Doxorubicin induces calcium release from terminal cisternae of skeletal muscle. A study on isolated sarcoplasmic reticulum and chemically skinned fibers

In this study, we investigated the effect of the anticancer drug doxorubicin on Ca2+ fluxes of isolated highly purified sarcoplasmic reticulum fractions (longitudinal tubules and terminal cisternae (Saito, A., Seiler, S., Chu, A., and Fleischer, S. (1984) J. Cell Biol. 99, 875-885] and of chemically skinned skeletal muscle fibers of the rabbit. In terminal cisternae, doxorubicin inhibits Ca2+ uptake (IC50 at 0.5 microM) and increases 2.6-fold Ca2+-dependent ATPase rate (half-maximal activation at 3 microM) and unidirectional Ca2+ efflux (8-fold stimulation at 25 microM). On the contrary, doxorubicin is without effect on longitudinal tubules. In skinned muscle fibers, doxorubicin induces rapid and transient Ca2+ release, as measured by tension development (half-maximal stimulation at 6 microM), which is completely and reversibly inhibited by ruthenium red, a known inhibitor of Ca2+ release from isolated terminal cisternae. Doxorubicin has no effect on the sarcoplasmic reticulum Ca2+ pump and on the contractile apparatus of skinned muscle fibers. It is concluded that doxorubicin activates Ca2+ release from sarcoplasmic reticulum and opens a Ca2+ efflux pathway (Ca2+ channel) selectively localized in terminal cisternae. Doxorubicin might interact with Ca2+ channels involved in physiological Ca2+ release.     

Frog brain expresses a 60 KDa Ca2+ binding protein similar to mammalian calreticulin

The present report was undertaken in an effort to characterize the nature of Ca2+ binding protein(s) in the central nervous system of less evolved vertebrates. In particular we investigated whether the brain microsomal fraction of Rana esculenta expresses calsequestrin, calreticulin and/or other related Ca2+ binding protein(s). We found that a 60 KDa protein having an NH2-terminal amino acid sequence similar to mammalian calreticulin is the major microsomal Ca2(+)-binding protein.     

Infections Caused by <Emphasis Type="Italic">Fusarium</Emphasis> Species in Pediatric Cancer Patients and Review of Published Literature

Fusarium species have emerged as responsible for a broad spectrum of infections, including superficial, locally invasive and disseminated ones, especially in the hospital environment. Since there are few reports of invasive and disseminated fusariosis in children, the aim of this study was to report four cases of nosocomial infection caused by this microorganism in children with cancer hospitalized in a public children’s hospital located in Brazil. Two of these patients were female and two were male. All patients presented febrile neutropenia, while three patients had acute lymphocytic leukemia and one patient had Wilms’ tumor as underlying disease. In two cases, fungi were isolated from blood and identified as Fusarium oxysporum species complex after phenotypic and genotypic studies, while in two other cases fungi were isolated from skin biopsies and identified as Fusarium solani species complex. One patient died 12 days after the onset of cutaneous lesions. All isolates, after susceptibility testing, presented high levels of minimum inhibitory concentration for itraconazole, voriconazole and amphotericin B. Considering the emergence of filamentous fungi as etiologic agents of nosocomial infections, health professionals should be aware of the problems these infections, especially fungal ones, may cause to debilitated patients.     

Melanocortins and opioids modulate early postnatal growth in rats

This study was undertaken to investigate the effects of melanocortins and opioids on rat early postnatal body and organ growth. Among melanocortins tested desacetyl-alpha-melanocyte-stimulating hormone (alpha-MSH) at dosages of 0.3 and 3 micrograms/g/day was effective in stimulating neonatal growth with a weight gain of 7 and 5.6%, respectively, after 2 weeks of treatment. Likewise, a weight rise of 4.2 and 3% was obtained with 3 micrograms/g/day of both alpha-MSH and Nle4-D-Phe7 alpha-MSH. As far as opioids were concerned, while N-acetyl-beta-endorphin (beta-End) was ineffective, the activity of beta-End was dependent on dosage. Indeed, newborns treated with 0.03 microgram/g/day showed a slight, but significant, increase in weight, whereas a marked decrease in growth followed treatment with 0.3 and, mainly, 3 micrograms/g/day, with a final weight loss of 3.4 and 5.5%, respectively. All melanocortins exerted a positive action on muscular and brain trophism and, in addition, desacetyl-alpha-MSH also induced a rise of fat deposits. On the contrary, while the 0.03 microgram/g/day beta-End dose caused an increase in muscular and brain weight, the higher dosages of the opioid were detrimental, not only for muscle and brain, but also for both liver and spleen weight. A slight, although significant (P < 0.05), enhancement of serum dehydroepiandrosterone sulfate (DHEAS) level was found after the injection of 0.3 microgram/g desacetyl-alpha-MSH, whereas both the 0.3 and 3 micrograms/g doses of desacetyl-alpha-MSH and the 3 micrograms/g dose of alpha-MSH determined the rise of plasma androstenedione (P < 0.05). All tested melanocortins and opioids failed to modify the concentrations of corticosterone. Our results suggest that melanocortins and opioids can modulate early postnatal growth in rats either by direct or indirect mechanisms.     

