Enzymatic Inactivation of Extracellular Carbonic Anhydrase and its Effect on K1/2 (CO2) for Photosynthesis in Chlorella ellipsoidea C-27

The ratio of the extracellular to the intracellular activityof carbonic anhydrase (CA) in cells of Chlorella ellipsoideaC-27, adapted to low levels of CO₂ for 24 h (low-CO₂ cells),was about one to one. Treatment of intact cells with PronaseP inactivated about one-half of the extracellular CA activitywithout affecting photosynthetic activity. The CA activity incell homogenates and in cell-wall ghosts liberated during celldivision was completely inactivated by the same treatment. Pretreatmentwith Glycosidase mix, Chitosanase and Macerozyme enhanced theinactivation of the CA activity in intact cells. These resultssuggest that extracellular CA is evenly distributed throughoutthe whole cell-wall region. The apparent K_1/2 for dissolved inorganic carbon (DIC) in low-CO₂cells doubled when extracellular CA was inactivated by treatmentwith Pronase P, but the K_1/2 obtained was still one-half ofthat in high-CO₂ cells. Photosynthetic ¹⁴CO₂-fixation in low-CO₂cells was enhanced by acetazolamide, whereas H¹⁴CO₃^–-fixationwas suppressed. The results suggest that CO₂ is a dominant substrateutilized by cells and that HCO₃^– is utilized after conversionto CO₂. The present results show that both intracellular andextracellular CA contribute to the increase in affinity forDIC during photosynthesis in low-CO₂ cells of Chlorella ellipsoideaC-27. (Received May 7, 1990; Accepted July 18, 1990)     

Fermentative production of 5′-purine ribonucleotide

Micrococcus sodonensis KY 3765 and Arthrobacter citreus KY 3155 were found capable of accumulating IMP in media supplemented with hypoxanthine as a precursor. High concentrations of phosphate and magnesium salts were required for high yields of IMP. Manganese deficiency in the media was also essential. Excessive Mn²⁺ effects were also seen in the IMP fermentation carried out with an adenineless mutant, of Cornynebacterium glutamicum. In M. sodonensis, R5P-like substances, 5-phosphoribose pyrophosphokinase and IMP pyrophosphorylase, were leaked out, of the cells grown in suboptimal Mn²⁺ levels. This excretion was inhibited by high levels of Mn²⁺. Such a phenomenon was not noted in A. citreus. An adenineless mutant (KY 7208) of Brevibacterium ammoniagenes was found to accumulate an appreciable amount of IMP. The chemical changes in this fermentation showed that, hypoxanthine was first produced de novo, excreted, and then reconverted into IMP by a salvage pathway. When hypoxanthine was added to 7208 culture, IMP yield was increased appreciably. In fact exogenous ¹⁴C-hypoxanthine was incorporated into ¹⁴C-IMP. Subsequent experiments showed that indeed Br. ammoniagenes ATCC 6872, a parent culture of KY 7208, was able to produce IMP, GMP, and AMP, in good yield from hypoxanthine, guanine, and adenine, respectively.     

Microenvironmental conditions for Japanese alder seedling establishment in a hummocky fen

In this article, we focused on hummocky microtopography as a prominent feature of mires and explored the microenvironmental conditions suitable for alder seedling establishment. Japanese alder (Alnus japonica) forest is widely distributed in wetlands in northern Japan. However, because alder seedlings are rare in mires, alder population dynamics and conditions that favor the establishment of alder seedlings are still unknown. The study was conducted in northern Japan at a site in mesotrophic mire. We surveyed the seedling density, the microenvironmental conditions (light, litter cover, and soil quality), and the density of dispersed seeds in alder forest and in the adjacent herbaceous fen. In addition, we performed a laboratory experiment to examine the germination characteristics of alder. Seedlings grew only on hummocks in alder forest. The percentage of litter cover on hummocks was lower than in hollows, and the density of dispersed seeds in alder forest was much higher than in herbaceous fen. Seeds of Japanese alder germinated under both light and dark conditions, and the germination rate were high under light and high-temperature conditions. Our results suggest that litter cover may inhibit seedling establishment and hummocks that characterized by less litter cover are suitable place for the establishment of seedlings. We conclude that hummocky microtopography and abundant seed rain in the mire enable the establishment of Japanese alder seedlings.     

