Tissue-specific expression of porphobilinogen deaminase. Two isoenzymes from a single gene

Porphobilinogen deaminase (hydroxymethylbilane synthase; EC 4.3.1.8), the third enzyme of the heme biosynthetic pathway, catalyzes the stepwise condensation of four porphobilinogen units to yield hydroxymethylbilane, which is in turn converted to uroporphyrinogen III by cosynthetase. We compared the apparent molecular mass of porphobilinogen deaminase from erythropoietic and from non-erythropoietic cells by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and immune-blotting. The results indicate that two isoforms of porphobilinogen deaminase can be distinguished and differ by 2000 Da. Analysis of cell-free translation products directed by mRNAs from human erythropoietic spleen and from human liver demonstrates that the two isoforms of porphobilinogen deaminase are encoded by distinct messenger RNAs. We cloned and sequenced cDNAs complementary to the non-erythropoietic form of porphobilinogen deaminase encoding RNA. Comparison of these sequences to that of human erythropoietic mRNA [Raich et al. (1986) Nucleic Acids Res. 14, 5955-5968] revealed that the two mRNA species differ by their 5' extremity. From the mRNA sequences we could deduce that an additional peptide of 17 amino acid residues at the NH2 terminus of the non-erythropoietic isoform of porphobilinogen deaminase accounts for its higher molecular mass. RNase mapping experiments demonstrate that the two porphobilinogen deaminase mRNAs are distributed according to a strict tissue-specificity, the erythropoietic form being restricted to erythropoietic cells. We propose that a single porphobilinogen deaminase gene is transcribed from two different promoters, yielding the two forms of porphobilinogen deaminase mRNAs. Our present finding may have some relevance for further understanding the porphobilinogen deaminase deficiency in certain cases of acute intermittent porphyria with an enzymatic defect restricted in non-erythropoietic cells.     

Anaerobic degradation of papermill sludge in a two-phase digester containing rumen microorganisms and colonized polyurethane foam

Summary The use of reticulated polyurethane foam as a support material for the immobilization of methanogenic associations and its application to the anaerobic treatment of fine particulate solid wastes was investigated. The colonization of polyurethane support particles in a continuous upflow reactor fed on a mixture of acetate, propionate and butyrate, was both rapid and dense. The combination of rumen microorganisms and colonized support particles in a two-phase digester resulted in an efficient anaerobic decomposition of papermill sludge.     

Characterization oftrans-acting regulatory elements affecting the expression of Mn-superoxide dismutase (sodA) inEscherichia coli

Escherichia coli strains harboringtrans-acting mutations affecting the expression of Mn-superoxide dismutase (SOD) gene (sodA) were used to studysodA regulation. Complementation studies revealed that eitherarc (aerobic respiratory control) orfur (ferric uptake regulation) loci independently complemented anaerobic expression of asodA::lacZ protein fusion in one mutant strain (UV16). This mutant exhibited phenotypes (i.e., elevated outer membrane proteins, enzyme activity, and dye sensitivity) typical offur andarc mutants. When these mutations were introduced into an otherwise wild-type background, anaerobicsodA expression occurred only when botharc andfur mutations were present simultaneously, suggesting cooperative roles of Fur and Arc insodA repression. The reconstructedfur arcA andfur arcB double mutants were still inducible by iron chelators, suggesting the possible involvement of another iron-containing repressor protein. A second independent mutant strain harboring atrans-acting regulatory mutation (UV14) was only partially complemented by multicopy plasmids carryingfur ⁺ orarc ⁺ genes, implicating other genetic elements insodA regulation.     

Morphology of the pericellular capsule in articular cartilage revealed by hyaluronidase digestion

To allow a more valid comparison between our previous ultrastructural data and the immunolocalization of type IX and other minor collagen species in cryosectioned cartilage, we examined both normal and testicular hyaluronidase-digested canine tibial cartilage by electron microscopy. Removal of matrix proteoglycans caused the pericellular capsule to collapse against the cell surface, suggesting that its normal anatomical position is mediated by pericellular matrix hydration. Detailed examination of the pericellular capsule and pericellular channel revealed fine, faintly banded fibrils and an amorphous component somewhat similar in structure to basement membrane collagens. Matrix vesicles and the electron-dense material of the interterritorial matrix were only partially digested by hyaluronidase. We propose that the pericellular capsule is composed of a "felt-like" network of minor collagen species which act synergistically to maintain both the composition of the pericellular matrix and the integrity of the chondrocyte/pericellular matrix complex during compressive loading.     

