Efficient Agrobacterium tumefaciens-mediated stable genetic transformation of green microalgae, Chlorella sorokiniana.

The green oleaginous microalgae, Chlorella sorokiniana, is a highly productive Chlorella species and a potential host for the production of biofuel, nutraceuticals, and recombinant therapeutic proteins. The lack of a stable and efficient genetic transformation system is the major bottleneck in improving this species. We report an efficient and stable Agrobacterium tumefaciens-mediated transformation system for the first time in C. sorokiniana. Cocultivation of C. sorokiniana cells (optical density at λ 680 = 1.0) with Agrobacterium at a cell density of OD600 = 0.6, on BG11 agar medium (pH 5.6) supplemented with 100 µM of acetosyringone, for three days at 25 ± 2 °C in the dark, resulted in significantly higher transformation efficiency (220 ± 5 hygromycin-resistant colonies per 106 cells). Transformed cells primarily selected on BG11 liquid medium with 30 mg/L hygromycin followed by selecting homogenous transformants on BG11 agar medium with 75 mg/L hygromycin. PCR analysis confirmed the presence of hptII, and the absence of virG amplification ruled out the Agrobacterium contamination in transformed microalgal cells. Southern hybridization confirmed the integration of the hptII gene into the genome of C. sorokiniana. The qRT-PCR and Western blot analyses confirmed hptII and GUS gene expression in the transgenic cell lines. The specific growth rate, biomass doubling time, PSII activity, and fatty-acid profile of transformed cells were found similar to wild-type untransformed cells, clearly indicating the growth and basic metabolic processes not compromised by transgene expression. This protocol can facilitate opportunities for future production of biofuel, carotenoids, nutraceuticals, and therapeutic proteins. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02750-7.

Activation of Nesfatin-1-Containing Neurones in the Hypothalamus and Brainstem by Peripheral Administration of Anorectic Hormones and Suppression of Feeding via Central Nesfatin-1 in Rats.

Peripheral anorectic hormones, such as glucagon-like peptide (GLP)-1, cholecystokinin (CCK)-8 and leptin, suppress food intake. The newly-identified anorectic neuropeptide, nesfatin-1, is synthesised in both peripheral tissues and the central nervous system, particularly by various nuclei in the hypothalamus and brainstem. In the present study, we examined the effects of i.p. administration of GLP-1 and CCK-8 and co-administrations of GLP-1 and leptin at subthreshold doses as confirmed by measurement of food intake, on nesfatin-1-immunoreactive (-IR) neurones in the hypothalamus and brainstem of rats by Fos immunohistochemistry. Intraperitoneal administration of GLP-1 (100 µg/kg) caused significant increases in the number of nesfatin-1-IR neurones expressing Fos-immunoreactivity in the supraoptic nucleus (SON), the area postrema (AP) and the nucleus tractus solitarii (NTS) but not in the paraventricular nucleus (PVN), the arcuate nucleus (ARC) or the lateral hypothalamic area (LHA). On the other hand, i.p. administration of CCK-8 (50 µg/kg) resulted in marked increases in the number of nesfatin-1-IR neurones expressing Fos-immunoreactivity in the SON, PVN, AP and NTS but not in the ARC or LHA. No differences in the percentage of nesfatin-1-IR neurones expressing Fos-immunoreactivity in the nuclei of the hypothalamus and brainstem were observed between rats treated with saline, GLP-1 (33 µg/kg) or leptin. However, co-administration of GLP-1 (33 µg/kg) and leptin resulted in significant increases in the number of nesfatin-1-IR neurones expressing Fos-immunoreactivity in the AP and the NTS. Furthermore, decreased food intake induced by GLP-1, CCK-8 and leptin was attenuated significantly by pretreatment with i.c.v. administration of antisense nesfatin-1. These results indicate that nesfatin-1-expressing neurones in the brainstem may play an important role in sensing peripheral levels of GLP-1 and leptin in addition to CCK-8, and also suppress food intake in rats.

