The pathogenicities of Cylindrocarpon tonkinense and Fusarium solani in the rabbit cornea

The pathogenicity of Cylindrocarpon tonkinense in the cornea was evaluated and compared with that of Fusarium solani in rabbits. F. solani was inoculated into the right eyes of 14 rabbits and C. tonkinense was into the left eyes of same rabbits. The corneal lesions of both eyes were examined carefully by slit lamp every day for three weeks and the severity of infections were compared each other. For histopathologic study, several eyes were enucleated periodically. C. tonkinense has a pathogenicity equally strong as F. solani in this inculum size (10⁴ microconidia per cornea) and produced severe infection in rabbit eyes.     

Enkephalin degrading enzymes in cerebrospinal fluid

Enkephalins were rapidly degraded by specific enzyme systems in vivo. In cerebrospinal fluid (CSF), however, it has been undefined whether these enzyme systems existed. Our experiments showed enkephalins were hydrolyzed by the enzymatic activity in both CSF of human and monkey. The results by the thin layer chromatography and the high performance liquid chromatography revealed the reaction products of CSF and enkephalin were tyrosine, tyrosyl-glycine and tyrosyl-glycyl-glycine. Therefore, the enzymes in CSF were considered to be an aminopeptidase, a dipeptidyl aminopeptidase and a dipeptidyl carboxypeptidase. Our results suggest that in the assay of enkephalin in CSF, the effects of these enzymes should be considered.     

Genetic Characterization of the SufJ Frameshift Suppressor in SALMONELLA TYPHIMURIUM

A new suppressor of +1 frameshift mutations has been isolated in Salmonella typhimurium. This suppressor, sufJ, maps at minute 89 on the Salmonella genetic map between the argH and rpo(rif) loci, closely linked to the gene for the ochre suppressor tyrU(supM). The suppressor mutation is dominant to its wild-type allele, consistent with the suppressor phenotype being caused by an altered tRNA species. The sufJ map position coincides with that of a threonine tRNA(ACC/U) gene; the suppressor has been shown to read the related fourbase codons ACCU, ACCC, ACCA.--The ability of sufJ to correct one particular mutation depends on the presence of a hisT mutation which causes a defect in tRNA modification. This requirement is allele specific, since other frameshift mutations can be corrected by sufJ regardless of the state of the hisT locus.--Strains carrying both a sufJ and a hisT mutation are acutely sensitive to growth inhibition by uracil; the inhibition is reversed by arginine. This behavior is characteristic of strains with mutations affecting the arginine-uracil biosynthetic enzyme carbamyl phosphate synthetase. The combination of two mutations affecting tRNA structure may reduce expression of the structural gene for this enzyme (pyrA).     

Host-controlled modification and restriction in Bacillus subtilis: Bsu 168-system and BsuR-system in B. subtilis 168.

Summary A Bsu168-specific restriction deficient (r ₁₆₈ ^- ) mutant of Bacillus subtilis Marburg 168 was transformed to be BsuR-specific restriction proficient (r _R ⁺ ) with B. subtilis R DNA as efficiently as the Bsu168-specific restriction proficient (r ₁₆₈ ⁺ ) parental strain (hsrM ⁺, hsdR ^-).We constructed r _R ⁺ m _R ⁺ r ₁₆₈ ⁺ m ₁₆₈ ⁺ strain (ISMR 4), r _R ⁺ m _R ⁺ r ₁₆₈ ^- m ₁₆₈ ⁺ strain (ISR 11) and r _R ⁺ m _R ⁺ r ₁₆₈ ^- m ₁₆₈ ^- strain (ISR 6) from strain 101 (r ₁₆₈ ⁺ m ₁₆₈ ⁺ ), strain 1012 (r ₁₆₈ ^- m ₁₆₈ ⁺ ) and strain RM125 (r ₁₆₈ ^- m ₁₆₈ ^- ), respectively by transformation with B. subtilis R DNA, and tested their restriction and modification activities on phage 105C. The results show that the sites recognized by Bsu168-specific restriction and modification enzymes and the sites recognized by BsuR-specific ones are not overlapping.We conclude that the Bsu168-modification and restriction system and the BsuR-modification and restriction system are controlled independently by two distinct sets of genes in the r _R ⁺ m _R ⁺ transformant of r ₁₆₈ ⁺ m ₁₆₈ ⁺ strain B. subtilis 168.     

Partial purification and characterization of glycylprolyl dipeptidyl aminopeptidase in porcine pancreas

This study reports the presence of glycylprolyl dipeptidyl aminopeptidase in porcine pancreas, and its partial purification and some properties. Crude enzyme preparation was obtained by extraction from acetone-dried powder of the pancreas at pH 7.6. For solubilization of enzyme, freezing and thawing were carried out. Crude enzyme extract was fractionated with ammonium sulfate precipitation, gel filtration on Sephadex G-200 column and ion-exchange chromatography on DEAE-cellulose. Partially purified enzyme showed 2897-folds purification. The enzyme activity on polyacrylamide gel electrophoresis showed good agreement with a main protein band stained with Coomassie brilliant blue. Molecular weight of this enzyme from the pancreas was estimated to be 300 000 by gel filtration on Sephacryl S-300 column. Optimum pH was between 8.5 and 9.0, and K_m value for glycylproline-p-nitroanilide tosilate was 0.33 mM. This enzyme from the pancreas was a serine enzyme and was relatively stable to heat at 60°C for 10 min.     

