Production of L-2,3-butanediol by a new pathway constructed in Escherichia coli.

AIMS: A metabolic pathway for L-2,3-butanediol (BD) as the main product has not yet been found. To rectify this situation, we attempted to produce L-BD from diacetyl (DA) by producing simultaneous expression of diacetyl reductase (DAR) and L-2,3-butanediol dehydrogenase (BDH) using transgenic bacteria, Escherichia coli JM109/pBUD-comb. METHODS AND RESULTS: The meso-BDH of Klebsiella pneumoniae was used for its DAR activity to convert DA to L-acetoin (AC) and the L-BDH of Brevibacterium saccharolyticum was used to reduce L-AC to L-BD. The respective gene coding each enzyme was connected in tandem to the MCS of pFLAG-CTC (pBUD-comb). The divided addition of DA as a source, addition of 2% glucose, and the combination of static and shaking culture was effective for the production. CONCLUSIONS: L-BD (2200 mg l(-1)) was generated from 3000 mg l(-1) added of DA, which corresponded to a 73% conversion rate. Meso-BD as a by-product was mixed by 2% at most. SIGNIFICANCE AND IMPACT OF THE STUDY: An enzyme system for converting DA to L-BD was constructed with a view to using DA-producing bacteria in the future.

Purification and characterization of L-2,3-butanediol dehydrogenase of Brevibacterium saccharolyticum C-1012 expressed in Escherichia coli.

The L-2,3-butanediol dehydrogenase produced in E. coli JM109/pLBD2-CTC was purified by 5 steps. The molecular mass of this enzyme was estimated at 110 kDa and the subunit was measured to be 30 kDa. The L-BDH had some differences from the BDHs from other sources in substrate specificity, pI value, pH stability, effects of divalent cations, and organic acids.

Stereochemical applications of the expression of the L-2,3-butanediol dehydrogenase gene in Escherichia coli.

The L-2,3-butanediol dehydrogenase (L-BDH) gene of Brevibacterium saccharolyticum was strongly expressed in Escherichia coli using the tac promoter. However, the stereospecificity of the resulting L-BDH was reduced. The region upstream of the meso-BDH gene of Klebsiella pneumoniae was also involved in the expression of the B. saccharolyticum gene. However, in this case, the resulting L-BDH exhibited more stable stereospecificity. A stereospecificity recognition region was located within the rear sequence (Hpa I site, carboxy terminal) of the BDH open reading frame. Using a transformed strain of E. coli, the conversion of L-acetoin (L-AC), in the commercially available racemic mixture of AC, to L-2,3-butanediol (L-BD) was attempted. As a result, 0.37% L-BD was formed from 1% AC added to the culture.

Crystal structure of meso-2,3-butanediol dehydrogenase in a complex with NAD+ and inhibitor mercaptoethanol at 1.7 A resolution for understanding of chiral substrate recognition mechanisms.

The crystal structure of a ternary complex of meso-2,3-butanediol dehydrogenase with NAD+ and a competitive inhibitor, mercaptoethanol, has been determined at 1.7 A resolution by means of molecular replacement and refined to a final R-factor of 0.194. The overall structure is similar to those of the other short chain dehydrogenase/reductase enzymes. The NAD+ binding site, and the positions of catalytic residues Ser139, Tyr152, and Lys156 are also conserved. The crystal structure revealed that mercaptoethanol bound specifically to meso-2,3-butanediol dehydrogenase. Two residues around the active site, Gln140 and Gly183, forming hydrogen bonds with the inhibitor, are important but not sufficient for distinguishing stereoisomerism of a chiral substrate.

[A case of meningioma of the jugular foramen without disturbance of lower cranial nerves].

We report a case of meningioma with extracranial extension via the jugular foramen not accompanied by any disturbances of the lower cranial nerves. A 32-year-old female suffered from a headache without any other neurological abnormalities. She had no particular family or past history. The extracranial extending meningioma was removed at the first operation. The meningioma of the jugular foramen was removed at the second staged operation by transcondylar approach. A meningioma of the jugular foramen is rare, and the meningioma with extracranial extension via the jugular foramen without showing disturbances of lower cranial nerves has not been reported so far.

