Cycling of soil carbon in a Japanese red pine forest II. Changes occurring in the first year after a clear-felling

Cycling of soil carbon in the first year after a clear-felling was compared with that before the felling in a Japanese red pine forest in Hiroshima Prefecture, west Japan. The daily mean temperature at the soil surface in summer was increased after the felling in comparison to that before felling, and the water content of both the A₀ layer and the surface mineral soil was decreased due to the loss of the forest canopy. The rate of weight loss of the A₀ layer was reduced after felling. However, accumulation of the A₀ layer rapidly decreased because of the lack of litter supply to the forest floor. Low soil respiration after felling was mainly caused by the cessation of root respiration. Analysis of annual soil carbon cycling was then conducted using a compartment model. The relative decomposition rate of the A₀ layer decreased whereas that of humus and dead roots in mineral soil increased to some extent after felling. The accumulation of carbon in mineral soil, however, increased slightly due to the supply of humus from roots killed by the felling.     

Chromatographic and mass spectrometric analysis of glutathione in biological samples

Biological thiol compounds are classified into high-molecular-mass protein thiols and low-molecular-mass free thiols. Endogenous low-molecular-mass thiol compounds, namely, reduced glutathione (GSH) and its corresponding disulfide, glutathione disulfide (GSSG), are very important molecules that participate in a variety of physiological and pathological processes. GSH plays an essential role in protecting cells from oxidative and nitrosative stress and GSSG can be converted into the reduced form by action of glutathione reductase. Measurement of GSH and GSSG is a useful indicator of oxidative stress and disease risk. Many publications have reported successful determination of GSH and GSSG in biological samples. In this article, we review newly developed techniques, such as liquid chromatography coupled with mass spectrometry and tandem mass spectrometry, for identifying GSH bound to proteins, or for localizing GSH in bound or free forms at specific sites in organs and in cellular locations.     

Developmental capacity of vitrified immature porcine oocytes following ICSI: effects of cytochalasin B and cryoprotectants

In the present study, effects of concentration and pretreatment time of cytochalasin B (CB), and of two types of cryoprotectant solutions on the nuclear maturation of vitrified-warmed porcine oocytes were examined. Also, the developmental capacity of vitrified immature porcine oocytes following intracytoplasmic sperm injection (ICSI) was investigated. The nuclear maturation rate (46.8%) of the vitrified-warmed oocytes treated with 7.5 microg/mL CB for 30 min was significantly higher (P < 0.05) than those (13.9-39.2%) of the vitrified-warmed oocytes treated with 0, 2.5, or 5.0 microg/mL CB for 10 or 30 min. Additionally, the nuclear maturation rate of oocytes treated with CB and vitrified in ethylene glycol (EG) (37.1%) was significantly higher (P < 0.05) than that of EG + dimethyl sulfoxide (Me(2)SO) (23.9%). However, no significant differences were observed in the cleavage and blastocyst development rates among the control (45.2 and 20.0%, respectively), the EG group (37.8 and 13.5%, respectively) and the EG + Me(2)SO group (39.3 and 14.3%, respectively). These results demonstrated that: (1) pretreatment with 7.5 microg/mL CB was beneficial for the vitrification of immature porcine oocytes; (2) the combination of EG and Me(2)SO as a cryoprotectant was not advantageous for in vitro maturation (IVM) of vitrified immature porcine oocytes; and (3) vitrified-warmed porcine oocytes matured after IVM, developed to the blastocyst stage without distinct differences compared to fresh oocytes following ICSI.     

Phenolic composition and radical scavenging activity of sweetpotato-derived shochu distillery by-products treated with koji

Phenolic composition and radical scavenging activity in the shochu distillery by-products of sweetpotato (Ipomoea batatas L.) treated with koji (Aspergillus awamori mut.) and cellulase (Cellulosin T2) were investigated to develop new uses. Koji and Cellulosin T2 treatment of shochu distillery by-products from sweetpotatoes, rice, and barley increased phenolic content. Caffeic acid was identified as a dominant phenolic component in the shochu distillery by-products of the sweetpotato. Adding koji and/or Cellulosin T2 to the shochu distillery by-product indicated that koji was involved in caffeic acid production. Caffeic acid was not detected in raw or steamed roots of "Koganesengan", the material of sweetpotato for shochu production, suggesting that it is produced during shochu fermentation. The phenolic content and radical scavenging activity the shochu distillery by-product treated with koji and Cellulosin T2 were superior to those of commercial vinegar. These results suggest that koji treatment of sweetpotato-derived shochu distillery by-products has potential for food materials with physiological functions. Further koji treatment of sweetpotato shochu-distillery by-products may be applicable to mass production of caffeic acid.     