In vitro anticancer activity of methyl caffeate isolated from Solanum torvum Swartz. fruit.

The present study was undertaken to investigate the anticancer activity of methyl caffeate isolated from Solanum torvum Swartz. fruit and to explore the molecular mechanisms of action in MCF-7 cells. Cytotoxic properties of hexane, ethyl acetate and methanol extracts were carried out against MCF-7 cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Ethyl acetate extract showed good cytototoxic activities compared to hexane and methanol extracts. Methyl caffeate was isolated from the ethyl acetate extract using column chromatography. Cytotoxic properties of methyl caffeate was investigated against MCF-7, A549, COLO320, HepG-2 and Vero cells. The compound showed potent cytotoxic properties against MCF-7 cells compared to A549, COLO320 and HepG-2 cells. Methyl caffeate significantly reduced cell proliferation and increased formation of fragmented DNA and apoptotic body in MCF-7 cells. Bcl-2, Bax, Bid, p53, caspase-3, PARP and cytochrome c release were detected by western blot analysis. The activities of caspases-3 and PARP gradually increased after the addition of isolated compound. Bcl-2 protein was down regulated; Bid and Bax were up regulated after the treatment with methyl caffeate. Molecular docking studies showed that the compound bound stably to the active sites of poly (ADP-ribose) polymerase-1 (PARP1), B cell CLL/lymphoma-2 (BCL-2), E3 ubiquitin-protein ligase (MDM2) and tubulin. The results strongly suggested that methyl caffeate induced apoptosis in MCF-7 cells via caspase activation through cytochrome c release from mitochondria.

Site-specific changes in cytokine response to septic dose of lipopolysaccharide in ovariectomized female rats.

PURPOSE: The immune response is altered according to hormonal and metabolic status. Obesity increases the inflammatory and fever response, whereas loss of gonadal steroid decreases behavioral response to immune stress. However, the immune systems of ovariectomized animals exhibiting obesity and gonadal steroid deficiency, particularly under septic conditions, have not been fully examined. In the present study, we evaluated the ovariectomy-induced changes of central and peripheral immune responses to life-threatening septic stimulus. METHODS AND RESULTS: Ovariectomized rats showed heavier body weight and lighter uterine weight when compared with gonadally intact rats. Fever response to septic dose of lipopolysaccharide (LPS) in ovariectomized rats was less evident when compared with that in gonadally intact rats. In addition, under LPS-injected septic conditions, hypothalamic gene levels of Interleukin-1ß (IL-1ß), Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) and serum protein levels of IL-1ß and TNF-α in ovariectomized rats were lower than those in gonadally intact rats. On the other hand, IL-6 levels in visceral fat under septic conditions were higher in ovariectomized rats than in gonadally intact rats. CONCLUSIONS: These findings indicate that ovariectomy-induced site-specific changes in cytokine response under septic conditions. As hypothalamic, but not peripheral, pro-inflammatory cytokines are directly involved in the fever response, the attenuation of fever response observed in ovariectomized rats may be caused by a reduction in central cytokine responses.

Tensile Ge microstructures for lasing fabricated by means of a silicon complementary metal-oxide-semiconductor process.

In this work we study, using experiments and theoretical modeling, the mechanical and optical properties of tensile strained Ge microstructures directly fabricated in a state-of-the art complementary metal-oxide-semiconductor fabrication line, using fully qualified materials and methods. We show that these microstructures can be used as active lasing materials in mm-long Fabry-Perot cavities, taking advantage of strain-enhanced direct band gap recombination. The results of our study can be realistically applied to the fabrication of a prototype platform for monolithic integration of near infrared laser sources for silicon photonics.

Efficacy of porcine placental extract on climacteric symptoms in peri- and postmenopausal women.

