Improved strains for the production of riboflavin (vitamin B2) were constructed through metabolic engineering using recombinant DNA techniques in Corynebacterium ammoniagenes. A C. ammoniagenes strain harboring a plasmid containing its riboflavin biosynthetic genes accumulated 17-fold as much riboflavin as the host strain. In order to increase the expression of the biosynthetic genes, we isolated DNA fragments that had promoter activities in C. ammoniagenes. When the DNA fragment (P54-6) showing the strongest promoter activity in minimum medium was introduced into the upstream region of the riboflavin biosynthetic genes, the accumulation of riboflavin was 3-fold elevated. In that strain, the activity of guanosine 5'-triphosphate (GTP) cyclohydrolase II, the first enzyme in riboflavin biosynthesis, was 2.4-fold elevated whereas that of riboflavin synthase, the last enzyme in the biosynthesis, was 44.1-fold elevated. Changing the sequence containing the putative ribosome-binding sequence of 3,4-dihydroxy-2-butanone 4-phosphate synthase/GTP cyclohydrolase II gene led to higher GTP cyclohydrolase II activity and strong enhancement of riboflavin production. Throughout the strain improvement, the activity of GTP cyclohydrolase II correlated with the productivity of riboflavin. In the highest producer strain, riboflavin was produced at the level of 15.3 g l(-1) for 72 h in a 5-l jar fermentor without any end product inhibition.

Abstract-A practical procedure for evaluating uricosuric agents was demonstrated using clearance experiments with potassium oxonate-treated rats. The fractional excretion value of uric acid showed a reabsorptive net flux of uric acid in the renal tubules of the animal, though the value was obviously higher than those of primates such as men, chimpanzees and cebus monkeys. However, the rats responded well to uricosuric drugs and diuretics. Probenecid and uricosuric diuretics such as tienilic acid induced hyper uricosuria due to the increase of fractional excretion of uric acid and/or the increase of the filtered amount of uric acid with the rise of plasma uric acid. On the other hand, furosemide had no effect on uric acid excretion at a low dose with moderate diuresis, while a higher dose decreased the fractional excretion of uric acid with elevation of plasma uric acid. Benzothiazides were also uricosuric at the lower doses, but the high dose, as in the case of the so-called uricosuric drugs, had no effect on the uric acid excretion and plasma uric acid level. Thus, oxonate-treated rats were useful for evaluating drug effects on uric acid excretion.Because of species differences in the metabolism and excretion of uric acid, animal experiments have contributed little to the evaluation of drug activities on the renal handling of uric acid, except for those using primates such as chimpanzees and cebus monkeys. The urinary excretion of uric acid, however, has been well studied with various nonhuman mammalia in order to find useful animals for the evaluation, resulting in some successful examples (1). The rat is such an animal because it shows a reabsorptive net flux of uric acid in the renal tubules, though the animal handles uric acid by both hepatic metabolism and renal excretion. On the other hand, useful inhibitors of urate oxidase have already been developed to create the disease model of hyperuricemia (2-4), but the inhibitor-treated animals have not been utilized enough for evaluating uricosuric agents. A practical procedure for evaluating the drugs with potassium oxonate treated rats is reported here. Materials and MethodsTen-week-old male Slc-Wistar rats were used. To determine uric acid and inulin clearance, the animals were given potassium oxonate, 250 mg/kg i.p., and then the right femoral artery, left femoral vein and urinary bladder were cannulated for blood collection, infusion and urine collection, respectively, under anesthesia with sodium pentobarbital, 50 mg/kg i.p., within 2 hr after the adminis tration of oxonate. The same dose of oxonate was given again just 2 hr after the first administration, and then 60% urethane, 2 ml/kg, and 15% inulin, 4 ml/kg, were administered subcutaneously. The animals were then infused with 4% mannitol-1.5% inulin-0.9% sodium chloride solution at the flow rate of 0.1 ml/min on a hot plate at

Abstract:The effects of intracisternal administration of endothelin-1 on blood-brain barrier permeability were examined in dogs and rats. Single doses of endothelin-1 elevated blood-brain barrier permeability about two times compared with control groups in both species. However, repeated dosing with endothelin-1 at a 24 hr intervals caused a highly enhanced disruption of blood-brain barrier permeability in dogs, but not in rats, whereas the repeated administration with a 48 hr interval markedly increased the blood-brain barrier permeability in both species of animals (dogs: 923%, rats: more than 661%). Moreover, this abnormally enhanced permeability of blood-brain barrier in dogs was completely blocked by pretreatment with the endothelin ET-A receptor selective antagonist, S-0139, administered prior to either the first or second dosing with endothelin-1. From these results, we conclude that a repeated attack of endothelin-1 from the adventitial site of brain blood vessels produces a severe disruption of blood-brain barrier permeability through an endothelin ET-A receptor-mediated process.

