Expression of PVT-1 and miR-29a/29b as reliable biomarkers for liver cirrhosis and their correlation with the inflammatory biomarkers profile.

BACKGROUND & AIMS: The liver is a vital organ responsible for numerous metabolic processes, which can be significantly impacted by long non-coding RNAs (lncRNAs) and microRNAs (miRNAs). These ribonucleic acid (RNA) molecules have been shown to play a crucial role in regulating gene expression, and their dysregulation has been implicated in numerous liver disorders. Our study aimed to investigate the diagnostic accuracy of plasmacytoma variant translocation-1 (PVT-1), microRNA-29a/29b (miR-29a/miR-29b), and inflammatory biomarkers [ interleukine-6 (IL-6), tumor necrosis factor-alpha (TNF-α), transforming growth factor-beta (TGF-ß), and insulin growth factor-1 (IGF-1)] as diagnostic and prognostic biomarkers for liver cirrhosis. Therefore, understanding the mechanisms by which lncRNAs and miRNAs influence liver metabolism is of paramount importance in developing effective treatments for liver-related diseases. METHODS: Serum samples were collected from 164 participants, comprising 114 cirrhotic patients with varying grades (35 grade I, 35 grade II, and 44 grade III) and 50 healthy controls. PVT-1 and miR-29a/miR-29b expression was analyzed by reverse transcription-quantitative polymerase chain reaction (RT-PCR), while the serum levels of inflammatory biomarkers were assessed using enzyme-linked immunosorbent assay (ELISA). RESULTS: The study participants exhibited notable differences in PVT-1 and miR-29a/miR-29b expression. ROC analysis revealed excellent discriminative power for PVT-1 and miR-29a/miR-29b in distinguishing cirrhotic patients from healthy controls. CONCLUSION: This study demonstrates the promising potential of PVT-1 and miR-29a/miR-29b as early diagnostic biomarkers for liver cirrhosis detection, requiring further validation in larger cohorts. Our findings also reinforce the diagnostic value of circulating inflammatory biomarkers (IL-6, TNF-α, TGF-ß, and IGF-1) levels for liver cirrhosis screening.

Tumor necrosis factor alpha stimulates osteoclast differentiation by a mechanism independent of the ODF/RANKL-RANK interaction.

Osteoclast differentiation factor (ODF, also called RANKL/TRANCE/OPGL) stimulates the differentiation of osteoclast progenitors of the monocyte/macrophage lineage into osteoclasts in the presence of macrophage colony-stimulating factor (M-CSF, also called CSF-1). When mouse bone marrow cells were cultured with M-CSF, M-CSF-dependent bone marrow macrophages (M-BMM phi) appeared within 3 d. Tartrate-resistant acid phosphatase-positive osteoclasts were also formed when M-BMM phi were further cultured for 3 d with mouse tumor necrosis factor alpha (TNF-alpha) in the presence of M-CSF. Osteoclast formation induced by TNF-alpha was inhibited by the addition of respective antibodies against TNF receptor 1 (TNFR1) or TNFR2, but not by osteoclastogenesis inhibitory factor (OCIF, also called OPG, a decoy receptor of ODF/RANKL), nor the Fab fragment of anti-RANK (ODF/RANKL receptor) antibody. Experiments using M-BMM phi prepared from TNFR1- or TNFR2-deficient mice showed that both TNFR1- and TNFR2-induced signals were important for osteoclast formation induced by TNF-alpha. Osteoclasts induced by TNF-alpha formed resorption pits on dentine slices only in the presence of IL-1alpha. These results demonstrate that TNF-alpha stimulates osteoclast differentiation in the presence of M-CSF through a mechanism independent of the ODF/RANKL-RANK system. TNF-alpha together with IL-1alpha may play an important role in bone resorption of inflammatory bone diseases.

Parallel enrichment of polyphenols and phytosterols from Pinot noir grape seeds with molecularly imprinted polymers and analysis by capillary high-performance liquid chromatography electrospray ionisation tandem mass spectrometry.

