Isolation and characterization of Arabidopsis thaliana ISU1 gene

We describe the isolation of a cDNA encoding Arabidopsis thaliana ISU1 (AtISU1), which regulates iron homeostasis in the mitochondria. The AtISU1 gene contained an open reading frame that encoded 167 amino acid residues. Northern blot analysis demonstrated that AtISU1 gene was ubiquitously expressed in plant tissues examined. The yeast seo5-1, which harbors a single base-pair deletion in ScISU1, is a suppressor of oxidative damage in sod1-deficient mutant. Based on comparative expression analyses using yeast ISU1 gene (ScISU1) in seo5-1 mutant, we found that AtISU1 acts as a counterpart of ScISU1.     

A novel, extraneuronal role for cyclin-dependent protein kinase 5 (CDK5): modulation of cAMP-induced apoptosis in rat leukemia cells

A number of cyclin-dependent protein kinase (CDK) inhibitors were tested for the ability to protect IPC-81 rat leukemic cells against cAMP-induced apoptosis. A near perfect proportionality was observed between inhibitor potency to protect against cAMP-induced apoptosis and to antagonize CDK5, and to a lesser extent, CDK2 and CDK1. Enforced expression of dominant negative CDK5 (but not CDK1-dn or CDK2-dn) protected against death, indicating that CDK5 activity was necessary for cAMP-induced apoptosis. The CDK inhibitors failed to protect the cells against daunorubicine-, staurosporine-, or okadaic acid-induced apoptosis. The inhibition of CDK5 prevented the cleavage of pro-caspase-3 in cAMP-treated cells. The cells could be saved closer to the moment of their onset of death by inhibitors of caspases than by inhibitors of CDK5. This suggested that the action of CDK5 was upstream of caspase activation. The cAMP treatment resulted in a moderate increase of the level of CDK5 mRNA and protein in IPC-81 wild-type cells. Such cAMP induction of CDK5 was not observed in cells expressing the inducible cAMP early repressor. The cAMP-induced increase of CDK5 contributed to apoptosis since cells overexpressing CDK5-wt were more sensitive for cAMP-induced death. These results demonstrate the first example of a proapoptotic CDK action upstream of caspase activation and of an extra-neuronal effect of CDK5.     

Culture of red beet hairy roots by considering variation in sensitivity of tip meristems to hydraulic stress

In the culture of red beet hairy roots in shaking flasks, a period for acclimation without lateral root generation existed at the early stage, and the root tip meristems containing growing points (GPs) were found to be damaged under an elevated shear stress condition. The loading experiments of shear stress to the hairy roots revealed that the GPs subjected to the acclimation acquired tolerance to shear stress, retaining relatively high viability of GPs up to 0.6N/m(2) of loaded shear stress. Next, the hairy roots after culture for 50h at 0.05N/m(2) of shear stress were exposed to conditions at various levels of shear stress in a single column reactor, and a relatively high growth rate was obtained in the vicinity of 1.0N/m(2) of shear stress. According to these results, two-stage cultures of hairy roots were then performed, which was comprised of a first stage for 50h at 0.05N/m(2) of shear stress for the prevention of decay of the GPs caused by hydraulic stress and a second stage for 110h at 1.0N/m(2) of shear stress for active elongation of the GPs with sufficient nutrient supply by regulation of the medium flow rate. The cell concentration ultimately reached 7.6kg dry cells/m(3), although no growth was observed in the case where the hairy roots did not undergo the first stage.     

An outbreak of staphylococcal food poisoning caused by enterotoxin H in mashed potato made with raw milk

Mashed potato made with raw bovine milk was suspected to have been the source of a food poisoning outbreak. Almost 8 x 10(8)Staphylococcus aureus CFU g(-1) were detected in the mashed potato. S. aureus was also found in bulk milk from the farm that had supplied milk for the mashed potato. Isolates from mashed potato and bulk milk were positive for the gene encoding staphylococcal enterotoxin H (seh), and the corresponding protein toxin, SEH, was detected by ELISA in the mashed potato. Production of SEH by S. aureus isolates from mashed potato (n = 4) and bulk milk (n = 4) was also demonstrated by ELISA. Sequencing of seh from one mashed potato isolate and one bulk milk isolate confirmed that the gene was a variant seh, and that the genes in both isolates were identical. Macrorestriction of chromosomal DNA with Sma1 followed by pulsed-field gel electrophoresis of seh-positive S. aureus from mashed potato and bulk milk revealed indistinguishable banding patterns between isolates from both sources. It seems likely that raw bovine milk used in the preparation of mashed potato contained S. aureus that subsequently produced sufficient SEH in the mashed potato to cause food poisoning.     

The cytoplasmic tail of invariant chain regulates endosome fusion and morphology

The major histocompatibility complex class II associated invariant chain (Ii) has been shown to inhibit endocytic transport and to increase the size of endosomes. We have recently found that this property has a significant impact on antigen processing and presentation. Here, we show in a cell-free endosome fusion assay that expression of Ii can increase fusion after phosphatidylinositol 3-kinase activity is blocked by wortmannin. In live cells wortmannin was also not able to block formation of the Ii-induced enlarged endosomes. The effects of Ii on endosomal transport and morphology depend on elements within the cytoplasmic tail. Data from mutagenesis analysis and nuclear magnetic resonance-based structure calculations of the Ii cytoplasmic tail demonstrate that free negative charges that are not involved in internal salt bridges are essential for both interactions between the tails and for the formation of enlarged endosomes. This correlation indicates that it is interactions between the Ii cytoplasmic tails that are involved in endosome fusion. The combined data from live cells, cell-free assays, and molecular dynamic simulations suggest that Ii molecules on different vesicles can promote endosome docking and fusion and thereby control endosomal traffic of membrane proteins and endosomal content.     

