Development and characterization of continuous avian cell lines depleted of mitochondrial DNA

Summary Populations of quail and chicken cells were treated with ethidium bromide, an inhibitor of mitochondrial DNA replication. After long-term exposure to the drug, the cell populations were transferred to ethidium bromide (EtdBr)-free medium, and cloned. Clones HCF7 (quail) and DUS-3 (chicken) were propagated for more than a year, and then characterized. Analysis of total cellular DNA extracted from these cells revealed no characteristic mitochondrial DNA molecule by Southern blot hybridization of HindIII- or AvaI-digested total cellular DNA probed with cloned mitochondrial DNA fragments. Reconstruction experiments, where a small number of parental cells was mixed with HCF7 cells and DUS-3 cells before extraction of total cellular DNA, further strengthen the notion that the drug-treated cells are devoid of mitochondrial DNA molecules. The cell populations were found to proliferate at a moderately reduced growth rate as compared to their respective parents, to be auxotrophic for uridine, and to be stably resistant to the growth inhibitory effect of EtdBr and chloramphenicol. At the ultrastructural level, mitochondria were considerably enlarged and there was a severe reduction in the number of cristae within the organelles and loss of cristae orientation. Morphometric analysis revealed a fourfold increase of the mitochondrial profile area along with a twofold decrease of the numerical mitochondrial profiles. Analysis of biochemical parameters indicated that the cells grew with mitochondria devoid of a functional respiratory chain. The activity of the mitochondrial enzyme dihydroorotate dehydrogenase was decreased by 95% and presumably accounted for uridine auxotrophy. This work was supported by a grant from the Medical Research Council of Canada.     

Species specificity of iron delivery in hybridomas

Summary Studies with Human x Human (HxH), Human x Mouse (HxM), and Mouse x Mouse (MxM) hybridomas have enabled us to define specific factors that affect hybridoma growth in a species-specific manner. Three transferrins and three lipophilic iron chelates have been tested for their ability to support hybridoma proliferation and antibody production. The results of these studies demonstrate that HxH hybridomas do not respond to bovine transferrin a⁺ concentrations up to 100 μg/ml and are approximately 100-fold less responsive to mouse transferrin than to human transferrin. HxM and MxM hybridomas respond equally to human or mouse transferrin but are 100-fold less sensitive to bovine transferrin. An antibody to the human transferrin receptor inhibited the growth-promoting activity of human or mouse transferrin on HxH hybridomas but was ineffective on HxM hybridomas. This semonstrated the functionality of the human transferrin receptor in HxH hybridomas and that human, mouse, and bovine transferrin were interacting through the mouse transferrin receptor in HxM hybridomas. HxH and HxM hybridomas respond similarly to three different iron chelates exhibiting 80 to 110% of the growth response to human transferrin. MxM hybridomas fail to respond to the iron chelates at similar concentrations, suggesting that the human genome present in the other hybridoma species confers a unique ability for utilizing iron when delivered in this form.     

Characteristics of dynamic postural reactions in the locust hindleg

Summary The use of the locust (Schistocerca americana) hindleg in postural control was examined in animals that stood on a repeatedly swayed vertical substrate. Myograms were recorded from leg muscles and the angle of the femoro-tibial joint was monitored photographically. Two discrete strategies were observed,; in compensatory reactions the hindleg was held in place, while in flexion reactions, the leg was moved, most often to complete flexion of the femoro-tibial joint. Tightly coupled, rhythmic bursting occurred in the flexor and levator muscles of the leg during compensatory reactions. Bursting was initiated repeatedly when the substrate was being pulled away from the animal. Bursting was correlated with subsequent decreases in the rate of change of the femorotibial joint angle. Compensatory and flexion reactions occurred preferentially in different ranges of joint angles: most often, compensatory reactions occurred in the midrange, while flexion reactions were elicited in the extremes of joint angle. These differences may be due to the mechanical advantages of the tibial muscles and the leg may be moved to full flexion because of a locking mechanism of the flexor muscle tendon. These reactions are compared with known reflexes of hindleg proprioceptors and contrasted with similar responses of vertebrates.     