Unilateral interhemispheric keyhole approach for anterior cerebral artery aneurysms.

A special midline interhemispheric keyhole approach to the anterior communicating artery aneurysms is described. A small trephine opening of 3 cm in diameter is used in most cases. The detailed microsurgical technique of the unilateral interhemispheric exposure of the anterior cerebral artery complex is presented. For the past 10 years, a total of 138 patients, 112 with Acom AN and 26 with distal ACA AN, were operated upon through this approach. There were 16 cases with non-ruptured aneurysms and the postoperative results were excellent in all of them. The overall results in 122 cases with ruptured aneurysms were excellent and good (working) in 95 cases, fair in 10 cases, poor in 9 and death supervened in 8 cases. The mortality rate in 71 acute operation cases was 8% and 4% in 51 delayed cases. The advantages of this approach include simple and rapid craniotomy, minimum brain retraction, accurate midline exposure of all parent arteries and the aneurysm. This anterior interhemispheric approach is, in our experience, much superior to the conventional pterional approach.

[A case of synchronous double primary malignant neoplasms (malignant astrocytoma, and lung cancer) diagnosed by autopsy].

Discussed is a deceased patient, a 54-year-old male, who was admitted to hospital with the chief complaint of motor aphasia. Computed assisted tomography in the cranium showed a massive lesion in the left brain. Further, chest X-ray showed an abnormal tumorous shadow with a pleural indentation in the left middle lung field. In spite of a negativity of cancer cells in a bronchial biopsy and a brushing, we decided that this case reflected a lung cancer with brain metastasis. However, on autopsy, the tumor in the lung histologically was determined as being an (adenocarcinoma alveolar cell type) and the brain tumor was found to be a malignant astrocytoma. We have reported on this case because a double primary neoplasm, a malignant astrocytoma and lung cancer, is rare in the literature.

[A case of asbestosis complicated with double cancer of the stomach and colon].

A case of a double cancer, a stomach and a colon cancer, in a 84-year-old man is reported. The patient had an occupational history of asbestos exposure while working at two shipyards. His chest X-ray revealed typical, pleural plaque with calcification. Further, a significant number of asbestos bodies in his autopsied lung tissue was detected. In spite of failure to detect asbestos bodies in the stomach and the colon, our findings suggest that these two cancers may have been induced by asbestos exposure.

[Evaluation of double cancers of the lung and stomach].

Five cases of double cancers involving the lung and the stomach cancer are reported. The final diagnosis were done by the autopsy. Through the use of sodium hypochlorite and a Millipore filter, many ferrous bodies were detected in all cases. Three of the subjects had occupational histories involving exposure to asbestos that may have had a connection with their cancers and many crocidolites were found in their autopsied lungs. The two other cases, however, did not have any such occupational history and few pathological asbestos bodies. Further, most subjects were heavy smokers. These findings suggested that a high percentage of double cancers of lung and stomach might have been induced by the expose to asbestos in this area.

Enzymic synthesis of leukotriene B4 in guinea pig brain.

Leukotriene B4 [5(S), 12(R)-dihydroxy-6, 14-cis-8,10-trans-eicosatetraenoic acid] was obtained from endogenous arachidonic acid when slices of the guinea pig brain cortex were incubated with the calcium ionophore A 23187. Enzymes involved in its synthesis, arachidonate 5-lipoxygenase [arachidonic acid to 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid and subsequently to leukotriene A4] and leukotriene A4 hydrolase (leukotriene A4 to B4), were present in the cytosol fraction. Arachidonate 5-lipoxygenase was Ca2+-dependent, and was stimulated by ATP and the microsomal membrane, as was noted for the enzyme from mast cells. The lipid hydroperoxides stimulated 5-lipoxygenase by four- to sixfold. The leukotriene A4 hydrolase activity was rich in brain, and the specific activity (0.4 nmol/min/mg of protein) was much the same as that of guinea pig leukocytes. High activities of these enzymes were detected in the olfactory bulb, pituitary gland, hypothalamus, and cerebral cortex. Since leukotriene B4 is enzymically synthesized in the brain, possible roles related to neuronal functions or dysfunctions deserve to be examined.