Generation of hypothalamic neural stem cell-like cells in vitro from human pluripotent stem cells.

When damaged, restoring the function of the hypothalamus is currently impossible. It is unclear whether neural stem cells exist in the hypothalamus. Studies have reported that adult rodent tanycytes around the third ventricle function as hypothalamic neural stem cell-like cells. However, it is currently impossible to collect periventricular cells from humans. We attempted to generate hypothalamic neural stem cell-like cells from human embryonic stem cells (ESCs). We focused on retina and anterior neural fold homeobox (RAX) because its expression is gradually restricted to tanycytes during the late embryonic stage. We differentiated RAX::VENUS knockin human ESCs (hESCs) into hypothalamic organoids and sorted RAX+ cells from mature organoids. The isolated RAX+ cells formed neurospheres and exhibited self-renewal and multipotency. Neurogenesis was observed when neurospheres were transplanted into the mouse hypothalamus. We isolated RAX+ hypothalamic neural stem cell-like cells from wild-type human ES organoids. This is the first study to differentiate human hypothalamic neural stem cell-like cells from pluripotent stem cells.

Recovery from the damage of cranial radiation modulated by memantine, an NMDA receptor antagonist, combined with hyperbaric oxygen therapy.

BACKGROUND: Radiotherapy is an important treatment option for central nervous system malignancies. However, cranial radiation induces hippocampal dysfunction and white matter injury; this leads to cognitive dysfunction, and results in a reduced quality of life in patients. Excitatory glutamate signaling through N-methyl-d-aspartate receptors (NMDARs) plays a central role both in hippocampal neurogenesis and in the myelination of oligodendrocytes in the cerebrum. METHODS: We provide a method for quantifying neurogenesis in human subjects in live brain during cancer therapy. Neuroimaging using originally created behavioral tasks was employed to examine human hippocampal memory pathway in patients with brain disorders. RESULTS: Treatment with memantine, a non-competitive NMDAR antagonist, reversed impairment in hippocampal pattern separation networks as detected by functional magnetic resonance imaging. Hyperbaric preconditioning of the patients just before radiotherapy with memantine mostly reversed white matter injury as detected by whole brain analysis with Tract-Based Spatial Statics. Neuromodulation combined with the administration of hyperbaric oxygen therapy and memantine during radiotherapy facilitated the restoration of hippocampal function and white matter integrity, and improved higher cognitive function in patients receiving cranial radiation. CONCLUSIONS: The method described herein, for diagnosis of hippocampal dysfunction, and therapeutic intervention can be utilized to restore some of the cognitive decline experienced by patients who have received cranial radiation. The underlying mechanism of restoration is the production of new neurons, which enhances functionality in pattern separation networks in the hippocampi, resulting in an increase in cognitive score, and restoration of microstructural integrity of white matter tracts revealed by Tract-Based Spatial Statics Analysis.

Mineralocorticoids induce polyuria by reducing apical aquaporin-2 expression of the kidney in partial vasopressin deficiency.

The symptoms of diabetes insipidus may be masked by the concurrence of adrenal insufficiency and emerge after the administration of hydrocortisone, occasionally at high doses. To elucidate the mechanism underlying polyuria induced by the administration of high-dose corticosteroids in the deficiency of arginine vasopressin (AVP), we first examined the secretion of AVP in three patients in whom polyuria was observed only after the administration of high-dose corticosteroids. Next, we examined the effects of dexamethasone or aldosterone on water balance in wild-type and familial neurohypophyseal diabetes insipidus (FNDI) model mice. A hypertonic saline test showed that AVP secretion was partially impaired in all patients. In one patient, there were no apparent changes in AVP secretion before and after the administration of high-dose corticosteroids. In FNDI mice, unlike dexamethasone, the administration of aldosterone increased urine volumes and decreased urine osmolality. Immunohistochemical analyses showed that, after the administration of aldosterone in FNDI mice, aquaporin-2 expression was decreased in the apical membrane and increased in the basolateral membrane in the collecting duct. These changes were not observed in wild-type mice. The present data suggest that treatment with mineralocorticoids induces polyuria by reducing aquaporin-2 expression in the apical membrane of the kidney in partial AVP deficiency.

