The transposition pattern of the Ac element in tobacco cultured cells.

We investigated physical distances and directions of transposition of the maize transposable element Ac in tobacco cultured cells. We introduced a T-DNA construct that carried a non-autonomous derivative of Ac (designated dAc-I-RS) that included sites for cleavage by restriction endonuclease MluI. Another cleavage site was also introduced into the T-DNA region outside of the dAc-I-RS transposable element. The tobacco cultured cell line BY-2 was transformed with the T-DNA and several transformed lines that had a single copy of the T-DNA at a different chromosomal location were isolated. These lines were co-cultured with Agrobacterium tumefaciens cells that carried a cDNA for the Ac transposase gene under the control of various promoters. Sublines of cultured cells in which dAc-I-RS had been transposed, were isolated. The genomic DNAs of these sublines were isolated and digested with MluI. Sizes of DNA segments generated by digestion were determined by pulse-field gel electrophoresis. Our results showed that 20 to 70% of transposition events had occurred within several hundreds kilo-base pairs (kb) on the same chromosome. These results demonstrate that the Ac-Ds element preferentially transposed to regions near the original site in a tobacco chromosome. In addition, the present results are an example of asymmetric transposition as demonstrated by the distance of transposition on the chromosome.

A zinc finger protein RHL41 mediates the light acclimatization response in Arabidopsis.

Arabidopsis thaliana plants showed an increased tolerance to high-intensity light when pre-exposed to medium-intensity light. This response, known as light acclimatization, depended on the quantity of light, the period of irradiation, and the quality of light. Among characterized acclimatization-induced cDNA clones, we identified a zinc finger protein rhl41 (responsive to high light) gene, that was rapidly up-regulated in proportion to the time of irradiation and the light intensity. Transgenic Arabidopsis plants over-expressing the rhl41 gene showed an increased tolerance to high-intensity light, and also morphological changes of thicker and dark green leaves. Interestingly, the palisade parenchyma was highly developed in the leaves of the transgenic plants, which is one of the long-term acclimatization responses in Arabidopsis plants. The anthocyanin content (a light protectant) as well as the chlorophyll content also increased. Antisense transgenic plants exhibited decreased tolerance to high irradiation. We propose that the RHL41 zinc finger protein has a key role in the acclimatization response to changes in light intensity.

A major root protein of carrots with high homology to intracellular pathogenesis-related (PR) proteins and pollen allergens.

We isolated the cDNA clone coding for a major root specific protein (CR16) of carrots. The CR16 protein (154a.a.) has a high homology to intracellular pathogenesis-related (PR) proteins, stress-induced proteins and also the major allergen protein of celery (Api g1). The CR16 protein gene formed a super gene family and transcripts of this CR16 protein gene are predominant in root tissue, not in leaves or flowers.

Characterization of the transposition pattern of the Ac element in Arabidopsis thaliana using endonuclease I-SceI.

We have investigated physical distances and directions of transposition of the maize transposable element Ac in Arabidopsis thaliana. We prepared a transferred DNA (T-DNA) construct that carried a non-autonomous derivative of Ac with a site for cleavage by endonuclease I-SceI (designated dAc-I-RS element). Another cleavage site was also introduced into the T-DNA region outside dAc-I-RS. Three transgenic Arabidopsis plants were generated, each of which had a single copy of the T-DNA at a different chromosomal location. These transgenic plants were crossed with the Arabidopsis that carried the gene for Ac transposase and progeny in which dAc-I-RS had been transposed were isolated. After digestion of the genomic DNA of these progeny with endonuclease I-SceI, sizes of segment of DNA were determined by pulse-field gel electrophoresis. We also performed linkage analysis for the transposed elements and sites of mutations near the elements. Our results showed that 50% of all transposition events had occurred within 1,700 kb on the same chromosome, with 35% within 200 kb, and that the elements transposed in both directions on the chromosome with roughly equal probability. The data thus indicate that the Ac-Ds system is most useful for tagging of genes that are present within 200 kb of the chromosomal site of Ac in Arabidopsis. In addition, determination of the precise localization of the transposed dAc-I-RS element should definitely assist in map-based cloning of genes around insertion sites.

