Amino acid alterations essential for increasing the catalytic activity of the nylon-oligomer-degradation enzyme of Flavobacterium sp

The structural genes of two homologous enzymes, 6-aminohexanoate-dimer hydrolase (EII; nylB) and its evolutionally related protein EII' (nylB') of Flavobacterium sp. KI72 have an open reading frame encoding a peptide of 392 amino acids, of which 47 are different, and conserved restriction sites. The specific activity of EII towards 6-aminohexanoate dimer is about 1000-fold that of EII'. Construction of various hybrid genes obtained by exchanging fragments flanked by conserved restriction sites of the two genes demonstrated that two amino acid replacements in the EII' enzyme, i.e. Gly181----Asp (EII type) and His266----Asn (EII type), enhanced the activity toward 6-aminohexanoate dimer 1000-fold.     

Expression level of enzymes related to in situ estrogen synthesis and clinicopathological parameters in breast cancer patients

In order to evaluate the importance of estrogen production in tumor and surrounding tissues, we measured mRNA expression levels of 5 enzymes participating to estrogen synthesis in situ and 4 breast cancer-related proteins in 27 pairs of tumor and non-malignant tissues. Steroid sulfatase (STS) mRNA was more frequently detected in tumor tissues rather than in their non-malignant counterparts. Estrogen sulfotransferase (EST) was constantly expressed with high level not only in tumor tissues but also in their surrounding non-malignant counterparts. In contrast, mRNA expression levels of aromatase, and 17β-hydroxysteroid dehydrogenase type I and II were relatively low and detected only in small proportion of the patients. We also measured the mRNA expression levels of the same nine genes in tumor tissues of 197 breast cancer patients, and analyzed relationship between the mRNA expression level and the clinicopathological parameters. The mRNA expression levels of STS, aromatase and erbB2 in tumor tissues increased as breast cancer progressed. The tumoral mRNA expression levels of STS, estrogen receptor β, and erbB2 in patients with recurrence were higher than those in patients without recurrence. Upregulation of STS expression plays an important role in tumor progression of human breast cancer and is considered to be responsible for estrogen production in tumor and surrounding tissues.     

Synthesis of a new inhibitor of the UDP-GalNAc:polypeptide galactosaminyl transferase

Reaction of UDP-glucose with 1-hexadecanesulfonyl chloride (C16H33SO2Cl) in pyridine gave a new inhibitor of O-glycosylation. This reaction product was purified by TLC and shown by 1H-NMR and by chemical analysis of phosphorus to be uridine 5'-phosphoric (1-hexadecanesulfonic) anhydride. This compound was tested against the GalNAc transferase. The UMP-hexadecanesulfonic-anhydride did inhibit this enzyme with 50% inhibition requiring 160 microM. The inhibition with respect to UDP-GalNAc concentration was of the competitive type. We also synthesized the UMP-1-octanesulfonic anhydride (C8) and the UMP-butanesulfonic anhydride (C4) to see way effect fatty acid had on activity. The inhibition was in the order C16:C8:C4.     

Temporal Variability of Preferred Illuminance Self-Selected by Women during Wakefulness

The primary purpose of this study was to test our hypothesis that the behavior of self-selecting the preferred ambient illuminance might be determined by a load error between the core temperature and its set-point. Seven female volunteers were asked to choose the preferred ambient light intensity by adjusting a rheostat from the time when they rose spontaneously in the morning to the time when they felt sleepy and retired. Rectal temperature was measured every 10 min. Their preferred ambient light intensity increased quickly and then gradually after they rose and rose to approximately 2000 lx over 3 hrs. It decreased gradually and then quickly to the time when they retired. We discussed these findings in terms of a load error between the actual core temperature and its set-point.     