Ultrastructural changes in the rat thyroid gland during iodine deficiency

Thyroid ultrastructure changes were studied during the course of a low iodine diet in rats. At day 20, follicles were normal, but a number of them contained cells of higher density and with greatly elongated microvilli. Endoplasmic reticulum cisternae were frequently dilated. From day 20 until day 80, the most characteristic changes in the thyroid cells were the progressive accumulation of subapical peroxidase-positive exocytotic vesicles. After 80 days of the low iodine treatment, Golgi apparatuses were very active. Cell division could be observed. At this stage, exocytotic vesicles were generally very abundant. These data suggest that the remarkable accumulation of subapical exocytotic vesicles between day 20 and day 120 might represent an adaptation to the moderate and gradual increase in TSH stimulation that occurs in the conditions of low iodine diet.     

Maintenance and reversibility of active albumin secretion by adult rat hepatocytes co-cultured with another liver epithelial cell type

When adult rat hepatocytes were co-cultured with another liver epithelial cell type in a medium supplemented or not with fetal calf serum (FCS), it was found that 1. They survived for more than 2 months 2. Albumin secretion levels remained high over the whole culture period 3. Decreased secretion might be reversed 4. This protein secretion activity appeared to be dependent upon both the presence of cell-cell contacts and the production of an extracellular material. The results demonstrate for the first time long-term stabilization and reversibility of a specific function (albumin secretion) at high levels by adult hepatocytes cultured in serum-free medium and suggest that both the presence of other liver cell type(s) and the production of an extracellular matrix are needed for the maintenance of specific functions in cultured hepatocytes.     

The inconsistencies of present-day mathematical-physical methods pertaining to current generators in volume conductors used in electrocardiography

The present-day practices of electrocardiography and vectorardiography are based upon the theory that the surface potential differences can be assumed to be due to a single dipole inside the body. It is shown in this paper that a dipole cannot account for all the surface potentials due to realistic current generators, and hence the determination of the current generator from surface potential measurements based upon such a theory will lead to inconsistent representations of the heart for one and the same subject. To demonstrate this point two eccentric dipoles of different strengths and locations representing two muscle fibers are taken to be the current generator in a homogeneous spherical conductor. The exact surface potentials are then expressed by means of the “interior sphere theorem” of the authors. With these expressions the magnitude, direction, and location of the resultant dipole are determined by the method of D. Gabor and C. V. Nelson (J. App. Physics,25, 413–16, 1954). The surface potentials due to this resultant dipole are again exactly expressed by means of the “interior sphere theorem” and compared with those due to the eccentric dipoles assumed. It can be seen that the differences can be considerable. It is suggested that the multipole model of the authors (Bull. Math. Biophysics,20, 203–16, 1958) be used as a more accurate and the only unique representation of the heart. This investigation was supported by the National Heart Institute under a research grant H-2263(c).     

Cleavage of farnesylated COOH-terminal heptapeptide of mouse N-ras by brain microsomal membranes: evidence for a carboxypeptidase which specifically removes the COOH-terminal methionine.

Brain microsomal membranes are capable of sequentially removing Met, Leu and Val from a chemically synthesized COOH-terminal heptapeptide (propionyl-Gly-Ser-Pro-(farnesyl-Cys)-Val-Leu-Met) of mouse N-ras protein. The carboxypeptidase generating Met displays maximum activity at neutral pH and shows high affinity for the farnesylated substrate (Km = 73 microM) as compared to its non farnesylated precursor (Km = 600 microM). The results of inhibitor action suggest that the membrane carboxypeptidase is a novel, probably thiol-dependent, serine type peptidase.