Characterization of four extensin genes in Arabidopsis thaliana by differential gene expression under stress and non-stress conditions.

From Arabidopsis thaliana we isolated four different cDNAs that encode extensins, a family of cell-wall hydroxyproline-rich glycoproteins (HRGPs). Putative proteins (AtExt2-5) contained one open reading frame and characteristic Ser-(Pro)4 sequences organized in a high-order repetitive motif. AtExt2-5 genes were strongly expressed during rehydration after dehydration. They were also expressed after treatment with various amino acids. In particular, AtExt3 and five mRNAs were abundantly accumulated after treatment with L-Ser, Hyp, and L-Pro, which are major components of extensin proteins. The AtExt transcripts were strongly expressed in root tissues of both unbolted and bolted plants. The transcripts of AtExt2, 3, and 5 were also detected in the lower stem and flower buds, and that of AtExt4 was detected in bolted flowers. Therefore, we suggest that these four AtExt genes are novel extensin genes in A. thaliana, because the expression of atExt1, which has already been isolated from A. thaliana, was different from these.

Antisense suppression of proline degradation improves tolerance to freezing and salinity in Arabidopsis thaliana.

Synthesis, degradation, and transport of proline (Pro) are thought to cooperatively control its endogenous levels in higher plants in response to environmental conditions. To evaluate the function of Pro degradation in the regulation of the levels of Pro and to elucidate roles of Pro in stress tolerance, we generated antisense transgenic Arabidopsis plants with an AtProDH cDNA encoding proline dehydrogenase (ProDH), which catalyzes Pro degradation. Several transgenic lines accumulated Pro at higher levels than wild-type plants, providing evidence for a key role of ProDH in Pro degradation in Arabidopsis. These antisense transgenics were more tolerant to freezing and high salinity than wild-type plants, showing a positive correlation between Pro accumulation and stress tolerance in plants.

Stress-responsive and developmental regulation of Delta(1)-pyrroline-5-carboxylate synthetase 1 (P5CS1) gene expression in Arabidopsis thaliana.

Delta(1)-Pyrroline-5-carboxylate synthetase 1 (P5CS1) is the rate-limiting enzyme in the biosynthesis of proline by Arabidopsis thaliana. Results of Northern analysis using aba1, abi1, and abi3 mutants of A. thaliana suggest that the expression of the P5CS1 gene under water stress is induced via abscisic acid (ABA)-biosynthesis-dependent and -independent pathways. Expression via ABA biosynthesis does not require protein synthesis. Analysis using transgenic A. thaliana containing a P5CS1 promoter/GUS fused gene indicated that the P5CS1 gene of A. thaliana is expressed in the whole plant under dehydration and in reproductive organs and tissues (flower buds and surrounding parts, pollen and pistils, and young siliques in the early stage of seed formation) under unstressed conditions. Cis-acting elements involved in dehydration-responsive gene expression are shown to be located in a 117-bp region between positions -621 and -504 upstream from the transcriptional initiation site.

Biological functions of proline in morphogenesis and osmotolerance revealed in antisense transgenic Arabidopsis thaliana.

Many organisms, including higher plants, accumulate free proline (Pro) in response to osmotic stress. Although various studies have focused on the ability of Pro as a compatible osmolyte involved in osmotolerance, its specific role throughout plant growth is still unclear. It has been reported that Pro is synthesized from Glu catalyzed by a key enzyme, delta 1-pyrroline-5-carboxylate synthetase (P5CS), in plants. To elucidate essential roles of Pro, we generated antisense transgenic Arabidopsis plants with a P5CS cDNA. Several transgenics accumulated Pro at a significantly lower level than wild-type plants, providing direct evidence for a key role of P5CS in Pro production in Arabidopsis. These antisense transgenics showed morphological alterations in leaves and a defect in elongation of inflorescences. Furthermore, transgenic leaves were hypersensitive to osmotic stress. Microscopic analysis of transgenic leaves, in which the mutated phenotype clearly occurred, showed morphological abnormalities of epidermal and parenchymatous cells and retardation of differentiation of vascular systems. These phenotypes were suppressed by exogenous L-Pro but not by D-Pro or other Pro analogues. In addition, Pro deficiency did not broadly affect all proteins but specifically affected structural proteins of cell walls in the antisense transgenic plants. These results indicate that Pro is not just an osmoregulator in stressed plants but has a unique function involved in osmotolerance as well as in morphogenesis as a major constituent of cell wall structural proteins in plants.