Severe progressive erosive arthritis in multicentric reticulohistiocytosis: possible involvement of cytokines in synovial proliferation.

A case of multicentric reticulohistiocytosis complicated by severe progressive erosive arthritis in a 40-year-old man is described. The diagnosis was confirmed by biopsies of the cutaneous nodule and the synovial tissue of the right knee joint. Destructive changes in the multiple joints rapidly progressed and resulted in severe progressive erosive arthritis. Immunohistochemical staining revealed that the histiocytes reacted positively to interleukin 1 beta and platelet derived growth factor B, leading to the speculation that these cytokines may play a role in the synovial proliferation seen in patients with multicentric reticulohistiocytosis.

Malignancy and interstitial pneumonitis as fatal complications in dermatomyositis.

By sending questionnaires to the major dermatological clinics in Japan, we examined the outcome and causes of death in 182 cases of dermatomyositis (DM) observed between 1983 and 1987. The mortality rate in adults was 26.7%. Associated malignancy occurred in 47 cases (25.8% of adults), and 16 of them died of neoplasms. In contrast to 43 carcinomas, only 1 patient died of lymphoma. Associated interstitial pneumonitis (IP) appeared in 31 cases. IP started simultaneously or within 6 months after the diagnosis of DM in the majority of these cases. Of the 13 fatal cases due to IP, 8 died of respiratory failure within two months of the onset of lung disease. In the malignancy group, periungual erythema was noted in a higher proportion. Raynaud's phenomenon was more frequently noted in the IP group. DM overlapped with other collagen diseases in 14 cases.

Dependency of microdissected nephron segments upon oxidative phosphorylation and exogenous substrates: a relationship between tubular anatomical location in the kidney and metabolic activity.

1. In order to examine the possibility of heterogeneity in the dependence of renal tubular cells upon oxidative phosphorylation and exogenous substrates, the effects of antimycin A and substrate deprivation on adenosine 5'-triphosphate (ATP) content were examined in isolated rat nephron segments in vitro at 37 degrees C. 2. Antimycin A (5 mumol/l) caused varying decrements in cell ATP level within 5 min in the following order: proximal tubules greater than cortical thick ascending limb of Henle's loop (cTAL) greater than cortical collecting duct (cCD) in the cortex, and thin descending limb of Henle's loop (TDL) greater than medullary thick ascending limb of Henle's loop (mTAL) greater than outer medullary collecting duct (omCD) in the inner stripe of the outer medulla. In the thick ascending limb and the collecting duct, the segments located in the cortex were more sensitive than those in the medulla. 3. Substrate deprivation for 30 min markedly decreased the cell ATP content in cortical and medullary proximal tubules and also in medullary TDL, whereas it caused only a slight decrease in cTAL and mTAL with no change in cCD and omCD. 4. Media made hypertonic by the addition of 200 mmol/l NaCl under aerobic conditions, increased the requirement for exogenous substrates in TDL and mTAL, but not in omCD. This stimulation was seen to a lesser extent in media made hypertonic by the addition of mannitol instead of NaCl.(ABSTRACT TRUNCATED AT 250 WORDS)

Factors influencing vascular and natriuretic responses to ANP.

To explore factors that modulate the magnitude of vasorelaxation and natriuretic/diuretic responses to exogenous ANP, clearance studies were performed in unilateral hydronephrotic rats with renal vasoconstriction complications. An increase in renal plasma flow associated with a decrease in calculated renal vascular resistance in response to ANP (2 micrograms.min-1.kg-1, i.v.) was obtained only in the hydronephrotic kidney. In contrast, the control kidney of the unilateral hydronephrotic rat had a decrease in glomerular filtration rate and an abolished natriuretic response following ANP administration. A prior renal denervation in the control kidney restored the natriuretic effect of ANP and abolished the lowering of glomerular filtration rate. These data suggest that the vasodilatory effect of ANP may depend on the vascular tone itself, and that renal responses to ANP are significantly modified by renal nerve activity.

