Sequence of the human erythrocyte phosphoglycerate mutase by microsequencer and mass spectrometry

We have previously reported the isolation in pure form of the human erythrocyte phosphoglycerate mutase isozyme B. We now report the sequence of the whole protein and the identification of its N-terminal blocking group. The protein tryptic peptides of phosphoglycerate mutase isozyme B were isolated by high performance liquid chromatography and their sequence determined by microsequencing. The sequence and the nature of the blocking group of the N-terminal tryptic peptide was shown to be N-acetyl-Ala-Ala-Tyr-Lys by mass spectrometry. Overlaps of the tryptic peptides were obtained by studying the V8 Staphylococcus aureus protease peptides of the aminoethylated phosphoglycerate mutase isozyme B either by microsequencing or by mass spectrometry. The procedure used allowed us to obtain the sequence on a very small amount of material and in a short period of time. Our data agree well with those derived from the cDNA nucleotide sequence described by Sakoda et al. (Sakoda, S., Shanske, S., DiMauro, S., and Schon, E. A. (1988) J. Biol. Chem. 263, 16899-16905). In addition, our data directly indicate that the initiation codon does not introduce a methionine as N-terminal amino acid and allowed the identification of the acetyl N-terminal group.     

The role of morphometry in the cytology of well-differentiated hepatocarcinoma and cirrhosis with atypia

A morphometric study was performed on 200 nuclei per case in six well-differentiated hepatocarcinomas and in six cirrhoses with cytologic atypia, using samples obtained by fine needle aspiration (FNA) biopsy of the liver. The parameters measured were the nuclear area, the nuclear perimeter and the maximum nuclear diameter. The nuclei of well-differentiated hepatocarcinomas could be distinguished from those of cirrhoses on the basis of the larger size and greater anisonucleosis of the former. A statistical analysis (using a two-sided t-test) of the means of the parameters showed significant differences between the two diagnostic groups. These results suggest that morphometric analysis can help in the differential diagnosis between well-differentiated hepatocellular carcinoma and cirrhosis with cytologic atypia in FNA biopsy samples.     

Long term incubation of cardiac myocytes with oleic acid and very-low density lipoprotein reduces heparin-releasable lipoprotein lipase activity

An exogenous [³H]triolein emulsion was hydrolyzed by intact cardiac myocytes with functional LPL located on the cell surface. This surface-bound LPL could be released into the medium when cardiac myocytes were incubated with heparin. Incubation of cardiac myocytes with VLDL, or the products of TG breakdown, oleic acid or 2-monoolein, did not increase LPL activity in the medium. However, incubation of cardiac myocytes with either VLDL or oleic acid for > 60 min did reduce heparin-releasable LPL activity. In the heart, this inhibitory effect of FFA could regulate the translocation of LPL from its site of synthesis in the cardiac myocyte to its functional site at the capillary endothelium.Abbreviations LPL lipoprotein lipase - TG triacylglycerol - FFA free fatty acids - VLDL very-low density lipoprotein     

Polyamines in liver and their influence on chromatin condensation after 17-β estradiol treatment of Atlantic salmon

Atlantic salmon (Salmo salar) were treated with 17- estradiol to induce vitellogenin synthesis in liver. This led to an increase in liver wet weight and total DNA. After incubation with micrococcal nuclease (EC 3.1.31.1) less soluble chromatin was obtained from nuclei of the estradiol treated than the control fish, but active gene regions were solubilized by the nuclease. Thus, in the estradiol treated fish soluble mononucleosomes contained hybridizable vitellogenin gene sequences. As a result of estradiol treatment the content in total liver of putrescine rose 3-fold, that of spermidine 2-fold, while spermine was unchanged. In muscle no significant changes were observed. The regulatory functions of polyamines during gene expression were investigated by binding (¹⁴C)spermine to isolated liver nuclei depleted of endogenous polyamines. The number of binding sites was higher in nuclei of estradiol treated than control fish. (^14C)spermine associated preferentially with micrococcal nuclease insensitive chromatin. Thus, the high content of putrescine and spermidine in liver supported the view of polyamine accumulation in proliferating tissues. The preferential binding to condensed chromatin indicated a stabilizing effect of polyamines on the organization of inactive chromatin structures.Abbreviations MNase micrococcal nuclease - PMSF phenylmethylsulfonylfluoride     