OBJECTIVES: Injections of human placental extract have long been used to treat menopausal symptoms. Recently, porcine placental extract (PPE), an oral supplement, has been developed for this purpose. The aim of this study was to assess whether PPE has an impact on climacteric symptoms in perimenopausal and postmenopausal women. METHODS: Seventy-six women with climacteric symptoms were enrolled into this open-label, randomized, controlled study. The control group (n = 38) underwent 24 weeks of open treatment with Toki-shakuyaku-san (TJ23), an oral herbal remedy used to alleviate climacteric symptoms. The PPE group (n = 38) received three capsules of PPE/day orally for the initial 12 weeks and six capsules/day for the next 12 weeks. Climacteric symptoms were evaluated in both groups using the Simplified Menopausal Index (SMI) score, Zung's Self-Rating Depression Scale (ZSDS) and the Spielberger State-Trait Anxiety Inventory (STAI) before commencing treatment, after 12 weeks of treatment and on completion of treatment. RESULTS: Treatment with PPE was significantly (p < 0.01) more effective in reducing the SMI, ZSDS and STAI measures at 12 and 24 weeks than TJ23 treatment alone. Treatment with PPE was also significantly effective (p < 0.01) in reducing the subscale scores of the SMI for items such as hot flushes, insomnia, irritability, depression, fatigue and joint pain. PPE treatment had no significant adverse effects. CONCLUSION: Oral PPE treatment is another possible option for treating perimenopausal and postmenopausal women with climacteric symptoms.

Efficacy of porcine placental extracts with hormone therapy for postmenopausal women with knee pain.

OBJECTIVES: Knee pain related to osteoarthritis increases with age and is more common in middle-aged women. Although hormone replacement therapy (HRT) improves knee pain, women unresponsive to HRT need an effective adjunctive therapy. The aim of this study was to assess whether oral porcine placental extracts (PPE) have an impact on patients with knee pain as an adjunctive therapy combined with HRT. METHODS: Forty-eight postmenopausal women with knee pain receiving HRT were enrolled into this open-label, randomized, controlled study. Subjects were randomized into Group 1 (n= 24) or Group 2 (n=24). Subjects in Group 1 were given 3 months open treatment with calcium (260 mg/day) as adjunctive therapy combined with HRT. Group 2 received PPE (9 capsules/day) as adjunctive therapy combined with HRT. Changes in the degree of knee pain were evaluated by the Visual Analog Scale (VAS). RESULTS: Treatment with PPE was significantly effective in reducing the VAS score for knee pain at 4 weeks (p < 0.05), at 8 weeks (p< 0.01) and at 12 weeks (p<0.01), compared with the control group. Interestingly, the effects continued for 4 weeks after cessation of treatment in the PPE group (p< 0.01) compared with the control group. The PPE treatment had no significant adverse effects on blood biochemical and metabolic profiles, especially related to the risk factors for cardiovascular disease. CONCLUSION: PPE is a possible option as an adjunctive oral supplement in the case of HRT-resistant, long-lasting knee pain.

Gene expression signature and response to the use of leucovorin, fluorouracil and oxaliplatin in colorectal cancer patients

PURPOSE: FOLFOX (a combination of leucovorin, fluorouracil and oxaliplatin) has achieved substantial success in the treatment of colorectal cancer (CRC) patients. However, about half of all patients show resistance to this regimen and some develop adverse symptoms such as neurotoxicity. In order to select patients who would benefit most from this therapy, we aimed to build a predictor for the response to FOLFOX using microarray gene expression profiles of primary CRC samples. PATIENTS AND METHODS: Forty patients who underwent surgery for primary lesions were examined. All patients had metastatic or recurrent CRC and received modified FOLFOX6. Responders and nonresponders were determined according to the best observed response at the end of the first-line treatment. Gene-expression profiles of primary CRC were determined using Human Genome GeneChip arrays U133. We identified discriminating genes whose expression differed significantly between responders and nonresponders and then carried out supervised class prediction using the k-nearest-neighbour method. RESULTS: We identified 27 probes that were differentially expressed between responders and nonresponders at significant levels. Based on the expression of these genes, we constructed a FOLFOX response predictor with an overall accuracy of 92.5%. The sensitivity, specificity, positive and negative predictive values were 78.6%, 100%, 100% and 89.7%, respectively. CONCLUSION: The present model suggests the possibility of selecting patients who would benefit from FOLFOX therapy both in the metastatic and the adjuvant setting. To our knowledge, this is the first study to establish a prediction model for the response to FOLFOX chemotherapy based on gene expression by microarray analysis (AU)