Differentiation of human induced pluripotent stem cells into hypothalamic vasopressin neurons with minimal exogenous signals and partial conversion to the naive state.

Familial neurohypophyseal diabetes insipidus (FNDI) is a degenerative disease of vasopressin (AVP) neurons. Studies in mouse in vivo models indicate that accumulation of mutant AVP prehormone is associated with FNDI pathology. However, studying human FNDI pathology in vivo is technically challenging. Therefore, an in vitro human model needs to be developed. When exogenous signals are minimized in the early phase of differentiation in vitro, mouse embryonic stem cells (ESCs)/induced pluripotent stem cells (iPSCs) differentiate into AVP neurons, whereas human ESCs/iPSCs die. Human ESCs/iPSCs are generally more similar to mouse epiblast stem cells (mEpiSCs) compared to mouse ESCs. In this study, we converted human FNDI-specific iPSCs by the naive conversion kit. Although the conversion was partial, we found improved cell survival under minimal exogenous signals and differentiation into rostral hypothalamic organoids. Overall, this method provides a simple and straightforward differentiation direction, which may improve the efficiency of hypothalamic differentiation.

Enhancement of lysine biosynthesis confers high-temperature stress tolerance to Escherichia coli cells.

Lysine, a nutritionally important amino acid, is involved in adaptation and tolerance to environmental stresses in various organisms. Previous studies reported that lysine accumulation occurs in response to stress and that lysine supplementation enhances stress tolerance; however, the effect of lysine biosynthesis enhancement on stress tolerance has yet to be elucidated. In this study, we confirmed that lysine supplementation to the culture medium increased intracellular lysine content and improved cell growth of Escherichia coli at high temperature (42.5 °C). Lysine-overproducing strains were then isolated from the lysine analogue S-adenosylmethionine-resistant mutants by conventional mutagenesis and exhibited higher tolerance to high-temperature stress than the wild-type strain. We identified novel amino acid substitutions Gly474Asp and Cys554Tyr on ThrA, a bifunctional aspartate kinase/homoserine dehydrogenase (AK/HSDH), in the lysine-overproducing mutants. Interestingly, the Gly474Asp and Cys554Tyr variants of ThrA induced lysine accumulation and conferred high-temperature stress tolerance to E. coli cells. Enzymatic analysis revealed that the Gly474Asp substitution in ThrA reduced HSDH activity, suggesting that the intracellular level of aspartate semialdehyde, which is a substrate for HSDH and an intermediate for lysine biosynthesis, is elevated by the loss of HSDH activity and converted to lysine in E. coli. The present study demonstrated that both lysine supplementation and lysine biosynthesis enhancement improved the high-temperature stress tolerance of E. coli cells. Our findings suggest that lysine-overproducing strains have the potential as stress-tolerant microorganisms and can be applied to robust host cells for microbial production of useful compounds. KEY POINTS: • Lysine supplementation improved the growth of E. coli cells at high temperature. • The G474D and C554Y variant ThrA increased lysine productivity in E. coli cells. • The G474D substitution in ThrA reduced homoserine dehydrogenase activity. • E. coli cells that overproduce lysine exhibited high-temperature stress tolerance.

Preventive Effect of the Japanese Traditional Herbal Medicine Boiogito on Posttraumatic Osteoarthritis in Rats.

