Purification and characterization of chick intestine brush border membrane Effects of 1α(OH) vitamin D3 treatment

A new technique has been developed for the isolation of membrane vesicles from the vitamin D-deficient and vitamin D-treated chick intestinal brush border membrane. The technique involves removal of nuclei from a low speed pellet by discontinuous sucrose gradient centrifugation. The resulting intact brush borders are then homogenized in 0.5 M Tris and the membrane fragments purified on a glycerol gradient. This preparation represents a 20-fold purification of the brush border marker sucrase. After 1α-hydroxyvitamin D₃ treatment there is a significant increase in membrane phospholipid phosphorous, an alteration in the fatty acid composition of the phosphatidylcholine fraction of membrane phospholipid, and a decrease in sucrase specific activity.     

Regression of blood vessels precedes cartilage differentiation during chick limb development

We have previously investigated distinct areas of vascular regression in the developing vascular system of the chick limb bud. Avascular areas appear in a characteristic spatial and temporal pattern, and are correlated with the position of developing cartilage. In the present study, we examined limb-bud sections which had been double labeled for endothelial cells and developing cartilage in order to determine the relationship between the appearance of cartilage and the disappearance of capillaries. Endothelial cells, which specifically take up acetylated low-density lipoprotein (acLDL), were labeled by intravenously injecting fluorescent acLDL (DiIacLDL) into chick embryos at Hamburger and Hamilton stages 26-30. Avascular zones, which correspond to the developing digits, were clearly visible within the fluorescently labeled distal vasculature. The same sections were labeled with monoclonal antibodies specific for cartilage. We found that progressing avascularity in the digital regions was followed by increased staining for cartilage antigens in the same areas. Zones of avascularity always developed earlier than morphologically and immunologically detectable cartilage in all planes of section and were always larger than the areas of cartilage. These results demonstrate that blood vessels disappear in predictable areas prior to the overt differentiation of cartilage.     

Genetic linkage of Beckwith-Wiedemann syndrome to 11p15.

Beckwith-Wiedemann syndrome (BWS), characterized by multiorgan developmental abnormalities and predisposition to cancer, usually occurs sporadically, but small apparently dominant pedigrees have been described. Since rare patients show varying karyotypic abnormalities on the short arm of chromosome 11, it has been suggested that BWS may be related to the Wilms tumor gene on 11p13 or, alternatively, to growth factor genes on 11p15. We performed genetic linkage analysis on two BWS kindreds, using RFLPs for loci on 11p. BWS was linked to the insulin gene (11p15.5), with an overall maximum lod score of 3.60 (recombination fraction = .00). Linkage to D11S16 (11p13) could be excluded for recombination fractions less than or equal to .03. These results suggest that BWS defines a tumor-predisposition gene on 11p15.     

Distinction between light-mediated and light-independent variations in phytoplankton production rates

An array of factors simultaneously controls phytoplankton photosynthesis and hence the primary production process. Because their relative importance shifts both with depth and with season, the significance of individual factors cannot be resolved by in situ incubations, even if all relevant environmental and biotic variables are measured.Here a procedure is described by which in addition to in situ measurements, photosynthesis is simultaneously assessed in identical subsamples under constant temperature (10 °C) and light (0.66 mol m^–2 h^–1 PAR conditions, in vitro). By calculating photosynthesis per unit of chlorophyll, effects of shifting biomass on photosynthesis can be eliminated but seasonal variations of light-saturated photosynthesis generated by temperature, and vertical changes of light-requirements (e.g. by light-shade adaptation) remain obscure. Quotients of in situ photosynthetic rates divided by in vitro rates allow the quantification of light-mediated changes. Provided that photosynthesis measured in vitro is light-saturated, quotients in situ: in vitro rates should never exceed unity. They are a measure for the degree of light-limitation. In vitro rates normalized to chlorophyll give information on temporal changes caused by variations in photosynthetic capacity. In Lake Constance, mean cell size appears to control light-saturated assimilation numbers.     