Mitotic cells form actin-based bridges with adjacent cells to provide intercellular communication during rounding

In order to achieve accurate chromosome segregation, eukaryotic cells undergo a dramatic change in morphology to obtain a spherical shape during mitosis. Interphase cells communicate directly with each other by exchanging ions and small molecules via gap junctions, which have important roles in controlling cell growth and differentiation. As cells round up during mitosis, the gap junctional communication between mitotic cells and adjacent interphase cells ceases. Whether mitotic cells use alternative mechanisms for mediating direct cell-cell communication during rounding is currently unknown. Here, we have studied the mechanisms involved in the remodeling of gap junctions during mitosis. We further demonstrate that mitotic cells are able to form actin-based plasma membrane bridges with adjacent cells during rounding. These structures, termed “mitotic nanotubes,” were found to be involved in mediating the transport of cytoplasm, including Rab11-positive vesicles, between mitotic cells and adjacent cells. Moreover, a subpool of the gap-junction channel protein connexin43 localized in these intercellular bridges during mitosis. Collectively, the data provide new insights into the mechanisms involved in the remodeling of gap junctions during mitosis and identify actin-based plasma membrane bridges as a novel means of communication between mitotic cells and adjacent cells during rounding.     

Nucleotide sequences and unusual electrophoretic behavior of the W chromosome-specific repeating DNA units of the domestic fowl,Gallus gallus domesticus

Nucleotide sequences of three independently cloned repeating units of the W chromosome-specific repettive DNA sequences (XhoI family) of the chicken were determined. All three units are 717 bp long with XhoI sites at both ends. There are only 21 sites out of 717 bases where a single base change occurs in one of the three clones. Each of these repeating units consists of 34 tandem repeats of about 21 bp. Sequences of some members of these internal repeats are not well conserved, but the majority of the repeats are characterized by the presence of (A)_3–5 and (T)_3–5 clusters separated by 6–7 relatively G+C-rich base pairs. One striking feature of the cloned 717 bp repeating units is that they migrate unusually slowly on electrophoresis in polyacrylamide gels. The same feature is also shown by a genomic population of the 0.7 kb repeating units recovered from XhoI digests of the genomic DNA of the female chicken. This anomalous behavior is attributed to the occurrence of DNA curvatures because of the above sequence characteristics and partial recovery of the electrophoretic mobility in the presence of distamycin A. Another feature of the 717 bp repeating unit is the presence of 438 and 159 nucleotide-long open reading frames (ORFs) at each end of the unit. A possible function of the XhoI family sequences in the heterochromatization of the W chromosome and the significance of the ORFs are discussed.     

Structure of human alpha 2-plasmin inhibitor deduced from the cDNA sequence

We have isolated three cDNA clones for human alpha 2-plasmin inhibitor (alpha 2-PI). Two clones are from human hepatoma cell line, Hep G2, and cover the entire protein coding region plus the 3'-flanking region up to the poly(A) sequence, and the other clone is from human liver and contains the carboxyl-terminal half. The total length of the cDNAs is 2.29 kb, corresponding to more than 95% of the full-length mRNA. alpha 2-PI seems to consist of 452 amino acid residues plus 39 amino acid residues for the signal peptide. The amino acid sequence shows 23 to 28% homology to those of five other protease inhibitors, plasminogen activator inhibitor (PAI), protein C inhibitor (PCI), alpha 1-antitrypsin (alpha 1-AT), antithrombin III (AT III), and alpha 1-antichymotrypsin (alpha 1-AC). alpha 2-PI seems to be the most distantly related among these inhibitors. Comparison of the phylogenetic trees of proteases and their inhibitors indicates that four proteases, namely elastase (or trypsin), chymotrypsin, plasminogen activator, and thrombin, may have evolved concurrently with the corresponding inhibitors. However, alpha 2-PI and PCI seem to have evolved asynchronously from their substrates. The data suggest that alpha 2-PI may originally have inhibited some protease other than plasmin, and protein C may have had an inhibitor different from the present one early in its evolutionary history.     

Sequencing and high expression of aminopeptidase P gene from Escherichia coli HB101

A plasmid pAPP1 with a 4 kbp insert at the PstI site of pBR322, encoding aminopeptidase P gene of Escherichia coli HB101 (Yoshimoto et al. (1988) J. Biochem. 104, 730-734), was subcloned into pUC18 and pUC19. The transformant of E. coli JM83 harboring pAPP4 with a 1.9 kbp fragment showed more than 50-fold higher enzyme activity than that of the host, after cultivation at 37 degrees C for 40 h in LB-medium containing ampicillin. When the gene DNA was inserted reversely in pAPP4, the enzyme productivity decreased markedly. The whole nucleotide sequence of the inserted fragment of plasmid pAPP4 was clarified by the dideoxy chain-terminating method. Within this sequence, the mature enzyme protein-encoding sequence was found to start just after an ATG codon, as judged by comparison with amino-terminal protein sequencing. Eleven bases upstream from the proposed initiation codon was an AGGAGA sequence which seemed to be a ribosome binding site. Thirty-four bases upstream from the proposed start codon was the 6-base sequence TACAAA, the so-called -10 region or Pribnow box. Further, the 6-base sequence TTTACT around 77 bases upstream from the start codon was deduced to be a putative -35 region consensus sequence. The inverted repeat at 1334 was tentatively assumed to be a terminator. The molecular weight of the enzyme was estimated to be 49,650 from the nucleotide sequence. The purified enzyme contained 0.2 gram atom of zinc per subunit. The enzyme activity was inhibited by EDTA and activated 5-fold by Mn2+.