Anti-helix-loop-helix domain antibodies: discovery of autoantibodies that inhibit DNA binding activity of human centromere protein B (CENP-B).

Centromere protein B (CENP-B) is one of the centromere DNA binding proteins constituting centromeric heterochromatin of human chromosomes. This protein was originally identified as the target antigen in autoimmune disease patients (often with scleroderma). In this study, we cloned a human CENP-B cDNA which was longer than the previously isolated one and expressed functional recombinant CENP-B in Escherichia coli. The DNA binding domain was finely located within the N-terminal 134-amino-acid residues covering a predicted helix-loop-helix (HLH) structure, by using a set of recombinant products with stepwise deletions from the C-terminus. From the analysis of their reactivity to anti-centromere sera from autoimmune disease patients, four epitopes were mapped on CENP-B antigen. In addition to two epitopes at the C-terminus, two were found on the HLH region at the N-terminus. In the analysis of the interaction between the antigen and autoantibodies, we found that the DNA binding activity of CENP-B was distorted by the attack of the anti-HLH domain antibodies in in vitro binding reactions. Our results suggest that the direct inhibition of the DNA binding activity by the autoantibodies might be involved in patients' autoimmune reactions in vivo.     

cDNA sequence and expression of a phosphoenolpyruvate carboxylase gene from soybean

A full-length cDNA encoding a subunit of phosphoenolpyruvate carboxylase (PEPC) was isolated from a developing seed expression library of the C₃ plant Glycine max. The corresponding mRNA is present at similar levels in leaf, stem, root and developing seed. Two potential start codons exist, and the activity of protein initiated from the first such codon could be subject to regulation by protein kinase. Sequence comparison shows a similar upstream start codon in the case of the Ppc2 gene from Mesembryanthemum crystallinum, previously assumed to lack the sequences necessary for phosphorylation. The soybean encoded protein tends to resemble other C₃-type PEPC proteins more closely than those implicated in C₄ or crassulacean acid metabolism.     

Effect of elevated extracellular calcium on the proliferation of osteoblastic MC3T3-E1 cells:its direct and indirect effects via monocytes.

There has been evidence that elevated calcium concentration at the resorptive site of the bone directly regulates osteoclast function. In the present study, in order to clarify the role of elevated calcium concentration at the resorptive site in the regulation of osteoblast function, not only direct but also indirect effect via human monocytes of the increase in extracellular calcium (Cae) on the proliferation of osteoblastic MC3T3-E1 cells have been investigated in serum-free condition. The increase in Cae enormously stimulated osteoblast proliferation at the concentration of 3 to 20 mM. When human monocytes were cultured at the elevated Cae concentration, monocyte-conditioned medium-induced stimulation of osteoblast proliferation was significantly amplified. Present data demonstrate that elevated Cae has pronounced stimulatory effect on osteoblast proliferation not only directly but also indirectly via monocytes. Calcium released from bone matrix at the resorptive sites might be linked to the coupling of osteoclast and osteoblast functions.     

The activation of cAMP-dependent protein kinase is directly linked to the inhibition of osteoblast proliferation (UMR-106) by parathyroid hormone-related protein

The present study was performed to compare the effect of parathyroid hormone-related protein (PTHrP) on the proliferation of osteoblastic osteosarcoma cells (UMR-106) with that of PTH and characterize the direct involvement of cAMP in the change of osteoblast proliferation by PTHrP. Human(h)PTHrP-(1-34) (10(-11)-10(-7)M) dose-dependently inhibited [3H]thymidine incorporation (TdR) in the same manner as hPTH-(1-34). The simultaneous addition of PTHrP and PTH at a maximal effective dose of 10(-7) M did not cause additive suppressive effect on cell proliferation. Rp-cAMPs, which has been recently shown to act directly as antagonist in the activation of cAMP-dependent protein kinase (PKA), dose-dependently (10(-6)-10(-4)M) antagonized PTHrP-induced suppression of TdR in the same manner as PTH. Present study indicated that PTHrP has the same effect on osteoblast proliferation as PTH and that the activation of PKA is directly linked to the change of osteoblast proliferation by PTHrP.     

Beta-angonists modulate ACh-inhibition of a K current in intestinal smooth muscle cells.

ACh causes a long-lasting inhibition of STOCs via G proteins in intestinal smooth muscle cells. We examined the effects of isoproterenol (Iso) on the ACh-induced inhibition of STOCs in isolated ileal smooth muscle cells using the G omega-seal whole cell clamp technique. In control, ACh (1 microM) completely suppressed STOCs, which did not desensitize over a period lasting 20 minutes. When Iso (10 microM) was added to the bath in the presence of ACh, the ACh-induced inhibition of STOCs was gradually removed. This effect of Iso was prevented by propranolol (10 microM). Application of Db-cAMP (500 microM) mimicked the Iso effects. Intracellulary applied GTP-gamma S (100 microM) gradually suppressed STOCs in the absence of ACh, which could not be removed by either Iso or Db-cAMP. These results suggest that beta-adrenergic stimulation causes a removal of the muscarinic inhibition of STOCs via a cAMP-dependent process.     

Replication origin of the Escherichia coli K-12 chromosome: The size and structure of the minimum DNA segment carrying the information for autonomous replication

Summary A DNA fragment containing the replication origin of the Escherichia coli K-12 chromosome was inserted in two orientations at either the BamHI or SalI site of pBR322 DNA. All the resulting hybrid plasmids were found to replicate in both polA and polA ⁺ cells, whereas pBR322 replicates only in polA ⁺ cells. This characteristic provided a method for assaying the autonomously replicating ability (Ori function) of the E. coli origin.In order to define the minimum DNA region (ori) that determines Ori function, deletions of various sizes were introduced from either side of the ori-containing segment in the hybrid plasmids by in vitro techniques, and the correlation between the Ori phenotype and nucleotide sequence of the deletion derivatives was analyzed. It was found that the left end of ori is between positions 23 and 35, and the right end is either position 266 or 267 in our nucleotide coordinate (Sugimoto et al., 1979). Therefore, ori is present within a region of minimum 232 base pairs and maximum 245 base pairs in length. The Ori⁺ and Ori^- phenotypes were clearly resolved at both sides of these boundaries by the above assay procedure.To obtain information about the effect of mutations in the internal region of the defined ori stretch, short sequences were inserted or deleted in vitro in the vicinity of several restriction sites within ori on the hybrid plasmids. Most of these plasmids carrying modified sequences showed Ori^- phenotype, suggesting that most parts of the ori stretch play important roles in Ori function.     

Scanning electron-microscopy of starch granules, with or without amylase attack

Scanning electron-microscopy (SEM) revealed that, for starch granules relatively susceptible to amylase, numerous pin holes could be observed on the surfaces of granules attacked by amylase. We also observed that the pores penetrated into the inner layers of granules during the enzyme action and some of the granules exhibited a terraced or step-shaped apperance in their inner portions. These internal characteristics are most probably indicative of layered internal structures of the granules. The other characteristic observations by SEM were striated structures on the surfaces of starch granules of banana, lily, and lotus attacked by pancreatin.