Hypervariability of intronic simple (gt)n(ga)m repeats in HLA-DRB genes

We have investigated the extent of DNA variability in intronic simple (gt)_n(ga)_m repeat sequences and correlated this to sequence polymorphisms in the flanking exon 2 of HLA-DRB genes. The polymerase chain reaction (PCR) was used to amplify a DNA fragment containing exon 2 and the repeat region of intron 2. The PCR products were separated on sequencing gels in order to demonstrate length hypervariability of the (gt)_n(ga)_m repeats. In a parallel experiment, the PCR products were cloned and sequenced (each exon 2 plus adjacent simple repeats) to characterize the simple repeats in relation to the HLA-DRB sequences. In a panel of 25 DRB1, DRB4, and DRB5 alleles new sequences were not detected. Restriction fragment length polymorphism (RFLP) subtyping of serologically defined haplotypes corresponds to translated DNA sequences in 85% of the cases, the exceptions involving unusual DR/DQ combinations. Many identical DRB1 alleles can be distinguished on the basis of their adjacent simple repeats. We found group-specific organization of the repeats: the DRw52 supergroup repeats differ from those of DRB1*0101, DRB4*0101, and DRB5*0101 alleles and from those of pseudogenes. Finally, we amplified baboon DNA and found a DRB allele with extensive similarity to DRB1 sequences of the DRw52 supergroup. The simple repeat of the baboon gene, however, resembles that of human pseudogenes. In addition to further subtyping, the parallel study of polymorphic protein and hypervariable DNA alleles may allow conclusions to be drawn on the relationships between the DRB genes and perhaps also on the theory of trans-species evolution.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M 34258.     

Background Factors of Reflux Esophagitis and Non-Erosive Reflux Disease: A Cross-Sectional Study of 10,837 Subjects in Japan

Background

Despite the high prevalence of gastroesophageal reflux disease (GERD), its risk factors are still a subject of controversy. This is probably due to inadequate distinction between reflux esophagitis (RE) and non-erosive reflux disease (NERD), and is also due to inadequate evaluation of adjacent stomach. Our aim is therefore to define background factors of RE and NERD independently, based on the evaluation of Helicobacter pylori infection and gastric atrophy.

Methods

We analyzed 10,837 healthy Japanese subjects (6,332 men and 4,505 women, aged 20–87 years) who underwent upper gastrointestinal endoscopy. RE was diagnosed as the presence of mucosal break, and NERD was diagnosed as the presence of heartburn and/or acid regurgitation in RE-free subjects. Using GERD-free subjects as control, background factors for RE and NERD were separately analyzed using logistic regression to evaluate standardized coefficients (SC), odds ratio (OR), and p-value.

Results

Of the 10,837 study subjects, we diagnosed 733 (6.8%) as RE and 1,722 (15.9%) as NERD. For RE, male gender (SC = 0.557, OR = 1.75), HP non-infection (SC = 0.552, OR = 1.74), higher pepsinogen I/II ratio (SC = 0.496, OR = 1.64), higher BMI (SC = 0.464, OR = 1.60), alcohol drinking (SC = 0.161, OR = 1.17), older age (SC = 0.148, OR = 1.16), and smoking (SC = 0.129, OR = 1.14) are positively correlated factors. For NERD, HP infection (SC = 0.106, OR = 1.11), female gender (SC = 0.099, OR = 1.10), younger age (SC = 0.099, OR = 1.10), higher pepsinogen I/II ratio (SC = 0.099, OR = 1.10), smoking (SC = 0.080, OR = 1.08), higher BMI (SC = 0.078, OR = 1.08), and alcohol drinking (SC = 0.076, OR = 1.08) are positively correlated factors. Prevalence of RE in subjects with chronic HP infection and successful HP eradication denotes significant difference (2.3% and 8.8%; p<0.0001), whereas that of NERD shows no difference (18.2% and 20.8%; p = 0.064).

Conclusions

Significantly associated factors of NERD are considerably different from those of RE, indicating that these two disorders are pathophysiologically distinct. Eradication of Helicobacter pylori may have disadvantageous effects on RE but not on NERD.     