This investigation describes an integrated workflow for the parallel extraction and recovery of polyphenols and phytosterols from Pinot noir grape seeds. Using (E)-resveratrol and stigmasterol as exemplars, the approach employs two different molecular imprinted polymers in tandem for the extraction of these compounds and their subsequent analysis by capillary high-performance liquid chromatography (capHPLC) interfaced with electrospray ionisation tandem mass spectrometry (ESI MS/MS). Information on the selectivity of the solid-phase extraction processes was obtained through analysis of the binding behaviour of (E)-resveratrol- and stigmasterol-imprinted polymers using structurally similar polyphenols or phytosterols with the extent of binding determined from the capHPLC-ion trap ESI MS/MS data. This study documents with Pinot noir grape seed extracts and optimised solid-phase extraction protocols that the (E)-resveratrol-templated MIP enabled a very high recovery (99%) of the health-beneficial polyphenol (E)-resveratrol with co-purification of procyanidin and catechin/epicatechin. Further, the stigmasteryl-3-O-methacrylate-templated polymer resulted in high recovery (96%) of the phytosterol stigmasterol with co-purification of campesteryl glycoside. The results also demonstrate that rapid and high-resolution capHPLC-ESI MS/MS methods can be used as part of the work flow for selectivity optimisation and monitoring of the performance of MIPs intended for use in the solid-phase extraction of bioactive molecules with nutraceutical properties from agricultural waste streams.

Recovery of ergosterol from the medicinal mushroom, Ganoderma tsugae var. Janniae, with a molecularly imprinted polymer derived from a cleavable monomer-template composite.

A semi-covalent imprinting strategy has been developed for the synthesis of molecularly-imprinted polymers specific for the fungal sterol, ergosterol, a biological precursor of vitamin D2. This imprinting approach involved a novel post-synthesis cleavable monomer-template composite, namely ergosteryl methacrylate, and resulted in the formation of an imprinted polymer that selectively and efficiently recognized ergosterol through non-covalent interactions. The derived molecularly-imprinted polymer and the corresponding non-imprinted polymer were systematically evaluated for their selectivity towards ergosterol via static and dynamic binding studies using various ergosteryl esters (e.g. ergosteryl-cinnamate, -ferulate, -coumarate, -ferulate acetate and -acetate, respectively) as competitors. Moreover, the binding capacity of the molecularly imprinted polymer for ergosterol was enhanced when the sample loading conditions involved the use of partially aqueous solvent mixtures, such as acetonitrile/water (9:1 (v/v) or 8:2 (v/v)). These attributes were exploited in a solid-phase extraction format, whereby ergosterol was obtained with excellent recoveries from an extract of the fruiting body powder of the medicinal fungus Ganoderma tsugae var. Janniae.

Selectivity mapping of the binding sites of (E)-resveratrol imprinted polymers using structurally diverse polyphenolic compounds present in Pinot noir grape skins.

This investigation describes a general procedure for the selectivity mapping of molecularly imprinted polymers, using (E)-resveratrol-imprinted polymers as the exemplar, and polyphenolic compounds present in Pinot noir grape skin extracts as the test compounds. The procedure is based on the analysis of samples generated before and after solid-phase extraction of (E)-resveratrol and other polyphenols contained within the Pinot noir grape skins using (E)-resveratrol-imprinted polymers. Capillary reversed-phase high-performance liquid chromatography (RP-HPLC) and electrospray ionisation tandem mass spectrometry (ESI MS/MS) was then employed for compound analysis and identification. Under optimised solid-phase extraction conditions, the (E)-resveratrol-imprinted polymer showed high binding affinity and selectivity towards (E)-resveratrol, whilst no resveratrol was bound by the corresponding non-imprinted polymer. In addition, quercetin-3-O-glucuronide and a dimer of catechin-methyl-5-furfuraldehyde, which share some structural features with (E)-resveratrol, were also bound by the (E)-resveratrol-imprinted polymer. Polyphenols that were non-specifically retained by both the imprinted and non-imprinted polymer were (+)-catechin, a B-type procyanidin and (-)-epicatechin. The compounds that did not bind to the (E)-resveratrol molecularly imprinted polymer had at least one of the following molecular characteristics in comparison to the (E)-resveratrol template: (i) different spatial arrangements of their phenolic hydroxyl groups, (ii) less than three or more than four phenolic hydroxyl groups, or (iii) contained a bulky substituent moiety. The results show that capillary RP-HPLC in conjunction with ESI MS/MS represent very useful techniques for mapping the selectivity of the binding sites of imprinted polymer. Moreover, this procedure permits performance monitoring of the characteristics of molecularly imprinted polymers intended for solid-phase extraction of bioactive and nutraceutical molecules from diverse agricultural waste sources.