Respiratory electron transport activity in plankton of the Weddell and Scotia Seas during late spring — early summer: relationships with other biological parameters

Summary As a means to estimate potential oxygen consumption, profiles of elctron transport system (ETS) activity were made along three transects across the Weddell-Scotia Confluence zone (WSC) and the marginal ice zone (which overlapped in part) during the EPOS leg 2 cruise of the RV Polarstern. The integrated ETS activity between 0 and 100 m depth (referred to in situ temperatures) ranged from 261 meq (mili-electron equivalents) m^–2 day^–1 in the WSC to 45 meq m^–2 day^–1 in the southernmost stations at 62° S. The temporal changes in the overall distribution of ETS activity were small compared with the spatial variations. The main feature of the ETS activity distribution was the presence of maxima located in the WSC, coinciding with peaks of phytoplankton biomass. Different relationships between ETS and chlorophyll a concentration in these maxima appeared to be related to diatom or flagellate dominance. Vertically integrated ETS activities were significantly correlated with chlorophyll a and paniculate organic carbon concentrations, primary production and bacterial thymidine uptake.Data presented here were collected during the European Polarstern Study (EPOS) sponsored by the European Science Foundation     

Effect of TSH in human thyroid cells: evidence for both mitogenic and antimitogenic effects.

The well-known mitogenic effects of TSH observed in vivo on the thyroid are not always reproducible of human thyroid cells in vitro where conflicting results have been obtained. In order to clarify this issue, we have used primary cultures of human thyroid cells obtained from normal tissue and maintained in serum-free medium for several days. In this in vitro model we have studied the effect of TSH on growth by measuring three different parameters: [3H]-thymidine incorporation, cell counts, and DNA measurement. Monolayer cultures were plated at both low and high cell density (2 x 10(4) and 8 x 10(4) cells/25 mm well, respectively). Although at either cell density cultures were equally able to functionally respond to TSH in terms of cAMP accumulation a significant growth response to TSH was observed only in low density cultures. In high density cultures TSH had an antimitogenic effect. Moreover, TSH potentiated the mitogenic effect of insulin only in low density cultures. In contrast to TSH, FCS induced a similar proliferative response at both high and low cell density. Following TSH stimulation, cAMP content was always increased, paralleling the effect of growth in low density but not in high density cultures. The cAMP analogues dibutyryl-cAMP and 8-bromo-cAMP, as well as cholera toxin and forskolin, did not mimic the mitogenic effect of TSH but had an antiproliferative effect. In addition, these agents blunted the proliferative effect of insulin. These data suggest that in thyroid cells TSH is able to elicit both a mitogenic and an antimitogenic effect depending on the environmental conditions such as cell density.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of complex alleles by direct analysis of DNA heteroduplexes

DNA molecules derived from three alleles of the HLA-DRB3 locus and differing from each other at several nucleotide sites were denatured and cross-hybridized. Each allelic combination was found to generate a pair of heteroduplexes of different mobility. Their retardation as compared to homoduplexes was proportional to the number of mismatches. In each heteroduplexes pair the component possessing the highest number of Pyr-Pyr oppositions was the most retarded. The results are those predicted by a theoretical model implying a correlation between base-pair opening and bending of the DNA double helix. These observations introduce a new HLA typing method at the genomic level and indicate an experimental approach to the analysis of the superhelical DNA conformation as related to different types of base oppositions.     

Anatomical differences involving the archicortex and the neocortex of male and female brains: A quantitative syudy of 20 brains of each sex

The relative sizes of the brain stem, cerebellum, diencephalon, archicertex and neocortex were investigated in twenty brains of each sex, by determining the percentages of average total brain weights in each case. The differences between the left and the right cortical regions were also compared. The neo-archicortical ratio was found to be smaller in females than in males. Significantly heavier right neocortex occurred in males than in females. Weight predominance of the right archicortex was found in both sexes. These findings are suggestive that anatomical differences may contribute to the behaviour pattern differences occurring between male and female.