A bioenergetic explanation for the selective vulnerability of renal medullary tubules to hypoxia.

1. In order to explain the vulnerability of medullary thick ascending limb of Henle's loop (mTAL) during hypoxia, adenosine 5'-triphosphate (ATP) content was measured in isolated rat mTAL cells during control conditions and chemically induced hypoxia and compared with those in medullary collecting duct (MCD) cells. 2. Basal ATP levels in mTAL and MCD were estimated as 3.6 and 2.1 mmol/l, respectively. Antimycin A (5 mumol/l) decreased the ATP content by 41% of the control value in the mTAL cells, but failed to reduce that of the MCD cells. Administration of sodium cyanide (5 mmol/l) drastically depleted ATP in the mTAL cells within 5 min (2-3% of control). On the other hand, ATP levels in MCD cells were sustained for at least 60 min after cyanide administration (64% of control). 3. When tubules were made permeable to sodium by the addition of nystatin, the effects of chemical hypoxia on the cell ATP levels were intensified in both segments, and this was partially blocked by pretreatment with ouabain, or by lowering the sodium concentration of the medium. 4. Higher doses of nystatin in mTAL caused a reduction in ATP levels even under control conditions, but its effect was prevented in low sodium medium. 5. The present study suggests that cell ATP levels can be altered by sodium, potassium-dependent adenosine triphosphatase activity, and that due to their high sodium-transporting activity, mTAL cells are more sensitive to reductions in ATP levels during hypoxia than are MCD cells.

A renorenal suppression in response to atrial natriuretic peptide in the unilateral ischaemic rat.

1. The present study was carried out to investigate the haemodynamic and natriuretic effects of atrial natriuretic peptide (ANP) in normal rats and in rats with unilateral ischaemia. 2. Twenty-four hour unilateral ureter occlusion gave rise to a marked vasoconstriction in the ipsilateral kidney after its release. Intravenous infusion of ANP doubled p-aminohippurate clearance and inulin clearance and elicited massive natriuresis in the hydronephrotic kidney, while in the contralateral kidney these clearance values were decreased and there was a lack of natriuresis. The responses in the latter kidney were also different from those in the normal rat, in which significant natriuresis was elicited by ANP even though a decrease in p-aminohippurate clearance occurred. 3. After the control kidney in the unilateral hydronephrotic rat was denervated either mechanically or by pretreatment with prazosin, ANP induced a natriuresis in both kidneys. Furthermore, the renal denervation prevented the decrease in inulin clearance in the control kidney after ANP administration. 4. These findings suggest that the renal response to ANP may depend on the vascular tone before administration, and that renal nerve activity may modify the effects of ANP on renal haemodynamics and sodium excretion.

Intrarenal localization of degradation of atrial natriuretic peptide in isolated glomeruli and cortical nephron segments.

Using isolated glomeruli and nephron segments obtained from collagenase treated rabbit kidneys, we examined the in vitro degradation of alpha-human atrial natriuretic polypeptide (alpha-hANP). The ANP-degrading activity was measured by the amount of immunoreactive ANP remaining after incubation of about 50 fmoles alpha-hANP with each tissue preparation for 7.5 min. The sequence of degrading activity among isolated nephron segments was as follows: proximal straight tubule greater than proximal convoluted tubule greater than cortical collecting tubule greater than distal convoluted tubule greater than cortical thick ascending limb. A single glomerulus exhibited the degrading activity which was comparable to approximately 50% of the activity of 1 mm proximal convoluted tubule. Phosphoramidon, an inhibitor of endopeptidase, prevented the degradation of ANP in proximal convoluted tubule and glomerulus by 68% and 89%, respectively, but not in cortical thick ascending limb and cortical collecting tubule. From these results, we conclude that the degradation of ANP by endopeptidase occurs mainly in the proximal tubule and glomerulus.

Tubular prostaglandin E2 production and its role in urinary hypotonicity after release of ureteral occlusion in the rat.