Role of Ca2+ in t-PA production by human embryonic lung diploid fibroblast, IMR-90 cells, stimulated by proteose peptone.

Proteose peptone (p.peptone) remarkably induced tissue plasminogen activator (t-PA) activity in the conditioned medium of confluently cultured human embryonic lung diploid fibroblast, IMR-90 cells, in a dose-dependent manner. t-PA activity correlated well with the amount of t-PA antigen found in the conditioned medium of IMR-90 cells stimulated by p.peptone. t-PA production by IMR-90 cells stimulated by p.peptone was dependent on extracellular Ca2+ concentration and maximum t-PA production required approximately 3.6 mM extracellular Ca2+. Conversely, elimination of Ca2+ from the culture medium by EGTA, Ca2+ chelate agent, strongly inhibited t-PA production induced by p.peptone. t-PA production induced by p.peptone was inhibited in a dose-dependent manner by Verapamil, which inhibits Ca2+ uptake through the slow channels and also by W-7, an inhibitor of calmodulin. These results suggested that influx of extracellular Ca2+ into IMR-90 cells was caused by p.peptone and induced t-PA production by the cells.

Transforming growth factor beta affects osteoclast differentiation via direct and indirect actions.

Transforming growth factor beta (TGF-beta) is abundant in bone and has complex effects on osteolysis, with both positive and negative effects on osteoclast differentiation, suggesting that it acts via more than one mechanism. Osteoclastogenesis is determined primarily by osteoblast (OB) expression of the tumor necrosis factor (TNF)-related molecule receptor activator of NF-kappaB ligand (RANKL) and its decoy receptor osteoprotegerin (OPG), which are increased and decreased, respectively, by osteolytic factors. A RANKL-independent osteoclastogenic mechanism mediated by TNF-alpha has also been shown. Therefore, we investigated TGF-beta effects on osteoclast formation in culture systems in which osteoclastogenic stimulus is dependent on OBs and culture systems where it was provided by exogenously added RANKL or TNF-alpha. Both OPG and TGF-beta inhibited osteoclast formation in hemopoietic cell/OB cocultures, but the kinetics of their action differed. TGF-beta also inhibited osteoclastogenesis in cocultures of cells derived from OPG null (opg-/-) mice. TGF-beta strongly decreased RANKL messenger RNA (mRNA) expression in cultured osteoblasts, and addition of exogenous RANKL to TGFbeta-inhibited cocultures of opg-/- cells partially restored osteoclastogenesis. Combined, these data indicate that the inhibitory actions of TGF-beta were mediated mainly by decreased OB production of RANKL. In contrast, in the absence of OBs, TGF-beta greatly increased osteoclast formation in recombinant RANKL- or TNF-alpha-stimulated cultures of hemopoietic cells or RAW 264.7 macrophage-like cells to levels several-fold greater than attainable by maximal stimulation by RANKL or TNF-alpha. These data suggest that TGF-beta may increase osteoclast formation via action on osteoclast precursors. Therefore, although RANKL (or TNF-alpha) is essential for osteoclast formation, factors such as TGF-beta may powerfully modify these osteoclastogenic stimuli. Such actions may be critical to the control of physiological and pathophysiological osteolysis.

