Molecular cloning of a chloroplastic proteinassociated with both the envelope and thylakoid membranes

Chloroplasts consist of six morphologically distinct compartments. Each compartment has a specific set of polypeptides that perform distinct biochemical functions. We report here the identification of a membrane-associated protein with a novel localization. This protein was synthesized as a 37 kDa precursor and was processed to a mature protein of 30 kDa after being imported into isolated pea chloroplasts. Fractionation of chloroplasts showed that the 30 kDa mature protein was associated with both of the envelope membranes as well as with thylakoid membranes. Immunocyto-chemical localization of the 30 kDa protein revealed that the protein occurred in clusters in the vicinity of both the envelope and the thylakoid. Possible functions of this 30 kDa protein, inferred from its novel localization pattern, are discussed.Abbreviations CAB light-harvesting chlorophyll a/b-binding protein of photosystem II - prCAB precursor protein to CAB - SS small subunit of ribulose-1,5-bisphosphate carboxylase - prSS precursor protein to SS - RCF relative centrifugation force     

Importance of purine-pyridine hydroxyl and purine amino groups for hammerhead ribozyme cleavage

The importance of the 2′-hydroxyl and 2-amino groups of guanosine residues for the catalytic efficiency of a hammerhead ribozyme has been investigated. The three guanosines in the central core of a hammerhead ribozyme were replaced by deoxyinosine, inosine, and deoxyguanosine, and ribozymes containing these analogues were chemically synthesized. Most of the modified ribozymes are drastically descreased in their cleavage efficiency. However. deletion of the 2-amino group at G₈ (replacement with inosine, deoxyguanosine, deoxyinosine) caused little alteration in the catalytic activity relative to that obtained with the unmodified ribozyme. Whereas, deletion of the 2′-amino group at G₁₂ and G₅ (replacement with inosine, deoxyinosine, and deoxyguanosine) resulted in ribozymes with drastic decrease in the catalytic activity relative to that obtained with the unmodified ribozyme. In contrast, two uridine residues, U⁷ and U⁴, in the ribozyne sequence were replaced by deoxyuridine (dU). The dU4 complex resulted in a decrease in the catalytic rate, with relative cleavage activity that ws about half that observed for the native complex. By comparison, the dU⁷ complex exhibited a relative cleavage activity within 3.3-fold of that observed with native ribozyme/substrate complex. This result suggests that the 2′-hydroxyl group at U ⁷ is not essential for activity.

The importance of the 2′-hydroxyl, and 2-amino groups of guanosine residues for the catalytic efficiency of a hammerhead roibozyme has been investigated. Most of the modified rybozymes are drastically decreased in their cleavage efficiency. However, deletion of the 2-amino group at G₈ or deletion of the 2′-hydroxyl group at G₁₂ caused little alteration in the catalytic activity relative to that obtained with the unmodified ribozyme. In contrast, two uridine residues, U₇ and U₄, in the ribozyme sequence were replaced by deoxyuridine (dU). The U4 complex resulted in a decrease in the catalytic rate, with relative cleavage activity that was about half that observed for the native complex.     

Synthesis of sulfated derivatives of curdlan and their anti-HIV activity

Sulfopropyl curdlan was synthesized, its structure was determined, and the anti-HIV activity was compared with that of standard curdlan sulfates obtained with piperidine N-sulfonic acid in dimethyl sulfoxide. It was shown that sulfopropyl curdlan exhibits weaker anti-HIV activity than curdlan sulfate. Curdlan sulfates were synthesized with a SO₃-pyridine complex in a heterogeneous phase. It was shown from ¹³C-NMR spectra of acetylated curdlan sulfates that they had a different substituent distribution from standard curdlan sulfate. The cytotoxicity of the curdlan sulfates was attributed to their heterogeneous structure.     

Cloning of the blasticidin S deaminase gene (BSD) from Aspergillus terreus and its use as a selectable marker for Schizosaccharomyces pombe and Pyricularia oryzae

Aspergillus terreus produces a unique enzyme, blasticidin S deaminase, which catalyzes the deamination of blasticidin S (BS), and in consequence confers high resistance to the antibiotic. A cDNA clone derived from the structural gene for BS deaminase (BSD) was isolated by transforming Escherichia coli with an Aspergillus cDNA expression library and directly selecting for the ability to grow in the presence of the antibiotic. The complete nucleotide sequene of BSD was determined and proved to contain an open reading frame of 393 bp, encoding a polypeptide of 130 amino acids. Comparison of its nulceotide sequence with that of bsr, the BS deaminase gene isolated from Bacillus cereus, indicated no homology and a large difference in codon usage. The activity of BSD expressed in E. coli was easily quantified by an assay based on spectrophotometric recording. The BSD gene was placed in a shuttle vector for Schizosaccharomyces pombe, downstream of the SV40 early region promoter, and this allowed direct selection with BS at high frequency, following transformation into the yeast. The BSD gene was also employed as a selectable marker for Pyricularia oryzae, which could not be transformed to BS resistance by bsr. These results promise that the BSD gene will be useful as a new dominant selectable marker for eukaryotes.     

