Cell-cycle-dependent changes in labelling of specific phosphoproteins by the monoclonal antibody MPM-2 in plant cells

The MPM-2 antibody, which recognizes a mitosis-specific phosphorylated epitope, has been used to study cell-cycle-related proteins in partially synchronized cell suspension cultures and root meristem cells. Immunofluorescence revealed that the epitope recognized by MPM-2 is located in the nucleus in interphase cells. In mitotic cells, MPM-2 labels the prophase nucleus, the spindle and some cytoplasmic components. The relative amount of the epitope changes significantly during the cell cycle. Labelling is lowest in G1 and S-phase cells and increases 2–3-fold during G2. Prophase and metaphase show four to five times the labelling of G1 cells. Labelling decreases rapidly after metaphase and is at a very low level by telophase. One- (1-D) and two-dimensional (2-D) immunoblots showed that MPM-2 labels a family of phosphorylated proteins. The labelling shows significant cell cycle dependence. Subfractionation shows at least one of these proteins is a component of the detergent-insoluble cytoskeleton cell fraction. This component is resolved on 2-D immunoblots to two to three spots of slightly different isoelectric point, possibly charge isomers, at a relative molecular mass of approximately 65 kDa. The same spots are labelled by IFA, an antibody against intermediate filament proteins. Another three of the spots at lower relative molecular mass are labelled on 2-D immunoblots of the nuclear matrix fraction.     

Reactive synaptogenesis following degeneration of photoreceptor terminals in the fly's optic lobe: a quantitative electron microscopic study.

Following photo-ablation of receptor cells in the retina of the housefly's compound eye, their synaptic terminals degenerate with a timecourse which we have followed over 8 d. Degeneration deprives the monopolar interneurons in the first optic neuropile, the lamina, of their main synaptic input. Simultaneously it deprives one monopolar interneuron (L2) of one of its synaptic targets, as L2 makes numerous feedback synaptic contacts at which it is pre-synaptic upon receptor terminals. Because the feedback synapses are dyadic, input still remains available to the second element post-synaptic at the dyad, which does not degenerate. This element is T1, a higher-order interneuron from the next most proximal neuropile (the medulla). Some of the original feedback synaptic sites soon disappear as a consequence of the photo-ablation, but their loss is partly offset by the production of new synaptic contacts. The new pre-synaptic ribbons resemble those at the original sites except for being smaller. The sites are, moreover, monadic, with T1 now the sole post-synaptic partner. These results show that interneurons in the fly's lamina retain a dynamic capacity for synaptogenesis throughout much of adult life, normally a few weeks in Musca, and that during this synaptogenesis they re-enact the same cell preferences expressed earlier in development.     

Nucleotide sequence analysis of a variant human T-cell leukemia virus (HTLV-Ib) provirus with a deletion in pX-I.

A variant of human T-cell leukemia virus subgroup I (HTLV-I), designated HTLV-Ib, has been isolated from a transformed T-lymphocytic cell line established from a Zairian patient with adult T-cell lymphoma. A recombinant phage clone of the variant provirus, denoted lambda MC-1, hybridizes under high stringency to HTLV-I DNA probes, but 17 of 43 restriction enzyme sites differ from those of HTLV-I, 10 of them clustering within 1.5 kilobases in the env-pX region. Since this variant virus retains its capacity to transform T-cells in vitro, and since a pX product is suspected to be important in transformation, we have determined the nucleotide sequence of the entire pX region of this virus for comparison to the prototype HTLV-I. In addition, the region between the gag and pol genes, parts of the pol and env genes, and a portion of the U3 region of the long terminal repeat sequence were also analyzed. We noted 141 single-base-pair changes among 3,897 base pairs, which were relatively well distributed over those portions of the provirus that were examined. In addition, an 11-base-pair deletion was found which included the potential initiator ATG codon of the first open reading frame of pX (pX-I). The next potential initiator codon predicted by the sequence is followed by 10 codons and then a termination codon. An identical deletion was also demonstrated in the only provirus present in another cell line established from the same patient on a different occasion after transformation in vitro of normal human umbilical cord blood cells. These results indicate that pX-I is not required for transformation.     