Identity of osteoclastogenesis inhibitory factor (OCIF) and osteoprotegerin (OPG): a mechanism by which OPG/OCIF inhibits osteoclastogenesis in vitro.

The morphogenesis and remodeling of bone depends on the integrated activity of osteoblasts that form bone and osteoclasts that resorb bone. We previously reported the isolation of a new cytokine termed osteoclastogenesis inhibitory factor, OCIF, which specifically inhibits osteoclast development. Here we report the cloning of a complementary DNA of human OCIF. OCIF is identical to osteoprotegerin (OPG), a soluble member of the tumor-necrosis factor receptor family that inhibits osteoclastogenesis. Recombinant human OPG/OCIF specifically acts on bone tissues and increases bone mineral density and bone volume associated with a decrease of active osteoclast number in normal rats. Osteoblasts or bone marrow-derived stromal cells support osteoclastogenesis through cell-to-cell interactions. A single class of high affinity binding sites for OPG/OCIF appears on a mouse stromal cell line, ST2, in response to 1,25-dihydroxyvitamin D3. An anti-OPG/OCIF antibody that blocks the binding abolishes the biological activity of OPG/OCIF. When the sites are blocked with OPG/OCIF, ST2 cells fail to support osteoclastogenesis. These results suggest that the sites are involved in cell-to-cell signaling between stromal cells and osteoclast progenitors and that OPG/OCIF inhibits osteoclastogenesis by interrupting the signaling through the sites.

Cloning and characterization of the gene encoding mouse osteoclast differentiation factor.

Osteoclast differentiation factor (ODF), a ligand for osteoclastogenesis inhibitory factor (OCIF)/ osteoprotegerin (OPG), is a member of the membrane-associated tumor necrosis factor (TNF) family and induces osteoclast-like cell formation in vitro. In the present study, mouse ODF genomic clones were isolated and sequenced to determine their gene structure. The mouse ODF gene is a single copy gene consisting of five exons and spans approximately 40kb of the mouse genome. The first exon encodes the intracellular and transmembrane domains. The extracellular region of ODF containing the TNF homologous domain is encoded by exons 1 through 5. The translation-termination codon and six polyadenylation signal residues are present in exon 5. A major transcription-initiation site is present 143 nucleotides upstream of the initiation-ATG codon. This genomic organization is similar to that of other members of the TNF family, especially the CD40 ligand.

A novel molecular mechanism modulating osteoclast differentiation and function.

Osteoclasts, the multinucleated giant cells that resorb bone, develop from hematopoietic cells of the monocyte/ macrophage lineage. Osteoblasts, as well as bone marrow stromal cells, support osteoclast development through a mechanism of cell-to-cell interaction with osteoclast progenitors. We recently purified and molecularly cloned osteoclastogenesis inhibitory factor (OCIF), which was identical to osteoprotegerin (OPG). OPG/OCIF, a secreted member of the tumor necrosis factor (TNF) receptor family, inhibited differentiation and activation of osteoclasts. A single class of high-affinity binding sites for OPG/OCIF appeared on a mouse bone marrow stromal cell line, ST2, in response to 1alpha,25-dihydroxyvitamin D3 [1,25(OH)2D3] and dexamethasone (Dex). When the binding sites were occupied by OPG/OCIF, ST2 cells failed to support the osteoclast formation from spleen cells. To identify an OPG/OCIF ligand, we screened a cDNA expression library of ST2 cells treated with 1,25(OH)2D3 and Dex using OPG/OCIF as a probe. The cloned molecule was found to be a member of the membrane-associated TNF ligand family, and it induced osteoclast formation from mouse and human osteoclast progenitors in the presence of macrophage colony-stimulating factor (M-CSF) in vitro. Expression of its gene in osteoblasts/stromal cells was up-regulated by osteotropic factors, such as 1,25(OH)2D3, prostaglandin E2 (P(GE2), parathyroid hormone (PTH), and interleukin (IL)-11. A polyclonal antibody against this protein, as well as OPG/OCIF, negated not only the osteoclastogenesis induced by the protein, but also bone resorption elicited by various osteotropic factors in a fetal mouse long bone culture system. These findings led us to conclude that the protein is osteoclast differentiation factor (ODF), a long sought-after ligand that mediates an essential signal to osteoclast progenitors for their differentiation into active osteoclasts. Recent analyses of ODF receptor demonstrated that RANK, a member of the TNF receptor family, is the signaling receptor for ODF in osteoclastogenesis, and that OPG/OCIF acts as a decoy receptor for ODF to compete against RANK. The discovery of ODF, OPG/OCIF, and RANK opens a new era in the investigation of the regulation of osteoclast differentiation and function.

