Differential gene expression in apoptotic 32Dcl3 cells: induction of metallothionein

Growth factor deprivation induced cell death of the hematopoietic cell line 32Dcl3 is widely used as a model system to study apoptotic signalling pathways. Here we show that the onset of cell death after IL-3 withdrawal can be strongly delayed by either cycloheximide or actinomycin D, indicating that de novo protein synthesis is required. Subtractive cDNA library hybridization was used to identify genes upregulated in apoptotic 32Dcl3 cells. Here we present data showing metallothionein-I (MT-I) mRNA transiently upregulated by a factor of three- to 20-fold. Increased levels of total MT-I+II protein after IL-3 withdrawal were demonstrated. An induction of MT-I RNA as well as of MT-I+II total protein was also observed in serum deprived NIH3T3 fibroblasts. Testing the effect of different inducers of apoptosis on 32Dcl3 cells we found that only IL-3 withdrawal and ethanol treatment led to an upregulation of MT-I mRNA level. Since MTs are believed to play a role in the metabolism of zinc, we tested the effect of zinc on induced cell death. When 32Dcl3 cells are treated with zinc (50-300 M) in the absence of IL-3, loss of viability as well as degradation of the cellular DNA were delayed, indicating that zinc represses apoptosis. On the other hand zinc pre-treatment induced MT expression and accelerated the onset of apoptosis. Our data, therefore, suggest that MT exerts a proapoptotic function.     

Hepatic myelolipoma in two Goeldi's monkeys from South America held captive

Two cases of hepatic myelolipoma in Goeldi's monkeys from South America are described. One was a female evaluated due progressive abdominal distension. Ultrasound and computed tomography detected hepatic mass. Partial hepatectomy was performed, but the monkey died after surgery. Case 2 was a male that died suddenly, showing non‐specific clinical signs.     

Kinetic and functional properties of human mitochondrial phosphoenolpyruvate carboxykinase

The cytosolic form of phosphoenolpyruvate carboxykinase (PCK1) plays a regulatory role in gluconeogenesis and glyceroneogenesis. The role of the mitochondrial isoform (PCK2) remains unclear. We report the partial purification and kinetic and functional characterization of human PCK2. Kinetic properties of the enzyme are very similar to those of the cytosolic enzyme. PCK2 has an absolute requirement for Mn²⁺ ions for activity; Mg²⁺ ions reduce the K_m for Mn²⁺ by about 60 fold. Its specificity constant is 100 fold larger for oxaloacetate than for phosphoenolpyruvate suggesting that oxaloacetate phosphorylation is the favored reaction in vivo. The enzyme possesses weak pyruvate kinase-like activity (k_cat=2.7 s^?1). When overexpressed in HEK293T cells it enhances strongly glucose and lipid production showing that it can play, as the cytosolic isoenzyme, an active role in glyceroneogenesis and gluconeogenesis.     

Structural and functional differentiation of two clinally distributed glucosephosphate isomerase allelic isozymes from the teleost Fundulus heteroclitus

The teleost Fundulus heteroclitus (L.) possesses two loci, Gpi-A and Gpi-B, for the glycolytic enzyme, glucose-phosphate isomerase (GPI; D- glucose-6-phosphate ketol-isomerase; E.C. 5.3.1.9). The Gpi-B locus is polymorphic in Fundulus, with two common alleles, Gpi-Bb and Gpi-Bc, distributed in a clinal manner in populations along the east coast of North America. Since this clinal distribution is strongly correlated with a temperature gradient, we asked whether the GPI-B2 allozymes were functionally adapted to the thermal environment in which a given phenotype predominated. The two major GPI-B2 allozymes were purified to homogeneity and were characterized as to molecular weight, isoelectric pH, thermal denaturation, and kinetic parameters. Both GPI-Bb2 and GPI- Bc2 allozymes have molecular masses of 110 kD, and they have isoelectric pHs of 6.4 and 6.6, respectively. The GPI-Bb2 allozyme was more stable to thermal denaturation than was the GPI-Bc2 enzyme. Kinetic properties of the allelic isozymes were investigated both as a function of pH and as a function of temperature. At 25 degrees C, over the pH range considered, there were no significant differences between allozymes, either in Km for fructose-6-phosphate or in Ki for 6- phosphogluconate, but apparent Vmax values differed between pH 7.5 and pH 8.5. All steady-state kinetic parameters showed strong temperature dependence, but the allozymes differed only in the Ki for 6- phosphogluconate at temperatures greater than 30 degrees C. On the basis of the observed structural and functional differences alluded to above, the hypothesis that the major allelic isozymes of the Gpi-B locus were functionally equivalent was rejected. However, it is not yet known whether these structural and functional differences have any significance at higher levels of biological organization.