BACKGROUND: Considering the anti-inflammatory properties of the Japanese traditional Kampo medicine Boiogito (BO), we aimed to investigate the therapeutic effect of BO to prevent the development of knee osteoarthritis (KOA) in rats with surgically induced KOA. METHODS: Destabilization of the medial meniscus (DMM) was performed to induce osteoarthritis in the right knees of 12-week-old Wistar rats under general anesthesia. The rats were orally administered 3% BO in standard powder chow for 4 weeks after surgery (controls: n = 6; sham group: n = 6; DMM group: n = 5; DMM + BO group: n = 5). During this period, the rotarod test was performed to monitor locomotive function. After 4 weeks, histological assessment was performed on the right knee. RESULTS: Oral administration of BO improved locomotive function in the rotarod test. Walking time on postoperative days 1, 14, or later was significantly longer in the DMM + BO group than in the DMM group. Histologically, the DMM group showed significant progression of KOA, which, in the DMM + BO group, was strongly suppressed, as assessed by the Osteoarthritis Research Society International score. CONCLUSIONS: Our results showed that oral administration of BO had a clinically preventive effect on early stage posttraumatic KOA.

Hypothalamic Contribution to Pituitary Functions Is Recapitulated In Vitro Using 3D-Cultured Human iPS Cells.

The pituitary is a major hormone center that secretes systemic hormones responding to hypothalamus-derived-releasing hormones. Previously, we reported the independent pituitary induction and hypothalamic differentiation of human embryonic stem cells (ESCs). Here, a functional hypothalamic-pituitary unit is generated using human induced pluripotent stem (iPS) cells in vitro. The adrenocorticotropic hormone (ACTH) secretion capacity of the induced pituitary reached a comparable level to that of adult mouse pituitary because of the simultaneous maturation with hypothalamic neurons within the same aggregates. Corticotropin-releasing hormone (CRH) from the hypothalamic area regulates ACTH cells similarly to our hypothalamic-pituitary axis. Our induced hypothalamic-pituitary units respond to environmental hypoglycemic condition in vitro, which mimics a life-threatening situation in vivo, through the CRH-ACTH pathway, and succeed in increasing ACTH secretion. Thus, we generated powerful hybrid organoids by recapitulating hypothalamic-pituitary development, showing autonomous maturation on the basis of interactions between developing tissues.

Chemical screening identifies an extract from marine Pseudomonas sp.-PTR-08 as an anti-aging agent that promotes fission yeast longevity by modulating the Pap1-ctt1+ pathway and the cell cycle.

Aging is a degenerative process characterized by progressive deterioration of cellular components, ultimately resulting in mortality, in which massive accumulation of reactive oxygen species (ROS) and advanced glycation end products (AGEs) are implicated as crucial factors. At the same time, natural products are rich sources from which to isolate and characterize potential anti-aging compounds. The current study was designed to extract compounds from the marine bacterium Pseudomonas sp. and investigate their in vitro antioxidant and anti-glycation activities, as well as their in vivo effects on aging in the model organism Schizosaccharomyces pombe. In vitro assays showed that a Pseudomonas sp. PTR-08 extract exhibited the best antioxidant and anti-glycation activities. Further, direct administration of the extract significantly increased yeast longevity, accompanied by induction of the yeast oxidative stress response. Molecular analyses indicated that selected extract dramatically up-regulated the expression of pap1+, which encodes the transcriptional factor Pap1 and ctt1+, which encodes catalase, following H2O2 treatment. In line with these results, catalase activity significantly increased, leading to a decrease in intracellular ROS. In addition, this extract may delay the G1 phase of the yeast cell cycle, leading to an extended lifespan. Moreover, our findings indicated that the extract contains pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-, which substantially promotes anti-aging activity in yeast. However, further research must be conducted to better understand the role of this compound in our system.

Inhibition of Calcineurin and Glycogen Synthase Kinase-3ß by Ricinoleic Acid Derived from Castor Oil.