A radiotherapeutic experience for localized extranodal non-Hodgkin's lymphoma: prognostic factors and re-evaluation of treatment modality.

Between 1966 and 1988, 149 patients were treated with radiotherapy for localized extranodal lymphoma. The average total dose given was 39.8 Gy for low grade disease and 48.7 Gy for all other disease. Of the 149 patients, 60 also received adjuvant chemotherapy. Twenty-four had low grade lymphoma, 109 had intermediate grade disease, and 16 had high grade disease, histologically. The distribution of histological grade and T/B phenotype varied with the primary site. Low grade lymphomas were found mainly in the orbit, and T-cell lymphomas were found in the nasal cavity and nasopharynx. The 5-year survival rates according to tumor location were 89% for oral cavity, 86% for paranasal sinus, 83% for thyroid, 69% for orbit, 47% for Waldeyer's ring (WAR), 44% for testis, 23% for CNS, 21% for nasal cavity and 60% for other sites. Histological grade and T/B phenotype both had prognostic importance. Combined chemotherapy significantly improved the survival rate only for disease with intermediate or high grade histology. Other prognostic factors according to the primary site were the bulk of lymph node for WAR disease, the radiation dose for CNS disease, bone erosion for orbital disease, stridor for disease of the thyroid, and the tumor stage for disease of both the testis and the thyroid.

Tumor cytotoxic activity of HGF-SF.

IMR-90 human embryonic lung fibroblasts secrete a tumor cytotoxic factor. This factor, termed F-TCF, is moderately cytotoxic in human tumor cell lines (KB, MCF-7, BG-1) and is very cytotoxic in mouse tumor cell lines (Sarcoma 180, Meth A sarcoma, P388). The cytotoxicity depends on the initial target cell number and is due to cytostasis rather than cytolysis. F-TCF was purified from conditioned medium by a combination of UF-concentration, CM sephadex C-50, Con A sepharose, Mono S cation-exchange and heparin sepharose chromatography and exhibited a molecular mass (M(r)) of 76 to 80 kD on SDS-PAGE under non-reducing conditions. F-TCF is a heterodimer composed of a large alpha-subunit with M(r) 52 to 56 kD and a small beta-subunit with M(r) 30 to 34 kD. F-TCF is a heparin-binding, heat-labile, basic glycoprotein (pI 7.4-8.6). Its activity is stable over the pH range of 6.0 to 9.0, but is completely lost after reduction with 2-mercaptoethanol. Protein sequencing indicates that the alpha-subunit is blocked at the aminoterminus. The primary amino acid sequences deduced from hepatocyte growth factor (HGF) cDNAs cloned from human placenta and liver cDNA libraries indicate that F-TCF is identical to the placenta type HGF in the aminoterminal sequence of the beta-subunit, but differs at two sites from the liver type HGF. Two forms of F-TCF cDNA were found in an IMR-90 human fibroblast cDNA library. One form was identical to placenta type HGF cDNA and the other was a variant with a 15 base pair deletion in the coding region. In addition, mRNA corresponding to the deleted form of cDNA was present in total RNA prepared from IMR-90 cells. F-TCF was thus identified as placenta type HGFs including a variant. The deleted form of recombinant HGF (rHGF) expressed in CHO cells had slightly lower heparin-binding affinity than did the intact form. Both rHGFs had almost the same dose-response curves for cytotoxicity in Sarcoma 180 or Meth A sarcoma cells. Moreover, rHGF (the deleted form) was cytotoxic in hepatocellular carcinoma cells (HepG2, Hep3B, H35). Dose-response curves for the stimulation of DNA synthesis in rat hepatocytes by HGFs were very similar up to about 12.5 ng/ml, but differed significantly at higher concentrations. The deleted form gave maximal activity in a dose range of 12.5 to 100 ng/ml and had about 1.4- to 1.9-fold higher specific activity in that range than the intact form did.(ABSTRACT TRUNCATED AT 400 WORDS)

