Quirks of dye nomenclature. 5. Rhodamines

Rhodamines were first produced in the late 19^th century, when they constituted a new class of synthetic dyes. These compounds since have been used to color many things including cosmetics, inks, textiles, and in some countries, food products. Certain rhodamine dyes also have been used to stain biological specimens and currently are widely used as fluorescent probes for mitochondria in living cells. The early history and current biological applications are sketched briefly and an account of the ambiguities, complications and confusions concerning dye identification and nomenclature are discussed.     

Type C Niemann-Pick disease. Lysosomal accumulation and defective intracellular mobilization of low density lipoprotein cholesterol

The intracellular accumulation of unesterified cholesterol was examined during 24 h of low density lipoprotein (LDL) uptake in normal and Niemann-Pick C fibroblasts by fluorescence microscopy with filipin staining and immunocytochemistry. Perinuclear fluorescence derived from filipin-sterol complexes was observed in both normal and mutant cells by 2 h. This perinuclear cholesterol staining reached its peak in normal cells at 6 h. Subsequent development of fluorescence during the remaining 18 h of LDL incubation was primarily limited to the plasma membrane region of normal cells. In contrast, mutant cells developed a much more intense perinuclear fluorescence throughout the entire 24 h of LDL uptake with little enhancement of cholesterol fluorescence staining in the plasma membranes. Direct mass measurements confirmed that internalized LDL cholesterol more readily replenishes the plasma membrane cholesterol of normal than of mutant fibroblasts. Perinuclear filipin-cholesterol fluorescence of both normal and mutant cells was colocalized with lysosomes by indirect immunocytochemical staining of lysosomal membrane protein. Abnormal sequestration of LDL cholesterol in mutant cells within a metabolically latent pool is supported by the finding that in vitro esterification of cellular cholesterol could be stimulated in mutant but not in normal cell homogenates by extensive disruption of the intracellular membranous structures of cells previously cultured with LDL. Deficient translocation of exogenously derived cholesterol from lysosomes to other intracellular membrane sites may be responsible for the delayed homeostatic responses associated with LDL uptake by mutant Niemann-Pick Type C fibroblasts.     

Identification of a novel sequence that governs both polyadenylation and alternative splicing in region E3 of adenovirus.

Region E3 encodes four major overlapping mRNAs with different splicing patterns. There are two poly(A) sites, an upstream site called E3A and a downstream site called E3B. We have analyzed virus mutants with deletions or insertions in E3 in order to identify sequences that function in the alternative processing of E3 pre-mRNAs, and to understand what determines which poly(A) sites and which splice sites are used. In previous studies we established that the 5' boundary of the E3A poly(A) signal is at an ATTAAA sequence. We now show, using viable virus mutants, that the 3' boundary of the E3A signal is located within 47-62 nucleotides (nt) downstream of the ATTAAA (17-32 nt downstream of the last microheterogenous poly(A) addition site). Our data further suggest that the spacing between the ATTAAA, the cleavage sites, and the essential downstream sequences may be important in E3A 3' end formation. Of particular interest, these mutants suggest a novel mechanism for the control of alternative pre-mRNA processing. Mutants which are almost completely defective in E3A 3' end formation display greatly increased use of a 3' splice site located 4 nt upstream of the ATTAAA. The mRNA that uses this 3' splice site is polyadenylated at the E3B poly(A) site. We suggest, for this particular case, that alternative pre-mRNA processing could be determined by a competition between trans-acting factors that function in E3A 3' end formation or in splicing. These factors could compete for overlapping sequences in pre-mRNA.     