Characteristic brain hypoperfusion by 99mTc-ECD single photon emission computed tomography (SPECT) in patients with the first-episode schizophrenia.

OBJECTIVE: In this study, we evaluated brain perfusion in patients with first-episode medicated schizophrenia using the new analytical method, statistical parametric mapping (SPM) applied to single photon emission computed tomography (SPECT). METHOD: We performed SPECT with 99-Tc-ethyl cysteinate dimer (99mTc-ECD) of the brain and magnetic resonance imaging (MRI) in patients with schizophrenia (n=30) and control subjects matched for age and gender (n=37). A voxel-by-voxel group analysis was performed using SPM2 (Z>3.0, P<0.001, uncorrected for multiple comparisons). RESULT: In comparison with control subjects, the volumes of the bilateral frontal areas were found to be decreased on MRI. Blood flow was found to be reduced in the bilateral temporal areas in the patients with schizophrenia on SPECT. CONCLUSION: In this study, patients with first-episode schizophrenia appeared to have significant bilateral temporal hypoperfusion, although temporal volumes were not significantly decreased in comparison with control subjects. Abnormality of temporal lobe blood flow in schizophrenia may show that functional changes occur earlier than structural changes, and may assist in the diagnosis of schizophrenia.

Improvement of motor functions by noisy vestibular stimulation in central neurodegenerative disorders.

Through the cerebellar vermis, the vestibular nerves are known to influence the basal ganglia and the limbic system. By means of noisy galvanic vestibular stimulation (GVS), it may be possible to ameliorate movement disorders, particularly akinesic symptoms, in patients with central neurodegenerative disorders. We evaluated the effect of 24-hour noisy GVS on a power-law temporal autocorrelation exponent of daytime wrist activity, separately for higher (local maxima) and lower (local minima) levels of activity, in 14 hospitalized patients. The power-law exponent for the local maxima was significantly (p < 0.002) lower with the noisy GVS than with sham stimulation, suggestive of more frequent switching behavior from low to high levels of activity or less severe akinesia. The noisy GVS may thus potentially improve certain motor dysfunctions in patients with distinct central neurodegenerative diseases.

Effects of long-term training on neutrophil function in male university judoists.

OBJECTIVES: To clarify the effects of high-intensity and high-frequency long-term/chronic training on neutrophil function and serum levels of myogenic enzymes in male university judoists. METHODS: The subjects were 24 male judoists who had stopped judo training for 6 months and then restarted their training. The following parameters were examined before and after a 2 h unified exercise loading (UEL) at the beginning of the restarted quotidian training (pre-training) and at 2 months, 4 months and 6 months thereafter: myogenic enzymes, neutrophil and leucocyte counts, and neutrophil phagocytic activity (PA) and oxidative burst activity as a measure of reactive oxygen species (ROS) production capability. RESULTS: Myogenic enzymes that were measured after UEL at all four points significantly increased except for creatine kinase at the 2-month point (p<0.01 in each) and neutrophil counts significantly increased after UEL at the pre-training, 2-month and 4-month points (p<0.01 in each), but these changes became smaller from the 2-month point. PA significantly decreased after UEL at the pre-training and 2-month points (p<0.01 in each), but no change was seen at the 4-month and 6-month points. On the other hand, no change in ROS production per cell after UEL was seen at the pre-training point, but it significantly increased after UEL at the 2-month, 4-month and 6-month points (p<0.01 in each). CONCLUSION: The changing rate of the levels of UEL-mediated myogenic enzymes, neutrophil mobilisation and neutrophil function was seen to decrease at the 2-month, 4-month and 6-month assessments, compared with the pre-training point: these may comprise at least some of the long-term training effects.

