Eclectic mechanisms of heme regulation of hematopoiesis

Regulatory features of heme (ferroprotoporphyrin IX) on hematopoietic growth/differentiation and related processes are reviewed. It is emphasized that expressions of specific erythroid and nonerythroid heme biosynthetic and degradatory enzymes are required, and the regulatory processes whereby this occurs is considered. The specificity of heme, relationship to cellular events such as differentiation, response to growth factors, oncogene and receptor expression, and how heme counteracts toxic effects such as viral growth are all discussed. The significance of heme in the hemopoietic bone marrow microenvironment and growth factor network are considered. Finally, the third pathway for arachidonic acid metabolism via the heme-cytochrome P450 monooxygenase system, in addition to cyclooxygenase and lipoxygenase, by bone marrow adherent cells and its role in cellular differentiation is briefly reviewed.     

Quantification and analysis of reverse mutations at the hgprt locus in Chinese hamster ovary cells

We describe an assay for the quantification of reverse mutations at the hypoxanthine-guanine phosphoribosyltransferase (hgprt) locus in Chinese hamster ovary cells utilizing the selective agent L-azaserine (AS). Conditions are defined in terms of optimal AS concentration, cell density, and phenotypic expression time. After treatment, replicate cultures of 10⁶ cells are allowed a 48-h phenotypic expression time in 100-mm plates. AS (10 μM) is then added directly to the growing culture and AS-resistant (AS^r) cells form visible colonies. This assay is used to quantify ICR-191-, ICR-170-, and N-ethyl-N-nitrosourea-induced reversion of independently isolated HGPRT^? clones. The AS^r phenotype is characterized both physiologically and biochemically. All AS^r clones isolated are stably resistant to AS and aminopterin but sensitive to 6-thioguanine. They also have re-expressed HGPRT enzyme. In addition, several revertants are shown to contain altered HGPRT. The data provide further evidence that ICR-191 and ICR-170 cause structural gene mutations in mammalian cells and also suggest that ICR-191, ICR-170, and N-ethyl-N-nitrosourea induce similar types of mutations in Chinese hamster ovary cells.     

Neue diterpene und neue dihydrochalkon-derivate sowie weitere inhaltsstoffe aus Helichrysum-arten

The investigation of 25 further Helichrysum species afforded in addition to known compounds a new diterpene, an oxo-geranyllinalol, a second one, most probably a cembrene derivative, and three dihydrochalkone derivatives. The distribution of the constituents in South African Helichrysum species is discussed briefly.     

Transfer of genes to Chinese hamster ovary cells by DNA-mediated transformation

We have transferred DNa to Chinese hamster ovary (CHO) cells by DNA-mediated transformation. CHO tk- cells were transformed with the clones gene for herpes simplex virus thymidine kinase (HSV-tk) and were found to have a 50-fold lower frequency of transformation than mouse Ltk- cells at the same DNA dosage. By altering the amount of tk gene and carrier DNA present, frequencies of up to 5 x 10(-5) were obtained. CHO HSV-tk+ transformants were very stable, and in several clones the HSV-tk gene copies integrated in higher-molecular-weight DNA. These cells also exhibited cotransformation for unselected markers. CHO lines were also transformed at a frequency of 10(-4) with the bacterial gene Ecogpt in a SV40-pBR322 vector. CHO tk-cells could be transformed at a frequency of 10(-7) with cellular DNA isolated from CHO tk+ cells. CHO cells offer a well-defined genetic system within which to transfer either cloned or whole cellular DNAs.     

Polypeptide synthesis by mitoplasts isolated from mouse liver

Polypeptide synthesis by mouse liver mitochondria was studied by incubating purified mitoplasts (mitochondria treated with digitonin) with [³⁵S]methionine. The products were separated either by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, or by isoelectric focusing, followed by SDS polyacrylamide gel electrophoresis. At least 14 distinct bands with molecular weights (mol. wt) ranging from about 8 000 to about 70 000 were found upon radioautography of the gels. When the samples were incubated in the presence of chloramphenicol, only a single weak band was found, whereas the protein pattern was unaffected by the presence of cycloheximide in the medium. The newly synthesized proteins were all acidic and evidence was obtained that they were hydrophobic in nature. Virtually all the labelled polypeptides were present in the membrane fraction, whereas the matrix showed little radioactivity. The data indicate that the proteins synthesized by mammalian mitochondria, like those in yeast, are components of the inner mitochondrial membrane. One protein of mol. wt 22 000 D was detected in the incubation medium. Since more of this component was present in the medium than in the pelleted mitoplasts and since this protein was not found in the matrix fraction of sonicated mitoplasts, it is believed that it had been excreted from the inner mitochondrial membrane. The finding that the number of proteins synthesized in mitoplasts isolated from mouse liver is considerably higher than that synthesized in yeast mitochondria reflects a most efficient utilization of the mammalian mitochondrial genome.     