A comparison of net and gross rates of oxygen production as a function of light intensity in some natural plankton populations and in a Synechococcus culture

In vitrorates of gross and net oxygen production were measuredas a function of light intensity in some plankton communitiescollected from Bedford Basin, Nova Scotia, and in a monoclonalculture of Synechococcus. The rate of gross oxygen productionwas measured by a technique in which the stable oxygen isotope,¹⁸O, serves as a photosynthetic tracer Net oxygen productionwas measured by automated Winkler technique. The rate of communityrespiration in the light was then determined by the differencebetween gross and net rates of oxygen production. In the naturalpopulations examined, neither gross nor net oxygen productionrates were significantly inhibited at the highest light intensitymeasured (500–800 µE m^–2 s^–1) In a samplein which the dark respiration rate was small relative to themaximal rate of production [P_max;sensu Platt et al (1980) JMar. Res., 38, 687–701] the rates of ‘light’respiration were 3 times greater. In two other communities,with high rates of dark respiration relative to P_maxthe ratesof ‘light’ respiration were closer to rates of darkrespiration. In the Synechococcus clone, both gross and netoxygen production rates were inhibited at high light intensities.Rates of ‘light’ respiration were found to varyas a function of light intensity. The greatest rates of respirationwere measured in samples incubated at light intensities thatwere just saturating (100 µE m^–2 s^–1). Therates of ¹⁴C production were also measured as a function oflight intensity The photosynthetic quotients, based on ¹⁴C productionrates and gross oxygen production rates, average 1 9     

Characterization of a human cervical carcinoma-associated antigen by lectin binding and immuno-electron microscopy

Summary The specific binding and nature of the epitope recognized by monoclonal antibody (Mab) 1H10, which binds an antigen expressed on human cervical tumors, was characterized by enzyme digestion, lectin competition assay and immuno-electron microscopy. Membrane homogenates of CaSki cervical carcinoma cells were digested with various enzymes, then analysed by SDS-PAGE and immunoblotting. Cells grown on coverslips were treated with various enzymes and in situ binding of Mab 1H10 to cells was analysed by electron microscopy. The ability of lectin-conjugates to block Mab 1H10 binding to CaSki cells was also examined. Treatment of samples with sodium periodate abrogated antigen recognition by Mab 1H10. Neuraminidase and hyaluronidase digestion decreased but did not eliminate Mab 1H10 binding to cells in situ. Chondroitinase ABC digestion, in contrast, removed Mab 1H10 binding sites both in vitro and in situ. Trypsin and chymotrypsin digestion of cell membrane homogenates decreased the molecular weight of the Mab 1H10 antigen but did not decrease the binding intensity. Wheat germ agglutinin (WGA) strongly bound to CaSki cells and partially blocked Mab 1H10 binding, indicating that the antigen contains N-acetyl-galactosamine residues at or near the epitope recognized by Mab 1H10. Ricinus communis agglutinin (RCA) exhibited a similar binding pattern to WGA. However, concanavalin A bound only weakly to CaSki cells and was ineffective at blocking Mab 1H10 binding. The tumor-associated antigen recognized by Mab 1H10 is concluded to be a chondroitin sulphate glycoprotein or proteoglycan rather than a mucopolysaccharide or lipoprotein.     

Properties of heart fibroblasts of adult rats in culture

Summary In the heart of the adult rat, fibroblasts are mainly responsible for the synthesis and deposition of the collagenous matrix. Because these cells in vitro may serve as an important model system for studies of collagen metabolism in heart tissue, we have cultured and characterized rat-heart fibroblasts from young adult and old animals. Conditions included use of media of different compositions with and without addition of ascorbate. Cell used were either cultured directly from fresh tissues or thawed previously frozen cells. Cultured cells were studied with respect to growth properties, morphology and ultrastructure and patterns of collagen. Heart fibroblasts generally resembled fibroblasts cultured from other tissues, but were more like skeletal muscle fibroblasts in that they deposited, in addition to type I collagen, type IV collagen and laminin. The fibroblasts showed a typical appearance in phase-contrast microscopy and electron microscopy. In the case of cells grown with added ascorbate, aligned collagen fibrils in the extracellular matrix showed a periodicity typical of type I collagen. The deposition of type I collagen occurred only in medium supplemented with ascorbate, and in that circumstance increased as a function of time past confluence; this was independent of the age of the animal from which the cells were obtained or of other changes of medium composition studied. Immunofluorescence studies with specific antibodies revealed that the cells deposited types I and IV collagens, laminin and fibronectin. In contrast to the case of type I collagen, the deposition of type IV collagen occurred in cells grown either with or without ascorbate. Direct observation of type IV collagen is consistent with the previous finding of type IV mRNA in cardiac fibroblasts in situ and in freshly isolated populations of these cells.     

