Direct solution hybridization of guanidine thiocyanate-solubilized cells for quantitation of mRNAs in hepatocytes

The sensitivity of direct solution hybridization of hepatocytes solubilized in guanidium thiocyanate (GuSCN) for detecting alpha 1-acid glycoprotein and albumin mRNAs was studied. The sensitivity of detection was inversely correlated with the DNA concentration. Raising the hybridization temperature from 20 to 37 or 50 degrees C (with formamide) increased the hybridization efficiency three- to fourfold in cell lysates with a high DNA concentration (1 microgram/microliter), whereas the hybridization efficiency was already maximal at 20 degrees C in diluted samples. It was most important to normalize all hybridization reactions with an internal standard, such as sense mRNA, because of the great variation in hybridization efficiency from one cell preparation to another depending on the DNA concentration. Direct hybridization of GuSCN cell lysates labeled in vivo with [6-14C]orotic acid was more efficient than hybridizing equivalent amounts of purified [6-14C]-labeled RNA, perhaps because of greater mRNA integrity and/or better recoveries of mRNA in GuSCN cell lysates. Therefore, direct solution hybridization of GuSCN-solubilized hepatocytes, which avoids the problem of RNA purification, appears to be a rapid, sensitive, and reliable method for quantifying mRNA in hepatocytes.     

Fibrolamellar carcinoma of the liver: composition of the extracellular matrix and expression of cell-matrix and cell-cell adhesion molecules

We have analyzed the composition of the tumor stroma and the expression of cell-matrix and cell-cell adhesion molecules in 11 cases of fibrolamellar carcinoma of the liver (FLC), in comparison with 34 cases of hepatocellular carcinoma and 8 cases of focal nodular hyperplasia. Fibrolamellar carcinoma was characterized by the presence of large amounts of tenascin in tumor stroma and by the scarce expression of basement membrane components at the contact of neoplastic clusters. Like normal hepatocytes, neoplastic cells constantly expressed the alpha1 integrin chain, lacked the beta4 integrin chain, and coexpressed E-cadherin and the hepatocyte N-related cadherin. Abnormalities in the expression of cell adhesion molecules, including altered cadherin expression, alphaV integrin chain induction, and CD44 expression, were detected in the majority of cases. The composition of the tumor stroma and the pattern of expression of cell adhesion molecules in fibrolamellar carcinoma were reminiscent of those observed in grade III and grade IV hepatocellular carcinomas. Our results therefore show that, despite its slow local growth and good prognosis, fibrolamellar carcinoma expresses many characteristics usually associated with clinically aggressive malignancies. Further studies are needed to identify the factors responsible for the apparent dissociation between clinical behavior and biological characteristics in this tumor.     

Multiexcitonic dual emission in CdSe/CdS tetrapods and nanorods

CdSe/CdS semiconductor nanocrystal heterostructures are currently of high interest for the peculiar electronic structure offering unique optical properties. Here, we show that nanorods and tetrapods made of such material combination enable efficient multiexcitonic emission, when the volume of the nanoparticle is maximized. This condition is fulfilled by tetrapods with an arm length of 55 nm and results in a dual emission with comparable intensities from the CdS arms and CdSe core. The relative intensities of the dual emission, originating from exciton phase-space filling and reduced Auger recombination, can be effectively modulated by the photon fluence of the pump laser. The results, obtained under steady-state detection conditions, highlight the properties of tetrapods as multiexciton dual-color emitters.     

Molecular cloning of CIF1, a yeast gene necessary for growth on glucose.

The cif1 mutation of Saccharomyces cerevisiae (Navon et al., Biochemistry 18, 4487-4499, 1979) causes inability to grow on glucose and absence of catabolite inactivation. We have cloned the CIF1 gene by complementation of function and located it in a 2.75 kb SphI-BstEII fragment situated at ca. 18 kb centromere distal of LYS2 and ca. 80 kb centromere proximal of TYR1 on chromosome II. Southern analysis demonstrated that CIF1 is present in a single copy in the yeast genome. Northern analysis revealed that the corresponding mRNA of 1.8 kb is more abundant in cells grown on galactose than in those grown on glucose. A protein of ca. 54 kDa was predicted from the open reading frame in the sequenced fragment. In strains carrying the cif1 mutation the intracellular concentration of ATP decreased immediately after addition of glucose while the intracellular concentration of cAMP did not increase. cAMP concentration increased in response to galactose or 2,4-dinitrophenol. Disruption of BCY1 or overexpression of CDC25 in a cif1 background did not restore growth on glucose, suggesting that the absence of cAMP signal is not the primary cause of lack of growth on glucose. Complementation tests showed that cif1 is not allelic to fdp1 although the two genes seem to be functionally related.