A randomized outcome evaluation of group exercise programs in long-term care institutions.

BACKGROUND: Physical activity programs in nursing homes typically consist of seated, range of motion (ROM) exercises, regardless of resident abilities. The Functional Fitness for Long-Term Care (FFLTC) Program was designed not only to maintain ROM, but also to improve strength, balance, flexibility, mobility, and function. In addition, it was tailored to meet the needs of both high and low mobility residents. METHODS: The feasibility and efficacy of the FFLTC Program were evaluated with 68 residents (mean age 80) from five institutions. Persons were classified as low or high mobility and randomized into either the FFLTC program or a seated ROM program. Classes were conducted in groups of 4 to 10 residents by trained facility staff for 45 minutes, three times per week. Assessments at baseline and 4 months consisted of mobility, balance, gait, flexibility, functional capacity, and several upper and lower extremity strength measures. RESULTS: Attendance averaged 86% for the FFLTC and 79% for the ROM classes. Four months of exercise led to significant improvements in mobility (16%), balance (9%), flexibility (36%), knee (55%), and hip (12%) strength for the FFLTC group. Shoulder strength was the only improvement found for the ROM group. The ROM group significantly deteriorated in some areas, particularly hip strength, mobility, and functional ability. CONCLUSIONS: Institutionalized seniors, even those who are physically frail, incontinent and/or have mild dementia, can respond positively to a challenging exercise program. The FFLTC program demonstrated clear benefits over typical, seated ROM exercises. Moreover, with minimal training, the program can be safely delivered at low cost by institutional staff and volunteers.

Ventilatory chemosensitive adaptations to intermittent hypoxic exposure with endurance training and detraining.

The present study was performed to clarify the effects of intermittent exposure to an altitude of 4,500 m with endurance training and detraining on ventilatory chemosensitivity. Seven subjects (sea-level group) trained at sea level at 70% maximal oxygen uptake (VO2 max) for 30 min/day, 5 days/wk for 2 wk, whereas the other seven subjects (altitude group) trained at the same relative intensity (70% altitude VO2 max) in a hypobaric chamber. VO2 max, hypoxic ventilatory response (HVR), and hypercapnic ventilatory response, as an index of central hypercapnic chemosensitivity (HCVR) and as an index of peripheral chemosensitivity (HCVRSB), were measured. In both groups VO2 max increased significantly after training, and a significant loss of VO2 max occurred during 2 wk of detraining. HVR tended to increase in the altitude group but not significantly, whereas it decreased significantly in the sea-level group after training. HCVR and HCVRSB did not change in each group. After detraining, HVR returned to the pretraining level in both groups. These results suggest that ventilatory chemosensitivity to hypoxia is more variable by endurance training and detraining than that to hypercapnia.

Cardiovascular response to hypoxia after endurance training at altitude and sea level and after detraining.

