Different expression levels of TNF receptors on the rheumatoid synovial macrophages derived from surgery and a synovectomy as detected by a new flow cytometric analysis

TNFα plays a crucial role in the pathogenesis of rheumatoid arthritis. It is very important to examine the expression of the TNF receptors, the ligand of TNFα. In this study, we developed a triple-color flow cytometric analysis using CD45 and CD14 monoclonal antibodies to simply detect the expression of the TNF receptors on the heterogeneous rheumatoid synovial cells. Using this system, we detected a higher population of macrophages and a greater TNF receptor expression on the synovial macrophages derived from a synovectomy in comparison to the findings obtained from knee joint replacement surgery.     

Possible involvement of caspase-6 and -7 but not caspase-3 in the regulation of hypoxia-induced apoptosis in tube-forming endothelial cells

We recently reported that a broad-spectrum caspase inhibitor zVAD-fmk failed, while p38 inhibitor SB203580 succeeded, to prevent chromatin condensation and nuclear fragmentation induced by hypoxia in tube-forming HUVECs. In this study, we investigated the reasons for zVAD-fmk's inability to inhibit these morphological changes at the molecular level. The inhibitor effectively inhibited DNA ladder formation and activation of caspase-3 and -6, but it surprisingly failed to inhibit caspase-7 activation. On the other hand, SB203580 successfully inhibited all of these molecular events. When zLEHD-fmk, which specifically inhibits initiator caspase-9 upstream of caspase-3, was used, it inhibited caspase-3 activation but failed to inhibit caspase-6 and -7 activation. It also failed to inhibit hypoxia-induced chromatin condensation, nuclear fragmentation and DNA ladder formation. Taken together, our results indicate that, during hypoxia, caspase-7 is responsible for chromatin condensation and nuclear fragmentation while caspase-6 is responsible for DNA ladder formation.     

Apoptotic Death of Hematopoietic Tumor Cells through Potentiated and Sustained Adhesion to Fibronectin via VLA-4

It has been postulated that inactivated β1-integrins are involved in the disordered growth of hematopoietic tumor cells. We recently found that TNIIIA2, a peptide derived from tenascin-C, strongly activates β1-integrins through binding with syndecan-4. We show here that Ramos Burkitt''s lymphoma cells can survive and grow in suspension but undergo apoptosis when kept adhering to fibronectin by stimulation with TNIIIA2. Other integrin activators, Mg²⁺ and TS2/16 (an integrin-activating antibody), were also capable of inducing apoptosis. The inactivation of ERK1/2 and Akt and the subsequent activation of Bad were involved in the apoptosis. The results using other hematopoietic tumor cell lines expressing different levels of fibronectin receptors (VLA-4 and VLA-5) showed that potentiated and sustained adhesion to fibronectin via VLA-4 causally induces apoptosis also in various types of hematopoietic tumor cells in addition to Ramos cells. Because TNIIIA2 requires syndecan-4 as a membrane receptor for activation of β1-integrins, it induced apoptosis preferentially in hematopoietic tumor cells, which expressed both VLA-4 and syndecan-4 as membrane receptors mediating the effects of fibronectin and TNIIIA2, respectively. Therefore, normal peripheral blood cells, such as neutrophils, monocytes, and lymphocytes, which poorly expressed syndecan-4, were almost insusceptible to TNIIIA2-induced apoptosis. The TNIIIA2-related matricryptic site of TN-C could contribute, once exposed, to preventing prolonged survival of hematopoietic malignant progenitors through potentiated and sustained activation of VLA-4.     

Construction of transgenic Ipomoea obscura that exhibits new reddish leaf and flower colors due to introduction of β-carotene ketolase and hydroxylase genes

Ipomoea obscura, small white morning glory, is an ornamental plant belonging to the family Convolvulaceae, and cultivated worldwide. I. obscura generates white petals including a pale-yellow colored star-shaped center (flower vein). Its fully opened flowers were known to accumulate trace amounts of carotenoids such as β-carotene. In the present study, the embryogenic calli of I. obscura, were successfully produced through its immature embryo culture, and co-cultured with Agrobacterium tumefaciens carrying the β-carotene 4,4′-ketolase (crtW) and β-carotene 3,3′-hydroxylase (crtZ) genes for astaxanthin biosynthesis in addition to the isopentenyl diphosphate isomerase (idi) and hygromycin resistance genes. Transgenic plants, in which these four genes were introduced, were regenerated from the infected calli. They generated bronze (reddish green) leaves and novel petals that exhibited a color change from pale-yellow to pale-orange in the star-shaped center part. Especially, the color of their withered leaves changed drastically. HPLC-PDA-MS analysis showed that the expanded leaves of a transgenic line (T₀) produced astaxanthin (5.2% of total carotenoids), adonirubin (3.9%), canthaxanthin (3.8%), and 3-hydroxyechinenone (3.6%), which indicated that these ketocarotenoids corresponded to 16.5% of the total carotenoids produced there (530 µg g⁻¹ fresh weight). Furthermore, the altered traits of the transgenic plants were found to be inherited to their progenies by self-crossing.     