Ricinoleic acid (RA) is the main fatty acid component of castor oil and was found to inhibit Ca2+ -signal transduction pathway-mediated cell cycle regulation in a yeast-based drug screening assay. RA is expected to have antidiabetic, antiallergy, and/or anticancer properties but its target molecule is unknown. To identify a novel pharmacological effect of RA, we investigated its target molecule in the Ca2+ -signal transduction pathway. RA inhibition of calcineurin (CN) was examined in a yeast-based CN inhibitor screening assay using the rsp5A401E mutant and in a phosphatase assay using recombinant human CN. RA showed growth-restoration activity at 5 µg/spot in the CN inhibitor screening assay with the rsp5A401E yeast strain. Furthermore, it directly inhibited CN without immunophilins at Ki = 33.7 µM in a substrate-competitive manner. The effects of RA on CN in mammalian cells were further evaluated by measuring ß-hexosaminidase (ß-HEX) release in RBL-2H3 cells. RA at 50 µM suppressed the release of ß-HEX from RBL-2H3 cells. Moreover, this compound was found to inhibit glycogen synthase kinase-3ß (GSK-3ß), as determined by a kinase assay using recombinant human GSK-3ß. RA inhibited GSK-3ß at Ki = 1.43 µM in a peptide substrate-competitive manner. The inhibition of GSK-3ß by this molecule was further assessed in mammalian cells by measuring the inhibition of glucose production in H4IIE rat hepatoma cells. RA at 25 µM suppressed glucose production in these cells. These findings indicate that RA and/or castor oil could be a useful functional fatty acid to treat allergy or type 2 diabetes.

Improved methods for the differentiation of hypothalamic vasopressin neurons using mouse induced pluripotent stem cells.

High differentiation efficiency is one of the most important factors in developing an in vitro model from pluripotent stem cells. In this report, we improved the handling technique applied to mouse-induced pluripotent stem (iPS) cells, resulting in better differentiation into hypothalamic vasopressin (AVP) neurons. We modified the culture procedure to make the maintenance of iPS cells in an undifferentiated state much easier. Three-dimensional floating culture was demonstrated to be effective for mouse iPS cells. We also improved the differentiation method with regards to embryology, resulting in a greater number of bigger colonies of AVP neurons differentiating from mouse iPS cells. Fgf8, which was not used in the original differentiation method, increased iPS differentiation into AVP neurons. These refinements will be useful as a valuable tool for the modeling of degenerative disease in AVP neurons in vitro using disease-specific iPS cells in future studies.

Tanycyte-Like Cells Derived From Mouse Embryonic Stem Culture Show Hypothalamic Neural Stem/Progenitor Cell Functions.

Tanycytes have recently been accepted as neural stem/progenitor cells in the postnatal hypothalamus. Persistent retina and anterior neural fold homeobox (Rax) expression is characteristic of tanycytes in contrast to its transient expression of whole hypothalamic precursors. In this study, we found that Rax+ residual cells in the maturation phase of hypothalamic differentiation in mouse embryonic stem cell (mESC) cultures had similar characteristics to ventral tanycytes. They expressed typical neural stem/progenitor cell markers, including Sox2, vimentin, and nestin, and differentiated into mature neurons and glial cells. Quantitative RT-PCR analysis showed that Rax+ residual cells expressed Fgf-10, Fgf-18, and Lhx2, which are expressed by ventral tanycytes. They highly expressed tanycyte-specific genes Dio2 and Gpr50 compared with Rax+ early hypothalamic progenitor cells. Therefore, Rax+ residual cells in the maturation phase of hypothalamic differentiation were considered to be more differentiated and similar to late progenitor cells and tanycytes. They self-renewed and formed neurospheres when cultured with exogenous FGF-2. Additionally, these Rax+ neurospheres differentiated into three neuronal lineages (neurons, astrocytes, and oligodendrocytes), including neuropeptide Y+ neuron, that are reported to be differentiated from ventral tanycytes toward the arcuate nuclei. Thus, Rax+ residual cells were multipotent neural stem/progenitor cells. Rax+ neurospheres were stably passaged and retained high Sox2 expression even after multiple passages. These results suggest the successful induction of Rax+ tanycyte-like cells from mESCs [induced tanycyte-like (iTan) cells]. These hypothalamic neural stem/progenitor cells may have potential in regenerative medicine and as a research tool.

