In vitro characterization of estrogen induced syrian hamster renal tumors: Comparison with an immortalized cell line derived from diethylstilbestrol-treated adult hamster kidney

Summary Primary diethylstilbestrol-induced kidney tumors from Syrian hamsters were grown in vitro and maintained in culture for 6 mo. Combined immunohistochemical studies using antibodies to intermediate filaments and ultrastructural studies of tumor cells in culture exhibited characteristics similar to tumor cells in vivo. Furthermore, the cells manifested transformed properties in culture; they grew both as multilayered colonies attached to the tissue culture substrate and as floating multicellular colonies (spheroids). When cultured cells were injected into diethylstilbestrol-treated recipient hamsters, tumors developed at the injection sites. In contrast, renal tubules or whole kidney cortex from control hamsters cultured in the same medium underwent only short-term growth, with senescence developing after approximately 1 mo. However, cell cultures of kidney cortex from animals treated in vivo for 5 mo. with diethylstilbestrol formed a cell line. This diethylstilbestrol-induced cell line has been maintained in culture for 1.5 yr and has the following characteristics: a) it is anchorage-dependent, b) it is negative in in vivo tumorigenicity tests, and c) cultured cells are histochemically and ultrastructurally similar to cultured tumor cells. This culture system should prove to be of use in studying hormonal carcinogenesis in vitro. This study was supported by the Medical Research Service, Department of Veterans Affairs, Washington, DC, and by grant CA-22008 from the National Cancer Institute, NIH, DHHS, Bethesda, MD.     

The Metabolism of Quinate in Pea Roots (Purification and Partial Characterization of a Quinate Hydrolyase)

A quinate (QA) hydrolyase was isolated from pea (Pisum sativum L.) roots. The enzyme converts QA into shikimate by elimination of water. The enzymatic reaction is independent of cofactors and divalent cations. The QA hydrolyase was purified about 1,600-fold to apparent electrophoretic homogeneity in three steps, including bovine serum albumin-affinity chromatography. The enzyme forms oligomers and/or complexes with bovine serum albumin and ovalbumin. The monomer molecular weight of the enzyme is about 15,000. The hydrolyase shows regular Michaelis-Menten kinetics with a Km, of 2.0 mM for QA. Compartmentation studies reveal that the QA hydrolyase is localized in plastids. The QA hydrolyase may function in channeling imported QA into the shikimate pathway to support aromatic amino acid biosynthesis in plastids.     

Expression of TRPC3 in Chinese Hamster Ovary Cells Results in Calcium-activated Cation Currents Not Related to Store Depletion

TRPC3 (or Htrp3) is a human member of the trp family of Ca²⁺-permeable cation channels. Since expression of TRPC3 cDNA results in markedly enhanced Ca²⁺ influx in response to stimulation of membrane receptors linked to phospholipase C (Zhu, X., J. Meisheng, M. Peyton, G. Bouley, R. Hurst, E. Stefani, and L. Birnbaumer. 1996. Cell. 85:661–671), we tested whether TRPC3 might represent a Ca²⁺ entry pathway activated as a consequence of depletion of intracellular calcium stores. CHO cells expressing TRPC3 after intranuclear injection of cDNA coding for TRPC3 were identified by fluorescence from green fluorescent protein. Expression of TRPC3 produced cation currents with little selectivity for Ca²⁺ over Na⁺. These currents were constitutively active, not enhanced by depletion of calcium stores with inositol-1,4,5-trisphosphate or thapsigargin, and attenuated by strong intracellular Ca²⁺ buffering. Ionomycin led to profound increases of currents, but this effect was strictly dependent on the presence of extracellular Ca²⁺. Likewise, infusion of Ca²⁺ into cell through the patch pipette increased TRPC3 currents. Therefore, TRPC3 is stimulated by a Ca²⁺-dependent mechanism. Studies on TRPC3 in inside-out patches showed cation-selective channels with 60-pS conductance and short (<2 ms) mean open times. Application of ionomycin to cells increased channel activity in cell-attached patches. Increasing the Ca²⁺ concentration on the cytosolic side of inside-out patches (from 0 to 1 and 30 μM), however, failed to stimulate channel activity, even in the presence of calmodulin (0.2 μM). We conclude that TRPC3 codes for a Ca²⁺-permeable channel that supports Ca²⁺-induced Ca²⁺-entry but should not be considered store operated.     

Identification of two homologs of the Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) in retroperitoneal fibromatosis of different macaque species.

Simian retroperitoneal fibromatosis (RF) is a vascular fibroproliferative neoplasm which has many morphological and histological similarities to human Kaposi's sarcoma (KS). Like epidemic KS in AIDS patients, RF is highly associated with an immunodeficiency syndrome (simian acquired immunodeficiency syndrome [SAIDS]) caused by a retrovirus infection. Recently, a new gammaherpesvirus, called Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV8), has been identified in KS tumors, suggesting that KS has a viral etiology. Our previous experimental transmission studies and epidemiological data suggest that RF also has an infectious etiology. In order to determine whether a similar virus is also associated with RF, we have assayed for the presence of an unknown herpesvirus using degenerate PCR primers targeting the highly conserved DNA polymerase genes of the herpesvirus family. Here we provide DNA sequence evidence for two new herpesviruses closely related to KSHV from RF tissues of two macaque species, Macaca nemestrina and Macaca mulatta. Our data suggest that KSHV and the putative macaque herpesviruses define a new group within the subfamily Gammaherpesvirinae whose members are implicated in the pathogenesis of KS and KS-like neoplasms in different primate species.     

