Improved methods for construction of carbon fibre electrodes for extracellular spike recording.

Microelectrodes using single carbon fibres as the conducting element have been used since 1979 for electrophysiological measurements in vivo and in vitro. However, there is still considerable discussion about the manufacture of these electrodes, and no overall agreement as to the best way to minimise electrode noise. In this article we describe some revised methods for carbon fibre electrode manufacture, which we believe, gives the lowest noise performance for spike recording in vivo.

New cationic fluorochromes from diaryloxazole scintillators: fluorescence of chromatin DNA induced by N-quaternary POPOP derivatives.

N-quaternary derivatives of the diaryloxazole scintillators POPOP and dimethyl-POPOP (dmPOPOP) in chloroform solution were obtained by methylation with dimethylsulfate. After drying, aqueous solutions of the corresponding oxazolium compounds (Q-POPOP and Q-dmPOPOP) revealed strong fluorescence (peaks at 485 and 493 nm, respectively). Under 365 nm excitation, both N-quaternary derivatives induced a bright greenish blue fluorescence in nuclei of chicken erythrocytes and human buccal cells, as well as in the kinetoplast DNA of Trypanosoma cruzi epimastigotes; mouse mast cell granules showed a green-yellow metachromatic emission. Chromatin fluorescence was dependent on the presence of DNA; it was abolished by washing with a 10 mM solution of the bisguanidine compound Phenformin, whereas 1 M NaCl or MgCl2 had no effect. The oxazolium derivatives were hydrophilic (log P: -6.409 and -5.373 for Q-POPOP and Q-dmPOPOP, respectively). Molecular modelling studies revealed that these cationic and non-rigid (cis) scintillator derivatives are well suited to locate along the convex floor of the narrow DNA minor groove from adenine-thymine regions.

Scanning electron microscopy of polytene chromosomes (I).

The structure of polytene chromosomes has been observed by conventional scanning electron microscopy and also with a high resolution 'in lens' field emission instrument. Surface imaging with secondary electron emission has characterized condensed chromatin and regions known for their RNA synthetic potential (nucleoli, Balbiani rings and puffs). In DNA-rich bands pairing of chromatids appears so perfect that individual chromomeres cannot be visualized as discrete units. In the interbands only chromatid bundles appear as elements, but not individual chromatids. High resolution scanning electron microscopy allows resolution at the nucleosome level. Improved localization of chromosomal structures is demonstrated, as in the case of the proposed separation of the prepupal 'Balbiani ring 1'. Surface images of the RNA synthetic centres of the salivary gland cell are presented. Transcripts of the Balbiani ring template can be observed in the condensed and gradually unfolded state, allowing measurement of unit ribonucleoparticles. The surfaces of puffs have been visualized and are characterized by multiple supercoiled loop structures, which are described according to size, conformation and distribution.

Metachromatic staining of human sperm nuclei after reduction of disulphide bonds.

Intra- and interprotamine cross-linking by disulphide bonds are the main factors responsible for the highly compact and stable structure of chromatin in mammalian spermatozoa. Unfixed or methanol fixed smears of human sperm and sperm suspensions from fertile donors and oligospermic patients were subjected to a reductive cleavage of disulphide bonds by using 2-mercaptoethanol (ME) or dithiothreitol (DTT). Untreated (control) and ME or DTT treated samples were stained with toluidine blue (TB) and examined in light microscopy; spectral characteristics of TB stained sperm nuclei were also analyzed. Untreated smears from fertile donors showed an orthochromatic (pale blue) staining of most sperm heads, while a variable proportion of metachromatic nuclei was found in spermatozoa from patients with oligospermia. After treatment with DTT followed by TB staining, fixed and unfixed smears showed metachromatic sperm heads. ME treatment only induced a scarce colour shift, whereas a striking metachromatic reaction and variable nuclear swelling were observed in DTT treated sperm suspensions. These results indicate that after cleavage of disulphide bonds, phosphate groups from chromatin DNA are unmasked and available for TB binding and metachromatic staining.

DNA cytochemistry in polytene chromosomes: electron contrasting agents for the ultrastructural detection of chromatin DNA after alkaline hydrolysis/methylation-acetylation.