Leaf photosynthesis, plant growth and nitrogen allocation in rice under different irradiances

The photosynthetic rates and various components of photosynthesis including ribulose-1,5-bisphosphate carboxylase (Rubisco; EC 4.1.1.39), chlorophyll (Chl), cytochrome (Cyt) f, and coupling factor 1 (CF₁) contents, and sucrose-phosphate synthase (SPS; EC 2.4.1.14) activity were examined in young, fully expanded leaves of rice (Oryza sativa L.) grown hydroponically under two irradiances, namely, 1000 and 350 μmol quanta · m⁻² · s⁻¹, at three N concentrations. The light-saturated rate of photosynthesis measured at 1800 μmol · m⁻² · s⁻¹ was almost the same for a given leaf N content irrespective of growth irradiance. Similarly, Rubisco content and SPS activity were not different for the same leaf N content between irradiance treatments. In contrast, Chl content was significantly greater in the plants grown at 350 μmol · m⁻² · s⁻¹, whereas Cyt f and CF₁ contents tended to be slightly smaller. However, these changes were not substantial, as shown by the fact that the light-limited rate of photosynthesis measured at 350 μmol · m⁻² · s⁻¹ was the same or only a little higher in the plants grown at 350 μmol · m⁻² · s⁻¹ and that CO₂-saturated photosynthesis did not differ between irradiance treatments. These results indicate that growth-irradiance-dependent changes in N partitioning in a leaf were far from optimal with respect to N-use efficiency of photosynthesis. In spite of the difference in growth irradiance, the relative growth rate of the whole plant did not differ between the treatments because there was an increase in the leaf area ratio in the low-irradiance-grown plants. This increase was associated with the preferential N-investment in leaf blades and the extremely low accumulation of starch and sucrose in leaf blades and sheaths, allowing a more efficient use of the fixed carbon. Thus, morphogenic responses at the whole-plant level may be more important for plants as an adaptation strategy to light environments than a response of N partitioning at the level of a single leaf. Received: 23 February 1997 / Accepted: 8 May 1997     

Simultaneous ultrastructural identification of growth hormone and its messenger ribonucleic acid using combined immunohistochemistry and non-radioisotopicin situ hybridization: A technical note

Summary The present electron microscopical study is concerned with the simultaneous visualization of messenger ribonucleic acid (mRNA) and its encoded protein in the same specimen. Pre-embedding electron microscopicalin situ hybridization (EM-ISH) on rat pituitary gland tissue localized growth hormone mRNA in the polysomes of the rough endoplasmic reticulum, and subsequent postembedding immunolabelling using protein A-colloidal gold particles identified growth hormone mainly in the secretory granules. We believe that our report provides the first simultaneous ultrastructural identification of mRNA and its encoded protein using combined pre-embedding EM-ISH and immunohistochemistry. In this method, the signals for mRNA were localized specifically as highly electron dense products on the polysomes of the endoplasmic reticulum, and those for its encoded protein were recognized as gold particles both in the cisternae of the reticulum and in the secretory granules. Our ultrastructural double labelling method for mRNA and protein may provide a tool to find important clues for elucidating the intracellular correlation of mRNA translation and secretion of translated protein, because of its high resolution, good morphological preservation, and the specific localization of the reaction products.     

Conformational difference of T cell antigen receptors revealed by monoclonal antibodies to mouse V beta 5 T cell receptor for antigen determinants.

The mAb MR9-4 and MR9-8 react with T cells expressing the V beta 5.1 and -5.2 chains of the TCR. T cells expressing V beta 5.1 TCR were stained by both antibodies with similar surface fluorescence intensity. For the T cell clones and hybridomas expressing V beta 5.2 TCR, staining intensity with MR9-8 varied from negative to comparable to that stained with the anti-pan V beta 5 mAb MR9-4, whereas every V beta 5-positive T cell can be activated with either MR9-4 or -9-8 mAb, suggesting a differential binding affinity of MR9-8 mAb to V beta 5 TCR molecules. Analysis of J beta segment and V alpha chain usage in the V beta 5-positive T cell hybridomas revealed that a differential binding of MR9-8 mAb to the V beta 5.2 chain is not dependent on either the J beta segment usage or the associating V alpha chain alone. These results suggest that the differential binding of MR9-8 mAb to V beta 5.2 TCR is due to the conformational change of the V beta chain created by a combination of the V alpha (possibly J alpha) and D beta-J beta segment associating with the V beta 5.2 chain.     

Enzyme specificity of proteinase inhibitor region in amyloid precursor protein of Alzheimer's disease: different properties compared with protease nexin I

Senile plaques, often surrounded by abnormally grown neurites, are characteristic of Alzheimer's diseased brain. The core of the plaque is mainly composed of amyloid beta protein (beta-AP), two of whose three precursors (APP) have serine proteinase inhibitor regions (APPI). APPI derivatives containing 60, 72 or 88 amino-acid fragments (APPI-60, APPI-72 and APPI-88, respectively) of the longest APP were produced in COS-1 cell culture medium, with the APPI cDNA ligated to the signal sequence of tissue plasminogen activator. The secreted APPIs were purified by sequential acetone precipitation followed by affinity chromatography using immobilized trypsin. These three APPIs and O-glycosylation-site-mutated APPI showed similar inhibitory activity against trypsin, chymotrypsin and plasmin. The purified APPI-72 was found to inhibit trypsin (Ki = 1.1 x 10(-10) M) and chymotrypsin (Ki = 5.8 x 10(-9) M) most strongly, and to inhibit leukocyte elastase (Ki = 7.9 x 10(-7) M) and several blood coagulation proteinases (Ki = 0.46-12 x 10(-7) M), but not urokinase or thrombin. The observed inhibition pattern was quite different from that of protease nexin I, one of serine proteinase inhibitors possessing neurite outgrowth activity. This suggests that the physiological roles of APPI are different from those of protease nexin I, and that APPI could not cause aberrant growth of neurite into the plaque. The presence of APPI having strong inhibitory activity in the brain might lead to the formation of amyloid deposits by preventing complete degradation of APPs.