Beneficial effect of ACE inhibitor in congestive heart failure

The effects of captopril, an angiotensin-converting enzyme inhibitor, on congestive heart failure (CHF) were investigated in animal and clinical studies. Congestive heart failure was induced in rats by a combination of pressure and volume overload. Cardiac pressure overload was induced by constricting one renal artery (Goldblatt rat) and volume overload was induced by aorto-caval fistula. Captopril (100 mg/kg/day) was then administered for 14 weeks. Isometric contraction was assessed using isolated left ventricular papillary muscles. The maximum developed tension and the maximum rate of increase in tension (dT/dtmax) were decreased in untreated rats with CHF and improved in captopriltreated rats. The left ventricular myosin isoenzyme pattern was shifted towards V3 in untreated rats with CHF, and was shifted back towards V1 in the captopril-treated rats. In the clinical study, captopril (37.5–75 mg/day) was administered to patients with cardiomyopathy for 12 months. There was no effect on left ventricular mass in hypertrophic cardiomyopathy, although systolic anterior motion of the mitral valve disappeared in one patient. In dilated cardiomyopathy, however, left ventricular mass tended to decrease. These results indicate that captopril has a beneficial effect in congestive heart failure.     

Abnormalities of ADP/ATP carrier protein in J-2-N cardiomyopathic hamsters

ADP/ATP carrier protein (AAC) is located in the mitochondrial inner membrane and has an important function in mitochondrial energy supply. This protein transports ATP to the cytoplasm and counter transports ADP into the mitochondria. J-2-N cardiomyopathic hamsters were investigated to determine the AAC content in cardiac mitochondria. After recording an electrocardiogram and collecting blood, the cardiac mitochondria were isolated. The mitochondrial membranes were labelled with eosin-5-maleimide (EMA) and separated on SDS polyacrylamide gels. The position of the AAC component was identified by exposing the gel under UV light, and the AAC content was determined by densitometry after staining with Coomassie blue. The AAC content ratio was significantly decreased in both 10-week-old and 1-year survived J-2-N hamsters when compared to control Golden hamster. Among 10-week-old J-2-N hamsters, the decrease in the AAC content ratio was more marked for the animals with more severe myocardial damage. The H⁺-ATPase activities of mitochondrial membrane were higher in 10-week-old J-2-N hamsters than in control hamsters. These results suggest that the decrease of AAC in J-2-N hamster plays an important role in the pathogenesis of cardiomyopathy in J-2-N hamsters.     

First finding of urease-positive thermophilic strains of Campylobacter in river water in the Far East, namely, in Japan and their phenotypic and genotypic characterization

Two strains of urease-positive thermophilic Campylobacter (UPTC), CF89–12 and CF89–14, which were identified as UPTC by biochemical characterization, were found for the first time in river water in the Far East, namely, in Japan. The biochemical characteristics were identical to those of strains described previously by Bolton and colleagues. Furthermore, these two strains were positive for arylsulphatase. Consequently, it was demonstrated that UPTC may possibly be differentiated phenotypically from Campylobacter lari by the arylsulphatase test, as well as urease and nalidixic acid tests. Analysis by pulsed-field gel electrophoresis (PFGE) after digestion with Apa I, Sal I and Sma I, which were found to produce distributions of DNA fragments to be suitable for analysis of the genomic DNA from the thermophilic Campylobacter , respectively, demonstrated that these three restriction enzymes produced distributions of a relatively limited number of genomic DNA fragments and also demonstrated that the PFGE profiles obtained with the three restriction enzymes were indistinguishable between the two strains, respectively. The PFGE analysis and conventional fixed-field agarose gel electrophoresis suggested that the both genomes were approximately 1862 kb in length. Even though the two isolates of UPTC were isolated from water in different rivers in Japan, the results suggested that a single strain. as opposed to two distinct strains, was isolated. PFGE profiles after digestion with Sal I and Sma I, respectively, were also demonstrated to be distinctly different among strains isolated in Japan and previously in Europe. This is the first example of the isolation of UPTC from natural sources in countries other than those in Europe.     

Compilation of All Genes Encoding Bacterial Two-component Signal Transducers in the Genome of the Cyanobacterium, Synechocystis sp. Strain PCC 6803

Bacteria have devised sophisticated signaling systems for elicitinga variety of adaptive responses to their environment, whichare generally referred to as the "two-component regulatory system."The widespread occurrence of the two-component systems in bothprokaryotes and eukaryotes implies that it is a powerful devicefor a wide variety of adaptive responses of cells to their environment.The two-component signal transducers contain one or more ofthree conserved and characteristic phosphotransfer signalingdomains, named the "transmitter, receiver, and alternative transmitter."The recently determined entire genomic sequence of Synechocystissp. strain PCC 6803 allowed us to compile systematically a completelist of genes encoding such two-component signal transductionproteins. The results of such an effort, made in this study,revealed that at least 80 ORFs were identified as members ofthe two-component signal transducers in this single speciesof cyanobacteria.     

Antithrombotic effect of topically applied 3-Oxa-Methano-prostaglandin I1 in the microcirculation and antiplatelet functions of its active form

The antithrombotic effect of topical application of the 3-oxamethano-prostaglandin (PG) I₁ analog, SM-10902 in the microcirculation and in vitro antiplatelet functions of its active form SM-10906 were estimated in comparison with PGI₂ and PGE₁. In rat platelets, SM-10906 evoked accumulation of intracellular cyclic adenosine 3′,5′-monophosphate, and exhibited antiaggregatory and disaggregatory activities, which were all enhanced by the phosphodiesterase inhibitor theophylline. Additionally, SM-10906 was shown to inhibit platelet adhesion to collagen in human platelet-rich plasma. PGI₂ and PGE₁ also showed in vitro antiplatelet effects in the order of PGI₂ > SM-10906 ≥ PGE₁. SM-10902 exhibited a dose-dependent antithrombotic effect in the guinea pig mesenteric arteriole by a topical application, and this activity might be exerted by the antiplatelet functions of SM-10906. Although SM-10906, PGI₂ and PGE₁ also showed the antithrombotic effects, SM-10902 was the most potent. In conclusion, the present studies indicate that an external topical preparation of SM-10902 may be useful for the therapy of peripheral circulatory insufficiency.