Characterization of the recombinant Clostridium pasteurianum ferredoxin and comparison of its properties with those of the native protein

Ferredoxins (Fds) constitute an important class of nonheme iron-sulfur proteins. One of the most studied Fds is the [8Fe-8S] Fd from Clostridium pasteurianum. The gene for this Fd has previously been cloned and sequenced. We report the expression of this Fd in Escherichia coli, and the characterization and comparison of this recombinant protein to the native Fd. We have found that the purified recombinant protein has the same enzymatic, redox, magnetic and electronic properties as the native Fd isolated from C. pasteurianum, which indicates that the two [4Fe-4S] clusters present in the Fd were correctly formed in E. coli.     

Endoproteolytic cleavage of gp160 is required for the activation of human immunodeficiency virus

The envelope protein of human immunodeficiency virus (HIV) is synthesized as a polyprotein (gp160) and cleaved intracellularly to a gp120-gp41 heterodimer. In this study, the tryptic-like endoproteolytic cleavage site was removed by site-directed mutagenesis and replaced with a chymotryptic-like site. The resultant mutant, RIP7/mut10, was found to be indistinguishable from wild-type HIV when analyzed at the level of proviral replication, RNA processing, protein expression, and viral assembly. However, the gp160 polyprotein was not cleaved and the mutated virions were biologically inactive, until and unless they were exposed to limiting concentrations of chymotrypsin. As is the case for other enveloped mammalian viruses, endoproteolytic cleavage of the HIV envelope protein and release of a unique hydrophobic domain appear to be necessary for the full expression of viral infectivity.     

Lipase production of Staphylococcus carnosus in a dialysis fermentor

Summary In a newly constructed one-vessel dialysis fermentor, a strain of Staphylococcus carnosus TM300 carrying the lipase secretion plasmid pLipPS1 was used to investigate exoenzyme and biomass production. The bacterial culture grows in an inner compartment of 21 volume, separated from a 101 nutrient broth compartment by a conventional dialysis membrane. In order to avoid substrate depletion and to prolong the growth phase, a highly concentrated nutrient broth was used. The biomass production reached 60 g cell dry weight/l. The increase in extracellular lipase concentration was directly coupled with the increase of cell mass and reached a value of 230 mg/l culture supernatant. Harvesting the cells in the late growth phase, the lipase content was about 30% of the total exoproteins in the supernatant.     

Phytoplankton and nutrients in the Helgoland region

During recent decades, phytoplankton stock on the one hand and inorganic nutrients (P and bound N) on the other have increased considerably in the southern North Sea, as demonstrated at a permanent station (since 1962) near the island Helgoland. This correlation between phytoplankton and inorganic P and N need not have anything to do with causality; exceptional algal blooms have been observed and reported in the literature since in the 19^th century. Furthermore, these increases (four-fold for phytoplankton and two-fold for nutrients) are in the same range as the fluctuations from year to year under different hydrographical conditions. A detailed investigation carried out in 1981 demonstrated the presence of a slowly growing phytoplankton population. Starting with a considerable stock of flagellates in spring, it reached a peak in cell numbers over a long reproduction period which contrasted with the normal duration of a spring bloom of diatoms. These processes were not related to a limited production by P or N. A considerable concentration of these nutrients was permanently available in the form of inorganic compounds. The total amount of nutrients surpassed by far the portion incorporated in the phytoplankton. This is a consequence of the fact that small organisms have a high metabolic rate. Therefore, the relation between stock and production (daily production ≈stock) is completely different from that known e.g. in agriculture. The nutrients exist during the vegetation period mainly in the form of dissolved organic matter that is accessible to plankton. The great dynamics of this system, including a phase shifting during the year between inorganic P, N, Si, and production, indicates the significance of permanent and fast remineralization. Calculations demonstrate that the natural nutrient content of seawater normally satisfies the demands of phytoplankton present in the North Sea area under study. Only in the more productive coastal region (salinity<30 associated with fresh water run-offs of low nutrient content — an unrealistic assumption in the German Bight) might some limitation be observed. For diatoms, silicate may represent a critical component, but a high dynamic force exists in the presence of small Si concentrations. Therefore, a lack of silicon must not represent any limitation; however, knowledge on the silicon system is insufficient up to now. Dedicated to Dr. Dr. h. c. Peter Kornmann on the occasion of his eightieth birthday.