in an activity-dependent fashion. A highly homologous counterpart was also cloned from mouse brain.

Long-term effects of muscle-derived protein with molecular mass of 77 kDa (MDP77) on nerve regeneration

The long‐term effects of the 77‐kDa muscle‐derived protein (MDP77) on motor and sensory nerve regeneration were examined in vivo. Fourteen‐millimeter bridge grafts of the right sciatic nerve of SD rats were carried out with silicone tubes containing a solution of type I collagen together with 0, 5, 10, or 20 μg/ml recombinant human MDP77 (N = 10 in each group). Recovery of motor and sensory function was evaluated monthly by the maximal toe‐spread index (TSI) and hot‐plate test, respectively, for 6 months after the operation. Electrophysiology (nerve conduction velocity), histology (diameter and total number of the regenerated myelinated axons in the tube), and immunohistochemistry (total number of Schwann cells in the tube), as well as measurement of soleus muscle weight, were also performed at this time. Motor, but not sensory, function recovered rapidly in the MDP77‐treated groups in a dose‐dependent manner. Electrophysiological measurements and the ratio of soleus muscle weight corroborated the positive effects of MDP77 on motor nerve regeneration, but no facilitation of sensory nerve recovery was observed. Furthermore, histological and immunohistochemical evaluations suggested that MDP77 treatment accelerates Schwann cell migration, followed by enhanced maturation of regenerating axons, resulting in functional recovery of both the nerves and the atrophied, denervated muscle.

Regulatory expression of MDP77 protein in the skeletal and cardiac muscles

The mdp77 gene was first cloned from the cDNA library of denervated chick muscles, while its role(s) in vivo was unknown. In the present study, using specific polyclonal antibodies against MDP77, we show that MDP77 was expressed specifically in the skeletal and cardiac muscle, and confirm its presence in the cytoplasm of the extrafusal muscle fibers. In mature muscles, MDP77 immunoreactivity was observed in a repetitive manner along the sarcomere. The onset of MDP77 expression occurred just after myotube formation both in vivo and in vitro. Furthermore, MDP77 was enriched in the intrafusal muscle fibers. Our findings suggest that MDP77 plays an important role(s) in the differentiation, maturation and function of both the skeletal and cardiac muscles.

Cloning and characterization of a novel synaptosome-enriched mRNA that encodes 31 kDa protein.

Although a subpopulation of mRNAs has been identified as translocated to the dendrites or the synaptic regions of neurons, the translocational mechanism has not been elucidated. To find mRNAs enriched in synapses, we compared the synaptosomal mRNAs with those from whole forebrain using differential display (DD). We cloned one of these mRNAs, which encoded a novel 31 kDa protein (PMES-2). PMES-2 mRNA was specifically transcribed in the brain and was present in the dendrites of the hippocampal neurons. PMES-2 protein was partly localized in the postsynaptic density. Although this protein is very similar to human NABC1 protein, its function is still unknown.

Muscle-specific protein MDP77 specifically promotes motor nerve regeneration in rats

This study has examined the effects of recombinant human MDP77 (rhMDP77) on sciatic motor nerve regeneration in vivo. We carried out bridge grafting (14 mm) into the sciatic nerve of Sprague-Dawley rats using silicone tubes containing a mixture of type-I collagen and 0, 5, 10, or 20 microg/ml of rhMDP77, or containing phosphate-buffered solution (N = 6 in each group). Electrophysiological and histological evaluations carried out 12 weeks after implantation suggest that rhMDP77 has a positive effect on terminal and collateral sprouting of regenerating nerves and thereby promotes motor nerve regeneration in a dose-dependent manner.

Construction of a divalent cell adhesive lysozyme by introducing the Arg-Gly-Asp sequence at two sites.

To increase the cell adhesion activity of 74RGD4, an RGDS‐inserted mutant between Val74 and Asn75 of human lysozyme, one more site for the RGD introduction was investigated in the lysozyme molecule. We found that 47RGD4 with RGDS in place of AGDR (residues 47 to 50) in a β‐turn region possesses the same level of adhesion activity as that of 74RGD4. The acceptance of the RGD introduction in the β‐turn region of human lysozyme is in good agreement with recent studies on the functional conformation of RGD. We constructed (47,74)RGD4, a mutant containing RGD at two sites, by combining the N‐terminal domain of 47RGD4 and the C‐terminal domain of 74RGD4. The (47,74)RGD4 lysozyme, with two functional RGD sequences, exhibits even higher cell adhesion activity than that of 74RGD4 or 47RGD4.

Neurocrescin is specifically cleaved after the sequence DESD in a caspase-3-independent manner

Abstract1. A neurite outgrowth factor, neurocrescin (NC), which we previously identified from an extract of denervated skeletal muscle, was endoproteolytically processed in cell transfectants. In addition to the processing site identified in NC (DESD358/F) being similar to the optimal recognition sequence of group II caspases, DExD, cleavage site mutations confirmed the involvement of caspase(s) in NC processing.2. However, both the recombinant NC and the synthetic octapeptide (YL DESDFG) were scarcely cleaved in vitro by caspase-3 or -7. Furthermore, transiently expressed NC was cleaved even in the caspase-3-deficient cell line, MCF-7 cells, and this efficiency was not altered by the transfectional expression of caspase-3.3. Using the fluorescent substrate (Ac-DESD-AMC), the characteristic proteolytic activities, which cleaved it more effectively than caspase-3 and whose pH dependences were different from those of caspase-3, were endogenously identified in the muscle extract. These findings indicate the presence of proteolytic activities that are distinguishable from caspase-3.

A novel protein predominantly expressed in chick muscle promotes neurite-outgrowth

‘Lab. for Developmental Gene RegulationRIKEN Brain Science Institute 2-l Hirosawa, Wake-shi, Saitama, ,Japan, 35 l0198; ‘Dept. of Physiol., and 3Rehabilitation Medicine, Keio Univ., School of Med. 35 Shinanomachi , Shinjuku-ku, Tokyo, Japan 160-00 16 Sensory neurons( trigminal and Rohon-Beard neurons) in zebrafish embryos extend their axons in two directions; the central axons run longitudinally in the CNS and fasciculate with each other, whereas the peripheral axons branch and spread on the epidermis. They express LIMhomeodomain-type transcription factor Islet-2. We have previously shown that overexpression of LIMdomains represses the normal function of LIMhomeodomain proteins by competing for the LIM-binding activating molecules. The functional repression of Islet-2 on the differentiation of the sensory neurons revealed that extension of the peripherally branching axons were selectively impaired while that of centrifugal axons remained intact, These results indicate that Islet-2 may selectively regulate the peripheral branching but not central projection of the multi-polar sensory neurons of zebrafish embryos. Caudal primary motoneuron(CaP) also expresses Islet-2 and extends the axon to the ventral myotome. The functional repression of Islet-2 caused the axon of CaP to turn aberrantly in the spinal cord. This result indicates that Islet-2 also regulates the axonal pathfinding of CaP. Chimera analysis revealed that abnormalities of the axonal extension and pathfinding in LIM-overexpressing embryos were due to defects cell-autonomous to sensory and motor neurons.