The gamma-secretase complex: machinery for intramembrane proteolysis

Gamma-secretase is a membrane protease complex that possesses presenilin as a catalytic subunit. Presenilin generates amyloid beta peptides in the brains of Alzheimer's patients and is indispensable to Notch signaling in tissue development and renewal. Recent studies have revealed how presenilin is assembled with its cofactor proteins and acquires the gamma-secretase activity: Aph-1 and nicastrin initially form a subcomplex to bind and stabilize presenilin, and then Pen-2 confers the gamma-secretase activity and facilitates endoproteolysis of presenilin. Understanding the mechanism of gamma-secretase cleavage will help to clarify how intercellular cell signaling through transmembrane proteins is regulated by intramembrane proteolysis, and will hopefully eventually lead to a cure for Alzheimer's disease.     

Molecular cloning and characterization of a cDNA encoding ent-cassa-12,15-diene synthase, a putative diterpenoid phytoalexin biosynthetic enzyme, from suspension-cultured rice cells treated with a chitin elicitor

We have isolated and characterized a cDNA encoding a novel diterpene cyclase, OsDTC1, from suspension-cultured rice cells treated with a chitin elicitor. OsDTC1 functions as ent-cassa-12,15-diene synthase, which is considered to play a key role in the biosynthesis of (-)-phytocassanes recently isolated as rice diterpenoid phytoalexins. The expression of OsDTC1 mRNA was also confirmed in ultraviolet (UV)-irradiated rice leaves. In addition, we identified ent-cassa-12,15-diene, a putative diterpene hydrocarbon precursor of (-)-phytocassanes, as an endogenous compound in the chitin-elicited suspension-cultured rice cells and the UV-irradiated rice leaves. The OsDTC1 cDNA isolated here will be a useful tool to investigate the regulatory mechanisms of the biosynthesis of (-)-phytocassanes in rice.     

Differential expression of alpha2D-adrenoceptor and eNOS in aortas from early and later stages of diabetes in Goto-Kakizaki rats

The aim of the present study was to compare vascular dysfunction between the early (12 wk old) and later (36 wk old) stages of spontaneous diabetes in Goto-Kakizaki (GK) rats. We also evaluated the aortic expression of the alpha(2D)-adrenoceptor and endothelial nitric oxide synthase (eNOS). Vascular reactivity was assessed in thoracic aortas from age-matched control rats and 12- and 36-wk GK rats. Using RT-PCR and immunoblots, we also examined the changes in expression of the alpha(2D-)adrenoceptor and eNOS. In aortas from GK rats (vs. those from age-matched control rats): 1) the relaxation response to ACh was enhanced at 12 wk but decreased at 36 wk; 2) the relaxation response to sodium nitroprusside was decreased at both 12 and 36 wk, 3) norepinephrine (NE)-induced contractility was decreased at 12 wk but not at 36 wk, 4) the expressions of alpha(1B)- and alpha(1D)-adrenoceptors were unaffected, whereas those of alpha(2D)-adrenoceptor and eNOS mRNAs were increased at both 12 and 36 wk; and 5) NE- and ACh-stimulated NO(x) (nitrite and nitrate) levels were increased at 12 wk, although at 36 wk ACh-stimulated NO(x) was lower, whereas NE-stimulated NO(x) showed no change. These results clearly demonstrate that enhanced ACh-induced relaxation and impaired NE-induced contraction, due to NO overproduction via eNOS and increased alpha(2D)-adrenoceptor expression, occur in early-stage GK rats and that the impaired ACh-induced relaxation in later-stage GK rats is due to reductions in both NO production and NO responsiveness (but not in eNOS expression).     

Molecular characterization of dextranase from Streptococcus rattus

The complete nucleotide sequence of the dextranase gene of Streptococcus rattus ATCC19645 was determined. An open reading frame of the dextranase gene was 2,760 bp long and encoded a dextranase protein consisting of 920 amino acids with a molecular weight of 100,163 Da and an isoelectric point of 4.67. The S. rattus dextranase purified from recombinant Escherichia coli cells showed dextran-hydrolyzing activity with optimal pH (5.0) and temperature (40 C) similar to those of dextranases from Streptococcus mutans and Streptococcus sobrinus. The deduced amino acid sequence of the S. rattus dextranase revealed that the dextranase molecule consists of two variable regions and a conserved region. The variable regions contained an N-terminal signal peptide and a C-terminal cell wall sorting signal; the conserved region contained two functional domains, catalytic and dextran-binding sites. This structural feature of the S. rattus dextranase is quite similar to that of other cariogenic species such as S. mutans, S. sobrinus, and Streptococcus downei.     

First isolation of Bartonella henselae type I from a cat-scratch disease patient in Japan and its molecular analysis

We isolated Bartonella henselae from an inguinal lymph node of a 36-year-old male patient with cat-scratch disease. The patient had many areas of erythema on his body, swelling of the left inguinal lymph nodes with pain and slight fever. The diagnosis was made on the basis of polymerase chain reaction for B. henselae DNA from the lymph node biopsies and blood sample, and isolation of the organism, histology of the lymph node and serology with an indirect immunofluorescent antibody test. We also analyzed the genome profiles for five strains of 90 isolates from the lymph node by pulsed-field gel electrophoresis after Not I endonuclease digestion. We found two different genomic profiles. These results suggest that the patient had been either co-infected or re-infected with two genetically different strains of B. henselae.     

Plant-growth inhibitory activity of heliannuol derivatives

In addition to (+)-, (-)- and (+/-)-heliannuol E, growth-inhibitory activities of five synthetic chromans and four tetrahydrobenzo[b]oxepins were examined against oat and cress. All heliannuol E isomers exhibited similar biological activities against cress, whereas when tested against oat roots, the unnatural optical isomer (+) showed no inhibitory activity. Four brominated chromans and two tetrahydrobenzo[b]oxepin derivatives also showed apparent inhibition against both cress and oat.     

Target proteins of the cytosolic thioredoxins in Arabidopsis thaliana

Possible target proteins of cytosolic thioredoxin in higher plants have been investigated in the cell lysate of dark-grown Arabidopsis thaliana whole tissues. We immobilized a mutant of cytosolic thioredoxin, in which an internal cysteine at the active site was substituted with serine, on CNBr activated resin, and used the resin for the thioredoxin-affinity chromatography. By using this resin, the target proteins for thioredoxin in the higher plant cytosol were efficiently acquired. The obtained proteins were separated by two-dimensional gel electrophoresis and analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Thus we have identified proteins of the anti-oxidative stress system proteins (ascorbate peroxidase, germin-like protein, and monomeric type II peroxiredoxin), proteins involved in protein biosynthesis (elongation factor-2 and eukaryotic translation initiation factor 4A), proteins involved in protein degradation (the regulatory subunit of 26S proteasome), and several metabolic enzymes (alcohol dehydrogenase, fructose 1,6-bis phosphate aldolase-like protein, cytosolic glyceraldehyde 3-phosphate dehydrogenase, cytosolic malate dehydrogenase, and vitamin B(12)-independent methionine synthase) together with some chloroplast proteins (chaperonin 60-alpha and 60-beta, heat shock protein 70, and glutamine synthase). The results in this study and recent proteomics studies on the target proteins of chloroplast thioredoxin indicate the versatility and the physiological significance of thioredoxin as reductant in plant cell.