Inhibitory effects of macrocarpals on the biological activity of Porphyromonas gingivalis and other periodontopathic bacteria.

BACKGROUND/AIMS: Macrocarpals, which are phloroglucinol derivatives contained in eucalyptus leaves, exhibit antimicrobial activity against a variety of bacteria including oral bacteria. This study examined effects of macrocarpals A, B, and C on periodontopathic bacteria, especially Porphyromonas gingivalis. METHODS: Macrocarpals A, B, and C were purified from a 60% ethanol-extract of Eucalyptus globules leaves. To investigate antibacterial activity, representative periodontopathic bacteria were cultured in media with or without various amounts of macrocarpals; subsequently, the optical density at 660 nm was measured. Macrocarpal inhibition of P. gingivalis Arg- and Lys-specific proteinases was assessed by spectrofluorophotometric assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The effect of macrocarpals on P. gingivalis binding to saliva-coated hydroxyapatite beads was examined with (3)H-labeled P. gingivalis. RESULTS: Growth of P. gingivalis was inhibited more strongly than growth of Prevotella intermedia or Prevotella nigrescens and Treponema denticola by macrocarpals, however, Actinobacillus actinomycetemcomitans and Fusobacterium nucleatum were much more resistant. Macrocarpals inhibited P. gingivalis Arg- and Lys-specific proteinases in a dose-dependent manner. The enzyme-inhibitory effect of macrocarpals was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis in which hemoglobin degradation by P. gingivalis proteinase was inhibited by macrocarpals. P. gingivalis binding to saliva-coated hydroxyapatite beads was also strongly attenuated by macrocarpals. CONCLUSIONS: Macrocarpals A, B and C demonstrated antibacterial activity against periodontopathic bacteria. Among tested bacteria, P. gingivalis displayed the greatest sensitivity to macrocarpals; additionally, its trypsin-like proteinase activity and binding to saliva-coated hydroxyapatite beads were inhibited by macrocarpals. These results indicate that eucalyptus leaf extracts may be useful as a potent preventative of periodontal disease.

Successful treatment with allogeneic peripheral blood stem cell transplantation and granulocyte transfusion for severe aplastic anemia with sinusitis.

A 43-year-old woman with severe aplastic anemia (SAA) received anti-thymocyte globulin and cyclosporin A (CyA) and achieved hematological remission. Although she had maintained hematological remission, the disease relapsed 10 months after arbitrary discontinuance of maintenance therapy with CyA. Resumption of CyA therapy was not effective, and her condition became complicated with progressive sinusitis with bone destruction, which was refractory to antibiotics, antifungal agents, granulocyte colony-stimulating factor, and surgical drainage. Because of the necessity for early neutrophil recovery (to resolve the infection), we proceeded with a combination therapy using allogeneic peripheral blood stem cell transplantation (PBSCT) promptly followed by granulocyte transfusion (GTX) from the same human leukocyte antigen-identical donor rather than carrying out a second immunosuppressive therapy. The patient showed temporal resolution of infection on the second day after a single GTX. Although the patient had pneumonia on day 11, it was resolved promptly after engraftment on day 16. This report suggests the clinical utility of a salvage therapy with allogeneic PBSCT followed by GTX in a particular case of recurrent SAA with refractory infections.

The "pulvinar sign" in a case of paraneoplastic limbic encephalitis associated with non-Hodgkin's lymphoma.

