GLI1 localization in the germinal epithelial cells alternates between cytoplasm and nucleus: upregulation in transgenic mice blocks spermatogenesis in pachytene.

The zinc finger transcription factor GLI1 is the mediator of signaling by members of the Hedgehog (Hh) family. Male mice in which Desert hedgehog (Dhh), an Hh homologue expressed in Sertoli cells of the testis, was knocked out are sterile, suggesting that the Dhh/GLI1 pathway plays a role in spermatogenesis. Using an antiserum raised against human GLI1, we found that during the first round of spermatogenesis, GLI1 expression is initially cytoplasmic, then shifts to the nuclei of Sertoli and germ cells, and finally shifts back to the cytoplasm. In the adult mouse testis, GLI1 expression localized to the nuclei of germ cells, beginning with pachytene cells and persisting through round spermatids. Localization of GLI1 in elongating spermatids shifted from the nucleus to the cytoplasm and became associated with microtubules. We also examined a line of transgenic mice that overexpressed human GLI1. Male mice in this line were sterile. Spermatogenesis was blocked at the pachytene stage, and a subset of the morphologically indistinguishable pachytene cells underwent apoptosis. Patched-2, which is a Dhh receptor, and Fused, another component of the signal transduction pathway, are expressed in Leydig cells and in primary and secondary spermatocytes. Expression of GLI1 in the same cell types as Patched-2 and Fused and the disruption of spermatogenesis by GLI1 overexpression suggest that GLI1 is the mediator of the Dhh signal in the testis, and that it may be a regulator of spermatogenesis.

Methylation of CpG dinucleotides alters binding and silences testis-specific transcription directed by the mouse lactate dehydrogenase C promoter.

The mouse lactate dehydrogenase c gene (mldhc) is transcribed only in cells of the germinal epithelium. Cloning and analysis of the mldhc promoter revealed that a 100-base pair fragment was able to drive testis-specific transcription in vitro and in transgenic mice. Several testis-specific genes are believed to be regulated at least in part through differential methylation of CpG dinucleotides. We investigated the possibility that transcriptional repression of the mldhc gene is mediated in somatic tissues by hypermethylation of CpG dinucleotides. The CpG dinucleotides within a fragment of the mldhc promoter containing a GC box and tandem activating transcription factor/cAMP-responsive element binding sites are hypermethylated in somatic tissues and hypomethylated in testis. Methylation of the activating transcription factor/cAMP-responsive elements altered the protein binding pattern observed in electrophoretic mobility shift assays using mouse liver but not testis nuclear extract. Furthermore, methylation of an extended mldhc promoter fragment driving lac Z silenced transcription from the promoter in a transient transfection assay. These data suggest that tissue-specific differential methylation plays a role in mldhc silencing in somatic tissues.

3'-untranslated region of SP-B mRNA mediates inhibitory effects of TPA and TNF-alpha on SP-B expression.

Surfactant protein-B (SP-B) is a small hydrophobic polypeptide that enhances spreading and stability of surfactant phospholipids in the alveolus of the lung. Decreased expression of SP-B is associated with respiratory failure in premature infants and in adult patients with acute respiratory distress syndrome (ARDS). Tumor necrosis factor-alpha (TNF-alpha) and 12-O-tetradecanoylphorbol-13 acetate (TPA) cause ARDS-like lung injury in vivo. Inhibitory effects of TPA and TNF-alpha on SP-B mRNA expression in vitro were mediated by decreased SP-B mRNA stability rather than by decreased rate of SP-B gene transcription. In the present study, a human pulmonary adenocarcinoma cell line, NCI H441-4, was stably transfected with expression vectors consisting of the thymidine kinase (TK) promotor and human growth hormone (hGH) gene, in which the hGH 3'-untranslated region (3'-UTR) was replaced by the 2.0-kb human SP-B cDNA [pTKGH(SP-B2.0)] or the 837-bp human SP-B 3'-UTR [pTKGH(SP-B.837)]. The mRNAs and cellular growth hormone protein generated from the chimeric TKGH(SP-B2.0) and TKGH(SP-B.837) genes were each inhibited by approximately 50% by TPA and TNF-alpha. Dexamethasone decreased the inhibitory effects of TPA and TNF-alpha. The inhibition of steady-state hGH-SP-B mRNA by TPA and TNF-alpha was mediated by a cis-active element located in the 3-UTR region of SP-B mRNA.