Intracavitary injection of OK-432 for malignant pericardial effusion, a case report.

The clinical efficacy of OK-432 has been evaluated favorably when administered for malignant effusion in pleural and peritoneal cavities. It may be effective also in pericardial space in response to inflammation or tumor, i.e., in weep fluid, but there are few reports of the clinical efficacy of a single administration of this agent in this space. We present here a case of spontaneous remission of malignant pericardial effusion after a single injection of OK-432 into the pericardial space.

Computed tomography and histologic appearance of pancreatic metastases from distant sources.

The computed tomographic and histologic appearances in 34 cases of autopsy-confirmed metastatic tumors of the pancreas including secondary malignant lymphoma are discussed. In 18 (53.8%) the pancreas appeared abnormal on computed tomography, the lesions being classified into three types: In 8 cases there was diffuse enlargement of the pancreas; in 9 cases a localized mass; and one patient had multiple low attenuated nodules within the organ. Histologic investigation revealed that metastatic carcinoma involved the pancreatic lobules. The degree of infiltration in the interlobular connective tissue was related to the invasiveness of the tumors. In the majority of cases with diffuse infiltration the pancreatic lobules were destroyed and varying degrees of proliferation of malignant cells into the interlobular septa were documented. In patients with localized infiltration there was extensive invasion of the carcinoma within the pancreatic lobule. Dilatation of the pancreatic duct and/or organ-related symptoms were occasionally seen in these cases.

Inflammatory pseudotumor of the liver.

A rare inflammatory pseudotumor of the liver diagnosed by US-guided needle biopsy is reported.

CT evidence of renal angiomyolipoma with subcapsular hemorrhage.

Renal angiomyolipoma (AML) composed of adipose tissue, smooth muscle, and vessels can be clearly visualized on computed tomography (CT). However, once hemorrhage occurs, it is difficult to distinguish this lesion from other solid tumors. While there are reviews of AML with hemorrhage, those with the subcapsular type are rare. We present a case of preoperatively diagnosed angiomyolipoma complicated with subcapsular hemorrhage, as evaluated on CT.

Actions of nipradilol (K-351), a new alpha- and beta-adrenoceptor blocker, on the rabbit portal vein.

Nipradilol but not desnitro nipradilol [N-) nipradilol) inhibited the norepinephrine (NE)-induced depolarization and contraction of the rabbit portal vein. The NE-induced contraction and depolarization were also blocked by prazosin, but not blocked by yohimbine. Therefore, nipradilol possesses an alpha 1-blocking action. The order of potency was prazosin greater than nipradilol greater than yohimbine greater than (N-)-nipradilol = 0. With applications of field stimulations to muscle tissues, the smooth muscle membrane was depolarized with a latency of several seconds, and the action potential was generated. These phenomena were blocked by tetrodotoxin (TTX), prazosin or nipradilol, but not by yohimbine. Isoproterenol (Isop) inhibited the 30 mM K-induced contraction, and this inhibitory action was blocked by (N-), nipradilol, nipradilol or propranolol, dose-dependently. The potency of beta-blocking actions of nipradilol was much the same as that observed by propranolol and (N-) nipradilol. When nipradilol (10(-5) M) was applied to the tissue, the amplitude of the 30 mM K contraction was slightly reduced. Such inhibitory action was not observed by application of (N-) nipradilol. The Ki values of nipradilol for blocking actions on the NE-induced contraction and Isop-induced relaxation were of the same order of 10(-7) M. Therefore, the potencies of alpha 1-blocking and beta-blocking actions of nipradilol may be the same in the rabbit portal vein. These findings suggest that the vasodilating action of nipradilol on the rabbit portal vein is mainly due to the alpha 1-blocking action and that the nitrate action of this agent may be weak.

Effects of noradrenaline and acetylcholine on electro-mechanical properties of the guinea-pig portal vein.