High Cell Density Upregulates Calcium Oscillation by Increasing Calcium Store Content via Basal Mitogen-Activated Protein Kinase Activity

Calcium releases of non-excitable cells are generally a combination of oscillatory and non-oscillatory patterns, and factors affecting the calcium dynamics are still to be determined. Here we report the influence of cell density on calcium increase patterns of clonal cell lines. The majority of HeLa cells seeded at 1.5 x 10⁴/cm² showed calcium oscillations in response to histamine and ATP, whereas cells seeded at 0.5 x 10⁴/cm² largely showed transient and sustained calcium increases. Cell density also affected the response of HEK293 cells to ATP in a similar manner. High cell density increased the basal activity of the mitogen-activated protein (MAP) kinase and calcium store content, and both calcium oscillation and calcium store content were down-regulated by a MAP kinase inhibitor, U0126. Thus, MAP kinase-mediated regulation of calcium store likely underlie the effect of cell density on calcium oscillation. Calcium increase patterns of HeLa cells were conserved at any histamine concentrations tested, whereas the overexpression of histamine H1 receptor, which robustly increased histamine-induced inositol phospholipid hydrolysis, converted calcium oscillations to sustained calcium increases only at high histamine concentrations. Thus, the consequence of modulating inositol phospholipid metabolism was distinct from that of changing cell density, suggesting the effect of cell density is not attributed to inositol phospholipid metabolism. Collectively, our results propose that calcium increase patterns of non-excitable cells reflect calcium store, which is regulated by the basal MAP kinase activity under the influence of cell density.     

Injection of the insulin receptor alpha subunit increases blood glucose levels in mice

Using the expression vector of the truncated human insulin receptor (hIR), we have constructed a stable Chinese hamster ovary (CHO) cell line which secretes the His-tagged alpha subunit (insulin-binding domain) of hIR into medium. To examine characteristics of the His-tagged hIRalpha, we purified the protein secreted from the CHO cells. The His-tagged hIRalpha was glycosylated and processed a dimer. The molecule bound insulin with an affinity similar to that of the intact hIR. The His-tagged full length of hIR was autophosphorylated by insulin stimulation in CHO cells. Injection of the purified His-tagged hIRalpha into veins of mice increased in the concentration of blood glucose within 30 min. The intraperitoneal glucose tolerance test (ipGTT) done after injection of the purified His-tagged hIRalpha showed evidence of a marked hyperglycemia. These findings provide direct evidence that the presence of hIRalpha in the blood stream inhibits insulin actions by binding with plasma insulin.     

Thermococcus kodakarensis Mutants Deficient in Di-myo-Inositol Phosphate Use Aspartate To Cope with Heat Stress

