Methylated DNA-binding protein is present in various mammalian cell types.

A DNA-binding protein from human placenta, methylated DNA-binding protein (MDBP), binds to certain DNA sequences only when they contain 5-methylcytosine (m5C) residues at specific positions. We found a very similar DNA-binding activity in nuclear extracts of rat tissues, calf thymus, human embryonal carcinoma cells, HeLa cells, and mouse LTK cells. Like human placental MDBP, the analogous DNA-binding proteins from the above mammalian cell lines formed a number of different low-electrophoretic-mobility complexes with a 14-bp MDBP-specific oligonucleotide duplex. All of these complexes exhibited the same DNA methylation specificity and DNA sequence specificity. From the extracts of rat and calf tissues, oligonucleotide protein complexes formed that also had the same specificity as human placental MDBP although they had a higher electrophoretic mobility probably due to digestion by proteases in the nuclear extracts. Although MDBP activity was found in various mammalian cell types, it was not detected in extracts of cultured mosquito cells and so may be associated only with cells with vertebrate-type DNA methylation.     

Ultrastructure of Babesia major vermicules from the tick Haemaphysalis punctata as demonstrated by negative staining.

The ultrastructure of Babesia major vermicules was studied in samples derived from the haemolymph of Haemaphysalis punctata adults and negatively stained with phosphotungstic acid. Most of the organelles observed were typical of those found in apicomplexan parasites. These were the apical complex with the polar ring and the ribs, micronemes and subpellicular microtubules. The number of ribs was 27 or 28. The outer membrane of the pellicle was composed of a large number of fibrils running along the length of the parasite. The inner membrane had large numbers of irregularly scattered holes. A cytoplasmic organelle similar to the granular body described in Theileria annulata ookinetes was seen for the first time in a B. major vermicule.     

Hybridization frequencies of different mammalian cell types by electrofusion

The efficiency of electrofusion of four types of cells: CHO, HeLa, mouse melanoma cells and human skin fibroblasts has been studied. The frequencies of fusion products were determined 1) directly in a closed flow-through fusion chamber after dielectrophoresis and pulsation; 2) after short-term postfusion cultivation period of 5 to 10 minutes; and 3) in various intervals up to 30 hours after fusion induction. No substantial differences were found in the rates of formation of heterokaryons and synkaryons between the individual cell types, and this confirmed the uniformity of the effects of electric fields on diverse cell membranes. After 5 hours of culture the yield of fusion products reached 15 to 35% in various cell combinations and the frequencies of synkaryons reached up to 7% in almost all the combinations studied 24 to 30 hours after fusion.     

Innervation pattern in jaw muscles of various mammalian chewing types

The intramuscular nerve branches of the masticatory muscles were investigated in different mammalian species such as Canis familiaris, Capreolus capreolus, Capra hircus africans, Bos primigenius forma taurus, Myocastor coypus MOLINA 1782, Sus scrofa domestica, Pan troglodytes, Homo sapiens. The ramifications of the nerves form a specific pattern that is adapted to the specialised muscle structures in all mammalian chewing type. The innervation pattern is fully consistent with the infrastructures of the muscles which are already fixed at birth. Differences between the ramification pattern in comparable muscles in different chewing types are only minor, not fundamental.     

Tyrosine hydroxylase positive perisomatic rings are formed around various amacrine cell types in the mammalian retina