Effects of acetylcholine (ACh) and noradrenaline (NA) on mechanical properties of smooth muscle cells of the guinea-pig portal vein were investigated using intact and skinned muscle preparations. In some preparations, the electrical activity was also recorded. In addition to ACh and NA, the effects of caffeine, procaine, excess concentrations of [K]o and MnCl2 were investigated. NA enhanced the mechanical response due to increase in the spike generation, receptor activated depolarization and release of Ca stored in the cell. ACh also enhanced the mechanical response, but this agent had little effect on the Ca release from the storage sites. Caffeine and procaine (10 mM) depolarized the membrane and enhanced the electrical activity. Caffeine enhanced the mechanical activity due to an increase in the membrane activity and the release of Ca stored in the cell, while procaine inhibited the contraction. Procaine inhibited and caffeine accelerated the Ca-induced Ca-release mechanism in the cell. NA released the Ca from the storage sites to a greater extent than did caffeine or ACh. MnCl2 inhibited the spontaneous membrane activity and contraction; however, low concentrations of MnCl2 increased the caffeine- or NA-induced contraction in Ca-free solution. When the Ca-tension relationship was observed in saponin-treated skinned muscles, the minimum concentration of Ca required to produce the contraction was 10(-7)M, as observed in other vascular tissues. Caffeine released the stored Ca, Na and ACh had no effect on the Ca release and procaine inhibited the caffeine-induced Ca release in skinned muscles. It was concluded that the membrane properties of smooth muscle cells in the portal vein are much the same as observed in other spontaneously active visceral muscles. Differences observed in the actions of NA and ACh on mechanical properties seems to be mainly due to different receptor-operated Ca release mechanisms.

Modulation of the cytotoxicity of the antitumor antibiotic peplomycin by membrane-interacting drugs and by increased levels of calcium ions.

The cytotoxic effect of peplomycin (PEP), a new derivative of bleomycin glycopeptide antibiotics, toward HeLa cells and mouse FM3A cells was markedly enhanced by combination of PEP with non-toxic doses of the membrane-interacting drugs verapamil (0.1-0.2mM) and dibucaine (0.15-0.25mM), or with CaCl2 (10-16 mM). Treatment with verapamil or CaCl2 following PEP treatment also effectively enhanced the cytotoxic effect of PEP, suggesting an interaction with PEP-induced damage. Cellular uptake of PEP did not increase in dibucaine-treated cells, suggesting no correlation between membrane permeability to PEP and enhanced cytotoxicity. Increased Ca2+ did not enhance the cytotoxic effects of adriamycin, mitomycin C, cis-diamminedichloroplatinum (II), vinblastine or macromomycin, thus suggesting a unique interaction with PEP. The enhancing action of verapamil and Ca2+ was greatly promoted by 41 degrees hyperthermia, although 41 degrees alone scarcely enhanced PEP cytotoxicity. Cytochalasin B and alpha-tocopherol, but not cytochalasin D, reversed the enhanced cytotoxicity produced by PEP and verapamil or dibucaine, but did not reverse the enhanced cytotoxicity by PEP combined with increased Ca2+. The enhancing action of Ca2+ was, however, antagonized by ruthenium red and lanthanum chloride, which are potent inhibitors of Ca2+ uptake by cells, or by magnesium chloride. Verapamil and increased Ca2+ promoted the decomposition of the DNA-membrane complex induced by PEP in HeLa cells, and also impaired the regeneration of the decomposed DNA complex.

Effects of calcium and manganese ions on mechanical properties of intact and skinned muscles from the guinea-pig stomach.