The purpose of this study was to elucidate 1) the effects of endurance exercise training during hypoxia or normoxia and of detraining on ventilatory and cardiovascular responses to progressive isocapnic hypoxia and 2) whether the change in the cardiovascular response to hypoxia is correlated to changes in the hypoxic ventilatory response (HVR) after training and detraining. Seven men (altitude group) performed endurance training using a cycle ergometer in a hypobaric chamber of simulated 4,500 m, whereas the other seven men (sea-level group) trained at sea level (K. Katayama, Y. Sato, Y. Morotome, N. Shima, K. Ishida, S. Mori, and M. Miyamura. J. Appl. Physiol. 86: 1805-1811, 1999). The HVR, systolic and diastolic blood pressure responses (DeltaSBP/DeltaSa(O(2)), DeltaDBP/DeltaSa(O(2))), and heart rate response (DeltaHR/DeltaSa(O(2)); Sa(O(2)) is arterial oxygen saturation) to progressive isocapnic hypoxia were measured before and after training and during detraining. DeltaSBP/DeltaSa(O(2)) increased significantly in the altitude group and decreased significantly in the sea-level group after training. The changed DeltaSBP/DeltaSa(O(2)) in both groups was restored during 2 wk of detraining, as were the changes in HVR, whereas there were no changes in the DeltaDBP/DeltaSa(O(2)) and DeltaHR/DeltaSa(O(2)) throughout the experimental period. The changes in DeltaSBP/DeltaSa(O(2)) after training and detraining were significantly correlated with those in HVR. These results suggest that DeltaSBP/DeltaSa(O(2)) to progressive isocapnic hypoxia is variable after endurance training during hypoxia and normoxia and after detraining, as is HVR, but DeltaDBP/DeltaSa(O(2)) and DeltaHR/DeltaSa(O(2)) are not. It also suggests that there is an interaction between the changes in DeltaSBP/DeltaSa(O(2)) and HVR after endurance training or detraining.

Intermittent hypoxia increases ventilation and Sa(O2) during hypoxic exercise and hypoxic chemosensitivity.

The purpose of this study was 1) to test the hypothesis that ventilation and arterial oxygen saturation (Sa(O2)) during acute hypoxia may increase during intermittent hypoxia and remain elevated for a week without hypoxic exposure and 2) to clarify whether the changes in ventilation and Sa(O2) during hypoxic exercise are correlated with the change in hypoxic chemosensitivity. Six subjects were exposed to a simulated altitude of 4,500 m altitude for 7 days (1 h/day). Oxygen uptake (VO2), expired minute ventilation (VE), and Sa(O2) were measured during maximal and submaximal exercise at 432 Torr before (Pre), after intermittent hypoxia (Post), and again after a week at sea level (De). Hypoxic ventilatory response (HVR) was also determined. At both Post and De, significant increases from Pre were found in HVR at rest and in ventilatory equivalent for O2 (VE/VO2) and Sa(O2) during submaximal exercise. There were significant correlations among the changes in HVR at rest and in VE/VO2 and Sa(O2) during hypoxic exercise during intermittent hypoxia. We conclude that 1 wk of daily exposure to 1 h of hypoxia significantly improved oxygenation in exercise during subsequent acute hypoxic exposures up to 1 wk after the conditioning, presumably caused by the enhanced hypoxic ventilatory chemosensitivity.

Synergistic effects of N-ethyl-N-nitrosourea (an alkylating agent with a low Swain-Scott substrate constant) and X-rays in the stamen hairs of Tradescantia clone BNL 4430.

The mutagenic interaction between N-ethyl-N-nitrosourea (ENU) and X-rays was tested in the stamen hairs of Tradescantia clone BNL 4430, a blue/pink heterozygote. ENU, a monofunctional alkylating agent with a low Swain-Scott substrate constant (s) of 0.26, exhibited a strong cytotoxicity. ENU-induced somatic pink mutation frequency per 10(4) hair-cell divisions increased with increasing ENU dose, with a slope of 1.243 on a log-log graph, the slope value being similar to that for X-ray-induced mutation frequency. Three out of five combined treatments with ENU and X-rays produced mutation frequencies significantly higher than those expected from the additive effects of the two mutagens. Clear synergistic effects were detected when relatively higher X-ray doses were applied, resembling those confirmed earlier between methyl methanesulfonate (MMS) and X-rays, although the s value for ENU is very much smaller than that (0.88) for MMS. It is therefore concluded that mutagenic interactions between alkylating agents and X-rays do not have any clear relationship with the s values.

The cII locus in the MutaMouse system.