Magnesium enhances function of postischaemic human myocardial tissue.

OBJECTIVES: The effect of Mg2+ on the developed force and concentrations of high energy phosphate metabolites in isolated human atrial trabeculae has been investigated. METHODS: Human atrial trabeculae, obtained from right atrial appendages of patients undergoing cardiac surgery requiring cardiopulmonary bypass, were dissected at room temperature in modified Krebs-Henseleit buffer containing 1.2 or 16 mM Mg2+, mounted on muscle stands, and rewarmed to 34 degrees C in the same buffer. After 30 minutes, their mechanical function was assessed. At the end of the protocol, trabeculae were fast frozen for measurement of concentrations of metabolites of high energy phosphates. RESULTS: Trabeculae collected and rewarmed in 16 mM Mg2+ Krebs-Henseleit buffer showed significantly higher mean developed force (0.59(SEM 0.10) g, p < 0.01) than those rewarmed in 1.2 mM Mg2+ Krebs-Henseleit buffer (0.32(0.03) g). Trabeculae that had a developed force > or = 0.8 g, a resting force < or = 0.7 g, and a cross sectional area < or = 1 mm2 ("functional" trabeculae) were selected for further comparison. New reverse phase high performance liquid chromatography techniques developed for the analysis of small samples (0.5-5 mg dry weight) were used to measure nucleotide, nucleoside, and creatine compounds. Total adenylate (ATP+ADP+AMP) concentrations in trabeculae revived in the presence of 16 mM Mg2+ (15.4(1.1) mumol.g-1 dry weight) were significantly higher (p < 0.01) than in those revived with 1.2 mM Mg2+ (11.8(1.0) mumol.g-1), but lower (p < 0.01) than in trabeculae fast frozen immediately after removal from the patient (22.6(1.0) mumol.g-1). There were no significant differences in NAD and total creatine (phosphocreatine+creatine) concentrations between the three groups. CONCLUSIONS: The presence of high Mg2+ during the rewarming of human atrial trabecular preparations maintains a significantly higher developed force and a significantly higher total adenylate pool than does collection and rewarming with normal concentrations of Mg2+.

A simple procedure for obtaining high-quality NMR spectra of semiquantitative value from small tissue specimens: cervical biopsies.

The handling of small tissue biopsy samples (< 100 microliters) for NMR investigations poses special problems. Optimal and stable positioning of the samples within the sensitive volume of the radiofrequency coil can be achieved by inserting the sample in a capillary. Methods for quantitation of the spectral information from samples requiring histological evaluation after the NMR experiment are discussed, in particular with respect to cervical biopsies.

Lipoprotein(a) and CA125 levels in the plasma of patients with benign and malignant ovarian disease.

Elevated plasma levels of apolipoprotein (a) have been reported earlier in cancer patients. In order to investigate the potential of apolipoprotein(a) as an ovarian tumour marker, plasma apolipoprotein(a) and CA125 levels were measured in healthy women and women with benign or malignant pelvic masses. Among women younger than 49 years, 80% of healthy controls and ovarian cancer patients had apolipoprotein(a) levels below 350 U/l. Among women aged 49 years or older, 46% of healthy controls but 73% of ovarian cancer patients and 77% of women with successfully treated ovarian cancer, had low apolipoprotein(a) levels. For both age groups, apolipoprotein(a) is not a suitable marker for ovarian cancer. No correlation was found between apolipoprotein(a), triglyceride or cholesterol in plasma. Healthy women younger than 49 years had significantly higher CA125 levels than women 49 years or older (20 +/- 14 U/ml vs. 13 +/- 12 U/ml, p less than 0.005). Levels of CA125 above 35 U/ml were found in 12% of the younger and 4% of the older healthy women, 73% of the younger and 61% of the older patients with untreated or residual tumours, and in 33% of the younger and 31% of older patients with no evidence of disease, as well as in 58% of women of both age groups with benign pelvic masses. The sensitivity and specificity of CA125 levels for the detection of cancer were 73% and 74% respectively for women younger than 49 years, and 62% and 78% respectively for women 49 years or older.

Evaluation of a simple line width test involving magnetic resonance spectroscopy of plasma in carcinoma of the ovary.

