Identification of crude-oil components and microorganisms that cause souring under anaerobic conditions.

Oil souring has important implications with respect to energy resources. Understanding the physiology of the microorganisms that play a role and the biological mechanisms are both important for the maintenance of infrastructure and mitigation of corrosion processes. The objective of this study was to identify crude-oil components and microorganisms in oil-field water that contribute to crude-oil souring. To identify the crude-oil components and microorganisms that are responsible for anaerobic souring in oil reservoirs, biological conversion of crude-oil components under anaerobic conditions was investigated. Microorganisms in oil field water in Akita, Japan degraded alkanes and aromatics to volatile fatty acids (VFAs) under anaerobic conditions, and fermenting bacteria such as Fusibacter sp. were involved in VFA production. Aromatics such as toluene and ethylbenzene were degraded by sulfate-reducing bacteria (Desulfotignum sp.) via the fumarate-addition pathway and not only degradation of VFA but also degradation of aromatics by sulfate-reducing bacteria was the cause of souring. Naphthenic acid and 2,4-xylenol were not converted.

Iron dependent degradation of an isothiazolone biocide (5-chloro-2-methyl-4-isothiazolin-3-one).

An isothiazolone biocide, 5-chloro-2-methyl-4-isothiazolin-3-one (CMI), was degraded in the presence of iron. According to the Fe-dependent degradation of CMI, stoichiometric production of chloride was observed. Copper and stainless steel did not enhance the physico-chemical degradation of CMI, whilst phosphate inhibited the Fe-dependent degradation. Neither aerobic nor anaerobic conditions influenced the Fe-dependent CMI degradation. Furthermore, FeO(OH)-powder and Fe(3)O(4)-powder did not lead to the physico-chemical degradation of CMI. Rapid disappearance of CMI was observed in an operating cooling water plant. CMI added to the cooling tower declined from 1.4 mg l(-1) to < 0.1 mg l(-1) in 2 d. This finding is important in optimising the use of CMI and combating resistance if encountered.

The survival response of Escherichia coli K12 in a natural environment.

To verify the hypothesis of cryptic growth and viable but nonculturable (VBNC) state, survival responses of Escherichia coli cells were examined under oligotrophic microcosm conditions for an extended period. In the case of filtered distilled water at 4 degrees C, E. coli cells definitely entered the VBNC state within 56 days. However, culturability and viability increased while the total number of cells declined after 110 days. This phenomenon can be explained by considering three possible states. The first is the existence of the VBNC state, the second is cryptic growth, and the third is the death of E. coli cells. In the case of artificial seawater at 4 degrees C, VBNC E. coli cells confirmed the existence of two log units of elongated VBNC cells. Moreover, elongated VBNC cells showed the most significant change among all the other transformed cells. Also, E. coli cells in microcosms at 28 degrees C indicated the entrance to the classical starvation survival state. In resuscitation tests, 1% diluted Luria-Bertani agar medium showed the highest level of resuscitation among amended agar media. To evaluate the survival ability of E. coli cells in the activated sludge samples, we used an E. colistrain XL-1 blue containing plasmids pQ2 including GFPcDNA (XL/GFP). In supernatant of activated sludge (SUP) at 28 degrees C, XL/GFP cells entered the VBNC state after 10 days, whereas existence of VBNC cells was not detectable in resuspended activated sludge (ACT) at 28 degrees C.

Toward rational control of Escherichia coli O157:H7 by a phage cocktail.

Twenty six phages infected with Escherichia coli O157:H7 were screened from various sources. Among them, nine caused visible lysis of E. coli O157:H7 cells in LB liquid medium. However, prolonged incubation of E. coli cells and phage allowed the emergence of phage-resistant cells. The susceptibility of the phage-resistant cells to the nine phages was diverse. A rational procedure for selecting an effective cocktail of phage for controlling bacteria was investigated based on the mechanism of phage-resistant cell conversion. Deletion of OmpC from the E. coli cells facilitated the emergence of cells resistant to SP21 phage. After 8 h of incubation, SP21-resistant cells appeared. By contrast, alteration of the lipopolysaccharide (LPS) profile facilitated cell resistance to SP22 phage, which was observed following a 6-h incubation. When a cocktail of phages SP21 and SP22 was used to infect E. coli O157:H7 cells, 30 h was required for the emergence of cells (R-C) resistant to both phages. The R-C cells carried almost the same outer membrane and LPS components as the wild-type cells. However, the reduced binding ability of both phages to R-C cells suggested disturbance of phage adsorption to the R-C surface. Even though R-C cells resistant to both phages appeared, this work shows that rational selection of phages has the potential to at least delay the emergence of phage resistance.