1. To investigate the mechanism of generation of contractions in tissues from the guinea-pig stomach, the effects of caffeine, procaine, acetylcholine (ACh), diltiazem or MnCl(2) on the contraction evoked from small bundles of intact or skinned muscles (50 mum in width and 250-300 mum in length) were observed.2. All these agents except for ACh blocked the spontaneously generated contraction. Diltiazem (1 x 10(-4)m) had no effect and MnCl(2) (3 mm) slightly reduced and caffeine enhanced the tonic contraction evoked in Na-free solution, whereas procaine relaxed the tissue. On the other hand, in the isotonic [K](o) solution, diltiazem, MnCl(2) and procaine relaxed the tissue, while caffeine enhanced the tonic contraction.3. Under pre-treatment with Ca-free solution (2 mm-EGTA-containing solution) after depletion of the stored Ca, application of 2.5 mm-Ca and subsequently applied 5 mm-caffeine produced contractions (Ca- and caffeine-induced contractions, respectively). In polarized (5.9 mm-K(o)) and depolarized (128 mm-K(o)) muscles, the various agents simultaneously applied with 2.5 mm-Ca modified the amplitude of the Ca-induced and the resulting caffeine-induced contractions. Thus, at least three different Ca influxes required to evoke the Ca- or caffeine-induced contraction were identified; diltiazem-sensitive Ca influx, diltiazem-insensitive but Mn-sensitive Ca influx and Mn-insensitive Ca influx.4. The Ca- and caffeine-induced contractions in Ca-free and 15.5 mm-Na-containing solutions were gradually reduced in amplitude, in proportion to the time of exposure. However, amplitude of the caffeine-induced contractions was inhibited to a greater extent and the duration of the contracts was less prolonged than the case of the Ca-induced contraction.5. In saponin-treated skinned muscles, the minimum concentration of Ca required to produce the contraction was 1 x 10(-7)m, and the maximum contraction was evoked by application of 1 x 10(-5)m-Ca. The effects of Na-free solution on the Ca accumulation and release to and from the storage site were also observed in these skinned muscles. The removal of Na from the cell seems to accelerate the Ca leakage, and depletes the stored Ca. In addition, Na-free solution inhibits to some extent the accumulation of Ca in the store site.6. In skinned muscles, Mn (over 2 x 10(-9)m) significantly enhanced the Ca-induced contraction and the pCa-tension relationship shifted to the left and upper directions. Mn seemed to possess the property of activating the contractile proteins, as determined from the pMn-tension relationship, and this agent may also inhibit leakage of Ca from the store sites. However, in relation to the latter two actions, the possible effects of Ca contaminations in the solution would have to be ruled out. Under physiological conditions, MnCl(2) may act at the level of the myoplasmic membrane and not actually penetrate the cell in this tissue.

Effects of 3,4-dihydro-8-(2-hydroxy-3-isopropylaminopropoxy)-3-nitroxy-2H-1-bezopyran (K-351) on smooth muscle cells and neuromuscular transmission in guinea-pig vascular tissues.

The effects of 3,4-dihydro-8-(2-hydroxy-3-isopropylaminopropoxy)-3-nitroxy-2H-1-benzopyran (K-351) on vascular smooth muscle cells and neuromuscular transmission were investigated by the microelectrode and mechanographic methods using intact and skinned smooth muscles from guinea pigs. K-351 markedly inhibited the norepinephrine-induced contraction and slightly reduced the K-induced contraction in the mesenteric artery, but did not affect the acetylcholine-induced contraction in the coronary artery. K-351 inhibited the contraction evoked by perivascular nerve stimulation to a greater extent than that evoked by exogenously applied norepinephrine in the pulmonary artery. However, these sequences were reversed in the mesenteric artery. In the mesenteric artery, K-351 (6 x 10(-7) M) suppressed and phentolamine (10(-6) M) enhanced the contraction evoked by perivascular nerve stimulation, at frequencies over 1.0 Hz. In the mesenteric artery, K-351 did not affect the spike evoked by an outward current pulse or the spike on the excitatory junction potential; in the portal vein, it did increase spontaneously generated spikes due to depolarization of the membrane. In the skinned mesenteric artery, K-351 did not modify the amplitude of Ca-induced contraction. The excitatory junction potential evoked by perivascular nerve stimulation (0.1-1.0 Hz) was markedly inhibited by K-351 with no effect on the facilitation process, whereas phentolamine enhanced both excitatory junction potentials and the facilitation process. These findings indicate that K-351 inhibited mainly intra- and extrajunctional alpha adrenoceptors and, as the former was not affected by phentolamine or prazosin, the action of this new agent differs from the actions of heretofore available alpha-1 and alpha-2 adrenoceptor antagonists.