A 160-bp palindrome is a Rad50.Rad32-dependent mitotic recombination hotspot in Schizosaccharomyces pombe

Palindromic sequences can form hairpin and cruciform structures that pose a threat to genome integrity. We found that a 160-bp palindrome (an inverted repeat of 80 bp) conferred a mitotic recombination hotspot relative to a control nonpalindromic sequence when inserted into the ade6 gene of Schizosaccharomyces pombe. The hotspot activity of the palindrome, but not the basal level of recombination, was abolished by a rad50 deletion, by a rad50S "separation of function" mutation, or by a rad32-D25A mutation in the nuclease domain of the Rad32 protein, an Mre11 homolog. We propose that upon extrusion of the palindrome the Rad50.Rad32 nuclease complex recognizes and cleaves the secondary structure thus formed and generates a recombinogenic break in the DNA.     

The role of the epidermis in kinetin-induced retardation of chlorophyll degradation in tobacco leaf discs during senescence

Three kinds of discs were taken from tobacco leaves whose lowerepidermis had been peeled off, half-peeled or unpeeled. Therole of the epidermis and its relation to the kinetin effecton chlorophyll degradation during senescence were studied. Ourresults follow.

1. Chlorophyll degradation due to kinetin was retarded only whenthe lower epidermis was present.
2. The decrease in chlorophyllcontent in leaf discs on water duringsenescence was nearlyproportional to the size of the lowerepidermis attached tothe discs; i.e., unpeeled discs>half-peeleddiscs>peeleddiscs.
3. Cellular fractions possessing activity which induceschlorophylldegradation were extracted from the isolated lowerepidermis(i, ii) and its acetone powder (iii): (i) L-2 fraction(1.14d1.16)was separated by stepwise sucrose density-gradientcentrifugationfrom the 10,000?g pellet of the cell homogenate.(ii) The A-fraction(M.W.5,000) was precipitated with 0–80%saturation ofammonium sulfate from 105,000 ? g supernatantof cell homogenateand eluted in the void volume by SephadexG-25 column chromatography.(iii) The fraction precipitatedwith 0–30% saturationof ammonium sulfate from the 105,000?gsupernatant, containeda large amount of DNA and its activityremained even if DNAwas removed.
4. Activity was not retainedwhen the fractions were obtained fromisolated lower epidermispretreated with 2?10^–5 M kinetinfor 2 hr in darknessat 25?C.

(Received June 3, 1976; )     

Possible role of prostaglandin F in blastocyst implantation

The synthesis and release of Prostaglandin F (PGF) by the rabbit blastocyst and endometrium were investigated on Day 6 and 7, using radioimmunoassay, autoradiography and conversion experiments. The following results were obtained:

• 1.1) The content of PGF in the blastocyst increased significantly (P < 0.01) form Day 6 and 7.
• 2.2) The content of PGF in the endometrium was significantly higher (P < 0.05) on Day 7 implantation sites compared to the other areas.
• 3.3) The in vitro synthesis and release of PGF by Day 6 balstocysts sharply increased after one and two hours of culture, respectively. Thereafter both values declined with time.
• 4.4) The in vitro synthesis and release of PGF by Day 6 endometria increased continuously with time.
• 5.5) ¹⁴C-arachidonic acid (¹⁴C-AA) was incorporated into Day 6 blastocysts in vitro and converted to PGF_2α. These results suggest that both the endometrium and the blastocyst are the sources of the PGs involved in implantation, and that PGF derived from the blastocysts may act as the trigger of implantation.

     

Immunogenic protein MPB57 from Mycobacterium bovis BCG: Molecular cloning, nucleotide sequence and expression

Using a single-probe method, we have cloned the gene for an immunogenic MPB57 protein of Mycobacterium bovis BCG. The nucleotide sequence includes an ORF of 300 base pairs encoding a protein of 99 amino acids with an M_r of 10 818. This cloned gene was expressed in an Escherichia coli expression vector to give a mature protein which reacted with a polyclonal antibody raised against MPB57.     

Microbial Production of Glyceric Acid,an Organic Acid That Can Be Mass Produced from Glycerol