A unique intracellular compartment formed during the oligotrophic growth of Rhodococcus erythropolis N9T-4.

Rhodococcus erythropolis N9T-4, isolated from stored crude oil, shows extremely oligotrophic features and can grow on a basal medium without any additional carbon, nitrogen, sulfur, and energy sources, but requires CO2 for its oligotrophic growth. Transmission electron microscopic observation showed that a relatively large and spherical compartment was observed in a N9T-4 cell grown under oligotrophic conditions. In most cases, only one compartment was observed per cell, but in some cases, it was localized at each pole of the cell, suggesting that it divides at cell division. We termed this unique bacterial compartment an oligobody. The oligobody was not observed or very rarely observed in small sizes under nutrient rich conditions, whereas additional carbon sources did not affect oligobody formation. Energy dispersive X-ray spectroscopy analysis revealed remarkable peaks corresponding to phosphorus and potassium in the oligobody. The oligobodies in N9T-4 cells could be stained by Toluidine blue, suggesting that the oligobody is composed of inorganic polyphosphate and is a type of acidocalcisome. Two genes-encoding polyphosphate kinases, ppk1 and ppk2, were found in the N9T-4 genome: ppk1 disruption caused a negative effect on the formation of the oligobody. Although it was suggested that the oligobody plays an important role for the oligotrophic growth, both ppk-deleted mutants showed the same level of oligotrophic growth as the wild-type strain.

Calcineurin inhibitors suppress the high-temperature stress sensitivity of the yeast ubiquitin ligase Rsp5 mutant: a new method of screening for calcineurin inhibitors.

The ubiquitin/proteasome system plays significant and important roles in the regulation of metabolism of various proteins. The dysfunction of this system is involved in several diseases, for example, cancer, neurogenic diseases and chronic inflammation. Therefore, the compounds, which regulate the ubiquitin/proteasome system, might be candidates for the development use as clinical drugs. The Saccharomyces cerevisiae mutant (rsp5(A401E)) has a single amino acid change, Ala401Glu, in the RSP5 gene, which encodes an essential E3 ubiquitin ligase, is hypersensitive to high-temperature stress. Here, we found that the immunosuppressants FK506 and cyclosporin A, both known as calcineurin inhibitors, complemented the high-temperature stress-induced growth defect of rsp5(A401E) strain. The defect of calcineurin pathway by disrupting the CNB1 and CRZ1 gene also partially complemented the high-temperature stress sensitivity of rsp5(A401E) cells. Thus, these results suggest that inhibition of the calcineurin pathway confers the tolerance to high-temperature stress on rsp5(A401E) cells. Furthermore, some diterpenoid compounds, which restore the growth of rsp5(A401E) cells, showed the activities of calcineurin inhibition and protein phosphatase 2C activation. These results indicate that calcineurin inhibitors suppress the high-temperature stress sensitivity of rsp5(A401E) cells and that analysis of their physiological function is effective for the screening of calcineurin inhibitors in yeast cells.

Proposed medications for taxane-induced myalgia and arthralgia (Review).

Taxanes inhibit the disassembly of microtubules, which are involved in mitosis and axoplasmatic transport, and may cause the degeneration of peripheral, mainly small, sensory nerves. Peripheral neurotoxicity is a dose-limiting side-effect of taxanes. Neuroprotective agents may aid in the reduction of neurotoxicity, thus allowing the intensification of cytostatic therapy in patients. An increasing number of medications for the prevention of taxane-induced arthralgia and myalgia are becoming available to oncology teams. The most widely studied medications include so-called analgesics such as Shakuyaku-kanzo-to (a herbal medicine), corticosteroids and antihistamines. Arthralgias and myalgias (muscle spasms, fasciculations and prolonged contractions) may be extremely distressing for patients. New anti-epileptic drugs, particularly gabapentin and pregabalin, have proven to be safe and effective in the treatment of taxane-induced neurotoxicity. The aim of this review was to examine the topical choices available for the protective management of taxane-induced neurotoxicity monitored in preliminary case studies and clinical trials. At present, there is no standard of care for the prevention of taxane-induced arthralgia and myalgia. In combination with taxane-based chemotherapeutic regimens, these medical agents may be crucial in the treatment of a variety of types of cancer.

