DIFFERENTIAL ENRICHMENT OF CELLS FROM EMBRYONIC RAT CEREBRA BY CENTRIFUGAL ELUTRIATION

—Centrifugal elutriation was used to obtain different populations of cells dissociated from 16-day-old rat embryo cerebra. The cell populations recovered were viable and could be maintained for several weeks in vitro. Sterile conditions were maintained throughout a preparation. Rat pups were removed by Caesarean section, the cerebra dissected and the cells dissociated by brief exposure to trypsin (0.125%, 6 min). An equivalent volume of elutriation medium (Dulbecco's medium containing 1% fetal calf serum, sodium bicarbonate, penicillin and streptomycin, EDTA, and deoxyribonuclease) was added to the trypsin-cell suspension, the dissociated cells pelleted, resuspended in elutriation medium and counted. Up to 4 x 10⁸ cells were injected into the previously sterilized elutriator. Seven fractions were usually recovered from a preparation. The first fraction contained primarily red blood cells and cell debris, which could not be maintained in vitro. Upon culture, fraction 2 consisted of predominantly non-neuronal cells, while fractions 3–6 contained neuronal and non-neuronal cells. The morphological characteristics of the neurons differed in these fractions. Fraction 7 contained cells that had reaggregated during the elutriation procedure and exhibited a variety of cell types in vitro.     

γ-Glutamylamine cyclotransferase

Summary -Glutamylamine cyclotransferase, an enzyme found in a number of animal tissues and cells, catalyzes the conversion of -(L--glutamyl)-L-lysine to free lysine and 5-oxo-L-proline as well as the release of free amines and the formation of 5-oxo-L-proline from a variety of other L--glutamylamines. Among its substrates are both the mono- and di--glutamyl derivatives of putrescine, spermidine and spermine, and a derivative of -(L--glutamyl)-L-lysine in which both the -amino group and the carboxyl group of the lysine moiety are blocked. The enzyme does not act on most -glutamyl--amino acids, nor is it active toward the -lysyl derivatives of L-aspartic acid or D-glutamic acid. Derivatives of -(L--glutamyl)-L-lysine in which the -amino or the -carboxyl function of the glutamyl moiety is blocked also do not serve as substrates. The specificity of -glutamylamine cyclotransferase is in accordance with the proposal that it functions biologically in the latter stages of the catabolism of products of the action of transglutaminases. Some suggestions as to the manner in which -glutamylamine cyclotransferase serves this function are made based on present knowledge of protein degradation.     

Fine structure of Merkel cells in lampreys

Summary The structure of Merkel cells occurring in the epidermis of adult and larval stages of Lampetra spp. is described; it is comparable to that reported from the gnathostome classes. The cells bear microvilli, grouped on the distal and proximal aspects, and are associated with sparsely branching and varicose nerve fibres. One branch of the neurite bears a spur-like process which indents the proximal side of the Merkel cell. Most of the specific Merkel granules are situated in the vicinity of this neurite projection; the cell membrane adjacent to the tip of the spur process bears structures resembling presynaptic densities. Occasionally, desmosome-like junctions are found between the neurite and the Merkel cell.The authors thank the Fresh Water Biological Association and the Department of Biological Sciences, University of Bath, for supplying the material, Dr. H. Fox for giving some prepared blocks of Lampetra planeri adults, Mr. B.L. Pirie for technical assistance, and the Science Research Council for support through grant GR/A/3740.6     

Transformation of Adenine to 8-Hydroxyadenine by Strains of Oerskovia xanthineolytica

The 8-hydroxy derivative of adenine (6-amino-1,7-dihydro-8H-purin-8-one) is produced from adenine by two Oerskovia xanthineolytica strains. This transformation by a microorganism has not been reported previously. No novel products of dissimilation of xanthine (3,7-dihydro-1H-purine-2,6-dione) or hypoxanthine (1,7-dihydro-6H-purin-6-one) were found. Xanthine was oxidized to uric acid, but intermediates in the breakdown of hypoxanthine could not be demonstrated.     