The amino acid sequence of equine milk lysozyme

The amino acid sequence of equine milk lysozyme has been elucidated. The study involves the determination of the sequence of the N-terminal region of the whole protein, cyanogen bromide fragments, tryptic and chymotryptic peptides and fragments produced by chemical cleavage after tryptophan residues. The protein consists of a single chain of 129 amino acid residues and has a Mr of 14647. While equine milk lysozyme has the essential features of a c(chick)-type lysozyme, there is only 51% sequence homology with human milk lysozyme and 50% with domestic hen egg white lysozyme. Some of the implications of the large number of differences are discussed.     

Metabolism and gastric acid secretion. Substrate-dependency of aminopyrine accumulation in isolated rat parietal cells.

The substrate-dependency of gastric acid secretion was investigated in isolated rat parietal cells by using the accumulation of the weak base aminopyrine as an index of acid secretion. Exogenous substrates enhanced accumulation of aminopyrine in rat parietal cells stimulated by secretagogues, and this effect was probably directly related to the provision of energy for acid secretion. At physiological concentrations, certain of the substrates (glucose, oleate, lactate, D-3-hydroxybutyrate, L-isoleucine, L-valine and acetoacetate) could support acid secretion, with glucose being the most effective. L-Leucine and acetate were only effective stimulators of parietal-cell aminopyrine accumulation at high concentrations (5mM). L-Glutamine was unable to stimulate aminopyrine accumulation even at high concentrations, and glutaminase activity in parietal cells was estimated to be low by comparison with small-intestinal epithelial cells. Variation in the concentrations of D-3-hydroxybutyrate and L-isoleucine, but not of glucose, within the physiological range affected their ability to support aminopyrine accumulation. The presence of 5 mM-L-isoleucine, 5 mM-lactate and combinations of certain substrates at physiological concentrations produced aminopyrine accumulation in stimulated parietal cells that was greater than that obtained in cells incubated with 5 mM-glucose alone. In conclusion, fulfillment of the metabolic requirements of the acid-secreting parietal cell under physiological circumstances requires a combination of substrates, and integration of the results with previous data [Anderson & Hanson (1983) Biochem. J. 210, 451-455; 212, 875-879] suggests that after overnight starvation in vivo metabolism of glucose, D-3-hydroxybutyrate and L-isoleucine may be of particular importance.     

A longitudinal study of tooth resorption in newly metamorphosed Xenopus laevis, with comments on tooth resorption in amphibians

Late larvae and newly metamorphosed specimens of Xenopus laevis (Daudin) were reared in such a way that their growth rates were assessed as similar. After the larvae had reached Nieuwkoop and Faber Stage 65,12 were killed each day for eight days, so that the events in resorption of the first teeth to develop could be studied histologically, and an absolute time scale applied. Resorption extended over several days and occurred in two phases, (a) erosion and (b) absorption. Erosion was a process lasting several days, and involved the destruction of a small quantity of dentine and adjacent bone at the base of a standing tooth. Osteoclasts were not seen inside the pulp, but were confined to the outer aspect of the dentine and bone, and were removing the hard tissues from without. Absorption was a rapid process lasting about 48 hours, during which the majority of the dental tissue was destroyed. Osteoclasts resorbed the dentine piecemeal from within along its full length from base to tip. As a result, the vast majority (probably all) of the calcified tissues would seem to have been available for reabsorption and recycling. It seemed that erosion occurred as a result of local mechanical factors (the growing successional tooth), but that absorption was controlled by a specific timing mechanism. It is suggested that gross internal resorption of teeth may be common in Amphibia, and that even in species where crowns are shed, extensive thinning of the dentine from its pulpal aspect is likely to have occurred beforehand.