This paper reports a 59 year old woman with paraneoplastic limbic encephalitis associated with diffuse large B cell lymphoma. Her brain magnetic resonance imaging scan showed bilateral posterior thalamic hyperintensities, similar to the "pulvinar sign". Her symptoms included progressive psychiatric disturbance and resembled the initial symptoms of variant Creutzfeldt-Jakob disease (vCJD). Clinicians should consider this treatable disorder in the differential diagnosis of vCJD.

Development of a phantom compatible for MRI and hyperthermia using carrageenan gel--relationship between dielectric properties and NaCl concentration.

A phantom has previously been developed containing carrageenan, agarose and gadolinium chloride (called CAG phantom) for MRI with 1.5 T. T(1) and T(2) relaxation times of this phantom are independently changeable by varying concentrations of relaxation-time modifiers to simulate relaxation times of the various types of human tissues. The CAG phantom has a T(1) value of 202-1904 ms and a T(2) value of 38-423 ms, when the GdCl(3) concentration is varied from 0-140 micromol/kg and the agarose concentration is varied from 0-1.6%. A new phantom has now been developed (called CAGN phantom), made by adding an electric conductive agent, NaCl, to the CAG phantom for use in the areas of MRI and hyperthermia research. Dielectric properties of the CAGN phantom were measured and the results of experiments were expressed by the Cole-Cole equation in the frequency range of 5-130 MHz. The relationship between the conductivity of the CAGN phantom and the concentration of NaCl was expressed by a linear function in the frequency range of 1-130 MHz. The linear function involves a parameter of frequency and, when the frequency is 10 MHz, the conductivity of the CAGN phantom can be changed from 0.27-1.26 Sm(-1) by increasing the NaCl concentration from 0-0.7%. The CAGN phantom developed can be employed in basic experiments for non-invasive temperature measurement using MRI and as a loading phantom for MRI with up to 3 T.

Additive antitumour effect of the epidermal growth factor receptor tyrosine kinase inhibitor gefitinib (Iressa, ZD1839) and the antioestrogen fulvestrant (Faslodex, ICI 182,780) in breast cancer cells.

A high expression level of epidermal growth factor receptor (EGFR)/HER1 has been suggested to lead to a shorter survival time and resistance to endocrine therapy in patients with breast cancer. To test the hypothesis that inhibition of the EGFR signalling pathway affects the antitumour effect of endocrine therapy, an EGFR tyrosine kinase inhibitor (EGFR-TKI), gefitinib, and an oestrogen receptor (ER) antagonist, fulvestrant, were administered to human breast cancer cells. A total of five human breast cancer cell lines were used. The effects of single or combined treatments with gefitinib and/or fulvestrant on cell growth, cell cycle progression and apoptosis were analysed. Changes in the expression levels of cyclin-dependent kinase inhibitors, p21 and p27, an antiapoptotic factor, Bcl-2, and a proapoptotic factor, Bax, were also investigated. All cell lines tested were sensitive to gefitinib (50% growth inhibitory concentration, 10-28.5 microM). Breast cancer cell lines with a high expression level of HER1 or HER2 were more sensitive to gefitinib than the others. Gefitinib induced a significant G1-S blockade in ER-positive KPL-3C cells. Gefitinib induced significant apoptosis in HER1-overexpressing MDA-MB-231 cells. Gefitinib additively increased the antitumour effect of fulvestrant in all three ER-positive cell lines in a medium supplemented with 17beta-oestradiol. The combined treatment promoted cell cycle retardation in KPL-3C cells, which is associated with an upregulation of p21 by fulvestrant and gefitinib, respectively. Apoptosis was associated with downregulation of Bcl-2 by gefitinib in MDA-MB-231 cells. These results suggest an additive interaction between the EGFR-TKI gefitinib and the antioestrogen fulvestrant in ER-positive breast cancer cells.

Adverse effects of energy restriction on changes in immunoglobulins and complements during weight reduction in judoists.

