Steady-State Effects of 2,5,2',5'-Tetrachlorobiphenyl on Growth, Photosynthesis, and P Uptake in Selenastrum capricornutum

The steady-state effect of 2,5,2',5'-tetrachlorobiphenyl (TCBP) on the green alga Selenastrum capricornutum was investigated in a P-limited two-stage chemostat system. The partition coefficient of this polychlorinated biphenyl congener was 5.9 x 10 in steady-state cultures. At a cellular TCBP concentration of 12.2 x 10 ng . cell, growth rate was not affected. However, photosynthetic capacity (P(max)) was significantly enhanced by TCBP (56 x 10 mumol of C . cell . h versus 34 x 10 mumol of C . cell . h in the control). Photosynthetic efficiency, or the slope of the photosynthesis-irradiance curve, was also significantly higher. There was little difference in the cell chlorophyll a content, and therefore the difference in these photosynthetic characteristics was the same even when they were expressed on a per-chlorophyll a basis. Cell C content was higher in TCBP-containing cells than in TCBP-free cells, but approximately 36% of the C fixed by cells with TCBP was not incorporated as cell C. The maximum P uptake rate was also enhanced by TCBP, but the half-saturation concentration appeared to be unaffected.     

Thermogenesis of white adipocytes: a novel method allowing long-term microcalorimetric investigations

A microcalorimetric method was developed to facilitate long-term assessment of energy balance in isolated fat cells. White rat adipocytes were primary cultured in glass ampoules with a matrix of agar-gel. Heat production was measured continuously over 3 days with an LKB BioActivityMonitor. In order to assist interpretation of the microcalorimetric measurements, glucose consumption and lactate and pyruvate production were determined in parallel cultures. Heat production, glucose consumption and lactate production were in an apparent steady state throughout the study whether employing aerobic (94 pW, 0.50 and 0.44 pmol/cell. h, respectively) or partial anaerobic experimental conditions (39 pW, 0.41 and 0.57 pmol/cell.h, respectively). However, oxygen availability influenced the apparent heat production and glucose and lactate metabolism. With partial anaerobiosis a 59% lower heat production, an 18% lower glucose consumption and a 30% higher lactate production than by employing aerobic experimental conditions were observed.     

Physical mapping of two Xp markers DXS16 and DXS143

Summary Lymphocyte karyotyping of an infant girl with the clinical features of microphthalmia, iridoschisis, goiter, hip joint dysplasia, labium synechia and craniotabes revealed an Xp deletion. The lymphocyte karyotypes of the parents were normal. Bromodeoxyuridine incorporation studies showed that, in 42 out of 43 metaphases, the deleted X chromosome was late replicating. In one metaphase, the normal X chromosome was observed to be allocyclic. Using DNA markers from the Xp22 region, the breakpoint was assigned distal to DXS16 (pXUT23) and proximal to DXS143 (dic56). Dosage intensity measurements confirmed that the STS gene and the DNA marker DXS31 were involved in the deleted area. Restriction fragment length polymorphism analysis revealed that the paternally derived X-chromosome was deleted.     

Purification and characterization of two oxidoreductases involved in the enantioselective reduction of 3-oxo, 4-oxo and 5-oxo esters in baker's yeast

Two NADPH-dependent oxidoreductases catalyzing the enantioselective reduction of 3-oxo esters to (S)- and (R)-3-hydroxy acid esters, [hereafter called (S)- and (R)-enzymes] have been purified 121- and 332-fold, respectively, from cell extracts of Saccharomyces cerevisiae by means of streptomycin sulfate treatment, Sephadex G-25 filtration, DEAE-Sepharose CL-6B chromatography, Sephadex G-150 filtration, Sepharose 6B filtration and hydroxyapatite chromatography. The relative molecular mass Mr, of the (S)-enzyme was estimated to be 48,000-50,000 on Sephadex G-150 column chromatography and 48,000 on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The enzyme was most active at pH 6.9 and reduced 3-oxo esters, 4-oxo and 5-oxo acids and esters enantioselectively to (S)- hydroxy compounds in the presence of NADPH. The Km values for ethyl 3-oxobutyrate, ethyl 3-oxohexanoate, 4-oxopentanoic and 5-oxohexanoic acid were determined as 0.9 mM, 5.3 mM, 17.1 mM and 13.1 mM, respectively. The Mr of the (R)-enzyme, estimated by means of column chromatography on Sepharose 6B, was 800,000. Under dissociating conditions of SDS/polyacrylamide gel electrophoresis the enzyme resolved into subunits of Mr 200,000 and 210,000, respectively. The enzyme is optimally active at pH 6.1, catalyzing specifically the reduction of 3-oxo esters to (R)-hydroxy esters, using NADPH for coenzyme. Km values for ethyl 3-oxobutyrate and ethyl 3-oxohexanoate were determined as 17.0 mM and 2.0 mM, respectively. Investigations with purified fatty acid synthase of baker's yeast revealed that the (R)-enzyme was identical with a subunit of this multifunctional complex; intact fatty acid synthase (Mr 2.4 X 10(6)) showed no activity in catalyzing the reduction of 3-oxo esters.     

