Multifactorial regulation of daily rhythms in expression of the metabolically responsive gene spot14 in the mouse liver.

Spot14 is a putative transcriptional regulator for genes involved in fatty acid synthesis. The Spot14 gene is activated in response to lipogenic stimuli such as dietary carbohydrate and is also under circadian regulation. The authors investigated factors responsible for daily oscillation of Spot14 expression. If mice were kept under a 12-h light/12-h dark cycle with ad libitum feeding, Spot14 mRNA levels in the liver reached a peak at an early dark period when mice, as nocturnal animals, start feeding. Under fasting, while Spot14 mRNA levels were generally decreased, the rhythmicity was still maintained, suggesting contribution of both nutritional elements and circadian clock factors on robust rhythmicity of Spot14 expression. Effects of circadian clock factors were confirmed by the observations that the circadian rhythm of Spot14 expression was seen also under the constant darkness and that the rhythmicity was lost in Clock mutant mice. When mice were housed in short-photoperiod (6-h light/18-h dark) and long-photoperiod (18-h light/6-h dark) cycles, rhythms of Spot14 mRNA levels were phase advanced and phase delayed, respectively, being concordant with the notion that Spot14 expression is under the control of the light-entrainable oscillator. As for nutritional mediators, in the liver of db/db mice exhibiting hyperinsulinemia-accompanied hyperglycemia, Spot14 mRNA levels were constantly high without apparent rhythmicity, consistent with previous observations for strong activation of the Spot14 gene by glucose and insulin. Restricted feeding during the 4-h mid-light period caused a phase advance of the Spot14 expression rhythm. On the other hand, restricted feeding during the 4-h mid-dark period led to damping of the rhythmicity, apparently resulting from the separation of phases between effects of the light/dark cycle and feeding on Spot14 expression. Thus, the daily rhythm of Spot14 expression in the liver is under the control of the light-entrainable oscillator, food-entrainable oscillator, and food-derived nutrients, in a separate or cooperative manner.

Dehydroepiandrosterone upregulates soluble guanylate cyclase and inhibits hypoxic pulmonary hypertension.

OBJECTIVE: It has been reported that dehydroepiandrosterone is a pulmonary vasodilator and inhibits chronic hypoxia-induced pulmonary hypertension. Additionally, dehydroepiandrosterone has been shown to improve systemic vascular endothelial function. Thus, we hypothesized that chronic treatment with dehydroepiandrosterone would attenuate hypoxic pulmonary hypertension by enhancing pulmonary artery endothelial function. METHODS AND RESULTS: Rats were randomly assigned to five groups. Three groups received food containing 0, 0.3, or 1% dehydroepiandrosterone during a 3-wk-exposure to simulated high altitude (HA). The other 2 groups were kept at Denver's low altitude (LA) and received food containing 0 or 1% dehydroepiandrosterone. Dehydroepiandrosterone dose-dependently inhibited hypoxic pulmonary hypertension (mean pulmonary artery pressures after treatment with 0, 0.3, and 1% dehydroepiandrosterone=45+/-5, 33+/-2*, and 25+/-1*# mmHg, respectively. *P<0.05 vs. 0% and # vs. 0.3%). Dehydroepiandrosterone (1%, 3 wks) treatment started after rats had been exposed to 3-wk hypoxia also effectively reversed established hypoxic pulmonary hypertension. Pulmonary artery rings isolated from both LA and HA rats treated with 1% dehydroepiandrosterone showed enhanced relaxations to acetylcholine and sodium nitroprusside, but not to 8-bromo-cGMP. In the pulmonary artery tissue from dehydroepiandrosterone-treated LA and HA rats, soluble guanylate cyclase, but not endothelial nitric oxide synthase, protein levels were increased. CONCLUSION: These results indicate that the protective effect of dehydroepiandrosterone against hypoxic pulmonary hypertension may involve upregulation of pulmonary artery soluble guanylate cyclase protein expression and augmented pulmonary artery vasodilator responsiveness to nitric oxide.

An mRNA amplification procedure with directional cDNA cloning and strand-specific cRNA synthesis for comprehensive gene expression analysis.

We developed an integrated system suitable for comprehensive gene expression studies including construction and analysis of cDNA microarrays starting from a small amount of mRNA. We amplified total mRNA first as cDNA mixtures by polymerase chain reaction and then as strand-specific cRNA mixtures by in vitro transcription. These amplified cDNA and cRNA enabled determination of mRNA levels by hybridization analyses such as Southern, Northern, reverse-Northern macroarray, and cDNA microarray analyses, as well as construction of the cDNA library with a unidirectional cDNA insert. By using strand-specific cRNA derived from rat primary-cultured hepatocytes, we detected putative antisense transcripts for the metallothionein gene. cDNA microarray analysis for genes regulated by glucocorticoids and glucagon in the hepatocytes revealed that a number of genes involved in signal transduction and transcriptional regulation were up- or down-regulated. The present system is widely applicable to gene expression analysis with limited amounts of RNA samples.

Two-peaked synchronization in day/night expression rhythms of the fibrinogen gene cluster in the mouse liver.

Genes expressed with day/night rhythms in the mouse liver were searched for by microarray analysis using an in-house array harboring mouse liver cDNAs. The rhythmic expression with a single peak and trough level was confirmed by RNA blot analysis for 3beta-Hsd and Gabarapl1 genes exhibiting a peak in the light phase and Spot14, Hspa8, Hspa5, and Hsp84-1 genes showing a peak in the dark phase. On the other hand, mRNA levels for all of the three fibrinogen subunits, Aalpha, Bbeta and gamma, exhibited two peaks each in the light and dark phases in a synchronized manner. This two-peaked rhythmic pattern of fibrinogen genes as well as the single peak-trough pattern of other genes was diminished or almost completely lost in the liver of Clock mutant mice, suggesting that the two-peaked expression is also under the control of oscillation-generating genes. In constant darkness, the first peak of the expression rhythm of fibrinogen genes was almost intact, but the second peak disappeared. Therefore, although the first peak in the subjective day is a component of the innate circadian rhythm, the second peak seems to require light stimuli. Fasting in constant darkness caused shifts of time phases of the circadian rhythms. Protein levels of the fibrinogen subunits in whole blood also exhibited circadian rhythms. In the mouse and human loci of the fibrinogen gene cluster, a number of sequence elements resembling circadian transcription factor-binding sites were found. The fibrinogen gene locus provides a unique system for the study of two-peaked day/night rhythms of gene expression in a synchronized form.

Report of a patient with a high level of parathyroid hormone-related peptide associated with malignant lymphoma involving multiple bony lesions.

Hypercalcemia in malignant lymphoma is not common. Our case of malignant lymphoma with multiple bony lesions showed hypercalcemia (13 mg/dl) at the time of bone marrow relapse. The serum level of parathyroid hormone-related peptide increased to 142 pmol/l, which may be secreted by malignant lymphoma cells. The course of the patient was aggressive and she died from bone marrow relapse after 6 months of treatment including high-dose methotrexate, which caused acute nonoliguric renal failure. At autopsy there was extensive calcium deposition in the lungs, kidneys and pancreas and prominent tubular damage of kidneys.