Synthesis of rat hepatic zinc thionein in response to the stress of sham operation

Following sham operation for adrenalectomy, a dramatic 30-fold increase in rat hepatic zinc thionein occurs, peaking at 18 hours after surgery. Hepatic cytosolic and serum zinc levels rise concomitantly with zinc thionein. Copper in hepatic thionein and cytosol rises only slightly and serum copper not at all during the period of observation. In the period 18 to 48 hours after surgery the content of hepatic zinc thionein decreases with a $\text{t}\text{1}\text{2}$ of 16.4 hours.Pretreatment with cycloheximide (1 mg/kg b.w.) two hours before surgery inhibits the rise in zinc thionein by 52%, the rise in cytosolic zinc by 56%, and actually causes a decrease in serum zinc by 33%. Pretreatment with the α-adrenergic receptor blocker, phetolamine (10 mg/kg b.w.), or the β-adrenergic receptor blocker, propranolol (10 mg/kg b.w.), 30 minutes before surgery also inhibited the rise in zinc thionein (82% and 60%, respectively) and cytosolic zinc (75% and 47%, respectively), and decreased serum zinc (38% and 44%, respectively) 19 hours after surgery.Treatment with corticosterone (40 mg/kg b.w.) alone or epinephrine (1–20 μg/kg b.w.) alone did not alter hepatic zinc thionein levels 18 hours late, although they each caused hypozincemia and epinephrine raised cytosolic zinc levels. Treatment with corticosterone and epinephrine together did, however, raise zinc thionein levels 3.2-fold (P<0.02).These experiments are consistent with the hypothesis that adrenal hormones are involved in the regulation of zinc metabolism, and, hence, zinc thionein in levels in rat liver following the stress of sham operation.     

Suppression subtractive hybridization identifies high glucose levels as a stimulus for expression of connective tissue growth factor and other genes in human mesangial cells

Accumulation of mesangial matrix is a pivotal event in the pathophysiology of diabetic nephropathy. The molecular triggers for matrix production are still being defined. Here, suppression subtractive hybridization identified 15 genes differentially induced when primary human mesangial cells are exposed to high glucose (30 mM versus 5 mM) in vitro. These genes included (a) known regulators of mesangial cell activation in diabetic nephropathy (fibronectin, caldesmon, thrombospondin, and plasminogen activator inhibitor-1), (b) novel genes, and (c) known genes whose induction by high glucose has not been reported. Prominent among the latter were genes encoding cytoskeleton-associated proteins and connective tissue growth factor (CTGF), a modulator of fibroblast matrix production. In parallel experiments, elevated CTGF mRNA levels were demonstrated in glomeruli of rats with streptozotocin-induced diabetic nephropathy. Mannitol provoked less mesangial cell CTGF expression in vitro than high glucose, excluding hyperosmolality as the key stimulus. The addition of recombinant CTGF to cultured mesangial cells enhanced expression of extracellular matrix proteins. High glucose stimulated expression of transforming growth factor beta1 (TGF-beta1), and addition of TGF-beta1 to mesangial cells triggered CTGF expression. CTGF expression induced by high glucose was partially suppressed by anti-TGF-beta1 antibody and by the protein kinase C inhibitor GF 109203X. Together, these data suggest that 1) high glucose stimulates mesangial CTGF expression by TGFbeta1-dependent and protein kinase C dependent pathways, and 2) CTGF may be a mediator of TGFbeta1-driven matrix production within a diabetic milieu.     

Involvement of cytoplasmic membranes in the non-lytic infection of K-562 cells by Semliki Forest virus

An analysis of the human leukemia cell line, K-562, infected with Semliki Forest virus, has been made with transmission electron microscopy. In contrast to the usual surface budding of the enveloped virus on the plasma membrane of vertebrate cells leading to cytolysis within 20 h, K-562 cells do not show surface budding, and the cells remain intact for periods of several months. Several unusual features of the infection include: 1) the rough endoplasmic reticulum arranges early into continuous perinuclear chains; 2) during the time of virus replication and release, the nucleocapsids aggregate on the cytoplasmic side of internal vesicles in the region of the cell where the Golgi complex is normally located; and 3) during this same time period, the vesicles are seen to contain enveloped virions and rod-like formations, a result suggesting that budding has occurred into these vesicles. Viruses are presumably released from the cell as these vesicles fuse with the plasma membrane. By 12 days post-infection and thereafter, the intact cells show electron-dense aggregates of chromatin, large vacuoles and lipid inclusions throughout the cytoplasm, and only a few virion-containing vesicles.