Dopaminergic neurons of the central nervous system are mainly found in nuclei of the midbrain and the hypothalamus that provide subcortical and cortical targets with a rich and divergent innervation. Disturbance of signaling through this system underlies a variety of deteriorating conditions such as Parkinson's disease and schizophrenia. Although retinal dopaminergic signaling is largely independent of the above circuitry, malfunction of the retinal dopaminergic system has been associated with anomalies in visual adaptation and a number of retinal disorders. Dopamine (DA) is released mainly in a paracrine manner by a population of tyrosine hydroxylase expressing (TH⁺) amacrine cells (AC) of the mammalian retina; thus DA reaches virtually all retinal cell types by diffusion. Despite this paracrine release, however, the so called AII ACs have been considered as the main targets of DA signaling owing to a characteristic and robust ring‐like TH⁺ innervation to the soma/dendritic‐stalk area of AII cells. This apparent selectivity of TH⁺ innervation seems to contradict the divergent DAergic signaling scheme of other brain loci. In this study, however, we show evidence for intimate proximity between TH⁺ rings and somata of neurochemically identified non‐AII cells. We also show that this phenomenon is not species specific, as we observe it in popular mammalian animal models including the rabbit, the rat, and the mouse. Finally, our dataset suggests the existence of further, yet unidentified post‐synaptic targets of TH⁺ dendritic rings. Therefore, we hypothesize that TH⁺ ring‐like structures target the majority of ACs non‐selectively and that such contacts are wide‐spread among mammals. Therefore, this new view of inner retinal TH⁺ innervation resembles the divergent DAergic innervation of other brain areas through the mesolimbic, mesocortical, and mesostriatal signaling streams.

     

Rapid flow cytofluorometric analysis of mammalian cell cycle by propidium iodide staining

A rapid method for the flow microfluorometric determination of the DNA content per cell is described. Incubation of cells in a hypotonic solution of propidium iodide results in disruption of the cell membrane and rapid staining of nuclear chromatin. DNA distribution histograms generated from cells stained by this method are identical to those generated after fixation and RNase digestion. In contrast to some earlier described methods, the present technique is rapid (5 min of processing), requires a minimal amount of material, and avoids formation of cell clumps.     

Acridine orange staining of the mammalian fibroblast cell coat

Summary Acridine orange selectively binds to glycos- and galactosaminoglycan (GAG) compunds in the presence of Na⁺ in low concentrations. We have worked out a cytochemical method, which is suitable for the specific demonstration of surface GAG components. The method consists of a glutaraldehyde prefixation, an acridine orange block-staining for 48 h and an OsO₄ postfixation for some hours. The specificity of the staining was verified with the help of fluorimetric-in vitro-measurements and enzymatic digestions, respectively.     

Acridine orange staining of the mammalian fibroblast cell coat

Acridine orange selectively binds to glycos- and galactosaminoglycan (GAG) compounds in the presence of Na+ in low concentrations. We have worked out a cytochemical method, which is suitable for the specific demonstration of surface GAG components. The method consists of a glutaraldehyde prefixation, an acridine orange block-staining for 48 h and an OsO4 postfixation for some hours. The specificity of the staining was verified with the help of fluorimetric--in vitro--measurements and enzymatic digestions, respectively.     

Effects of butyrate on cell cycle progression and polyploidization of various types of mammalian cells.

We studied the effect of butyrate on cell cycle progression and polyploidization in three fibroblast (rat 3Y1, human IMR-90, and human embryo lung HEL) and two epithelial (human embryo kidney HEK and monkey kidney BSC-1) cells. In these cells, except for 3Y1, G1 arrest with butyrate was incomplete, and the production of tetraploid cells was detectable in the presence of butyrate. G2 arrest with butyrate was also incomplete in HEL and BSC-1 cells, and the number of HEL cells increased in the presence of butyrate. On the contrary, most BSC-1 cells that divided in the presence of butyrate were unstable and the number of attached cells decreased. These results indicate that the effect of butyrate on cell cycle progression varies with the cell type and that polyploidization can be induced by a single treatment with butyrate.     