Lysophosphatidic acid enhances contractility of isolated airway smooth muscle

Toews, M. L., E. E. Ustinova, and H. D. Schultz.Lysophosphatidic acid enhances contractility of isolated airwaysmooth muscle. J. Appl. Physiol.83(4): 1216-1222, 1997.The effects of the simple phospholipidmediator lysophosphatidic acid (LPA) on the contractile responsivenessof isolated tracheal rings from rabbits and cats were assessed. In bothspecies, LPA increased the contractile response to the muscarinicagonist methacholine, but LPA did not induce contraction on its own.Conversely, LPA decreased the relaxation response to the-adrenergic-agonist isoproterenol in both species. Concentrations ofLPA as low as 10⁸ M wereeffective, and the effects of LPA were rapidly reversed on washing.Phosphatidic acid was much less effective, requiring higherconcentrations and producing only a minimal effect. Contractions induced by serotonin and by substance P also were enhanced by LPA, butKCl-induced contractions were unaffected. LPA inhibited theisoproterenol-induced relaxation of KCl-precontracted rings, similar toits effects on methacholine-precontracted rings, and relaxation inducedby the direct adenylyl cyclase activator forskolin was inhibited in amanner similar to that induced by isoproterenol. Epithelium removal didnot alter the contraction-enhancing effect of LPA. The ability of LPAto both enhance contraction and inhibit relaxation of airway smoothmuscle suggests that LPA could contribute to airway hypercontractilityin asthma, airway inflammation, or other types of lung injury.

     

Variation in heat shock proteins within tropical and desert species of poeciliid fishes

The 70-kilodalton heat shock protein (hsp70) family of molecular chaperones, which contains both stress-inducible and normally abundant constitutive members, is highly conserved across distantly related taxa. Analysis of this protein family in individuals from an outbred population of tropical topminnows, Poeciliopsis gracilis, showed that while constitutive hsp70 family members showed no variation in protein isoforms, inducibly synthesized hsp70 was polymorphic. Several species of Poeciliopsis adapted to desert environments exhibited lower levels of inducible hsp70 polymorphism than the tropical species, but constitutive forms were identical to those in P. gracilis, as they were in the confamilial species Gambusia affinis. These differences suggest that inducible and constitutive members of this family are under different evolutionary constraints and may indicate differences in their function within the cell. Also, northern desert species of Poeciliopsis synthesize a subset of the inducible hsp70 isoforms seen in tropical species. This distribution supports the theory that ancestral tropical fish migrated northward and colonized desert streams; the subsequent decrease in variation of inducible hsp70 may have been due to genetic drift or a consequence of adaptation to the desert environment. Higher levels of variability were found when the 30- kilodalton heat shock protein (hsp30) family was analyzed within different strains of two desert species of Poeciliopsis and also in wild-caught individuals of Gambusia affinis. In both cases the distribution of hsp30 isoform diversity was similar to that seen previously with allozyme polymorphisms.      

The aspartate aminotransferase gene family of Arabidopsis encodes isoenzymes localized to three distinct subcellular compartments

Here, a complete study is described of all the genes and isoenzymes for aspartate aminotransferase (AspAT) present in Arabidopsis thaliana . Four classes of cDNAs representing four distinct AspAT genes ( ASP1—ASP4 ) have been cloned from Arabidopsis . Sequence analysis of the cDNAs suggests that the encoded proteins are targeted to different subcellular compartments. ASP1 encodes a mitochondrial form of AspAT, ASP3 encodes a chloroplastic/plastidic form of AspAT, whereas ASP2 and ASP4 each encode cytosolic forms of AspAT. Three distinct AspAT holoenzymes (AAT1—AAT3) were resolved by activity gel analysis. Organelle isolation reveals that AAT1 is mitochondrial-localized, AAT3 is plastid-localized, and AAT2 is cytosolic. Gene-specific Northern analysis reveals that each Asp mRNA accumulates differentially with respect to organ-type. However, the individual Asp mRNAs show no dramatic fluctuations in response to environmental stimuli such as light. Southern analysis reveals that four distinct nuclear genes probably represent the entire AspAT gene family in Arabidopsis . These molecular studies shed light on the subcellular synthesis of aspartate in Arabidopsis and suggest that some of the AspAT isoenzymes may play overlapping roles in plant nitrogen metabolism.     

Problems in Fitting a Cosine Curve: Short Communications

In fitting of cosine curves latent experimental inequalities due to a serial effect have to be excluded. Though cosinor analysis may be sufficient then, inclusion of biological time, i.e. not fitting values to time but to a function of time, will lead to further improvement.