Red wine activates plasma membrane redox system in human erythrocytes

In the present study, we report that polyphenols present in red wine obtained by a controlled microvinification process are able to protect human erythrocytes from oxidative stress and to activate Plasma Membrane Redox System (PMRS). Human plasma obtained from healthy subjects was incubated in the presence of whole red wine at a concentration corresponding to 9.13–73?μg/ml gallic acid equivalents to verify the capacity to protect against hypochlorous acid (HOCl)-induced plasma oxidation and to minimize chloramine formation. Red wine reduced hemolysis and chloramine formation induced by HOCl of 40 and 35%, respectively. PMRS present on human erythrocytes transfers electrons from intracellular molecules to extracellular electron acceptors. We demonstrated that whole red wine activated PMRS activity in human erythrocytes isolated from donors in a dose-dependent manner with a maximum at about 70–100?μg/ml gallic acid equivalents. We also showed that red wine increased glutathione (GSH) levels and erythrocytic antioxidant capacity, measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH) quenching assay. Furthermore, we reported that GSH played a crucial role in regulating PMRS activity in erythrocytes. In fact, the effect of iodoacetamide, an alkylating agent that induces depletion of intracellular GSH, was completely counteracted by red wine. Bioactive compounds present in red wine, such as gallic acid, resveratrol, catechin, and quercetin were unable to activate PMRS when tested at the concentrations normally present in aged red wines. On the contrary, the increase of PMRS activity was associated with the anthocyanin fraction, suggesting the capacity of this class of compounds to positively modulate PMRS enzymatic activity.     

DNaseI-hypersensitive sites at promoter-like sequences in the spacer of Xenopus laevis and Xenopus borealis ribosomal DNA.

We have detected a DNAseI hypersensitive site in the ribosomal DNA spacer of Xenopus laevis and Xenopus borealis. The site is present in blood and embryonic nuclei of each species. In interspecies hybrids, however, the site is absent in unexpressed borealis rDNA, but is present normally in expressed laevis rDNA. Hypersensitive sites are located well upstream (over lkb) of the pre-ribosomal RNA promoter. Sequencing of the hypersensitive region in borealis rDNA, however, shows extensive homology with the promoter sequence, and with the hypersensitive region in X. laevis. Of two promoter-like duplications in each spacer, only the most upstream copy is associated with hypersensitivity to DNAaseI. Unlike DNAaseI, Endo R. MspI digests the rDNA of laevis blood nuclei at a domain extending downstream from the hypersensitive site to near the 40S promoter. Since the organisation of conserved sequence elements within this "proximal domain" is similar in three Xenopus species whose spacers have otherwise evolved rapidly, we conclude that this domain plays an important role in rDNA function.     

Relation of C-6 glial cells in culture to myelin.

C-6 glial cells were shown to contain enriched in their plasma membranes an enzyme, 2':3'-cyclic nucleotide 3'-phosphohydrolase, characteristic of myelin, and, in addition, proteolipid protein and two basic proteins that are identical in their electrophoretic mobilities with the respective proteins found in myelin. The data indicate that C-6 cells exhibit features of myelin-producing glia as well as astrocytes.     

Regulation of hepatic fatty acid synthetase in the obese-hyperglycemic mutant mouse.

