Rabbit β-glucuronidase. Purification and properties, and the existence of multiple forms

1. beta-Glucuronidase (EC 3.2.1.31) was purified from rabbit liver by a procedure involving autolysis, (NH(4))(2)SO(4) fractionation, chromatography on DEAE-cellulose and hydroxyapatite, gel filtration, sedimentation in a sucrose gradient, and isoelectric focusing. 2. Electron microscopy revealed ferritin as the major contaminant in later stages of purification and also showed aggregates of enzyme molecules. Particular attention was paid to the removal of ferritin. 3. The purified enzyme was homogeneous in polyacrylamide-gel electrophoresis both in non-dissociating conditions and in the presence of sodium dodecyl sulphate, and in Ouchterlony gel diffusion and immunoelectrophoresis against polyspecific antisera. 4. Sedimentation in sucrose gradients gave a molecular weight of 300000, whereas gel filtration indicated 440000. 5. Subunits of 75000 molecular weight were observed in gel electrophoresis in the presence of sodium dodecyl sulphate and in gel filtration in the presence of urea. 6. The K(m) value for p-nitrophenyl beta-d-glucuronide was 0.6mm, and the enzyme was extremely sensitive to lactone inhibitors. It was also inhibited by Hg(2+) ions. 7. Multiple forms were observed in the pure enzyme by isoelectric focusing, with pI values of 4.5-5.8. Subunits showed similar heterogeneity. The origin of the multiple forms was investigated in detail, and the possibility of artifact generation largely excluded. Some of the forms of lowest pI disappeared after neuraminidase digestion. The nature of the residual heterogeneity remains to be elucidated.     

Science,philosophy, and politics in the work of J. B. S. Haldane, 1922–1937

This paper analyzes the interaction between science, philosophy and politics (including ideology) in the early work of J. B. S. Haldane (from 1922 to 1937). This period is particularly important, not only because it is the period of Haldane's most significant biological work (both in biochemistry and genetics), but also because it is during this period that his philosophical and political views underwent their most significant transformation. His philosophical stance first changed from a radical organicism to a position far more compatible with mechanical materialism. The primary intellectual influence that was responsible for this shift was that of F. G. Hopkins. Later, Haldane came to accept Marxism and its official metaphysics, dialectical materialism, a move that let him accept the materialist conception of the world while still maintaining a resolute distance from mechanism. Throughout all these changes, what is most obvious is the influence of science on Haldane's philosophical views. An influence in the opposite direction is far less apparent.Parts of this paper are extracted from a longer work which concerns the interactions between philosophy and science throughout Haldane's scientific career (Sarkar forthcoming). The general conclusions reached here, from a consideration of Haldane's work only from 1922 to 1937 (see Section 6), remain the same for the rest of his life, as is detailed in the longer work. Thanks are due to R. S. Cohen, J. F. Crow, A. R. Fersht, J. Maynard Smith, R. C. Olby, D. Paul, M. Ruse, J. Stachel and S. Sturdy for helpful discussions and comments and criticism of the positions outlined in this paper. This is Contribution No. BTBG-92-4 from the Theoretical Biology Group, Boston University.     

Free‐running rhythmicity in the life spans of hydranths of the marine cnidarian,Campanularia flexuosa

Abstract

The adrenergic agonists and antagonists, norepinephrine, phenylephrine, iso‐proterenol, phentolamine, and propranolol, were administered to rats in different phases of the diurnal cycle, and their effects on tyrosine aminotransferase activity were studied. All substances tested, regardless whether being typically α‐ or ß ‐adrenergic or whether being agonists or antagonists, elevated tyrosine aminotransferase during the minimum of enzyme activity. The effects at the enzyme maximum, however, were generally more or less depressive. The extent of the depressions by norepinephrine and by propranolol highly depended on the duration of treatment. The hepatic concentration of cyclic AMP did not exhibit a significant rhythmicity. The results do not favour the idea of an adrenergic control of the diurnal rhythm in tyrosine aminotransferase activity.     