Magnetic resonance spectroscopic (MRS) measurement of human plasma has been reported as a generally applicable marker for malignancy: patients with malignancy had a MRS line width significantly different from patients with benign diseases or healthy controls. The authors investigated the value of this test in 213 women with ovarian carcinoma, benign pelvic masses, benign nongynecologic diseases, and healthy controls. The MRS measurements were performed on plasma samples at 21 degrees C or 27 degrees C. The line width parameters were obtained by averaging the width at half the height of the methyl and methylene peaks on the resonance spectra. At 27 degrees C using 33 Hz as the threshold for an abnormal result, there was a significant correlation between the result of the test and the presence or absence of malignancy. However, the study demonstrates that the specificity (0.44) and positive predictive value (0.42) are too low for the test to be useful in the management of patients with carcinoma of the ovary. At 21 degrees C no correlation between the results of the test and the clinical status of women with carcinoma of the ovary were observed. In 47 patients the test did not predict preoperatively the benign or malignant nature of a pelvic mass.

1H and 13C NMR studies of tissue from normal and diseased mice. Analysis of T1 and T2 relaxation profiles of triglycerides in liver.

The study was initiated to gain a better understanding of the manifestation of disease in the lipid dynamics in tissue. We have performed high resolution 13C and 1H NMR relaxation measurements on the observable lipid resonances in liver and adipose tissue, excised from mice which were normal fed, normal fasted, and infected with the malarial parasite Plasmodium berghei. We have observed that, although the parasite does not invade the hepatocytes, the composition of the liver lipids changes along with nutritional status and disease state. Analysis of the liver lipids using gas chromatography showed that in all cases studied (normal or malarial, fed or fasted) the phospholipid content of the liver remains constant. The triglyceride content, however, shows an increase of up to fourfold in both 24-hour fasted controls and highly parasitized mice. The fatty acid compositions of the triglycerides in 24-hour fasted normal and highly parasitized mice are altered, when compared with fed controls or with mice having a low level of infection. The unsaturation index increases twofold. The 13C T1 experiments on the methylene and olefinic resonances of liver give a single exponential decay. The value for a particular carbon is independent of the origin of the tissue, the nutritional status or the disease state of the animal. In contrast, both 1H and 13C T2 relaxation of the methylene and olefinic carbon resonances can generally be best analyzed as the sum of two exponential decays (based on an F-test). The 13C T2 values and the pre-exponential weighting factors are independent of the origin of the tissue examined. The T2 data could also be analyzed in terms of a distribution of relaxation times, a physically more realistic model. Lastly, our results suggest that the 1H and 13C resonances of lipids in the cells of diseased or normal liver tissues do not give rise to "long" T2 values, as have been observed in cancer cells with metastatic potential.

Large-scale selection synchrony of Tetrahymena thermophila.

A method is described, based on the phagocytosis of colloidal ferrite particles, which gives highly synchronous populations of Tetrahymena thermophila. To ensure a successful synchrony, the cell culture doubling time, the limits of the phagocytic period and the distribution of cell stages must first be determined. Once these parameters are known, synchrony can be achieved under a variety of growth conditions and with cultures ranging in volume from a few millilitres to 12 litres or more. The main advantages of the method are that the apparatus required is simple, large volumes of cells can be handled easily, and the synchronous populations can be prepared within a few hours. In principle, the method should be applicable to any cell population in which phagocytosis occurs discontinuously over the cell cycle.

NMR studies of malaria. 31P nuclear magnetic resonance of blood from mice infected with Plasmodium berghei.

High resolution 31P-NMR has been used for the non-invasive observation of metabolites and metabolic rates in blood of normal mice and of mice infected with Plasmodium berghei, the causative agent of malaria. 31P-NMR was used to quantitate levels of 2,3-diphosphoglycerate in whole cells as a function of the degree of parasitemia and yielded good agreement with the results of enzymatic assays. The time-dependence of 31P metabolites was monitored in both normal and infected erythrocytes, greater rates of decay of 2,3-diphosphoglycerate being observed in malarial blood which correlate with the level of parasitemia. Very high metabolic rates of infected cells render measurement of intracellular pH unreliable on freshly drawn whole blood. When appropriate measures are taken to avoid this complication, no difference is observed in the intracellular pH of parasitized and non-parasitized erythrocytes from infected animals. In both normal and parasitized mice the intraerythrocytic pH is more acidic than that of the suspending medium by 0.15 pH unit at 25 degrees C. Unlike free-living protozoa, the parasitic protozoan Plasmodium does not contain detectable levels of phosphonates or polyphosphates, in either whole cells or perchloric acid extracts thereof.