cDNA cloning and predicted primary structure of scallop sarcoplasmic reticulum Ca-ATPase

Sarcoplasmic reticulum (SR) Ca²⁺-ATPase of the scallop cross-striated adductor muscle was purified with deoxycholate and digested with lysyl endopeptidase for sequencing of the digested fragments. Overlapping cDNA clones of the ATPase were isolated by screening the cDNA library with an RT-PCR product as a hybridization probe, which encodes the partial amino acid sequence of the ATPase. The predicted amino acid sequence of the ATPase contained all the partial sequences determined with the proteolytic fragments and consisted of the 993 residues with 70% overall sequence similarity to those of the SR ATPases from rabbit fast-twitch and slow-twitch muscles. An outline of the structure of the scallop ATPase molecule is predicted to mainly consist of ten transmembrane and five ‘stalk’ domains with two large cytoplasmic regions as observed with the rabbit ATPase molecules. The sequence relationship between scallop and other sarco/endoplasmic reticulum-type Ca²⁺-ATPases is discussed.     

Expression and localization of collagen-binding stress protein Hsp47 in mouse embryo development: comparison with types I and II collagen

Heat shock protein (Hsp)47 is a collagen-binding stress protein localized in the endoplasmic reticulum and is thought to have chaperone-like functions that are specific to procollagen biosynthesis. In previous papers, we reported that the expression of Hsp47 is closely correlated with that of various types of collagen in various cell lines and also in the progression of experimental liver fibrosis. In the present study, the expression of Hsp47 was examined during the development of mouse embryos by immunostaining with an anti-Hsp47 antiserum. The spatio-temporal correlation of the expression of Hsp47 with those of types I and II collagen was also examined using specific antisera. Hsp47 expression during embryogenesis was observed mainly in mesoderm and in tissues that are derived from mesoderm, such as connective tissue, cartilage, bone, notochord and somites. Hsp47 was also detected in tissues derived from the neural crest mesenchyme. In the central nervous system, Hsp47 was detected in some restricted regions where cells proliferate, such as the ventral area of the neural tube and choroid plexus. Immunostaining for types I and II collagen revealed the spatial and temporal correlations of the expression of these proteins with that of Hsp47. These results suggest the biological importance of Hsp47 as a collagen-specific molecular chaperone in the mouse developmental program.     

Age Associated Changes in the Distribution of Ipr Gene-Induced B220-Positive T Cells in Lymphoid Organs of MRL/Mp-Ipr/Ipr Mice Using Dual Exposure Microphotographs of Double Immunofluorescence Staining

Homozygous MRL/Mp-1pr/1pr (MRL/1pr) mice, which have an autosomal recessive mutant 1pr gene and exhibit defects in Fas antigen, spontaneously develop autoimmune disease with progressive expansion and accumulation of characteristic abnormal CD4-CD8-double negative T cells that express B220 surface antigen, a B cell-specific surface marker in normal mice. We analyzed the distribution and age related changes of lpr gene-induced abnormal T cells (B220-positive lpr T cells) In the lymphoid organs of MRL/1pr mice. We studied cryostat sections of the spleen, peripheral lymph nodes, mesenteric lymph nodes, and Peyer's patches at different stages using FITC (fluorescein isothiocyanate)-conjugated monoclonal antibodies directed against B220 (RA3-6B2) and PE (phycoerythrin)-conjugated anti-mouse CD3 (2C11) monoclonal antibody, examining dual-exposure microphotographs of double-immunofluorescence stained preparations. We observed that in aged MRL/lpr mice, B220-positive abnormal 1pr T cells were not present in the thymus-dependent area, and the majority of the follicular area cells were displaced by 1pr T cells. These findings suggest that the cellular trafficking of B220-positive lpr T cells differs from that of conventional T cells and that these 1pr-derived T cells play a role in the follicle.     

Ionic conditions affecting the release and absorption of an alkaline protease associated with the occlusion bodies of insect baculoviruses

Nearly all of the alkaline protease found in the occlusion bodies of baculoviruses (polyhedra for nuclear polyhedrosis and capsules for granulosis viruses) (Baculovirus, subgroup A and B, family Baculoviridae) can be specifically extracted under high ionic concentration. The extraction is directly proportional to the concentrations of NaCl up to 0.25 m. It is not dependent on pH, species of ions, temperature, and incubation time. The protease is reabsorbed under low ionic concentration by protease-extracted and by heat-treated capsules and polyhedra. The protease from Streptomyces griseus is not absorbed. This indicates that the occlusion body proteins have distinct affinity for certain alkaline proteases.     

Complete amino acid sequence of Scytalidium lignicolum acid protease B

The acid protease B (SLB) of Scytalidium lignicolum was reduced and carboxymethylated and then subjected to tryptic digestion. Five fragments were isolated and some of them were further digested with alpha-chymotrypsin, thermolysin, and dilute acetic acid. The sequence analysis of these fragments and the peptides by conventional methods established the complete amino acid sequence of SLB. The enzyme was composed of 204 amino acid residues with threonine and valine as its amino- and carboxyl-termini, respectively. Locations of three disulfide bridges were also established to be Cys47-126, Cys140-163, and Cys192-201 by enzymatic fragmentation of the denatured and unmodified SLB. Only a slight homology was found in the sequences of SLB and other acid proteases hitherto reported.     