A New Efficient Technology for the Isolation of Human α-Fetoprotein and the Status of Free Sulfhydryl and Amino Groups in the Resulting Preparation

A new preparative method for the isolation of human -fetoprotein (AFP) from cord blood serum was described. This involves the precipitation of high-molecular compounds with polyethylene glycol and affinity, ion exchange, and gel permeation chromatographies. Up to several tens of milligrams of the homogeneous AFP can be rapidly (48 h) prepared in a high yield per one isolation cycle. The native structure of the isolated AFP was proved by immunochemical analysis. No free sulfhydryl groups were found in the purified AFP, and its reduction with dithiothreitol at 4°C led to the formation of two sulfhydryl groups probably belonging to the Cys18 and Cys67 residues in domain I of the protein molecule. Up to ten amino groups in the purified AFP were shown to be accessible to a mild modification by 3-(2-pyridyldithio)propionic acid N-succinimide ester.     

Ontogenesis of the α-MSH, β-MSH and ACTH cells in the foetal hypophysis of the rat. Correlation with the growth of the adrenals and adrenocortical activity

Pituitary sections from 15 to 21 day-old rat foetuses have been studied with the immunofluorescence technique, using antibodies anti alpha-MSH, anti beta-MSH and anti beta (1-24) ACTH. The first ACTH cells appear on day 17 of pregnancy in the pars distalis of the hypophysis and only on day 18 in the pars intermedia. beta-msh cells have been observed on day 16 in the pars anterior and on day 17 in the pars intermedia, while alpha-MSH cells appear only on day 18 and exclusively in the pars intermedia. The cytodifferentiation of the beta-MSH and ACTH cells occurs in the pars intermedia with about a 24 hours delay in comparison to that of the pars distalis. The first revealed cells are always located in the posterior half of the pituitary gland. The corticostimulating activity of the hypophysis has been tested with the fluorescence intensity of the corticotrophs, the adrenal weight, the adrenal content in corticosterone and the plasma corticosterone level. The fluorescence of the corticotrophs increases on day 18, shows a maximum on day 19 and decreases until term. The adrenal weight rises regularly between day 16 to day 20, thereafer the increase subsides. Adrenal and plasma corticosterone concentrations reach a peak on day 19 of pregnancy. These data suggest that hypophyseal corticostimulating activity is very high between days 18 and 19 and decreases between days 19 and 21.     

Reconstruction of the Allerød vegetation of the Neuwied Basin, western Germany, and its surroundings at 12,900 cal b.p.

During the late-glacial eruption of the Laacher See volcano 12,900 b.p. the whole area of the Neuwied Basin, middle Rhine region, was covered by several metres of the Laacher See Tephra (LST) damaging but also partly conserving the former vegetation. This gave the unique opportunity to study plant remains in situ and to estimate the spatial biodiversity of plant taxa within the area investigated at the end of the last glacial as if in a snapshot of 12,900 years ago. Different kinds of plant material can be found because of different preservation conditions namely imprints, charcoal and waterlogged remains. Investigations of this material from several places within the Neuwied Basin and the Brohl valley north of it allowed the reconstruction of the former vegetation at different scales from local to regional. This resulted in a large scale draft of a vegetation map of the studied area also based on the distribution and the stage of soil development at that time. As to whether thermophilous trees were already present is discussed but largely rejected. Electronic Supplementary Material Supplementary material is available for this article at     

Review of the fauna of Rotatoria,Cladocera, and Copepoda of the basin of the Anadyr’ River

The zooplankton composition is studied in the thermokarst, glacial and meteorite lakes, channels, former riverbeds, and hollows in the basin of Anadyr’. We found 174 taxa: 78, Rotatoria, 55, Cladocera, and 41, Copepoda. The most diverse is the lake fauna: 51 taxa of Rotatoria, 48, Cladocera, and 37, Copepoda. The thermokarst Lake Maiorskoe hosts 68 taxa: 31, Rotatoria, 14, Cladocera, and 23, Copepoda, wheras the cold ultraoligotrophic Lake El’gygytgyn features only one species of Cyclop of the group scutifer Cyclops neymanae Strel., and Rotatoria and Cladocera are present as allochtonous forms. The Copepoda illustrate the relations of the Anadyr’ fauna with those of Europe, North America, and Japan.     