Glyceric acid (GA), an unfamiliar biotechnological product, is currently produced as a small by-product of dihydroxyacetone production from glycerol by Gluconobacter oxydans. We developed a method for the efficient biotechnological production of GA as a target compound for new surplus glycerol applications in the biodiesel and oleochemical industries. We investigated the ability of 162 acetic acid bacterial strains to produce GA from glycerol and found that the patterns of productivity and enantiomeric GA compositions obtained from several strains differed significantly. The growth parameters of two different strain types, Gluconobacter frateurii NBRC103465 and Acetobacter tropicalis NBRC16470, were optimized using a jar fermentor. G. frateurii accumulated 136.5 g/liter of GA with a 72% d-GA enantiomeric excess (ee) in the culture broth, whereas A. tropicalis produced 101.8 g/liter of d-GA with a 99% ee. The 136.5 g/liter of glycerate in the culture broth was concentrated to 236.5 g/liter by desalting electrodialysis during the 140-min operating time, and then, from 50 ml of the concentrated solution, 9.35 g of GA calcium salt was obtained by crystallization. Gene disruption analysis using G. oxydans IFO12528 revealed that the membrane-bound alcohol dehydrogenase (mADH)-encoding gene (adhA) is required for GA production, and purified mADH from G. oxydans IFO12528 catalyzed the oxidation of glycerol. These results strongly suggest that mADH is involved in GA production by acetic acid bacteria. We propose that GA is potentially mass producible from glycerol feedstock by a biotechnological process.A shift from petroleum to bio-based feedstocks will be necessary for a sustainable industrial society and effective management of greenhouse gas emissions (2, 20). Biodiesel fuel (BDF) is produced from vegetable oils and animal fats and can replace the diesel in diesel engine motors. Although the European Union currently produces 82% of the BDF produced in the world (7), the use of BDF will probably continue to grow worldwide, because petroleum is a limited resource. Massive amounts of glycerol can be obtained as a by-product of BDF production (approximately 10% by weight) through transesterification with alcoholysis generally catalyzed by NaOH or KOH. As the use of this glycerol is an important component of the economics of the BDF industry, there is a worldwide demand for the efficient use of glycerol (24).Among the recent developments in the conversion of glycerol into valuable chemicals, epichlorohydrin (ECH) and 1,2-propanediol (propylene glycol) are now commercially synthesized from glycerol by chemical processes, and 1,3-propanediol (1,3-PDO) and dihydroxyacetone (DHA) are produced from glycerol by biotechnological processes (4, 5, 17, 18, 24, 25). ECH, propylene glycol, and 1,3-PDO are used mainly as intermediates for resins and polymers. However, an increase in the price of glycerol, such as that which occurred due to the collapse of the BDF market (its price increased nearly threefold in Germany by the end of 2007 [24]), can have a large negative effect on the production of such low-price commodity chemicals. Hence, research on the production of more value-added and functional chemicals from (raw) glycerol is important.Recently, we have focused on the production of a glycerol derivative, glyceric acid (GA), using a bioprocess (Fig. ​(Fig.1)1) (9, 10). GA from an extract of Cynara scolymus leaves has liver stimulant and cholesterolytic activity in dogs (11), and d-GA calcium salt accelerates ethanol and acetaldehyde oxidation in rats (8). GA-based esters also exhibit antitrypsin activity (12), and novel oligoesters based on GA derivatives may be useful for pharmaceutical purposes, such as drug delivery systems (23). These reports suggest that GA is a promising chemical, but it is very expensive as a reagent for investigational use.Open in a separate windowFIG. 1.Proposed pathway for the conversion of glycerol to GA (glyceric acid) by acetic acid bacteria. The bioconversion of glycerol to DHA (dihydroxyacetone) is also represented.Before we began our research, little was known about GA as a biotechnological product, except for one Japanese patent from 25 December 1987 (Daicel Chemical Industries, Japanese patent application 51069) and a report on its by-production during DHA production by Gluconobacter oxydans (3, 21). According to the patent, resting cells of Gluconobacter cerinus IFO3262 (later Gluconobacter frateurii NBRC3262) converted 100 g/liter of glycerol to 57 g/liter of d-GA in a fermentor over a 2-day incubation. Recently, we revealed that Acetobacter tropicalis NBRC16470 produced 22.7 g/liter of optically pure d-GA from 200 g/liter of glycerol in a fermentor over a 4-day incubation (9). However, because this method of GA production is far from practical, we are attempting to develop a GA manufacturing bioprocess based on strain, fermentation, and process development.In this study, we searched for a GA producer among 162 acetic acid bacterial strains and investigated the GA productivity and enantiomeric composition of 88 selected strains. We also investigated oxidative fermentation conditions in a 5-liter jar fermentor and applied electrodialysis (ED) to recover glycerate from culture broth. Furthermore, we clarified the gene and enzyme involved in GA production from glycerol for the first time.     

Globular adiponectin induces adhesion molecule expression through the sphingosine kinase pathway in vascular endothelial cells

The signaling pathways that couple adiponectin receptors to functional, particularly inflammatory, responses have remained elusive. We report here that globular adiponectin induces endothelial cell activation, as measured by the expression of adhesion proteins such as vascular adhesion molecule-1 (VCAM-1), intracellular adhesion molecule-1 (ICAM-1), E-selectin and MCP-1, through the sphingosine kinase (SKase) signaling pathway. Treatment of human umbilical vein endothelial cells with globular adiponectin resulted in NF-kappaB activation and increased mRNA levels of VCAM-1, ICAM-1, E-selectin and MCP-1. Sphingosine 1-phosphate (S1P), but not ceramide or sphingosine, was a potent stimulator of adhesion protein expression. As S1P is generated from sphingosine by SKase, we treated cells with siRNA for SKase to silence the effects of S1P in the endothelial cells. Treatment with SKase siRNA inhibited globular adiponectin-induced NF-kappaB activation and markedly decreased the globular adiponectin-induced mRNA levels of adhesion protein. Thus, we demonstrated that the SKase pathway, through the generation of S1P, is critically involved in mediating globular adiponectin-induced endothelial cell activation.