Cell Cycling of Astrocytes and Their Precursors in Primary Cultures: A Mevalonate Requirement Identified in Late G1, but Before the G1/S Transition, Involves Polypeptides

The relationship between mevalonate and cell cycling was investigated in developing glial cells. Primary cultures of newborn rat brains were serum-depleted (0.1%, vol/vol) for 48 h on days 4-6 in vitro, then returned to 10% calf serum (time 0). After 48 h, 70-80% of the cells were glial fibrillary acidic protein (GFAP)-negative by indirect immunofluorescence; 79 +/- 7% were GFAP-positive after an additional 3 days. Serum shift-up resulted in 12 h of quiescence, and then by 20 h (S phase) in increased proportions of cells synthesizing DNA (from 15 +/- 6% to 75 +/- 4% by bromodeoxyuridine immunofluorescence at 12 h and 20 h, respectively) and rates of DNA synthesis (42 +/- 6 versus 380 +/- 32 cpm/micrograms of protein/h of [3H]thymidine uptake). Additional mevalonate (25 mM) for 30 min at 10 h reversed the inhibition of DNA synthesis apparent with mevinolin (150 microM), an inhibitor of mevalonate synthesis, present from time 0. Cycloheximide added simultaneously with mevalonate prevented this reversal of inhibition. To cause arrest at G1/S, cultures were exposed to hydroxyurea between 10 and 22 h. By 3 h after hydroxyurea removal, bromodeoxyuridine-labeled nuclei increased from 0% to 75 +/- 9%, and DNA synthesis increased 10-fold. Mevinolin failed to inhibit these increases. Thus, primary astroglial precursors stimulated to progress through the cell cycle express a mevalonate requirement in late G1, but before the G1/S transition. The effect of mevalonate was characterized further as being brief (30 min) and as requiring polypeptides.     

Regulation of the final phase of mammalian melanogenesis. The role of dopachrome tautomerase and the ratio between 5,6-dihydroxyindole-2-carboxylic acid and 5,6-dihydroxyindole.

The regulation of the final steps of the melanogenesis pathway, after L-2-carboxy-2,3-dihydroindole-5,6-quinone (dopachrome) formation, is studied. It is shown that both tyrosinase and dopachrome tautomerase are involved in the process. In vivo, it seems that tyrosinase is involved in the regulation of the amount of melanin formed, whereas dopachrome tautomerase is mainly involved in the size, structure and composition of melanin, by regulating to the incorporation of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) into the polymer. Moreover, using L-3,4-dihydroxyphenylalanine (dopa) and related compounds, it was shown that the presence of dopachrome tautomerase mediates an initial acceleration of melanogenesis since L-dopachrome is rapidly transformed to DHICA, but that melanin formation is inhibited because of the stability of this carboxylated indole compared to 5,6-dihydroxyindole (DHI), its decarboxylated counterpart obtained by spontaneous decarboxylation of L-dopachrome. Using L-dopa methyl ester as a precursor of melanogenesis, it is shown that this carboxylated indole does not polymerize in the absence of DHI, even in the presence of tyrosinase. However, it is incorporated into the polymer in the presence of both tyrosinase and DHI. Thus, this study suggests that DHI is essential for melanin formation, and the rate of polymerization depends on the ratio between DHICA and DHI in the medium. In the melanosome, this ratio should be regulated by the ratio between the activities of dopachrome tautomerase and tyrosinase.     

Quantification of a decapeptide anticoagulant in rat and monkey plasma by high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method was developed to quantify a decapeptide anticoagulant in rat and monkey plasma. The compound and internal standard, a nonapeptide analogue, were extracted from plasma with an amino solid-phase extraction column with an extraction efficiency in the range 75–90%. A C₁₈ analytical column was used to separate the analytes by gradient elution followed by ultraviolet detection at 215 nm. Quantification of the decapeptide over the concentration range 0.1–10.1 μg/ml resulted in an assay relative error and relative standard deviation both less than 10%. The anticoagulant decapeptide was stable in both rat and monkey plasma frozen at −20°C.     

Stage-specific cuticular proteins of Ostertagia circumcincta and Ostertagia ostertagi

In this study we have shown that NHS-biotin and I¹²⁵-streptavidin can detect cuticular polypeptides of Ostertagia spp. The labelled polypeptide profile of intact nematodes is simple compared to the profile obtained by labelling homogenates. None of the major internal polypeptides are labelled and the subset of proteins labelled in intact nematodes appears to be mainly surface associated. The results presented here demonstrate that NHS-biotin may be used as a reagent for the analysis of surface polypeptides. The surface polypeptide profiles of the five major developmental stages (L1, L2, L3, L4 and adult) of Ostertagia circumcincta show a series of stage-specific molecules with no polypeptides common to all stages, indicating that the cuticle is a dynamic structure which changes throughout the life cycle. Similarity comparison of Ostertagia ostertagi L3 and L4 stage surface profiles showed that each stage is clearly distinct; comparison of these stages between the two species shows an overall similarity.