We examined the effect of intracisternal application of endothelin-1 (ET-1) on the permeability of fluorescein into the cerebrospinal fluid (CSF) in beagle dogs in order to evaluate its role in disruption of blood-brain barrier (BBB) permeability seen in the subarachnoid hemorrhage animal model. Intracisternal application of their autologous blood for producing a canine two-hemorrhage model revealed an enhanced fluorescein permeability into the CSF together with the development of cerebral vasospasm. A single dose of ET-1 (40 pmol/animal) significantly increased penetration of fluorescein compared with that in normal dogs. Although its magnitude was much less than that in the two-hemorrhage model after the first administration of ET-1, the second challenge of the same dose of ET-1 with a 48-h interval produced marked disruption of BBB permeability similar to those in the animal model. Moreover, the ET-1-induced enhancement of fluorescein permeability into the CSF was completely prevented by intracisternal pretreatment with an endothelin ET(A)-receptor selective antagonist, S-0139 (0.03 mg/kg), as were the ET-1-induced cerebral vasoconstriction and behavioral changes as previously reported. Thus, we conclude that ET-1 acting on the adventitial site of brain in dogs contributes to the disruption of BBB permeability via endothelin ET(A)-receptor mediation.

Abstract-Effects of antihypertensive and uricosuric drugs were studied on the plasma and urinary levels of uric acid in oxonate-treated rats. We made use of animals with a catheterized aorta to successively collect blood samples and this procedure simplified the evaluation of progressive changes of plasma uric acid, under successive loading with potassium oxonate.The plasma uric acid level of the oxonate-treated rats was increased even with a single administration of diuretic chlorothiazides, furosemide, diazoxide and also uricosuric drugs such as tienilic acid and probenecid.On the other hand, a wellmaintained plasma uric acid level was also produced by exogenously administered uric acid in rats which had been given allopurinol and potassium oxonate.Diazoxide, tienilic acid and probenecid increased the plasma uric acid, while diuretic chlorothiazides did not. Furosemide tended to decrease the plasma uric acid level at the early stage of administration to rats treated with allopurinol, oxonate and uric acid, but increased these levels several hours later when the effect was studied by uric acid loading test with rats treated with allopurinol and oxonate. These effects also appeared as changes in the urine-excreted uric acid. Thus, the oxonate-treated rats demonstrated an acutely induced hyperuricemia not only with certain antihypertensives, but also with uricosuric drugs. The utility of these procedures for evaluating the hyperuricemic and uricosuric effects of drugs is discussed.Since selective inhibitors of urate oxidase were reported by Fridovich (1) and Iwata et al.(2), the utility of inhibitor-treated animals has enabled a better understanding of various problems related to hyperuricemia (3).However, it has not been clarified whether such an approach is feasible for predicting the hyperuricemic or uricosuric effects of drugs; in particular, problems arising from species differences in the renal functions for uric acid excretion should be further investigated.Hyperuricemia is sometimes reported to be an undesirable side effect of some anti hypertensives (4-8), and a number of agents have been practically utilized as uricosuric drugs.However, the characterization of these drugs has been mainly developed in the clinical field, and therefore a useful model for evaluating characteristics in animal studies has long been awaited. The present study was undertaken to evaluate the utility of potassium oxonate-treated rats as models for such a purpose. Acutely induced hyperuricemia was demonstrated by some antihypertensive and uricosuric drugs. MATERIALS AND METHODS Animals:Nine-week-old male Wistar strain rats were maintained on a solid diet CA-1 (Japan CLEA Co., Tokyo) and tap water ad libitum. Experiments were done using rats