Characterization and Modeling of Intermittent Locomotor Dynamics in Clock Gene-Deficient Mice

The scale-invariant and intermittent dynamics of animal behavior are attracting scientific interest. Recent findings concerning the statistical laws of behavioral organization shared between healthy humans and wild-type mice (WT) and their alterations in human depression patients and circadian clock gene (Period 2; Per2) mutant mice indicate that clock genes play functional roles in intermittent, ultradian locomotor dynamics. They also claim the clinical and biological importance of the laws as objective biobehavioral measures or endophenotypes for psychiatric disorders. In this study, to elucidate the roles of breakdown of the broader circadian regulatory circuit in intermittent behavioral dynamics, we studied the statistical properties and rhythmicity of locomotor activity in Per2 mutants and mice deficient in other clock genes (Bmal1, Clock). We performed wavelet analysis to examine circadian and ultradian rhythms and estimated the cumulative distributions of resting period durations during which locomotor activity levels are continuously lower than a predefined threshold value. The wavelet analysis revealed significant amplification of ultradian rhythms in the BMAL1-deficient mice, and instability in the Per2 mutants. The resting period distributions followed a power-law form in all mice. While the distributions for the BMAL1-deficient and Clock mutant mice were almost identical to those for the WT mice, with no significant differences in their parameter (power-law scaling exponent), only the Per2 mutant mice showed consistently and significantly lower values of the scaling exponent, indicating the increased intermittency in ultradian locomotor dynamics. Furthermore, based on a stochastic priority queuing model, we explained the power-law nature of resting period distributions, as well as its alterations shared with human depressive patients and Per2 mutant mice. Our findings lead to the development of a novel mathematical model for abnormal behaviors in psychiatric disorders.     

Exopectic Acid Transeliminase of an Erwinia

A species of Erwinia was found to produce no other pectolytic enzyme than the two transeliminases of exo-types, namely, an oligogalacturonide transeliminase and an exopectic acid transeliminase. Of the two enzymes, the exopectic acid transeliminase was isolated and its properties were investigated. The results are as follows: (1) Pectic acids having an unsaturated galacturonic acid residue at the non-reducing end of the molecule are susceptible but oxidized or reduced pectic acids resistant to the enzyme action. (2) The enzyme has no activity toward pectinic acid and polymethylpolygalacturonate methyl glycoside. The limit of the enzymatic degradation for citrus pectic acid is 43.8%. (3) The rate of the enzyme activity was maximal with tetragalacturonic acid and followed by acid-soluble pectic acid, acid-insoluble pectic acid, pectic acid and trigalacturonic acid. Unlike the oligogalacturonide transeliminases of Pseudomonas sp. (strain S2) and Erwinia aroideae, the present enzyme shows a considerably high activity toward pectic acids of high molecular weight. (4) The pH-activity curves vary with the buffer employed. (5) The enzyme is activated by Co²⁺ and Mn²⁺ but powerfully inhibited by Cu²⁺ and Hg²⁺. Ca²⁺ has no significant effect on the enzyme activity.     

Plant glucosidase II catalyzes a transglucosylation reaction in addition to the hydrolytic reaction

When the purified plant glucosidase II was incubated with [3H]Glc2Man9GlcNAc in the presence of glycerol and the products were analyzed by gel filtration, a large peak of radioactivity emerged just before the glucose standard. The formation of this peak was dependent on both the presence of Glc2Man9GlcNAc and the presence of glycerol, and the amount of this product increased with time of incubation and amount of glucosidase II in the incubation. When the incubation was performed with [3H]Glc2Man9GlcNAc plus [14C]glycerol, the product contained both 14C and 3H. Strong acid hydrolysis of the purified product gave rise to [14C]glycerol and [3H]glucose. Various other chemical treatments and chromatographic techniques showed that the product was glucosyl----glycerol. Since the glucose was released by alpha-glucosidase, the product must be glucosyl-alpha-glycerol. This study demonstrates that the processing glucosidase II catalyzes a trans-glycosylation reaction in the presence of acceptors like glycerol. Since this transglycosylation reaction may give rise to unexpected products, investigators should be aware of its possible occurrence.     

