Carotenoid production in <Emphasis Type="Italic">Bacillus subtilis</Emphasis> achieved by metabolic engineering

The carotenoid synthetic genes, crtM and crtN, derived from Staphylococcus aureus, were introduced into B. subtilis, resulting in yellow pigmentation. Absorption maxima of pigments and MALDI-TOF mass spectrometry demonstrated that the pigmented strain accumulated two C₃₀ carotenoids, 4,4′-diapolycopene and 4,4′-diaponeurosporene. A survival test using H₂O₂ revealed that the pigmented strain was more resistant to oxidative stress than the strain harboring an empty-vector. These findings indicate that B. subtilis can produce carotenoids, and the strain accumulating the carotenoids, CarotenoBacillus, will become a basal host for production of C₃₀ carotenoids and evaluation of their antioxidative effects.     

Participation of caspase-3-like protease in oxidation-induced impairment of erythrocyte membrane properties

Erythrocytes are very susceptible to oxidative stress, having a high content of intracellular oxygen and hemoglobin. In the present study, exposure to oxidative stress resulted in a significant impairment of erythrocyte membrane functions, such as deformability and anion exchange. Band 3 protein, also known as anion exchanger-1, plays an important role in these two functions. We show that oxidative stress activated caspase-3 inside the erythrocytes, which resulted in band 3 protein cleavage. Interestingly, inhibition of the caspase-3 with its specific inhibitor not only suppressed the digestion of band 3 protein, but also blunted the functional damage to erythrocytes, such as deformability and anion exchange, without changing the level of peroxidation of membrane lipids. These results provide experimental evidence that activation of caspase-3 plays an important role in the oxidative stress-induced impairment of membrane functions of erythrocytes.     

Maximization of hydrogen production ability in high-density suspension of Rhodovulum sulfidophilum cells using intracellular poly(3-hydroxybutyrate) as sole substrate

Growth of and hydrogen production by wild-type (WT) Rhodovulum sulfidophilum were compared with those by one of its mutants lacking the poly(3-hydroxybutyrate) (PHB) biosynthesis ability (PNM2). During phototrophic growth under aerobic conditions with fixed illumination, changes in the extinction coefficient and PHB content of WT and PNM2 cells revealed interference of light penetration by PHB. WT cells synthesized PHB at an early stage of the cultivation. PHB degradation after exhaustion of acetate during the cultivation of WT resulted in a decrease of the extinction coefficient. The hydrogen production rate under anaerobic conditions with fixed illumination was examined in WT and PNM2 cell suspensions at different densities. The hydrogen production rate was determined not by the light penetration but by the kinds of hydrogen donors and the density of suspension. The highest value of the rate of hydrogen production from PHB, 33.0 ml/l/h, was improved compared with 26.6 ml/l/h, which was the highest value in hydrogen production from succinate. Under the same illumination, conversion to hydrogen from PHB is more efficient than that from succinate, which is one of the best substrates for hydrogen production. These results suggest that the hydrogen production rate can be maximized in the hydrogen production system based on PHB degradation, which is achieved in high-density suspension under external-substrate-depleted conditions after aerobic cultivation in the presence of an excess amount of acetate.     

Novel 4-amino-furo[2,3-d]pyrimidines as Tie-2 and VEGFR2 dual inhibitors

A novel class of furo[2,3-d]pyrimidines has been discovered as potent dual inhibitors of Tie-2 and VEGFR2 receptor tyrosine kinases (TK) and a diarylurea moiety at 5-position shows remarkably enhanced activity against both enzymes. One of the most active compounds, 4-amino-3-(4-((2-fluoro-5-(trifluoromethyl)phenyl)amino-carbonylamino)phenyl)-2-(4-methoxyphenyl)furo[2,3-d]pyrimidine (7k) is <3 nM on both TK receptors and the activity is rationalized based on the X-ray crystal structure.     

Development of Neospora caninum cultured with human serum in vitro and in vivo

Because there has been no report of symptomatic Neospora caninum infection in humans, we examined the effect of human serum on the parasite's growth in either a bovine angioendothelial cell or Caco-2 cell culture in vitro and in immunocompromised mice in vivo. There was no difference in intracellular parasite numbers between cells incubated with human serum at 24 hr after challenge and those incubated with fetal bovine serum (FBS), which has no titer for the anti-N. caninum agglutination antibody test. Serum of sheep infected with N. caninum, which has the anti-N. caninum antibody, reduced the numbers of the intracellular parasite significantly. We also showed that there was no inhibitory effect on the intracellular multiplication of the parasite in cells incubated with human serum through incorporation of 3H-uracil. CB-17 scid mice administered human serum daily and challenged with N. caninum died on day 20 or 22 after challenge, when large numbers of parasite clusters were found in the brain, oviduct, adrenal gland, lung, stomach, spleen, skeletal muscle, pancreas, and mesenteric lymph nodes. Scid mice administered FBS survived until the end of the experiment. These results suggest that adult human serum may have no inhibitory effect on the development of N. caninum in vitro and in vivo.     