Detection and differentiation of glycoconjugates in various cell types by lectin histochemistry

Knowledge of chemical structure of glycoconjugates (GCs) at precise loci has increased through histochemical use of a battery of horseradish peroxidase-conjugated lectins, each possessing affinity for a specific terminal sugar or internal sugar linkage. Lectin histochemistry has shown tremendous variability among GCs in different histologic sites, reflecting known chemical diversity of these substances. GCs differ in structure among various cell types in an animal and differ at a given histologic site between species or between individuals in outbred but not inbred species. Lectin conjugates react with and detect GCs not otherwise demonstrable histochemically and, because of low concentration in tissue, not identified biochemically. Lectin-HRP conjugates have visualized unique GC with terminal GalNAc in primordial germ cells of rat embryos, with terminal Gal in epithelial basal cells of rodents and nodes of Ranvier in rats and with terminal GalNAc in a cell population in the thymus, Peyer's patches and intestinal lamina propria of some but not other mice.     

Protection after x-irradiation by 1,4-dithiothreitol of two mammalian cell types in vitro

1,4-Dithiothreitol (DTT) was found to diminish the amount of chromosomal breakage in leucocyte cultures of the marsupial Potorous tridactylus and in the Don strain of Chinese hamster cells (LHI). The maximum decrease of damage in Potorous lymphocytes was to 54% of that of the irradiated cells without DTT treatment, and to 64% in the case of the Chinese hamster cells. For both cell types, it was shown that the most significant effect of DTT was due to its presence in the first 30 min post irradiation. The protective action of this sulphydryl compound occurred even when DTT was not present during irradiation but was introduced as soon as possible after the X-ray dose was delivered.     

An 18000-dalton protein metabolically labeled by polyamines in various mammalian cell lines

The possible role of polyamines in the covalent modification of cellular protein(s) was investigated by studying the metabolic labeling of NB-15 mouse neuroblastoma cells by [14C]putrescine in fresh Dulbecco's medium followed by separation of cellular proteins through sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under such incubation conditions, a single protein band with an apparent molecular weight of 18000 was radioactively labeled. [14C]Spermidine also specifically labeled this protein. The majority of the radioactivity covalently linked to the 18-kDa protein was recovered as hypusine. The radioactive labeling of this protein was stimulated 1.3-fold by 1 mM dibutyryl cAMP and 2.8-fold by 4% fetal calf serum. Fetal calf serum also stimulated the labeling of many other cellular proteins. This may be due to the conversion of putrescine to amino acids via the formation of gamma-aminobutyric acid. Aminoguanidine, a potent inhibitor of diamine oxidase, completely inhibited the fetal calf serum-stimulated labeling of these cellular proteins but had no effect on the labeling of the 18-kDa protein. The specific labeling of the 18-kDa protein by [14C]putrescine occurred in various mammalian cells examined including the N-18 mouse neuroblastoma cells, 3T3-L1 murine preadipocytes, and H-35 rat hepatoma cells. The specificity of labeling of the apparently ubiquitous 18-kDa protein and the stimulation of this labeling by fetal calf serum suggest that this protein may be important in mediating some of the actions of polyamines in cell growth regulation.     

Capturing time-resolved changes in molecular structure by negative staining

Imaging structural intermediates of biological processes is a key step in understanding biological function. Because intermediates are commonly short-lived, lasting only milliseconds, the main methods used to capture them have been conventional imaging of analog or inhibited states, having extended lifetimes, or rapid (millisecond timescale) freezing of intermediates with subsequent observation by cryo-EM. We have developed a simpler method that fixes structure on the millisecond timescale. The procedure consists of briefly (milliseconds) exposing the macromolecular structure of interest on an EM grid to conditions that initiate the structural change, then immediately fixing with uranyl acetate or tannic acid. Specimens are then observed by negative staining. The key finding that validates this approach is our demonstration that uranyl acetate, and in some cases tannic acid, fixes protein molecular structure on the millisecond timescale. This is demonstrated by our observation that exposure of actin and myosin filaments to these fixatives for as little as 10 ms is sufficient to fully preserve them against changes that normally induce rapid and major alteration in their molecular structure. Fixation appears to stabilize both ionic and hydrophobic bonds. This approach should be of general utility for studying transient molecular changes in many systems.