Does conditioned taste aversion learning in the pond snail Lymnaea stagnalis produce conditioned fear?

In conditioned taste aversion (CTA) training performed on the pond snail Lymnaea stagnalis, a stimulus (the conditional stimulus, CS; e.g., sucrose) that elicits a feeding response is paired with an aversive stimulus (the unconditional stimulus, US) that elicits the whole-body withdrawal response and inhibits feeding. After CTA training and memory formation, the CS no longer elicits feeding. We hypothesize that one reason for this result is that after CTA training the CS now elicits a fear response. Consistent with this hypothesis, we predict the CS will cause (1) the heart to skip a beat and (2) a significant change in the heart rate. Such changes are seen in mammalian preparations exposed to fearful stimuli. We found that in snails exhibiting long-term memory for one-trial CTA (i.e., good learners) the CS significantly increased the probability of a skipped heartbeat, but did not significantly change the heart rate. The probability of a skipped heartbeat was unaltered in control snails given backward conditioning (US followed by CS) or in snails that did not acquire associative learning (i.e., poor learners) after the one-trial CTA training. These results suggest that as a consequence of acquiring CTA, the CS evokes conditioned fear in the conditioned snails, as evidenced by a change in the nervous system control of cardiac activity.     

Presenilin-1 mutation activates the signaling pathway of caspase-4 in endoplasmic reticulum stress-induced apoptosis

In the previous reports, we showed that the familial Alzheimer's disease (AD)-linked presenilin-1 (PS1) mutation induced the fragility to the endoplasmic reticulum (ER) stress and that caspase-4 mediates ER stress-induced- and beta-amyloid induced-apoptotic signaling in human cells. These results suggest the involvement of ER stress and caspase-4 in the cell death observed in AD. In this report, we studied the activation of caspase-4 in the familial AD-linked PS1 mutation (DeltaE9). Cleavage of caspase-4 under ER stress was enhanced by the overexpression of the familial AD-linked mutation (DeltaE9), showing that caspase-4 is a key caspase involved in the apoptotic signaling of AD. We also showed that the overexpression of caspase-4 induced cleavage of caspase-9 and caspase-3 without releasing cytochrome-c from the mitochondria. Thus, caspase-4 activates downstream caspases independently of mitochondrial apoptotic signaling and this might contribute to the pathogenesis of AD. To sum up our data, the familial AD-linked PS1 mutation accelerates the cleavage of caspase-4 under the ER stress and results in the activation of caspase-9 and caspase-3, apoptosis signal, without releasing cytochrome-c.     

Environmental factors determining the distribution of highland plants at low-altitude algific talus sites

Algific talus is a micro-scale habitat type where highland plants (subalpine and alpine species) are found, disjunct from their typical range, in lowland forests. On algific talus, cold airflows from the interstices between talus fragments create a local microclimate colder than surrounding forests. Despite of the widely-known occurrence of unique vegetation on algific talus, critical environmental factors determining the distribution of highland species in this habitat type are unclear. In order to reveal the environmental factors enabling highland species to inhabit algific talus, we investigated the vegetation and environments of 26 algific talus sites and four reference (non-algific talus) sites in Hokkaido, northern Japan. Several algific talus sites were dominated by highland species, while some algific talus sites and all non-algific talus sites were dominated by lowland species. Community analysis based on detrended correspondence analysis (DCA) and canonical corresponding analysis (CCA) revealed that the algific talus sites dominated by highland species had lower ground temperature, more acidic soil, larger canopy openness, and less diverse vegetation than the sites dominated by lowland species. Highland plants might be maintained under conditions stressful for lowland plants, resulting in less competitive situation. Generalized linear models (GLM), used to evaluate the response of individual highland species to environmental factors, revealed that preferable environmental conditions for highland plants are highly species specific. These results indicate that the maintenance of diverse environments is crucial for the conservation of the unique vegetation and local populations of highland species in algific talus areas.     