Sol-Gel transition behavior of pure iota-carrageenan in both salt-free and added salt states.

This paper describes how strongly the gelation process of iota-carrageenan is affected by addition of metallic ions from the creep and creep recovery, dynamic viscoelasticity (DVE) and DSC measurements. Creep results at T = 25 degrees C indicate that below a polymer concentration C of 3.0 wt % the salt-free system behaves as a viscous solution, and it starts to exhibit viscoelasticity as C exceeds 3.0 wt %. In the range C = 5.0-7.0 wt %, the salt-free system shows gellike behavior whereas the added salt system, measured in the low C range 1.0-2.5 wt %, showed gellike behavior at the same temperature. The sol-gel transition temperature T(c) was determined using Winter's criterion as the temperature at which both G'(omega) and G' '(omega) follow power law behavior with the same exponent n. DSC measurements reveal that salt-free and added salt systems take different types of thermal behavior within the same temperature range. The temperature T(c) is quite close to the gelation temperature T(m) determined from DSC measurement. The Eldrige-Ferry plot was performed to estimate activaton enthalpy, which shows that physical cross-links in the salt-free iota-carrageenan is not strong in comparison with those of samples which contains metal ions. We conclude from the data analysis of C dependence of the plateau modulus using the theory developed by Jones and Marques for rigid networks based on the fractal theories that addition of metallic ions gives rise to a rigid fiber like structure even at low C of iota-carrageenan in contrast to the salt-free system for which a flexible structure has been maintained at higher C.

Serum platelet-activating factor acetylhydrolase (PAF-AH) activity in more than 3000 healthy Japanese.

BACKGROUND: A spectrophotometric assay for platelet-activating factor acetylhydrolase (PAF-AH) activity differs from the radioisotopic assay in its value because of a difference in substrate specificity. The spectrophotometric assay is more precise than the radioisotopic assay, providing information that is not clear with the radioisotopic assay. METHODS: We measured the serum PAF-AH activity in 3106 healthy Japanese, utilizing the spectrophotometric assay with an Hitachi 7170 automatic analyzer. We also measured the serum PAF-AH activity in 18 healthy volunteers to investigate the effect of diet and the change in activity in a day and over 6 weeks. Changes were examined at 0 (day 1), 1, 2, 4 and 6 weeks. RESULTS: The mean value for females was significantly lower than that of males at the 5% level and both male and female activity had a tendency to increase with advancing age. It is known that the PAF-AH is primarily associated with LDL in blood and the PAF-AH activity correlated with the total cholesterol (r=0.52, n=126) and the LDL cholesterol (r=0.60, n=126) concentrations. In the diet study, there was no observable effect on activity. No difference in PAF-AH activity was observed between serum and plasma sample types. The serum PAF-AH activity was stable at 7 degrees C for at least 7 days and at -20 degrees C for at least 2 months. CONCLUSIONS: The serum PAF-AH activity in women was lower than in men until the menopausal age was reached. We could use not only fresh fasting serum, but also plasma sample, non-fasting sample and stored sample to estimate the PAF-AH activity.

Molecular analysis of plasma alpha 1,3-fucosyltransferase deficiency and development of the methods for its genotyping.

Four patients with mental illness were found to be deficient in plasma alpha1,3-fucosyltransferase for the first time in Japan [Exp Clin Immunogenet 1999;16:125-130]. Complete sequencing of FUT6 genes in these individuals revealed the presence of two point mutations, i.e., G739 to A (Glu-->247 to Lys) and C945 to A (Tyr-->315 to stop). In addition to two reported alleles having G739 to A (pf1) and G739 to A and C945 to A (pf3), a new mutated allele having C945 to A (pf2) was found to be present and all the individuals who lack alpha1,3-fucosyltransferase activity in plasma were found to possess pf genes homozygously (pf/pf). In order to detect such lethal mutations in FUT6 genes easily, PCR-RFLP methods have also been developed and applied for the screening of FUT6 deficiency in a large number of samples which resulted in the demonstration of three additional FUT6-deficient individuals. The absence of alpha1,3-fucosylated molecules on alpha(1)-acid glycoprotein in plasma from all the 7 individuals was confirmed to result from the plasma alpha1,3-fucosyltransferase deficiency.

Quantitative determination of cultured strawberry-cell heterogeneity by image analysis: effects of medium modification on anthocyanin accumulation.