Developmental expression and intracellular location of P400 protein characteristic of Purkinje cells in the mouse cerebellum

The developmental expression and intracellular localization of a cerebellum-characteristic 250-kDa glycoprotein, P400 protein, were studied by immunohistochemical and immunoblot methods using a monoclonal antibody against P400 protein. In the cerebellum of normal mouse, the expression of P400 protein increased from Postnatal Day 3 to Day 21. This enhancement of P400 protein expression occurred only in the Purkinje cells and proceeded with the growth of their dendritic arborization. Electron microscopic analysis indicated that P400 protein is present at the plasma membrane, the endoplasmic reticulum, and the postsynaptic densities of Purkinje cells. Immunohistochemistry of the cerebella of neurological mutant mice indicated that the Purkinje cells of reeler, weaver, and pcd mutant mice retain the ability to produce a large amount of P400 protein. However, the Purkinje cells of staggerer mutant mouse proved to be incapable of enhanced P400 protein expression. These results indicate that P400 protein is a Purkinje cell-characteristic plasma membrane-associated glycoprotein, which is also present at the postsynaptic density and endoplasmic reticulum and that the expression of P400 protein in Purkinje cells is closely associated with the growth of their dendritic arborization.     

Changes in important apoptosis-related molecules in the endothelin-1-induced hypertrophied cardiomyocytes: effect of the pretreatment with eicosapentaenoic Acid

Human heart failure is preceded by a process called cardiac remodeling, in which heart chambers progressively enlarge and contractile function deteriorates. Programmed cell death (apoptosis) of cardiac muscle cells has been identified as an essential process in the progression to heart failure. The execution of the apoptotic program entails complex interactions between and execution of multiple molecular subprograms. Endothelin (ET)-1, a potent vasoconstrictor peptide, is synthesized and secreted by cardiomyocytes and induces hypertrophy of cardiomyocytes. The cardiovascular benefit of fish oil containing eicosapentaenoic acid (EPA) in humans and experimental animals was reported. Recently, we found that ET-1-induced cardiomyocytic remodeling could be prevented by pretreatment with EPA. The aim of the present study is to investigate whether there would be any alteration in the expression of important apoptosis-related molecules in ET-1-administered hypertrophied cardiomyocytes. We also sought to determine, if there are alterations in apoptotic molecules, what type of role for EPA would then exist. Ventricular cardiomyocytes were isolated from 2-day-old Sprague-Dawley rats and were cultured for 3 days. At Day 4 of culture, the cardiomyocytes were divided into three groups: control, the ET-1 (0.1 nM)-treated group, and the ET-1 group pretreated with EPA (10 microM). Twenty-four hours after the treatment, the gene expressions of three important molecules related to apoptosis (caspase-3, Bax, and Bcl-2) in three experimental groups were evaluated by real-time polymerase chain reaction. The present study could not demonstrate any significant or representative alteration in any of the above three apoptosis-related important markers in either ET-1-induced hypertrophied cardiomyocytes with or without EPA pretreatment. The present study would at least be able to exclude the involvement of some representative molecules related to apoptosis in ET-1-induced hypertrophied cardiomyocytes. In addition, the present study demonstrates that the antihypertrophic effect of EPA to ET-1-administered cardiomyocytes appears not to modulate the apoptosis signaling cascade.     

Isolation and characterization of the complete complementary and genomic DNA sequences of human serum amyloid P component

Complementary and genomic DNA clones corresponding to the human serum amyloid P component (SAP) mRNA have been isolated and analyzed. The nucleotide sequences of the cDNA and the corresponding regions of the genomic SAP DNA reported here were identical, and revealed that after coding for a signal peptide of 19 amino acids and the first two amino acids of the mature SAP protein, there is one small intron of 115-base pairs (bp), followed by a nucleotide sequence coding for the remaining 202 amino acid residues. The SAP gene has an ATATAAA sequence 29-bp upstream from the cap site, but there is no CAAT box-like sequence. A possible polyadenylation signal sequence, ATTAAA, was found to be located 28-bp upstream from the polyadenylation site. A comparison of the genomic SAP DNA sequence with that of human C-reactive protein (CRP) revealed a striking overall homology which was not uniform: several highly conserved regions were bounded by non-homologous regions. This comparison provides further support for the hypothesis that SAP and CRP are products of a gene duplication event.     

Amyloid beta protein (25-35) phosphorylates MARCKS through tyrosine kinase-activated protein kinase C signaling pathway in microglia

Myristoylated alanine-rich C kinase substrate (MARCKS) is a widely distributed specific protein kinase C (PKC) substrate and has been implicated in membrane trafficking, cell motility, secretion, cell cycle, and transformation. We found that amyloid beta protein (A beta) (25-35) and A beta (1-40) phosphorylate MARCKS in primary cultured rat microglia. Treatment of microglia with A beta (25-35) at 10 nM or 12-O-tetradecanoylphorbol 13-acetate (1.6 nM) led to phosphorylation of MARCKS, an event inhibited by PKC inhibitors, staurosporine, calphostin C, and chelerythrine. The A beta (25-35)-induced phosphorylation of MARCKS was inhibited by pretreatment with the tyrosine kinase inhibitors genistein and herbimycin A, but not with pertussis toxin. PKC isoforms alpha, delta, and epsilon were identified in microglia by immunocytochemistry and western blots using isoform-specific antibodies. PKC-delta was tyrosine-phosphorylated by the treatment of microglia for 10 min with A beta (25-35) at 10 nM. Other PKC isoforms alpha and epsilon were tyrosine-phosphorylated by A beta (25-35), but only to a small extent. We propose that a tyrosine kinase-activated PKC pathway is involved in the A beta (25-35)-induced phosphorylation of MARCKS in rat microglia.