Biochemical studies of the chromaffin granule. III. Redistribution of lipid phosphate,dopamine-β-hydroxylase and chromogranin a after freezing and thawing of the isolated granule membranes

Freezing and thawing a dialysed suspension of lysed chromaffin granules and sedimented membrane preparations resulted in redistribution of lipid phosphate and protein. By this treatment the high ratios of lipid phosphate/protein in the membrane fragments, isolated on sucrose density gradient from the dialysed suspensions and the sedimented membrane preparation, were reduced from 1.56 to 1.03 μmoles/mg and from 1.97 to 0.83 μmoles/mg, respectively.Multilamellar, liposomal structures could be isolated from the frozen and thawed membrane preparations and were found to sediment in the 0.4 M sucrose layer by density gradient centrigugation. This fraction was without morphological resemblance to the intact chromaffin granules or their membranes and was found to account for 53% of the lipid phosphate, 35% of chromogranin A, 21% of dopamine-β-hydroxylase activity and 12% of the protein of the total preparation. The specific activities of chromogranin A and dopamine-β-hydroxylase in the artificially formed liposomal structures closely resembled that of the solubilized protein and was significantly higher than in the lipid phosphate-depleted membrane fragments recovered in the 1.1 M sucrose layers.It is concluded that freezing and thawing as a means of purifying the isolated granule membranes lead not only to the solubilization of chromogranin A, but also to removal of dopamine-β-hydroxylase activity and lipid phosphate from the labile membrane fragments.     

Behaviour of selected PCBs,PAHs and γ-HCH in the Scheldt estuary,S.W. Netherlands

The behaviour of some organic micropollutants in the abiotic compartments water, particulate matter and bottom sediments of the Scheldt estuary, S.W. Netherlands, was studied between 1986 and 1989. Special attention was given to two individual PolyChlorinated Biphenyl congeners (PCB 52 and PCB 153), two Polycyclic Aromatic Hydrocarbons (PAHs) fluoranthene and benzo(a)pyrene and γ-HCH. Dissolved PCBs and γ-HCH behave conservatively during estuarine mixing. The concentration of γ-HCH in the fresh-water is extremely high in spring compared to the other seasons. Dissolved fluoranthene does not behave conservatively. The concentration of dissolved fluoranthene in the freshwater endmember shows a seasonal dependence with highest concentrations in winter and lowest in summer. Benzo(a)pyrene could hardly be detected in the dissolved phase with the methods used. PCBs and PAHs in particulate matter and sediments behave essentially conservatively as a result of the mixing of riverine particulates with high, and marine particulates with low organic micropollutant contents. Particulate fluoranthene is removed at low salinities under (nearly) anoxic conditions probably by microbial degradation. The rivers Scheldt and Rupel appear to be the major sources for the compounds studied. However, in particular the organic micropollutant contents of the sediments indicate that important emissions along the estuary were or are still present. Measurements of individual PCBs and PAHs in the <63 μm fraction of a dated sediment core from a salt marsh in the eastern part of the Scheldt estuary show that the recent input of PCBs and PAHs into the estuaryis probably 2–3 times lower than the maximum input in the mid-1960s. The PCB profile shows postdepositional congener-selective mobilization caused by advective transport. The PAH assemblage is remarkably uniform in the sediment core. The PAH ratios indicate that combustion of coal is the main source of PAHs in the Scheldt estuary.     

Free serosal cells originating from the embryo of the wasp Diadromus pulchellus in the pupal body of parasitized leek-moth,Acrolepiosis assectella. Are these cells teratocyte-like?

In braconid species, teratocytes are derived from a serosal cell membrane which envelops the developing parasitoid embryo. On hatching, this membrane dissociates into individual cells, the teratocytes, which then circulate in the haemolymph of the host. We describe herein such a membrane, surrounding the embryo in eggs of the ichneumonid parasitoid wasp, Diadromus pulchellus. This membrane consisted of a single sheet of tightly packed cells with large 12+/-1.4 &mgr;m nuclei. These cells were released after hatching in vitro and cells of the same size were detected in vivo, in the vicinity of the D. pulchellus embryo. The number of nuclei detected suggests that the serosal membrane consists of about 450+/-150 cells. These cells did not grow after hatching of the parasitoid egg in the parasitized host, Acrolepiosis assectella, during the development of the parasitoid wasp larva. Southern blot experiments, using D. pulchellus satellite DNA or the ribosomal genes as probes, showed that free-living floating cells of wasp origin were present in the body of the parasitized host. This is the first time that free-floating teratocyte-like cells have been described in species of the Ichneumonidae.