Cultured plant cells are often highly heterogeneous in terms of secondary metabolite production. We have developed a quantitative determination method that uses an image processing system to estimate such individual cell characteristics as content of the secondary metabolite, anthocyanin. In this study, strawberry cells producing anthocyanins were grown in modified Linsmaier-Skoog medium. Anthocyanin accumulation profiles of individual cells depended on medium compositions and were quantitatively determined using the new method. The modified medium supplemented with riboflavin and high sugar concentration showed a markedly higher anthocyanin accumulation profile and pigmented cell ratio than the other modified media. The maximum content was about 11mg (g-fresh cell weight)(-1), which was three times higher than that in the control medium. Moreover, the anthocyanin accumulation profiles in the individual cells cultured in all modified media could be approximated to the parts of the normal distribution curves with the constant variance.

Declining of memory functions of normal elderly persons.

Two studies examined the declining of memory functions in normal elderly persons using the Yokota memory test (YMT), which includes 15 items concerning verbal and non-verbal memory functions. In the first study, 552 subjects over 40 years of age in five age groups were examined. Factor analysis revealed that YMT consisted of two factors pertaining to short-term/working memory, and two factors pertaining to long-term memory. It is suggested that the former was more affected than the latter, with aging. In the second study, YMT was examined in relation to the revised version of Hasegawa dementia scale (HDS-R), which was the most popular intelligence scale for the elderly in Japan. As a result, memory functions differentially declined with the decreasing score of HDS-R, which suggests that memory functions differentially declined with progressive risk of dementia.

Analysis of pigment accumulation heterogeneity in plant cell population by image-processing system.

Plant-cultured cells are often highly heterogeneous in secondary metabolite productivity. The industrial application for large-scale metabolite production requires establishment of a stable high-producing cell line. In this study, image analysis of the individual cell is investigated as a method for evaluation of a heterogeneous cell population, and compared with the conventional method of estimation, which is based on average-cell productivity. Among strawberry cells producing anthocyanins, cells with a wide-range of pigment concentration were observed and maximum anthocyanin content was 10 times higher than the average value. In addition, a change of the frequency distribution was revealed in batch cultivation.

Two autopsy cases with Pelizaeus-Merzbacher disease phenotype of adult onset, without mutation of proteolipid protein gene.

We report the autopsy cases of two brothers which are pathologically compatible with Pelizaeus-Merzbacher disease (PMD). Both patients had a late onset (at the ages of 29 and 42 years) and chronic neurological symptoms including tremor, ataxia and dementia. The T2-weighted magnetic resonance imaging of the younger brother demonstrated increased signal areas with sparing of small areas in the cerebral white matter. The postmortem examinations, obtained at the ages of 45 and 61 years, showed similar neuropathological findings. Histologically, a cardinal finding was a lack of myelin in large parts of white matter with the preservation of islands of intact myelin, resulting in a "tigroid" appearance. Only small amounts of sudanophilic material were present. The axons were relatively well preserved, but oligodendrocytes were numerically reduced. Ultrastructurally, myelin sheaths in the white matter were markedly thin. Immunohistochemistry showed that proteolipid protein (PLP) was reduced in the affected white matter. However, genetic studies did not reveal exonic mutations or duplications of the PLP gene. We conclude that the two cases are a rare type of dysmyelinating disorder with PMD phenotype of adult onset and could be caused by previously unrecognized abnormalities of the PLP gene or other genes.

In vitro processing of amyloid precursor protein by cathepsin D.

The formation of beta A4 amyloid in the brains of individuals with Alzheimer's disease requires the proteolytic cleavage of amyloid precursor protein. Several lines of evidence suggest that cathepsin D, the major lysosomal/endosomal aspartic protease, may be involved in this process. In this work, we used a sensitive in vitro method of detection to investigate the role of cathepsin D in the proteolytic processing of a 100-amino acid C-terminal fragment (C100) inclusive of beta A4 and cytoplasmic domain of APP. Digestion of C100 with cathepsin D resulted in cleavage at the amyloidogenic gamma-cleavage sites. This occurred preferentially at Thr43-Val44 and at Ala42-Thr43, generating full length beta A4 43 and beta A4 42 amyloid peptides, respectively. Cathepsin D was also found to cleave the substrate at the following nonamyloidogenic sites; Leu34-Met35, Thr48-Leu49 and Leu49-Val50. A high concentration of cathepsin D resulted in cleavage also occurring at Phe19-Phe20, Phe20-Ala21 and Phe93-Phe94 of the C100, suggesting that these sites are somewhat less sensitive to the action of cathepsin D. Digestion of C100 using different solublizing agents indicated that the cleavage of C100 by cathepsin D is greatly influenced by the structural integrity of the substrate. However, our results suggest that cathepsin D could generate the pathogenic beta A4 amyloid peptides from its precursor in vitro, which may indicate a role in the amyloidogenesis of Alzheimer's disease.