Here, we report the first application and characterization of the cII locus as a mutational target for use with the Muta(trade mark)Mouse system for quantifying somatic mutations in vivo. This locus can be analyzed for mutations using positive selection and is identical in sequence to the cII in the Big Blue((R)) Mouse. The cII displays similar spontaneous (5.5 x 10(-5)) and induced mutation frequencies when compared to the lacZ gene in the small intestine of MutaMice treated with ENU (N-ethyl-N-nitrosourea). After acute treatment with 250 mg/kg ENU (ip) the mutant frequencies were 127 x 10(-5) at the cII and 147 x 10(-5) at the lacZ loci, reaching a maximal mutant frequency 10 days posttreatment and remaining constant thereafter. These data prove that this transgene is genetically neutral, conferring neither selective advantage nor disadvantage on the host cells. The cII dose response curve was linear (R(2) = 0.93) comparable to the lacZ after treatments with 0, 50, 150, or 250 mg/kg ENU. Use of the cII locus (0.3 kb) addresses the single most significant drawback associated with the MutaMouse system, namely the inability to obtain sequence spectra efficiently, due to the large size of the lacZ gene (3.0 kb). Moreover, a less obvious application, but nevertheless of considerable importance, is the easy identification of jackpot mutations, without sequencing. The cII, identical in both sequence and origin on the transgenic constructs used in producing the Big Blue and MutaMouse systems, provides the first transgenic locus common to the two widely used in vivo mutagenesis assays.

Mutagenic synergism detected between dimethyl sulfate and X-rays but not found between N-methyl-N-nitrosourea and X-rays in the stamen hairs of Tradescantia clone BNL 4430.

Mutagenic interactions with X-rays of two monofunctional alkylating agents, dimethyl sulfate (DMS) and N-methyl-N-nitrosourea (MNU), were studied in the stamen hairs of Tradescantia clone BNL 4430, a blue/pink heterozygote. The young inflorescence-bearing shoots with roots cultivated in the nutrient solution circulating growth chamber were used as tester plants. Synergism between two different mutagens was judged to have occurred when the mutation frequency observed after applying the two mutagens concurrently was statistically significantly higher than the mutation frequency expected from the additive effects of the two mutagens. Clear synergistic effects in inducing somatic pink mutations were detected with all combinations of doses of DMS and X-rays examined, even in a relatively low X-ray dose range (down to 299 mGy), resembling those confirmed earlier between ethyl methanesulfonate (EMS) and X-rays, but somewhat differing from the synergisms observed earlier between methyl methanesulfonate (MMS) and X-rays. On the other hand, no mutagenic synergism was detected between MNU and X-rays, even in a relatively high X-ray dose range (up to 862 mGy). The presence or absence of mutagenic synergisms of these alkylating agents with X-rays could be related to the action mechanism of each alkylating agent.

Somatic mutation in the mammary gland: influence of time and estrus.

A critical factor in the quantitation of mutation induction in vivo is the time interval between treatment and sampling. In order to study mutagenesis in the mammary epithelium, the cell type in which breast cancer arises, we have measured the manifestation time, the minimum time required for the maximum mutant frequency to be achieved, in this tissue. The F1 LacZ transgenic mice (Muta MousexSWR) were treated with N-ethyl-N-nitrosourea (ENU) at 50 mg/kg for five consecutive days and then sampled at 1, 2, 4, 6, 9, or 12 weeks after the last treatment. The LacZ- mutant frequency reached a maximum at 4 weeks post-treatment and did not vary significantly thereafter. Dlb-1- mutations in the small intestine reached a maximum at 2 weeks after treatment and did not vary significantly thereafter. Since the stage of estrus cycle during carcinogen exposure influences the mammary tumor incidence and latency, it was expected that it would also affect mutation induction. To test this, F1 LacZ mice in the estrus or di-estrus stage were treated with an acute dose of 250 mg/kg ENU and sampled 10-13 weeks post-treatment. No statistical difference between the two groups was found, indicating that the effect of estrus on carcinogenesis is not due to variation in the sensitivity of the stage of the mammary gland to mutation.