Hyperpolarization-activated currents in gonadotropin-releasing hormone (GnRH) neurons contribute to intrinsic excitability and are regulated by gonadal steroid feedback.

Pulsatile release of gonadotropin-releasing hormone (GnRH) is required for fertility and is regulated by steroid feedback. Hyperpolarization-activated currents (I(h)) play a critical role in many rhythmic neurons. We examined the contribution of I(h) to the membrane and firing properties of GnRH neurons and the modulation of this current by steroid milieu. Whole-cell voltage- and current-clamp recordings were made of GFP-identified GnRH neurons in brain slices from male mice that were gonad-intact, castrated, or castrated and treated with estradiol implants. APV, CNQX, and bicuculline were included to block fast synaptic transmission. GnRH neurons (47%) expressed a hyperpolarization-activated current with pharmacological and biophysical characteristics of I(h). The I(h)-specific blocker ZD7288 reduced hyperpolarization-induced sag and rebound potential, decreased GnRH neuron excitability and action potential firing, and hyperpolarized membrane potential in some cells. ZD7288 also altered the pattern of burst firing and reduced the slope of recovery from the after-hyperpolarization potential. Activation of I(h) by hyperpolarization increased spike frequency, whereas inactivation of I(h) by depolarization reduced spike frequency. Castration increased I(h) compared with that in gonad-intact males. This effect was reversed by in vivo estradiol replacement. Together, these data indicate I(h) provides an excitatory drive in GnRH neurons that contributes to action potential burst firing and that estradiol regulates I(h) in these cells. As estradiol is the primary central negative feedback hormone on GnRH neuron firing in males, this provides insight into the mechanisms by which steroid hormones potentially alter the intrinsic properties of GnRH neurons to change their activity.

Insufficiency of copper ion homeostasis causes freeze-thaw injury of yeast cells as revealed by indirect gene expression analysis.

Saccharomyces cerevisiae is exposed to freeze-thaw stress in commercial processes, including frozen dough baking. Cell viability and fermentation activity after a freeze-thaw cycle were dramatically decreased due to freeze-thaw injury. Because this type of injury involves complex phenomena, the injury mechanisms are not fully understood. We examined freeze-thaw injury by indirect gene expression analysis during postthaw incubation after freeze-thaw treatment using DNA microarray profiling. The results showed that genes involved in the homeostasis of metal ions were frequently contained in genes that were upregulated, depending on the freezing period. We assessed the phenotype of deletion mutants of the metal ion homeostasis genes that exhibited freezing period-dependent upregulation and found that the strains with deletion of the MAC1 and CTR1 genes involved in copper ion homeostasis exhibited freeze-thaw sensitivity, suggesting that copper ion homeostasis is required for freeze-thaw tolerance. We found that supplementation with copper ions during postthaw incubation increased intracellular superoxide dismutase activity and intracellular levels of reactive oxygen species were decreased. Moreover, cell viability was increased by supplementation with copper ions. These results suggest that insufficiency of copper ion homeostasis may be one of the causes of freeze-thaw injury.

Control of interstitial pneumonia by drip infusion of megadose vitamin C, dehydroepiandrosterone and cortisol. A short review of our experience.