Plasma alpha1,3-fucosyltransferase deficiency in schizophrenia.

Levels of plasma alpha1,3-fucosyltransferase (alpha1,3FT) were assayed in 44 patients with schizophrenia and in 50 healthy controls. Significantly reduced enzyme activities were observed in patients (p < 0.05) and 4 unrelated patients were found, for the first time in Japan, to be deficient in the enzyme activity. Two point mutations in the coding region of the FUT6 gene encoding plasma alpha1,3FT that were responsible for the inactivation of the enzyme activity were detected in those patients. Genotyping of the Le gene (FUT3) in these patients demonstrated that 2 of them were also FUT3 deficient and were grouped as Lewis- individuals whereas the rest were Lewis+.

Genotype-phenotype correlation of patients with multiple endocrine neoplasia type 2 in Japan.

BACKGROUND: Multiple endocrine neoplasia type 2 (MEN 2) is a hereditary syndrome characterized by medullary thyroid carcinoma (MTC), pheochromocytoma and hyperparathyroidism. MEN 2 is caused predominantly by germ-line mutations of the RET proto-oncogene. This study aimed to clarify the genotype-phenotype correlation in MEN 2 patients in Japan in order to modify the clinical management according to the genotype. METHODS: Constitutive DNA of 64 MEN 2 patients (48 kindreds) were searched for mutations at exons 10, 11, 13, 14 and 16 of the RET proto-oncogene using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP), direct sequencing and restriction enzyme digestion. The clinical characteristics of the patients were obtained from a previous nationwide questionnaire survey. RESULTS: Overall, 62 (96.9%) out of 64 patients had a germ-line point mutation at the hot spots. MTC and pheochromocytoma occurred equally in every genotype except C630S. Specific genotype had a correlation between tumor size and age at the operation for MTC or extent of MTC, i.e. C618S developed late onset type of MTC as compared with that of C634R, C634Y and M918T. Small MTC in C634R may be less aggressive than those in C634Y and M918T. CONCLUSIONS: DNA testing has good clinical implications for the management of patients with MEN 2 and the timing and operative procedures of thyroidectomy can be modified according to the genotype.

CHARGE association with congenital glaucoma due to maldevelopment of the anterior chamber angle.

PURPOSE: We examined the clinicopathologic features of a male infant with the CHARGE association and bilateral congenital glaucoma. METHODS: Trabeculectomy specimens were obtained from the anterior chamber angle and examined by light and electron microscopy. RESULTS: Histopathologic examination of the trabeculectomy specimens showed immature development of the trabecular meshwork that was covered by the ciliary muscles. There were few intertrabecular spaces because the meshwork was almost completely filled with cells and extracellular substances. Deposition of a granular and/or homogeneous substance was observed in the subendothelial area of Schlemm's canal. CONCLUSIONS: Our patient exhibited features typical of the CHARGE association, but also had congenital glaucoma. We hypothesize that these clinical findings are all mediated by a neurocristopathic mechanism. Our findings suggest that the CHARGE association may predispose to anterior chamber angle maldevelopment, which can lead to congenital glaucoma.

Persistent synchronous periodic discharges caused by anoxic encephalopathy due to cardiopulmonary arrest.

We report a patient with vegetative state induced by anoxic encephalopathy due to cardiopulmonary arrest who showed synchronous periodic discharges for a prolonged period. The patient was a 47-year-old man admitted to our hospital for depression, who suddenly developed cardiopulmonary arrest of unknown etiology, and entered chronic vegetative state as a result of anoxic encephalopathy. Electroencephalography (EEG) revealed that synchronous periodic discharges were present for up to 5 months. The wave pattern, periodicity and duration of appearance of synchronous periodic discharges were similar to those of synchronous periodic discharges in Creutzfeldt-Jakob disease (CJD). The periodicity of synchronous periodic discharges was gradually prolonged, with the course again similar to the discharges in CJD. The mechanism of occurrence is considered to be similar to that of synchronous periodic discharges in CJD.

[Porcine trabecular meshwork organ culture].