Salivary glands from Chironomus tentans larvae were fixed in glutaraldehyde and either subjected to alkaline hydrolysis followed by methylation-acetylation, or dehydrated without these treatments as controls. Ultrathin sections from Durcupan-embedded samples were contrasted by means of uranyl acetate, ruthenium red, indium trichloride, or the complex indium (III)-hematoxylin. Electron microscopic observations revealed a general contrasting pattern in control sections, while after the hydrolytic and blocking procedure only chromatin from polytene chromosomes appeared selectively contrasted. The nucleolus, Balbiani ring granules and puff materials showed weak or no electron opacity. After toluidine blue staining of semithin sections, an orthochromatic blue colour was found in chromatin bands from treated samples. These results indicate that alkaline hydrolysis/methylation-acetylation followed by contrasting with cationic heavy compounds is a valuable procedure to visualize chromatin DNA in polytene chromosomes.

X-ray microanalysis of toluidine blue stained chromosomes: a quantitative study of the metachromatic reaction of chromatin.

The stoichiometry of metachromatic staining of chromatin by toluidine blue was investigated in isolated metaphase chromosomes from L929 cells using X-ray microanalysis. Microspectrophotometric measurements revealed that a hypsochromic shift (from 595 to 570 nm) occurs in toluidine blue stained chromosomes in relation to the staining solution. Under the electron microscope, stained chromosomes. After toluidine blue staining, X-ray microanalysis of chromosomes revealed a large increase for sulphur counts and a considerable increase for Fe and Cu counts, while the signal of Mg, Ca, Cl, K and Zn was reduced. After subtraction of the intrinsic sulphur signal, S/P ratios of 0.82--for euchromatic arms--and 0.85--for centromeric heterochromatin--were obtained. They are considered representative of dye/DNA phosphate ratios. These results indicate the occurrence of a nearly stoichiometric binding of toluidine blue to chromatin DNA and suggest that an external dye stacking is responsible for the metachromatic staining of metaphase chromosomes.

Cell-free synthesis and immunological characterization of salivary proteins from Chironomus tentans.

We have isolated the salivary proteins of the larva of the harlequin fly Chironomus tentans, and characterized its constituents by gel electrophoresis and immunological techniques. The detailed composition of saliva from individual animals is shown to be very variable, but four main protein groups can be defined. The largest, Fraction A, comprises up to five species, with molecular weights of between 820,000 and 700,000 Daltons. It includes at least two distinct antigenic species. This finding is discussed in the context of the known heterogeneity of the 75S RNA fraction which is transcribed in the Balbiani rings 1 and 2. -- The other prominent protein classes in isolated saliva range in size from 230,000 down to less than 20,000 Daltons. -- We have also employed antiserum against salivary proteins to investigate the products of in vitro translation of salivary gland RNA in the rabbit reticulocyte lysate system. A broad spectrum of polypeptide species is obtained which are immunologically related to salivary components, including species of over 300,000 Daltons. These latter are interpreted as unfinished Fraction A polypeptides resulting from incomplete translation of 75S RNA from BR1 and BR2. Evidence is presented to demonstrate that other salivary proteins, apart from Fraction A, are faithfully translated in the reticulocyte lysate.

Incomplete dosage compensation in an evolving Drosophila sex chromosome.

Cellular autoradiography was used to measure relative rates of chromosomal RNA synthesis and to examine the regulatory phenomenon of X-linked dosage compensation in Drosophila miranda, a species containing two distinct, nonhomologous X chromosomes (X1 and X2). The X1 chromosome was found to be dosage-compensated, since the rate of RNA synthesis along the single X1 chromosome in males equaled that of both X1 chromosomes in females. Unlike other sex chromosomes that have been studied, the more recently evolved X2 heterochromosome exhibited regional differences in transcriptional activity when males and females were compared. The distal 10% of the X2 was not dosage-compensated, whereas the majority of an interior segment, representing 30% of the X2 chromosome's length, was found to be dosage-compensated. Our data are consistent with the idea that the evolution of X2 dosage compensation has paralleled the differentiation of the X2 sex chromosome. In addition, gene rearrangement seems to have accompanied the acquisition of a dosage-compensory mechanism in the X2.