Interstitial pneumonia can be controlled by the combined use of a prophylactic antibiotic system and the drip infusion system including megadose vitamin C, dehydroepiandrosterone (D) and cortisol (F), a fortified substitute of 3 adrenocortical elements. The response of patients was satisfying with few side-effects of F. It was shown that an excess of vitamin C improved the therapeutic efficacy of D-F complex, and that D and F improved the immunodeficient state of the host. The benefit of D as an adrenal androgen in immunology found another example in the combined use of cyclosporine A (CS) and glucocorticoid (G) in the kidney transplantation clinic: CS and G helps improve graft take by creating a state of androgen excess, as testified in both humans and mice--an alleviation of immune conflict.

Functional analysis of genes encoding putative oxidoreductases in Aspergillus oryzae, which are similar to fungal fructosyl-amino acid oxidase.

We found 11 genes (FAO1-11) encoding putative oxidoreductases in the Aspergillus oryzae genome, which are similar to fungal fructosyl-amino acid oxidases. The cDNAs corresponding to the genes were cloned and expressed in Escherichia coli. rFao2 had fructosyl-amino acid oxidase activity, whereas rFao1 did not show any enzyme activity, even though the deduced amino acid sequence of Fao1 is identical to that of one of the fructosyl-amino acid oxidase isozymes from Aspergillus oryzae. rFao7 and rFao8 showed oxidase activity toward sarcosine, L-pipecolate, and L-proline. rFao10 was active toward only sarcosine, of the substrates tested. The functions of the other proteins were also predicted from a phylogenetic analysis.

An extremely oligotrophic bacterium, Rhodococcus erythropolis N9T-4, isolated from crude oil.

Rhodococcus erythropolis N9T-4, which was isolated from crude oil, showed extremely oligotrophic growth and formed its colonies on a minimal salt medium solidified using agar or silica gel without any additional carbon source. N9T-4 did not grow under CO(2)-limiting conditions but could grow on a medium containing NaHCO(3) under the same conditions, suggesting that the oligotrophic growth of N9T-4 depends on CO(2). Proteomic analysis of N9T-4 revealed that two proteins, with molecular masses of 45 and 55 kDa, were highly induced under the oligotrophic conditions. The primary structures of these proteins exhibited striking similarities to those of methanol: N,N'-dimethyl-4-nitrosoaniline oxidoreductase and an aldehyde dehydrogenase from Rhodococcus sp. These enzyme activities were three times higher under oligotrophic conditions than under n-tetradecane-containing heterotrophic conditions, and gene disruption for the aldehyde dehydrogenase caused a lack of growth on the minimal salt medium. Furthermore, 3-hexulose 6-phosphate synthase and phospho-3-hexuloisomerase activities, which are key enzymes in the ribulose monophosphate pathway in methylotrophic bacteria, were detected specifically in the cell extract of oligotrophically grown N9T-4. These results suggest that CO(2) fixation involves methanol (formaldehyde) metabolism in the oligotrophic growth of R. erythropolis N9T-4.

Long-term effect of 5-fluorouracil enhanced by intermittent administration of polysaccharide K after curative resection of colon cancer. A randomized controlled trial for 7-year follow-up.

BACKGROUND AND AIMS: The efficacy of a biological response modifier polysaccharide K in adjuvant immunochemotherapy was evaluated in primary colon cancer patients with macroscopic Dukes' C after curative resection. PATIENTS AND METHODS: Employing the minimization method using three factors (lymph node metastases, preoperative serum CEA level, and institution), 446 patients were allocated into groups P and C. Group P received immunochemotherapy, oral PSK (3 g per day) followed by oral 5-FU (200 mg/body per day), while group C received only intermittent chemotherapy, oral 5-FU (200 mg per day) followed by 4-week rest. Both groups received ten courses. RESULTS: Survival for cancer death was significantly higher in group P than in group C, but there was no difference in 7-year disease-free survival or overall survival had. CONCLUSION: Repeated alternating administration with PSK followed by 5-FU can improve survival for cancer death.