The codon 408 mutation associated with haplotype 2 is predominant in Polish families with phenylketonuria

Summary The incidence of phenylketonuria (PKU) in the western part of Poland is 1 in 5000 live births. Restriction fragment length polymorphism (RFLP) haplotypes at the phenylalanine hydroxylase locus have been analysed in 46 Polish families with PKU. Among 43 fully-informative families 16 RFLP haplotypes were identified. Haplotype 2 is the most frequently (62%) associated with Polish PKU alleles, and the codon 408 mutation is in complete linkage disequilibrium with this haplotype in Poland. This finding is in agreement with observations in other eastern European countries (German Democratic Republic, Czechoslovakia, and Hungary) and in contrast to the genotype distribution observed in western European countries. The present observation suggests the spread of classical PKU, due to the codon 408 mutation associated with haplotype 2, from east to west in European populations. Perhaps more important for genetic counselling, 62% of all PKU chromosomes in the Polish population can now be detected using only one mutantspecific oligonucleotide probe.     

Glycine betaine and proline are the principal compatible solutes ofStaphylococcus aureus

The foodborne pathogenStaphylococcus aureus is distinguished by its ability to grow within environments of extremely high osmolarity (e.g., foods with low water activity values). In the present study, we examined the accumulation of intracellular organic solutes withinS. aureus strain ATCC 12600 when cells were grown in a complex medium containing high concentrations of NaCl. Consistent with previous reports [Measures JC (1975) Nature 257:398–400; Koujima I, et al. (1978) Appl Environ Microbiol 35:467–470; and Anderson CB, Witter LD (1982) Appl Environ Microbiol 43:1501–1503], intracellular proline was found to accumulate to high concentrations. However, NMR spectroscopy of cell extracts revealed glycine betaine to be the predominant intracellular organic solute accumulated within cells grown at high osmolarity. In additional experiments, we examined the growth rate ofS. aureus in a defined medium of high osmolarity and found it to be stimulated significantly by the presence of either exogenous proline or glycine betaine. Highest growth rates were obtained when the defined medium was supplemented with glycine betaine.     

Postnatal expression of glutamate decarboxylases in developing rat cerebellum

The recent identification of two genes encoding distinct forms of the GABA synthetic enzyme, glutamate decarboxylase (GAD), raises the possibility that varying expression of the two genes may contribute to the regulation of GABA production in individual neurons. We investigated the postnatal development the two forms of GAD in the rat cerebellum. The mRNA for GAD₆₇, the form which is less dependent on the presence of the cofactor, pyridoxal phosphate (PLP), is present at birth in presumptive Purkinje cells and increases during postnatal development. GAD₆₇ mRNA predominates in the cerebellum. The mRNA for GAD₆₅, which displays marked PLP-dependence for enzyme activity, cannot be detected in cerebellar cortex by in situ hybridization until P7 in Purkinje cells, and later in other GABA neurons. In deep cerebellar nuclei, which mature prenatally, both forms of GAD mRNA can be detected at birth. The amounts of immunoreactice GAD and GAD enzyme activity parallel changes in mRNA levels. We suggest that the delayed appearance of GAD₆₅ is coincident with synapse formation between GABA neurons and their targets during the second postnatal week. GAD₆₇ mRNA may be present prior to synaptogenesis to produce GABA for trophic and metabolic functions.Special issue dedicated to Dr. Eugene Roberts.