Accurate estimation of nitrogen concentration in deciduous tree leaves in a field study using a portable non-destructive nitrogen detector

A non-destructive nitrogen (N) detector [Agriexpert PPW-3000 (PPW-3000)] is a useful device for rapid and non-destructive measurement of leaf N content. However, some studies find a poor correlation between the PPW-3000 reading and the actual leaf N content; the R2 value of the approximate equation was low. To improve the accuracy of N estimation, we determined the approximate equation taking into account the leaf development stage (maturing and mature leaves) and leaf flush type (early and late leaves). For the leaf development stage, we determined approximate equations for maturing leaves (Ya), mature leaves (Yb), and "maturing+mature" leaves (Yc) in species having simultaneous leaf emergence. The resulting accuracy of Ya, Yb, and Yc was quite high. For leaf flush species, we determined approximate equations for early leaves (Y1), late leaves (Y2), and "early+late" leaves (Y3) in species having heterophyllous leaf emergence. The accuracy of Y1 and Y2 was relatively high, but that of Y3 was low. We conclude that, when using a PPW-3000, we can determine an approximate equation for maturing and mature leaves jointly, but should treat early and late leaves separately.     

14-3-3 Protein, Total Tau and Phosphorylated Tau in Cerebrospinal Fluid of Patients with Creutzfeldt-Jakob Disease and Neurodegenerative Disease in Japan

1.Sporadic Creutzfeldt-Jakob disease (CJD) is a rapidly progressive and fatal disease. Patients with CJD usually become akinetic mutism within approximately 6 months. In addition, clinical signs and symptoms at early stage of sporadic CJD may not be easy to distinguish from other neurodegenerative diseases by neurological findings. However, diagnostic biochemical parameters including 14-3-3 protein, S100, neuron-specific enorase in cerebrospinal fluid (CSF) have been used as diagnostic markers, elevated titers of these markers can also be observed in CSF in other neurodegenerative diseases. Therefore, we examined other biochemical markers to discriminate CJD from other neurodegenerative diseases in CSF. 2.We analyzed CSF samples derived from 100 patients with various neurodegenerative disorders by Western blot of 14-3-3 protein, quantification of total tau (t-tau) protein, and phosphorylated tau (p-tau) protein. All patients with CJD in this study showed positive 14-3-3 protein and elevated t-tau protein (>1000 pg/mL) in CSF. We also detected positive 14-3-3 protein bands in two patients in non-CJD group (patients with dementia of Alzheimer's type; DAT) and also detected elevated t-tau protein in three patients in non-CJD group. Elevated t-tau protein levels were observed in two patients with DAT and in one patient with cerevrovascular disease in acute phase. 3.To distinguish patients with CJD from non-CJD patients with elevated t-tau protein in CSF, we compared the ratio of p-tau and t-tau proteins. The p-/t-tau ratio was dramatically and significantly higher in DAT patients rather than in CJD patients. 4.Therefore, we concluded that the assay of t-tau protein may be useful as 1st screening and the ratio of p-tau protein/t-tau protein would be useful as 2nd screening to discriminate CJD from other neurodegenerative diseases.     

Cloning and nucleotide sequence of cDNA for the plastid glycerol-3-phosphate acyltransferase from squash

The partial amino acid sequence and amino acid composition of acyl-(acyl-carrier-protein):glycerol-3-phosphate acyltransferase purified from squash cotyledons were determined. cDNAs encoding this enzyme were isolated from lambda gt 11 cDNA libraries made from poly(A)+ RNA of squash cotyledons by immunological selection and cross-hybridization. One of the resultant clones contained a cDNA insert of 1426 base pairs and an open reading frame of 1188 base pairs. The amino acid sequence deduced from the nucleotide sequence matched the partial amino acid sequence determined for the enzyme. The results suggest that a precursor protein of 396 amino acid residues is processed to the mature enzyme of 368 amino acid residues, losing a leader peptide of 28 amino acid residues. Relative molecular masses of the precursor and mature proteins were calculated to be 43,838 and 40,929 Da, respectively.     

Purification and characterization of human urine-derived digitalis-like factor

We were able to purify a digitalis-like factor to apparent homogeneity from human urine based on the inhibitory effect on [3H]-ouabain binding to intact human erythrocytes. This ouabain displacing compound closely resembles ouabain in its polarity, molecular weight, non-peptidic nature and mode of action except for its UV absorbance spectrum. This compound sharing many biological activities of ouabain may be the endogenous ligand for the Na+, K+-ATPase and serve as a specific regulator of the sodium pump.     

The expression of melanosomal matrix protein in the transdifferentiation of pigmented epithelial cells into lens cells

A monoclonal antibody (MC/1) was constructed against melanosomes purified from the chicken pigmented epithelial cells (PECs) in order to characterize the differentiative phenotypes of PEC in the process of transdifferentiation into lens cells. Immunofluorescent studies revealed that MC/1 antibody specifically stains both retinal PECs in the eye and melanocytes in the skin, of chicken embryos. Immunoelectron microscopy showed that the antigen molecules are located on the peripheral region of the melanosomal matrix. A single protein band with an apparent molecular weight of 115,000 was labelled by MC/1 in Western blotting. The 115 kDa polypeptide identified by MC/1 is considered to be a member of the melanosomal matrix proteins. The maintenance of specificity of pigment cell nature is followed in the system of transdifferentiation of PEC into lens in vitro, utilizing 115 kDa protein as a marker. In the dedifferentiated PECs, this protein was undetectable.