1. In order to explore the involvement of endogenous prostaglandin E2 (PGE2) in the urine concentration defect after ureteral occlusion, PGE2 production by isolated collecting ducts in vitro and effects of indomethacin on urine osmolality in vivo were examined. 2. Twenty-four hours ureter obstruction caused increased PGE2 production by the medullary collecting ducts, which was maintained at a high level on the day after release of obstruction (0.8 +/- 0.2 pg/mm normal, 8.1 +/- 0.9 pg/mm 24 h obstruction, and 6.6 +/- 1.0 pg/mm post-obstruction, mean +/- SEM). An enhanced PGE2 production was also observed for papillary collecting duct on the day after release of 24 h ureteral occlusion (3.9 +/- 0.5 pg/mm normal and 7.7 +/- 1.2 pg/mm post-obstruction). 3. Administration of indomethacin to the unilateral post-obstructive rats slightly raised the urine osmolality of the post-obstructed kidney (from 339 +/- 17 to 390 +/- 22 mosmol/kg H2O), while it had a greater effect on the contralateral intact kidney (from 1569 +/- 138 to 2567 +/- 198 mosmol/kg H2O). 4. Our data may indicate that the urine concentration defect after 24 h ureteral occlusion is ascribable mainly to a mechanism other than increased endogenous PGE2.

Effect of phenylisopropyladenosine on vasopressin-dependent cyclic AMP generation in defined nephron segments from rat.

To test the possibility that adenosine may be involved in a urine concentrating mechanism, effects of 1-phenylisopropyladenosine (PIA) on cyclic AMP levels have been examined in medullary thick ascending limb (mTAL) and medullary collecting duct (MCD) isolated from the rat. Low and high doses of PIA did not alter basal cyclic AMP levels in both segments. However, PIA depressed vasopressin-dependent cyclic AMP production in MCD in a dose-dependent manner: this effect of PIA was maximum at 10(-6) M. 8-Phenyltheophylline, a competitive inhibitor for adenosine receptor, completely abolished this inhibitory effect of PIA. This finding may suggest an existence of adenosine receptor on the MCD. In mTAL, PIA also suppressed vasopressin-mediated cyclic AMP generation. The present study shows an interaction between PIA and vasopressin in both MCD and mTAL. This interaction may contribute in part to urinary-concentrating disturbance in renal ischemia.

Renal response to atrial natriuretic factor in rats without intact papillae.

In order to examine the contribution of renal papilla to atrial natriuretic factor (ANF)-induced diuresis, clearance studies were done in rats with intact papillae and bromoethylamine hydrobromide (BEA)-induced papillary necrosis. Papillary necrosis did not attenuate the diuretic and natriuretic response to ANF. In a moderately hydropenic state, urine flow and fractional water excretion were rather more increased in papillary necrosis rats than in papilla-intact ones. In a volume-expanded state, both groups had a similar diuretic response to ANF. Present data indicate that the existence of intact renal papilla is not essential for ANF-induced diuresis. Furthermore, it is suggested that absolute magnitude of ANF-induced diuresis may be dependent on its extrapapillary action(s).

Modulation by verapamil of hormonal action on the Henle's loop of mice.

In order to know the role of cytosolic calcium in the modulation of the hormone action on sodium chloride transport across the thick ascending limbs of Henle's loop, we examined whether verapamil, a blocker of cellular calcium entry, can modulate the effects of arginine vasopressin (AVP) or glucagon in stimulating transepithelial voltage (Vt) and cyclic AMP generation in the mouse medullary thick ascending limb (MAL). The pretreatment of the renal tubule with 5 X 10(-5)M verapamil reduced the Vt stimulated with 200 microU/mliter AVP from 1.7 +/- 0.3 mV to 0.4 +/- 0.4 mV (N = 7, P less than 0.05). The changes in Vt were well correlated with those of unidirectional Cl flux from the lumen to the bath. However, verapamil did not influence the Vt stimulated with 10(-3) M dibutyryl cyclic AMP. The pretreatment of the MAL with 10(-5) M verapamil also inhibited the cyclic AMP generation in the MAL from 72.1 +/- 17.9 to 50.6 +/- 13.6 fmoles/mm/7 min (N = 7, P less than 0.05) as well as in the medullary collecting tubule from 147.6 +/- 46.6 to 121.2 +/- 41.6 fmoles/mm/7 min (N = 4, P less than 0.05). The effect of verapamil in inhibiting the AVP-stimulated cAMP was dose-dependent: the cAMP generation was inhibited by 28.9 +/- 6.8 and 61.1 +/- 9.3% with 10(-5) M and 10(-4) M verapamil, respectively. When verapamil was added to the medium simultaneously with AVP, the generation of cyclic AMP was unaffected.(ABSTRACT TRUNCATED AT 250 WORDS)