Effect of long term starvation on the biochemical composition of the muscles and hepatopancreas in the crab Menippe rumphii (Fabricius) (Crustacea: Brachyura)

During the process of long term starvation both muscles and hepatopancreas are affected in their biochemical composition at different rates. During early days of starvation an increase in the muscular and hepatopancreatic glycogen is observed. At the same time a simultaneous decrease in the muscular lipid content is also observed. At a slightly later period a decrease in the hepatopancreatic lipid content is also noticed. This amount of decrease is slow in the early days of starvation and rapid in later days. Decrease in the muscular and hepatopancreatic protein content is observed when there is not an adequate quantity of hepatopancreatic lipid to be consumed.     

On the chromatographic heterogeneity of human fetal hemoglobin.

Minor fetal hemoglobins in red cell hemolysates of newborn and adults with elevated levels of Hb F have been separated and quantitated by Biorex 70 column chromatography. In addition to Hb F1, other minor hemoglobin zones eluting before F1, pre-F1, and after F1, post-f1 have been observed. The relative amounts of the two pre-F1 zones and F1 are higher in the red cells of adults with 97--100% Hb F (homozygous hereditary persistence of fetal hemoglobin, homozygous deltabeta-thalassemia and homozygous beta0-thalassemia) than in the red cells of an adult with homozygous beta+-thalassemia with 66% Hb F, a child with a trisomy-D-13 having 38% Hb F, and in two newborn. Hb F was glycosylated in vitro with [14C]glucose or [14C] glucose 6-phosphate, and was acetylated using chicken reticulocyte lysate or a crude acetyltransferase preparation isolated from the same lysate with [14C]acetyl-CoA as substrate. Chromatographic analyses indicated that the Hb F1 zone can be formed both by glycosylation and acetylation of Hb F, and that pre-F1 zones can be products of the reaction of Hb F with phosphorylated glycolytic intermediates. Biosynthesis of minor hemoglobins in reticulocytes was studied with [14C]leucine in the presence and absence of cycloheximide and by pulse-chase. The resulting data indicate that Hb F1 synthesis is dependent upon Hb F synthesis and that the posttranslational modification may take place at an early stage in Hb F synthesis.     

Incorporation of 3H from delta-(L-alpha-amino (4,5-3H)adipyl)-L-cysteinyl-D-(4,4-3H)valine into isopenicillin N.

1. delta-(L-alpha-Amino[4,5-3H]adipyl)-L-cysteinyl-D-[4,4-3H]valine has been synthesized from its constituent amino acids, the L-alpha-amino[4,5-3H]adipic acid being obtained by reduction with 3H2 of methyl 5-acetamido-5,5-diethoxycarbonylpent-2-enoate and subsequent decarboxylation and hydrolysis. 2. In a cell-free system prepared by lysis of protoplasts of Cephalosporium acremonium 3H was incorporated from the doubly labelled tripeptide into a compound that behaved like penicillin N or isopenicillin N. The relative specific radioactivities of the alpha-aminoadipyl and penicillamine moieties of the penicillin were the same (within experimental error) as those of the alpha-aminoadipic acid and valine residues respectively of the tripeptide. 3. The behaviour of the labelled alpha-aminoadipic acid from the penicillin to the L-amino acid oxidase of Crotalus adamanteus venom showed that it was mainly L-alpha-aminoadipic acid. 4. The results are consistent with the hypothesis that the carbon skeleton of the LLD-tripeptide is incorporated intact into the penicillin molecule and that the first product is isopenicillin N.     

The synergism of cardiotoxin and phospholipase A2 in hemolysis

The synergistic effect of exogenous cobra phospholipase A₂ on the hemolysis rate of guinea pig erythrocytes by highly purified snake venom cardiotoxins was investigated. In the presence of phospholipase A₂ the reaction was not only faster and had a lower activation energy but followed a sigmoidal instead of a linear time course. Similar results were obtained using porcine pancreatic phospholipase A₂. Significantly, addition of even a trace of cobra phospholipase A₂ (approx. 0.1%, w/w) was sufficient to bring about the full synergistic effect, emphasizing the stringent purity requirements for any meaningful investigation of cardiotoxin's own action. The possibility that the action of cardiotoxin on its own may involve the stimulation of an endogenous phospholipase is discussed in the light of the results obtained with exogenous cobra enzyme.