Relationship between gyrA mutations and quinolone resistance in Flavobacterium psychrophilum isolates

Flavobacterium psychrophilum is the causative agent of the fish diseases called bacterial cold-water disease and rainbow trout fry syndrome. It has been reported that some isolates of F. psychrophilum are resistant to quinolones; however, the mechanism of this quinolone resistance has been unexplained. In this study, we examined the quinolone susceptibility patterns of 27 F. psychrophilum strains isolated in Japan and the United States. Out of 27 isolates, 14 were resistant to both nalidixic acid (NA) and oxolinic acid (OXA), and the others were susceptible to NA and OXA. When amino acid sequences deduced from gyrA nucleotide sequences of all isolates tested were analyzed, two amino acid substitutions (a threonine residue replaced by an alanine or isoleucine residue in position 83 of GyrA [Escherichia coli numbering] and an aspartic acid residue replaced by a tyrosine residue in position 87) were observed in the 14 quinolone-resistant isolates. These results strongly suggest that, as in other gram-negative bacteria, DNA gyrase is an important target for quinolones in F. psychrophilum.     

Early behavioral changes and quantitative analysis of neuropathological features in murine prion disease: Stereological analysis in the albino Swiss mice model

Behavioral and neuropathological changes have been widely investigated in murine prion disease but stereological based unbiased estimates of key neuropathological features have not been carried out. After injections of ME7 infected (ME7) or normal brain homogenates (NBH) into dorsal CA1 of albino Swiss mice and C57BL6, we assessed behavioral changes on hippocampal-dependent tasks. We also estimated by optical fractionator at 15 and 18 weeks post-injections (w.p.i.) the total number of neurons, reactive astrocytes, activated microglia and perineuronal nets (PN) in the polymorphic layer of dentate gyrus (PolDG), CA1 and septum in albino Swiss mice. On average, early behavioral changes in albino Swiss mice start four weeks later than in C57BL6. Cluster and discriminant analysis of behavioral data in albino Swiss mice revealed that four of nine subjects start to change their behavior at 12 w.p.i. and reach terminal stage at 22 w.p.i and the remaining subjects start at 22 w.p.i. and reach terminal stage at 26 w.p.i. Biotinylated dextran-amine BDA-tracer experiments in mossy fiber pathway confirmed axonal degeneration and stereological data showed that early astrocytosis, microgliosis and reduction in the perineuronal nets are independent of a change in the number of neuronal cell bodies. Statistical analysis revealed that the septal region had greater levels of neuroinflammation and extracellular matrix damage than CA1. This stereological and multivariate analysis at early stages of disease in an outbred model of prion disease provided new insights connecting behavioral changes and neuroinflammation and seems to be important to understand the mechanisms of prion disease progression.Key words: prion disease, optical fractionator, neuropathology, behavioral changes, albino Swiss mice     

Purification and Properties of Penicillin Acylase from Kluyvera citrophila

Penicillin acylase (EC 3.5.1.11) of Kluyvera citrophila KY7844 was purified approximately 120-fold by DEAE-cellulose chromatography, hydroxyapatite chromatography and isoelectro-focusing fractionation. The purified enzyme, with an approximate molecular weight of 63,000, appeared to be homogeneous in disc electrophoretic analysis, and showed isoelectric point (Ip) 8.12 and 13.0 units/mg of specific activity for cephalexin hydrolysis. The Michaelis constant (Km) for cephalexin and for 7-[1-(1H)-tetrazolylacetamido]-desacetoxycephalosporanic acid ((1H) T-7ADCA) was 1.4 mM and 3.6 mM, respectively. This enzyme was capable of producing (1H) T-7ADCA in 80% yield from 1-(1H)-tetrazolylacetate methylester and 7-aminodesacetoxycephalosporanic acid.     

Histochemical studies of mucosubstances in metaplastic epithelium of the stomach,with special reference to the development of intestinal metaplasia

Summary Processes in the development of intestinal metaplasia of the stomach were investigated from the morphological and histochemical approaches using light and electron microscopic techniques. The specimens taken from 38 gastric carcinomas and 15 gastric and/or duodenal ulcers were subjected to this study. Morphological appearances of the intestinal metaplasia observed in routine examination with hematoxylin and eosin staining was able to be divided into complete and incomplete metaplasia by the light and electron microscopic histochemical stainings of the mucosubstances. The columnar cells at the area of the incomplete metaplasia had both the properties of the intestinal epithelia and the gastric foveolar epithelia. The incomplete as well as the complete metaplasia arose from the generative cells at the isthmus of the gland. The generative cells, however, sometimes gradually transformed to produce the complete metaplastic cells. The two processes of the development of the intestinal metaplasia were proposed and discussed.