Additive effects of atrial natriuretic polypeptide and of renal vasodilating agents in the anesthetized dog.

In order to examine the contribution of an increase in renal papillary plasma flow to the mechanism of natriuresis by atrial natriuretic polypeptide (ANP), we compared the natriuretic effects of ANP administered into the renal artery of the dog together with secretin or acetylcholine (ACh). At an equivalent renal vasodilating dose, ACh increased urinary excretion of sodium (UNaV) to 212 +/- 36% of the control associated with a decrease in urine osmolality (62 +/- 6%), whereas secretin did not change UNaV (113 +/- 12%) or urine osmolality (101 +/- 14%). This result was compatible with the view that ACh causes natriuresis mainly by increasing papillary plasma flow. Combined administration of ANP with secretin caused a marked increase in UNaV to 407 +/- 55%, in association with a decrease in urine osmolality to 55 +/- 9%, suggesting that ANP may cause natriuresis by a mechanism similar to that of ACh. Combined administration of ANP with ACh further increased UNaV to 323 +/- 67% and decreased urine osmolality to 50 +/- 6%. These observations suggest that ANP and ACh share common but not identical mechanisms of natriuretic action since ANP caused additional natriuresis during ACh infusion. These findings, however, do not necessarily exclude the possibility that ANP also inhibits renal sodium reabsorption by a direct action.

Localization of binding sites for alpha-rat atrial natriuretic polypeptide in rat kidney.

To determine the intrarenal localization of receptors for atrial natriuretic polypeptide (ANP) in the rat, we performed autoradiography and binding assay by using 125I-labeled alpha-rat ANP (alpha-rANP). Autoradiography at the slice and microscopic level in the kidney and binding assay in isolated glomeruli demonstrated that receptors in the renal cortex were distributed mainly in glomeruli. Although dense silver grains were distributed diffusely both in inner medulla and outer stripe of outer medulla, a marked displacement of the grains was observed only in the inner medulla. Autoradiography at the microscopic level also showed that silver grains were distributed in the renal artery, renal pelvis, and inner medullary collecting tubule (IMCT) prepared by the microdissection method, but not in the arcuate artery, interlobular artery, and afferent or efferent arterioles. Specific binding was demonstrated in the isolated glomeruli and the preparation was rich in fragments of IMCT. Apparent binding affinity (Kd) and receptor density (R) for 125I-labeled alpha-rANP in isolated glomeruli and IMCT were Kd = 3.2 and 21 X 10(-9) M, and R = 320 and 420 fmol/mg protein, respectively. These observations suggest that alpha-rANP has a physiological action on glomeruli and possibly on the inner medullary collecting tubules in addition to the renal artery.

Localization of thyroxine 5'-monodeiodinase activity in the renal proximal tubules in rabbits and rats.

To determine the localization of T4 5'-monodeiodinase activity in rabbit and rat nephron segments, the formation of tri-iodothyronine (T3) from thyroxine (T4) was measured in kidney homogenate and in isolated nephron segments obtained by the microdissection method. In order of decreasing activity, homogenates of rabbit renal cortex, outer medulla and inner medulla were capable of converting T4 to T3. In the isolated nephron segments of the rabbit cortex, the activities were noted in both proximal convoluted and proximal straight tubules. On the other hand, the activities were not detected in segments including the cortical thick ascending limb of Henle's loop, the distal convoluted tubule, the connecting tubule, and the cortical collecting tubule. It is concluded that both the convoluted and the straight tubules are the sites of T3 production in the kidney.