Many of the marine microorganisms which are adapted to grow at temperatures above 80°C accumulate di-myo-inositol phosphate (DIP) in response to heat stress. This led to the hypothesis that the solute plays a role in thermoprotection, but there is a lack of definitive experimental evidence. Mutant strains of Thermococcus kodakarensis (formerly Thermococcus kodakaraensis), manipulated in their ability to synthesize DIP, were constructed and used to investigate the involvement of DIP in thermoadaptation of this archaeon. The solute pool of the parental strain comprised DIP, aspartate, and α-glutamate. Under heat stress the level of DIP increased 20-fold compared to optimal conditions, whereas the pool of aspartate increased 4.3-fold in response to osmotic stress. Deleting the gene encoding the key enzyme in DIP synthesis, CTP:inositol-1-phosphate cytidylyltransferase/CDP-inositol:inositol-1-phosphate transferase, abolished DIP synthesis. Conversely, overexpression of the same gene resulted in a mutant with restored ability to synthesize DIP. Despite the absence of DIP in the deletion mutant, this strain exhibited growth parameters similar to those of the parental strain, both at optimal (85°C) and supraoptimal (93.7°C) temperatures for growth. Analysis of the respective solute pools showed that DIP was replaced by aspartate. We conclude that DIP is part of the strategy used by T. kodakarensis to cope with heat stress, and aspartate can be used as an alternative solute of similar efficacy. This is the first study using mutants to demonstrate the involvement of compatible solutes in the thermoadaptation of (hyper)thermophilic organisms.Hyperthermophilic bacteria and archaea isolated from saline environments accumulate unusual organic solutes in response to osmotic as well as heat stress. Mannosylglycerate, mannosylglyceramide, di-myo-inositol phosphate, mannosyl-di-myo-inositol phosphate (DIP), diglycerol phosphate, and glycero-phospho-myo-inositol are examples of compatible solutes highly restricted to thermophiles and hyperthermophiles (27, 31). Our team has, over several years, examined the compatible solute composition in a large number of hyperthermophiles and their accumulation under stressful conditions. The data reveal a trend toward specialization of roles in thermoadaptation and osmoadaptation. Indeed, mannosylglycerate and diglycerol phosphate typically accumulate in response to increased NaCl concentration in the growth medium, whereas the levels of DIP and derivatives consistently increase at supraoptimal growth temperatures (11, 16, 17, 27, 31).DIP is widespread among extreme archaeal hyperthermophiles, such as Methanotorris igneus, Aeropyrum pernix, Stetteria hydrogenophila, Pyrodictium occultum, Pyrolobus fumarii, Archaeoglobus spp., and all the members of the Thermococcales examined thus far, except Palaeococcus ferrophilus (5, 7, 11, 13, 16, 18, 31). This organic solute has also been found in representatives of the two hyperthermophilic bacterial genera, Aquifex and Thermotoga (14, 17, 22).The specific chemical nature of solutes encountered in hyperthermophiles, together with their accumulation in response to elevated temperatures, led to the hypothesis that they play a role in thermoprotection of cellular components in vivo. However, there is a lack of convincing experimental evidence, such as that obtained with suitable mutants. Progress toward understanding the physiological functions of these solutes critically depends on two conditions: the availability of genetic tools to manipulate hyperthermophilic organisms and knowledge about the genes and enzymes implicated in the synthesis of these unusual solutes.Thermococcus kodakarensis (formerly Thermococcus kodakaraensis) is a member of the order Thermococcales with an optimal growth temperature of 85°C and is able to grow at temperatures up to 94°C in batch cultures. The NaCl concentration for optimal growth matches that of seawater (1). T. kodakarensis is the only marine hyperthermophile for which a number of genetic tools have been developed, including Escherichia coli-T. kodakarensis shuttle vectors and a reliable gene disruption system (19, 29, 32, 34). The genome of T. kodakarensis possesses a gene encoding CTP:inositol-1-phosphate cytidylyltransferase/CDP-inositol:inositol-1-phosphate transferase (IPCT/DIPPS), a key enzyme in DIP synthesis (2, 25, 26). This enzyme catalyzes the synthesis of CDP-inositol from CTP and inositol-1-phosphate as well as the transfer of the inositol group from CDP-inositol to a second molecule of inositol-1-phosphate to yield a phosphorylated form of DIP (2). Therefore, we set out to investigate whether DIP was involved in thermoadaptation of T. kodakarensis. A DIP-deficient mutant was constructed by deleting the IPCT/DIPPS gene; subsequently, this strain was complemented in this activity by inserting the gene under the control of a constitutive promoter, resulting in a construct with restored ability to synthesize DIP. The effects of heat and osmotic stress on the pattern of solute accumulation and on the growth profiles of the two mutants provided evidence for the involvement of DIP in thermoprotection.     

Detection of chemiluminescence in lipid peroxidation of biological systems and its application to HPLC

A combined system of chemiluminescence detection and high performance liquid chromatography (CL–HPLC) was developed to determine primary peroxidation products in biological tissues, such as phosphatidylcholine hydroperoxide (PCOOH). The CL–HPLC assay consists of separation of lipid classes with HPLC and detection of hydroperoxide-specific chemiluminescence. Hydroperoxides react with heme compounds to produce oxidants as suggested by our early studies on tissue low-level chemiluminescence in which singlet molecular oxygen is generated as one of the excited species in several biological systems involving free radical events. In the CL–HPLC method, a cytochrome c–luminol mixture was used as a hydroperoxide-specific luminescent reagent, and the quantification of hydroperoxide was performed by detecting chemiluminescence due to the luminol oxidation caused by the oxidant produced during the lipid hydroperoxides with heme. The detection limit of PCOOH was 10 pmole hydroperoxide–O₂. PCOOH in normal human blood was found to be 10–500 pmol/ml plasma and significantly higher levels of PCOOH were observed in some hospitalized patients.