Incorporation of bile acid of low concentration into model and biological membranes studied by 2H and 31P NMR

We have analyzed the manner of incorporation of bile acid into lipid bilayers and resultant perturbation of the bilayer structure with lower bile acid/lipid ratios relevant to the physiological conditions (approximately 1 mM) by 2H and 31P NMR methods, as an aid to understanding the possible role as an endogenous tumor promoter in colon cancer besides the primary physiological function of solubilizing lipids. On the basis of the 2H quadrupole splittings of [6,6,7,7,8-2H5]deoxycholate and [11,11,12,12-2H4]chenodeoxycholate in the presence of lamellar multibilayers of egg yolk lecithin, these bile acids were found to be incorporated in such a manner that the B-D rings lie parallel with the normal of the bilayers when the ratio of the bile acid to lipid is low (less than 0.11). When the ratio is increased, these bile acid molecules are not dispersed entirely in the bilayer but aggregate to form micelles with lipids. Further, we studied the resultant perturbation of the multibilayers of egg yolk lecithin analyzed by using the 2H quadrupole splitting of [18,18,18-2H3]stearic acid as a probe and by 31P chemical shift anisotropy. We found that the bilayer structure is retained even at the bile acid-to-lipid ratio of 0.25, although a small amount of an isotropic phase appeared such as small vesicles and micelles. The molecular ordering of fatty acyl chains was rather enhanced by the presence of 1 mM deoxycholate in erythrocyte ghosts as seen from the 2H quadrupole splitting of [16,16,16-2H3]palmitic acid, although deoxycholate caused hemolysis in this condition. The former observation can be explained by the way the lipid-protein interaction is modified by deoxycholate located in the interface between the lipids and proteins.     

Phosphorylation of the myosin heavy chain. Its effect on actin-activated Mg2+-stimulated ATPase in leukaemic myeloblasts.

Myosin purified from a murine myeloid leukaemia cell line (M1) that had been incubated with [32P]orthophosphate incorporated 32P into the heavy, but not the light, chain. When the heavy chain was dephosphorylated by bacterial alkaline phosphatase, myosin that had low actin-activated ATPase activity gained higher activity only in the presence of the light-chain kinase. In the absence of the light-chain kinase, however, the Mg2+-stimulated ATPase activity of myosin was not activated by actin, regardless of phosphatase treatment. These results indicate that the activity of M1 myosin ATPase is regulated by phosphorylation of both the light and heavy chains. A scheme for this regulation by phosphorylation is presented and discussed.     

The effect of PGF2 alpha on parathyroid hormone-stimulated cyclic AMP production in mouse osteoblastic cell, MC3T3E1.

The effect of prostaglandin F2 alpha (PGF2 alpha) was investigated in MC3T3E1 cells on the succeeding cAMP response to parathyroid hormone (PTH). PGF2 alpha increased the membrane-associated protein kinase C (PKC) activity, indicating the activation of this enzyme. The effect of PTH to increase cAMP production was enhanced by pretreatment with PGF2 alpha. Phorbol 12-myristate 13-acetate also enhanced cAMP production stimulated by PTH, and PKC inhibitor H7 attenuated the enhancement of PGF2 alpha. A23187 did not reproduce the PGF2 alpha effect, and this effect was not antagonized by the calmodulin antagonist W7. PGF2 alpha did not change the ED50 nor the maximally responsive dose of PTH in stimulating cAMP production. The effect of PGF2 alpha was not affected by pertussis toxin, and PGF2 alpha also enhanced cholera toxin- or forskolin-stimulated cAMP production. In accordance with the response of cAMP to PTH, the resorption of mouse limb bones stimulated submaximally by PTH was enhanced by the concomitant presence of PGF2 alpha. These results indicate that PGF2 alpha modulates cAMP response through the activation of PKC, the target of which might be the catalytic unit of adenylate cyclase. Such interaction between signal transduction systems may have significance in modulating the effect of PTH on bone, i.e., bone resorption.     

Analyses of acute graft-versus-host-like reaction in [MRL/lpr----MRL/+] chimeric mice using MRL/lpr-Thy-1. 1 congenic mice.

When MRL/Mp(-)+/+(MRL/+) mice are lethally irradiated and then reconstituted with MRL/Mp-lpr/lpr (MRL/lpr) bone marrow and/or spleen cells, these MRL/+ mice develop "lpr-GVHD" which is similar to acute graft-versus-host disease (GVHD). Using a Thy-1 congenic strain of MRL/lpr mice (MRL/lpr-Thy-1.1), we analyzed T cell subpopulations in the thymus and spleen of MRL/+ mice suffering from lpr-GVHD. lpr-GVHD was induced in MRL/+ mice by transplantation of bone marrow cells (BMC) from MRL/lpr-Thy-1.1 mice; severe lymphocyte depletion associated with fibrosis was observed in the spleens after 7 weeks of bone marrow transplantation (BMT). Thymocytes of the host MRL/+ thymus were replaced with donor-derived cells from the early stage of lpr-GVHD, whereas in the spleen, a small number of host T cells (Thy-1.2+) (4-5%) were retained until the late stage of lpr-GVHD. Donor-type (Thy-1.1+) T cell subsets were not different from those of nontreated MRL/+ mice in the thymus, whereas in the spleen. CD8+ T cells (Thy-1.1+) reached a peak at 5 weeks after BMT, and CD4+ T cells (Thy-1.1+), a peak at 6 weeks. The elimination of T cells from MRL/lpr BMC had no evident effect on the prevention of lpr-GVHD. T cell subpopulations showed a similar pattern to GVHD elicited by MHC differences. Analyses of autoreactive T cells expressing V beta 5 or V beta 11 revealed that autoreactive T cells were deleted from the peripheral lymph nodes. Interestingly, the levels of IgG anti-ssDNA antibodies markedly increased, and both IgM and IgG rheumatoid factors slightly increased 5 to 7 weeks after BMT. These findings are discussed in relation to not only GVHD elicited by MHC differences but also autoimmune diseases.