Isolation to homogeneity and partial characterization of a histo-blood group A defined Fuc alpha 1----2Gal alpha 1----3-N-acetylgalactosaminyltransferase from human lung tissue

The soluble histo-blood group A glycosyltransferase (Fuc alpha 1----Gal alpha 1----3-N-acetylgalactosaminyltransferase) was purified approximately 600,000-fold to homogeneity from human lung tissue. The enzyme was solubilized in 1% Triton X-100, partially purified by affinity chromatography on Sepharose 4B, and eluted with UDP. Final purification was obtained by twice repeated fast protein liquid chromatography ion exchange (Mono STM) with NaCl gradient elution and reverse-phase chromatography (proRPC) with acetonitrile gradient elution. Identity of the purified protein was established by (i) demonstration of the putative A transferase protein only in affinity-purified extracts of A but not O individuals, and (ii) specific immunoprecipitation of enzyme activity and putative protein with monoclonal antibodies. Sodium dodecyl sulfate electrophoresis revealed a single protein band with apparent Mr of approximately 40,000 under both reducing and nonreducing conditions. Digestion with N-glycanase yielded a reduction in Mr of approximately 6,000 (estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis), suggesting that the A transferase is a glycoprotein with N-linked carbohydrate chains. Amino acid composition and N-terminal amino acid sequence of the intact transferase, as well as of peptides released by endolysyl peptidase digest or cyanogen bromide cleavage, are presented.     

Mathematical model analysis of mouse epidermal cell kinetics measured by bivariate DNA/anti-bromodeoxyuridine flow cytometry and continuous [3H]-thymidine labelling

In a previous study the epidermal cell kinetics of hairless mice were investigated with bivariate DNA/anti-bromodeoxyuridine (BrdU) flow cytometry of isolated basal cells after BrdU pulse labelling. The results confirmed our previous observations of two kinetically distinct sub-populations in the G2 phase. However, the results also showed that almost all BrdU-positive cells had left S phase 6-12 h after pulse labelling, contradicting our previous assumption of a distinct, slowly cycling, major sub-population in S phase. The latter study was based on an experiment combining continuous tritiated thymidine [( 3H]TdR) labelling and cell sorting. The purpose of the present study was to use a mathematical model to analyse epidermal cell kinetics by simulating bivariate DNA/BrdU data in order to get more details about the kinetic organization and cell cycle parameter values. We also wanted to re-evaluate our assumption of slowly cycling cells in S phase. The mathematical model shows a good fit to the experimental BrdU data initiated either at 08.00 hours or 20.00 hours. Simultaneously, it was also possible to obtain a good fit to our previous continuous labelling data without including a sub-population of slowly cycling cells in S phase. This was achieved by improving the way in which the continuous [3H]TdR labelling was simulated. The presence of two distinct subpopulations in G2 phase was confirmed and a similar kinetic organization with rapidly and slowly cycling cells in G1 phase is suggested. The sizes of the slowly cycling fractions in G1 and G2 showed the same distinct circadian dependency. The model analysis indicates that a small fraction of BrdU labelled cells (3-5%) was arrested in G2 phase due to BrdU toxicity. This is insignificant compared with the total number of labelled cells and has a negligible effect on the average cell cycle data. However, it comprises 1/3 to 1/2 of the BrdU positive G2 cells after the pulse labelled cells have been distributed among the cell cycle compartments.     

A quantitative and qualitative study of blood monocytes in patients with bronchogenic carcinoma