Characterization of dimer subunits of intermediate filament proteins

The fundamental subunit of the various types of intermediate-sized filaments (IF) has been shown to be a tetramer that is thought to represent a double dimer, i.e. an array of two laterally packed coiled-coils of alpha-helices. The two-chain state of intact IF proteins had up to this point not been isolated and characterized as has been done for other fibrous alpha-helical coiled-coil proteins. Using buffers containing 3 M-guanidinium hydrochloride we prepared dimers by depolymerization of IF or by reconstitution from fully denatured molecules. Dimers of desmin (from chicken gizzard), vimentin (from bovine lens tissue and cultured human fibroblasts) and the neurofilament protein NF-L (from bovine brain) as well as in vitro formed homodimers of human and rat cytokeratins numbers 8 (A), 18 (D) and 19 ("40K"), are characterized by ultracentrifugation techniques (sedimentation velocity and equilibrium), electron microscopy and chemical cross-linking. The results show that IF proteins from discrete complexes of two polypeptide chains in parallel orientation and probably in coiled-coil configuration, which apparently have a high tendency to further associate into double dimers. Implications of these results for concepts of IF organization and IF protein assembly are discussed.     

Trypanosoma cruzi cells undergo an alteration in protein N-glycosylation upon differentiation

Trypanosoma cruzi epimastigotes (insect gut stage) incubated with [U-14C]glucose synthesized Man9GlcNAc2-P-P-dolichol as practically the sole dolichol-P-P derivative. On the other hand, amastigotes (intracellular stage) of the same parasite synthesized four to five times more Man7GlcNAc2-P-P-dolichol than Man9GlcNAc2-P-P-dolichol. Evidence is presented indicating that, whereas in epimastigotes only Man9GlcNAc2 was transferred to proteins, in amastigotes both Man7GlcNAc2 and Man9GlcNAc2 were transferred in direct proportion to their respective amounts bound to dolichol-P-P. The change in the mechanism of protein N-glycosylation could be observed upon in vitro differentiation of amastigotes to epimastigotes. The dissimilar size of the main oligosaccharides transferred to proteins in epimastigotes and amastigotes was responsible for differences in two structural features of high mannose-type oligosaccharides present in mature glycoproteins of both forms of the parasite, namely the average size of the compounds and the structure of the main species of some isomer oligosaccharides.     

Structural model of the collagen-like region of C1q comprising the kink region and the fibre-like packing of the six triple helices

A detailed three-dimensional model of the collagenous part of C1q was derived by model building and computer-aided energy refinement calculations. The proposed structure is based on the collagen-like (-Gly-Xaa-Yaa-) repeating sequence of 78 to 81 residues in the N-terminal regions of the constituent A, B and C chains, on the mode of disulphide linkage between the 18 chains of C1q, and on its electron microscopically derived gross structure. It is demonstrated that the interruptions of the repeating sequence about half-way along the length of the collagenous regions (Gly36-Ile37-Arg38-Thr39 in the A chain and Ala36-Ile37-Hy138 in the C chain) do not lead to a disruption of the triple helical conformation but rather to a bend of about 60 degrees in an otherwise continuous triple helix. These features are consistent with a flexibility comparable with that of regular triple helices and with the observed low proteolytic susceptibility of the kink region. The azimuthal orientation of the kink is defined approximately by ArgA38 being located in the cap of the knee. Because of this extra residue between two glycine residues, a bad contact that would arise between the methyl group of AlaC36 and the peptide carbonyl of IleA37 in a straight triple helix is relaxed. The model features also a cluster of hydrophobic contacts between large hydrophobic side-chains in the interaction edges between the six collagen triple helices aligned with their about 10 nm long N-terminal regions in the fibril-like endpiece of C1q. The azimuthal orientations of the triple helices were derived by energy calculations of side-chain interactions previously applied to fibre-forming collagens. Independently, the same orientations and interaction edges were derived from the azimuthal orientation of the kink and the electron microscopically observed orientations of the triple helical arms that emerge from the endpiece, and which carry the C-terminal globular binding domains. The structural model has a number of implications for the assembly of the first component of complement from C1q and the zymogen complex C1r2C1s2 and possible mechanisms of its activation.     

Polymorphism of reconstituted human epidermal keratin filaments: determination of their mass-per-length and width by scanning transmission electron microscopy (STEM)

We have determined the mass-per-length (MPL) and the width of unstained freeze-dried reconstituted human epidermal keratin filaments by scanning transmission electron microscopy (STEM). Filaments were reassembled from keratins extracted from four different sources: cultured human epidermal cells (CHEC), human callus (CAL), and the living layers (LL) and stratum corneum (SC) of normal human epidermis. MPL histograms of all four keratin filament types could be fitted by a superposition of two or three Gaussians, with their respective major peaks located between 17 and 20 kDa/nm. We interpreted the multiple MPL peaks to represent different polymorphic forms of the reconstituted filaments. The number of subunits per filament cross section calculated from MPL peak positions, average subunit molecular weight, and an axial repeat of the subunits within the filament of 46.5 nm revealed an average difference between polymorphic variants of 7.5 +/- 0.9 subunits. These data suggest that reconstituted human epidermal keratin filaments are made of two to four 8-stranded "protofibrils" (i.e., made of two laterally aggregated 4-stranded protofilaments), in agreement with earlier observations. The average widths of unstained freeze-dried keratin filaments were larger than those of negatively stained filaments: 12.6 nm (9.6 nm) for CHEC, 12.3 nm (9.7 nm) for CAL, 11.6 nm (8.3 nm) for LL, and 11.3 nm (7.9 nm) for SC keratin filaments, with the values in brackets corresponding to negatively stained samples. Assuming the MPL to be proportional to the square of the filament width, there is a good correlation between the MPL and width measurements both for filaments within a given type as well as among those reconstituted from different types of keratin extracts.