Regulation of fatty acid synthetase has been studied in the obese-hyperglycemic mouse and compared with regulation in non obese, littermate control animals. The mechanisms underlying the regulatory changes were defined by immunochemical techniques. Several major conclusions are justified from the data obtained: (1) Although the hepatic specific activity of fatty acid synthetase is higher in obese than in non obese animals pair-fed chow, no difference in hepatic activities is apparent in animals pair-fed the fat-free diet; (2) The higher enzymatic activity in obese animals fed chow is related to a higher content of enzyme, and this higher content is associated with a higher rate of enzyme synthesis; (3) The decrease in hepatic synthetase activity with starvation is distinctly more striking in non obese than in obese animals, and the changes in activity reflect changes in content of enzyme; (4) With starvation there is a decrease in synthesis of enzyme in obese and non obese animals, but only in non obese animals is there also a marked increase in the rate of synthetase degradation (t1/2 = 24 h during starvation, t1/2 = 76 h during normalfeeding); (5) Refeeding starved mice a fat-free diet results in a more striking increase in hepatic synthetase activity in non obese than in obese animals; (6) Administration of triiodothyronine causes a more marked increase in hepatic synthetase activity in non obese than in obese animals. The data thus define a variety of differences in regulation of hepatic fatty acid synthetase in mutant and normal animals. The roles of enzyme synthesis and degradation in the etiology of these differences are defined, and possible mechanisms underlying regulation of synthetase synthesis and degradation in normal mammalian liver are suggested by the observations.     

Oligodendroglial Differentiation in Glial Primary Cultures: Requirement for Mevalonate

The oligodendroglial enzyme, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP), is a valuable marker for expression of oligodendroglial differentiation in glial primary cultures, and the inducibility of this enzyme by dibutyryl-3',5'-cyclic AMP (dBcAMP) appears to be limited to immature or developing oligodendroglia. To investigate the relationship between the induction of CNP and the sterol biosynthetic pathway, primary cultures of glia dissociated from the brains of newborn rats were maintained in 10% fetal calf serum (FCS) and exposed to 1 mM dBcAMP on day 7 in culture. Cultures so treated for either 48 h or 72 h demonstrated a three- to fourfold induction of CNP specific activity. The magnitude of this induction was not affected when the cholesterol content of the culture medium was reduced by greater than 95% by placing the cultures in 10% lipoprotein-poor serum rather than 10% FCS during the exposure to dBcAMP. Mevinolin (10 microM), a specific inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, the rate-limiting enzyme of the sterol biosynthetic pathway, completely inhibited the induction of CNP by dBcAMP, while not affecting either the accumulation of cellular protein per flask or rate of protein synthesis. Simultaneous addition of mevalonate (20 mM) prevented the inhibition of the induction of CNP by mevinolin. However, simultaneous addition of low-density lipoprotein sufficient to increase the cholesterol content of the medium 80-fold failed to correct mevinolin's inhibition of the induction of CNP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dolichol-Linked Glycoprotein Synthesis in G1 Is Necessary for DNA Synthesis in Synchronized Primary Cultures of Cerebral Glia

Primary cultures of newborn rat cerebrum, which are composed of glial cells (principally astroglia), were used for examining the relationship between dolichol-linked glycoprotein synthesis and DNA synthesis in developing cerebral glia. The cells were synchronized by reducing the content of fetal calf serum in the culture medium from 10 to 0.1% (vol/vol) for 48 h between days 4 and 6 in culture. Reversal of the quiescent state by return of the cultures to 10% serum causes a marked increase in DNA synthesis 12-24 h later. A sharp increase in glycoprotein synthesis (incorporation of [3H]mannose) occurred in the first 12 h after serum repletion, preceding the increase in DNA synthesis. Tunicamycin, an inhibitor of the dolichol-linked pathway to glycoprotein synthesis at the first committed step in oligosaccharide formation, promptly and completely prevented the increase in glycoprotein synthesis and, in addition, the subsequent increase in DNA synthesis. The effects of tunicamycin on glycoprotein and DNA syntheses were reversible, and no comparable effect on total protein synthesis was observed. When tunicamycin was added only during a temporally circumscribed period in G1, i.e., from 3 to 9 h after serum repletion, the increase in DNA synthesis between 12 and 24 h after repletion was still markedly inhibited, i.e., to approximately 45% of the value in untreated cultures. The data thus show that there is a requirement for dolichol-linked glycoprotein synthesis for the subsequent occurrence of DNA synthesis and that this requirement is expressed late in the G1 phase of the cell cycle.