AIM: A preliminary study to investigate the combined effects of dietary restriction and weight reduction through exercise on markers of immune function in college judoists before and after a single competition. METHODS: Forty-nine judoists participated in the study. Thirty-eight athletes combined exercise and dietary restriction (WR group), and 11 athletes did not require dietary restriction (EX group). Changes in anthropometric parameters, energy intake, concentrations of serum immunoglobulins and complements, and white blood cell counts were assessed at 4 time points: 20 days (pre-values), 4 days and 1 day before the competition, and 7 days after the competition. RESULTS: Compared with pre-values, the WR group exhibited significant decreases in body weight (-2.8 kg at 1 day before) and fat free mass (-1.7 kg at 1 day before); there were no changes in these variables in the EX group. The WR group exhibited significant decreases in IgG, IgM and C3 at 7 days after the competition (all p<0.01). In the EX group, significant decreases in IgM and C3 (both p<0.05) were observed at 7 days after the competition, though to a lesser degree than in the WR group. CONCLUSIONS: Energy restriction seemed to exacerbate alterations in immune markers such as immunoglobulin and complement induced by vigorous exercise at 7 days after a competition. Although the changed values were still within normal limits, we hypothesize that the potential cumulative effect of these changes over many competitions in 1 year might well induce abnormal levels with a possibly harmful clinical effect on judoists.

Two types of rhythm in the respiratory network output in the isolated ventrolateral medulla in the neonatal rats.

Effects of substance P and extracellular [K(+)](o) on respiratory motor activity in the ventrolateral medulla in neonatal rat (0-4 days old) brainstem-spinal cord preparation were studied. In addition to fictive eupneic rhythm (8-13 bursts/minute), the respiratory motor output was composed of biphasic bursts which might underlie the sigh pattern in vivo. These bursts had considerably lower frequency (0.15-0.86 bursts/minute) and appeared when inspiratory neurons generated augmented biphasic discharges. The two rhythms were differently affected when the respiratory network excitability was increased by substance P or decreased by lowering external [K(+)](o), the effects on biphasic burst frequency being considerably greater. The augmented bursts could suppress inspiratory, but not pre-inspiratory neuron discharge, suggesting that pre-inspiratory neurons formed a supplementary rhythmic network which was not directly affected by biphasic burst generation.

Aberrant growth plate development in VDR/RXR gamma double null mutant mice.

VDR forms heterodimers with one of three RXRs, RXR alpha, RXR beta, and RXR gamma, and it is thought that RXR ligands can also modulate the trans-activation function of VDR/RXR heterodimers. In the present study we generated VDR/RXR gamma double null mutant mice to examine the convergent actions of vitamin D and vitamin A signaling and to explore the possibility of a functionally redundant VDR. Although RXR gamma(-/-) mice exhibited no overt abnormalities, VDR(-/-)/RXR gamma(-/-) mice appeared similar to VDR(-/-) mice, showing features typical of vitamin D-dependent rickets type II, including growth retardation, impaired bone formation, hypocalcemia, and alopecia. However, compared to VDR(-/-) mice, growth plate development in VDR(-/-)/RXR gamma(-/-) mutant mice was more severely impaired. Normalizing mineral ion homeostasis through dietary supplementation with high calcium and phosphorous effectively prevented rachitic abnormalities, except for disarranged growth plates in VDR(-/-)/RXR gamma(-/-) mutant mice, and alopecia in both VDR(-/-) and VDR(-/-)/RXR gamma(-/-) mutant mice. Histological analysis of VDR(-/-)/RXR gamma(-/-) growth plates revealed that development of the hypertrophic chondrocytes was selectively impaired. Thus, our findings indicated that the combined actions of VDR- and RXR gamma-mediated signals are essential for the normal development of growth plate chondrocytes, and raised the possibility that a functionally redundant VDR is present on chondrocytes as a heterodimer with RXR gamma.