In order to investigate whether porcine eyes could be useful for trabecular meshwork (TM) organ culture, changes in ultrastructure and synthesis of glycosaminoglycans (GAGs) of organ-cultured porcine TM were studied. The presence of thrombospondin was also detected in porcine TM, using an immunohistochemical method. Explants were cultured for periods of 7 to 21 days and subsequently some of them were labeled with 3H-glucosamine and 35S-sulfate for 48 hours. There was very little change in the ultrastructure of the explants during our culture periods. The total incorporation of 3H-glucosamine and 35S-sulfate into trabecular GAGs for up to 21 days showed similar patterns. Immunoreactivity for thrombospondin was found in the trabecular sheets. This organ culture system suggests a model for studying the various drug and hormone induced alterations in this tissue.

Differential control of activin, inhibin and follistatin proteins in cultured rat granulosa cells.

Follistatin, activin and inhibin proteins are produced by granulosa cells, but the mechanisms controlling their production remain unclear. Here, we examined how the protein kinase A (PKA) and protein kinase C (PKC) pathways act and interact to regulate production of these proteins. Granulosa cells from immature rats were cultured with activators of the PKA pathway (100 ng/ml FSH, 10 microM forskolin) and/or activators of the PKC pathway (100 nM GnRH agonist, 100nM 2-0-tetradecanoyl-phorbol-13-acetate, TPA). Conditioned media were assayed for inhibin and activin by ligand blotting using recombinant human 125I-follistatin and for follistatin by double ligand blotting using cold activin plus 125I-follistatin. FSH and forskolin stimulated inhibin but not activin production. In contrast, GnRH and TPA stimulated activin, and to a lesser degree, inhibin production; significantly, this is the first demonstration of activin dimer production by granulosa cells. Activators of the PKA pathway antagonized the actions of PKC effectors and vice versa. All agents increased follistatin protein production, and the PKA and PKC activators interacted to generate further increases in follistatin production. These results show that the FSH-PKA signalling pathway favors formation of alpha beta inhibin dimers while the GnRH-PKC pathway favors formation of beta-subunit activin dimers. Both pathways act to increase follistatin protein production.

Structural and functional characterization of the rat follistatin (activin-binding protein) gene promoter.

Follistatin was originally identified as a specific inhibitor of follicle stimulating hormone secretion and later characterized as a binding protein for activin. Since activin regulates hormone secretion and cell differentiation, the importance of understanding the mechanisms regulating the synthesis of its binding protein, follistatin, is evident. To study the regulation of follistatin gene expression, we first determined the transcription start site (cap site) of the rat follistatin gene using primer extension and ribonuclease protection assay. Our results led to the identification of multiple cap sites located at three different positions of the promoter. DNA sequence analysis revealed that each cap site was located at approximately 30 nucleotide (nt) downstream of three distinct TATA-like sequences. In primary cultures of rat granulosa cells, transfection studies using 5'-flanking regions of follistatin gene fused to the chloramphenicol acetyltransferase (CAT) reporter gene revealed the presence of two DNA segments that act to suppress basal transcriptional activity. The promoter activity of the CAT construct containing 2.6 kilo base pairs (kb) of 5'-flanking region was induced 2.5-fold above basal activity by forskolin (10 microM), and 1.6-fold by 12-O-tetradecanoylphorbol 13-acetate (TPA, 100 nM). Co-treatment with forskolin and TPA resulted in a 6.4-fold induction in its promoter activity, suggesting that two distinct signal transduction pathways, the cAMP-dependent protein kinase-A pathway and diacylglycerol-dependent protein kinase-C pathway, act coordinately to modulate follistatin gene transcription. Experiments using a series of 5'-flanking region deletion constructs located the regulatory regions responsive to these two pharmacological agents at nt -312 to -32 and -35 to +139.

Corticotroph cell hyperplasia in a patient with Addison disease: case report.

A woman with Addison disease developed hyperpigmentation, headache, and nausea despite conventional replacement therapy with cortisone. Excessively elevated plasma adrenocorticotropic hormone (ACTH) with absence of response to administration of corticotropin-releasing factor (CRF), and roentgenological evidence of enlargement of the sella turcica, as well as detection of enlarged pituitary gland on magnetic resonance images, led to a diagnosis of ACTH-producing microadenoma, which was removed by transsphenoidal microsurgery. The specimen obtained at surgery evidenced corticotroph hyperplasia, as demonstrated by immunohistochemical staining for ACTH. Fine structure exhibited densely granulated cells with a few bundles of microfilaments and an abundance of large lysosomal bodies. Surgical removal of the hyperplasia alleviated the patient's symptoms, and hyperpigmentation faded remarkably. Her plasma ACTH level returned to normal, has remained normal for more than 3 years, and responds adequately to CRF administration.