Summary Absolute circulating number and functions of blood monocytes (i.e., pinocytosis, phagocytosis, and chemotaxis) were studied in 25 patients with untreated bronchogenic carcinoma and in 28 control subjects. The absolute circulating monocyte count was increased in 20 (80%) of the patients. There was no difference in the pinocytic and phagocytic activity of patient and control monocytes. In contrast, patient monocytes showed depressed chemotactic responsiveness. This defect was more severe in small cell anaplastic carcinoma than in the other histologic types of bronchogenic carcinoma (P=0.001), and may explain the difference in macrophage infiltration seen in solid tumours of the lung. There was no correlation between chemotaxis and clinical stage. Depressed chemotaxis may be related to a plasma factor, since patient plasma inhibited the chemotaxis of control monocytes as well as the activity of chemotactic agents. The defective chemotaxis and the presence of plasma inhibitory activity may interfere with the ability of blood monocytes to accumulate as macrophages in tumour sites. Abbreviations used in this paper are: MCR, monocyte chemotactic response; SAC, small cell anaplastic bronchogenic carcinoma; OBC, non-small cell bronchogenic carcinoma MEM, Eagle's minimal essential medium; CFI, chemotactic factor inhibitor(s); HSA, human serum albumin     

Suppressive Effect of Triethyllead on Entry of Proteins into the CNS Myelin Sheath In Vitro

Incorporation of [¹⁴C]leucine into the myelin sheath was studied in brain stem slices prepared from 22-day-old rats. Individual major myelin proteins were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. There was a time lag before incorporation of the label into proteolipid protein (PLP) and intermediate protein (IP) reached maximal rates. Labelling of basic proteins (BP) and Wolfgram proteins (WP) revealed a much shorter lag in entry. Appearance of radioactive proteins in the myelin sheath was significantly hampered by triethyllead (PbEt₃) added to the incubation medium at micromolar concentrations. Inhibition values were highest in the case of PLP and were closely followed by the values for IP. BP and WP were less inhibited, although incorporation of these proteins into myelin was still suppressed more than was synthesis of total homogenate protein. Thus, myelin-forming cells seem to be unduly vulnerable to the toxin relative to the rest of the tissue. Furthermore, the results indicate an interference of PbEt₃ with certain posttranslational processes involved in furnishing of integral myelin proteins.     

On the distribution of Rostkovia magellanica (Juncaceae), a species newly rediscovered in Ecuador

Rostkovia magellanica has hitherto been considered a species of Patagonia and the subantarctic islands from New Zealand to South Georgia. An old report from Ecuador has been considered erroneous, but it has now been rediscovered there. Brief notes on the ecology of the species are given, and the possible origin of its distribution is discussed. Long-distance dispersal by birds is suggested as having caused the disjunct distribution on mainland South America.     

REGENERATIVE PROLIFERATION OF MOUSE EPIDERMAL CELLS FOLLOWING ADHESIVE TAPE STRIPPING

The proliferating cells of mouse epidermis (basal cells) can be separated from the non-proliferating cells (differentiating cells) (Laerum, 1969) and brought into a mono-disperse suspension. This makes it possible to determine the cell cycle distributions (e.g. the relative number of cells in the G^ S and (G₂+ M) phases of the cell cycle) of the basal cell population by means of micro-flow fluorometry. To study the regenerative cell proliferation in epidermis in more detail, changes in cell cycle distributions were observed by means of micro-flow fluorometry during the first 48 hr following adhesive tape stripping. ³H-TdR uptake (LI and grain count distribution) and mitotic rate (colcemid method) were also observed. An initial accumulation of G₂ cells was observed 2 hr after stripping, followed by a subsequent decrease to less than half the control level. This was followed by an increase of cells entering mitosis from an initial depression to a first peak between 5 and 9 hr which could be satisfactorily explained by the changes in the G₂ pool. After an initial depression of the S phase parameters, three peaks with intervals of about 12 hr followed. The cells in these peaks could be followed as cohorts through the G₂ phase and mitosis, indicating a partial synchrony of cell cycle passage, with a shortening of the mean generation time of basal cells from 83-3 hr to about 12 hr. The oscillations of the proportion of cells in G₂ phase indicated a rapid passage through this cell cycle phase. The S phase duration was within the normal range but showed a moderate decrease and the Gj phase duration was decreased to a minimum. In rapidly proliferating epidermis there was a good correlation between change in the number of labelled cells and cells with S phase DNA content. This shows that micro-flow fluorometry is a rapid method for the study of cell kinetics in a perturbed cell system in vivo.