Possible activity of acidic fibroblast growth factor as a progression factor rather than a transforming factor.

Acidic and basic fibroblast growth factors (aFGF and bFGF) are mitogens for mesoderm- and neuroectoderm-derived cells. The facts that FGF-related proteins are oncogenic and that FGFs are expressed in a variety of tumor cell lines or tumor tissues suggest the transforming activities of FGFs. To examine such an activity of aFGF, we introduced a human aFGF expression vector into two populations of Rat-1 cells; one was non-transformed (nRat-1), the other was partially-transformed (tRat-1). tRat-1 cells transfected with aFGF cDNA formed larger colonies in soft agar and produced larger and more malignant tumors in nude mice than those of parental cells. In contrast, nRat-1 cells transfected with aFGF cDNA neither formed colonies in soft agar nor produced tumors in nude mice. Our results suggest that high expression of aFGF may enhance a tumorigenic potential of Rat-1 cells rather than confer such a potential de novo.     

Quantifying the Uncertainty of Spontaneous Ca Oscillations in Astrocytes: Particulars of Alzheimer's Disease

The quantification of spontaneous calcium (Ca²⁺) oscillations (SCOs) in astrocytes presents a challenge because of the large irregularities in the amplitudes, durations, and initiation times of the underlying events. In this article, we use a stochastic context to account for such SCO variability, which is based on previous models for cellular Ca²⁺ signaling. First, we found that passive Ca²⁺ influx from the extracellular space determine the basal concentration of this ion in the cytosol. Second, we demonstrated the feasibility of estimating both the inositol 1,4,5-trisphosphate (IP₃) production levels and the average number of IP₃ receptor channels in the somatic clusters from epifluorescent Ca²⁺ imaging through the combination of a filtering strategy and a maximum-likelihood criterion. We estimated these two biophysical parameters using data from wild-type adult mice and age-matched transgenic mice overexpressing the 695-amino-acid isoform of human Alzheimer β-amyloid precursor protein. We found that, together with an increase in the passive Ca²⁺ influx, a significant reduction in the sensitivity of G protein-coupled receptors might lie beneath the abnormalities in the astrocytic Ca²⁺ signaling, as was observed in rodent models of Alzheimer's disease. This study provides new, to our knowledge, indices for a quantitative analysis of SCOs in normal and pathological astrocytes.     

‘Ecologically complex carbon’– linking biodiversity values,carbon storage and habitat structure in some austral temperate forests

Aim We assessed how avian biodiversity and above‐ground carbon storage were related in different forest age‐classes, including mature stands (> 100 years), in a managed, mixed‐species eucalypt forest. Location Gippsland, south‐eastern Australia. Methods In 50 2‐ha stands ranging in age from ≤ 5 years to mature stands > 100 years, we undertook repeated avian surveys, performed detailed habitat measurements and estimated amounts of above‐ground carbon. Extensive wildfire reduced the number of sites to 28 (seven in each of four age classes) upon which analyses and inferences were made. We also analysed data on carbon storage and some bird responses from previously published studies. Results Mature vegetation (> 100 years) had the greatest richness, abundance and biomass of birds. Key ecological resources, such as tree‐hollows for nesting, generally occurred mostly in stands > 60 years. Avian richness per unit of above‐ground carbon storage was relatively low for stands of 20–60 years. While above‐ground carbon storage appeared to increase in a monotonic fashion as stands age and mature, there were quantum increases in all measures of avian biodiversity in mature stands (> 100 years). Main conclusions Our results suggest that carbon is organized in a different way, with substantially greater biodiversity benefits, in very old stands. Mature vegetation simultaneously maximizes both avian biodiversity and above‐ground carbon storage. These results bolster arguments for allocating highest priorities to the preservation of old‐growth forest stands rather than alternative investments (e.g. reafforestation for carbon sequestration).