Crystals of the NC1 domain of human type IV collagen

Crystals of the non-collagenous C-terminal region (NC1) of type IV collagen have been obtained from human placenta. These crystals diffract to 2.0 A, and belong to space group P22(1)2(1), with cell dimensions a = 81 A, b = 158 A, c = 138 A, alpha = beta = gamma = 90 degrees. The crystals contain one hexamer in the asymmetric unit; they are very stable with respect to X-rays.     

Effects of the Introduction of Agrobacterium tumefaciens T-DNA ipt Gene in Nicotiana tabacum L. cv. Petit Havana SR1 Plant Cells

Transformation of tobacco leaf discs with the ‘cytokinin’ipt gene yielded several transgenic callus tissue lines, respectiveto the kind of ipt construction present in the A. tumefacienscointegrates. Those calli containing an active ipt gene wereable to grow hormone-autotrophically and showed an increasedendogenous cytokinin level in comparison with controls. Analysisof endogenous IAA level did not allow any quantitative correlationwith the cytokinin content. However, a minimal level of auxinseems to be necessary to obtain hormone-autotrophic growth.Exogenously supplied NAA significantly reduced the endogenouscytokinin content without modifying growth characteristics. The varying chlorophyll content in the different callus lineselicited the study of the ultrastructure of the plastids. Thecontrols contained small plastids, often filled with starchor accumulated vesicles that did not allow observation of theinternal membrane system. The ‘Pssu-ipt’ line, havinga higher cytokinin content, showed plastids with an internalmembrane system consisting of stroma and grana thylakoids, butthis structure was lost during subculture. Swollen thylakoidsappeared, the amount of starch was reduced and vesicles wereaccumulating. (Received November 15, 1990; Accepted March 4, 1991)     

Binding-protein expression is subject to temporal,developmental and stress-induced regulation in terminally differentiated soybean organs

Binding protein (BiP) is a widely distributed and highly conserved endoplasmic-reticulum luminal protein that has been implicated in cotranslational folding of nascent polypeptides, and in the recognition and disposal of misfolded polypeptides. Analysis of cDNA sequences and genomic blots indicates that soybeans (Glycine max L. Merr.) possess a small gene family encoding BiP. The deduced sequence of BiP is very similar to that of other plant BiPs. We have examined the expression of BiP in several different terminally differentiated soybean organs including leaves, pods and seed cotyledons. Expression of BiP mRNA increases during leaf expansion while levels of BiP protein decrease. Leaf BiP mRNA is subject to temporal control, exhibiting a large difference in expression in a few hours between dusk and night. The expression of BiP mRNA varies in direct correlation with accumulation of seed storage proteins. The hybridization suggests that maturing-seed BiP is likely to be a different isoform from vegetative BiPs. Levels of BiP protein in maturing seeds vary with BiP mRNA. High levels of BiP mRNA are detected after 3 d of seedling growth. Little change in either BiP mRNA or protein levels was detected in maturing soybean pods, although BiP-protein levels decrease in fully mature pods. Persistent wounding of leaves by whiteflies induces massive overexpression of BiP mRNA while only slightly increasing BiP-protein levels. In contrast single-event puncture wounding only slightly induces additional BiP expression above the temporal variations. These observations indicate that BiP is not constitutively expressed in terminally differentiated plant organs. Expression of BiP is highest during the developmental stages of leaves, pods and seeds when their constituent cells are producing seed or vegetative storage proteins, and appears to be subject to complex regulation, including developmental, temporal and wounding.The mention of vendor or product does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over vendors of similar products not mentioned.Abbreviations BiP binding protein The sequences reported in this paper have been submitted to Gen-Bank and are identified with the accession numbers BiP-A (U08384), BiP-B (U08383), BiP-C (U08382) and -1,3 glucanase (U08405).     

The impact of acidification on diatoms and chemistry of Dutch moorland pools

Summary Moorland pools are shallow oligotrophic soft water lakes on poorly buffered sandy soils. Diatom assemblages of samples from 16 pools taken in 1920 and 1978 were compared by analysis of pH-spectra, diversity, dissimilarity and multivariate statistical techniques.The pH-spectra of pools in the southern (S) and central (C) part of the country indicate a fall in pH from 4.5–6.0 in the old samples to 3.7–4.6 in the recent ones. The pH-spectra of the northern pools (N) do not indicate a significant shift from the original pH (ca 4.5).The number of species in the count and the diversity (indices of Simpson and Shannon) decreased significantly in S+C, and that goes also for the dissimilarity index of Dyer. No changes were found in N.The first component (PC 1) of the principal component analysis explains 61% of total variance. PC 1 is correlated with log [SO₄] (r=0.83, p<0.001) and even better (r=0.95, p<0.001) with the relative sulphate concentration,i.e. the ratio of sulphate to all major anions (sulphate, chloride, bicarbonate). All old samples have low scores on PC 1, recent samples have low scores on the second (PC 2) and third (PC 3) principal component. Old samples have high scores on PC 2 and PC 3, explaining 9 and 6% of total variance, respectively.The orginal variation, caused by regional factors, is replaced by a SO₄ ^2– controlled variation. PC 1 is nearly completely determined by the relative abundance ofEunotia exigua. This species, which is known to be very resistant to pollution by sulphur, aluminium and heavy metals, increased largely from 1920 to 1978.In spite of the rather homogeneous distribution of wet sulphate deposition in the Netherlands, substantial differences in SO₄ ^2– content in the pools are observed, being lowest in N (0.13–0.48 meq.l^–1) and highest in S+C (0.38–1.65 meq.l^–1). Sulphate is positively correlated with calcium, aluminum and magnesium but negatively with factors that characterize humic acid waters (e.g. permanganate-consumption, iron and the ratio of univalent to divalent cations). Sulphate concentration depends on the intensity of sulphate reduction, accumulation by dry deposition in surrounding forests of Scots pine, drought and atmospheric deposition.     

Expression,Purification and Low-Resolution Structure of Human Vitamin C Transporter SVCT1 (SLC23A1)

Expression and purification of human membrane proteins for structural studies represent a great challenge. This is because micro- to milligram amounts of pure isolated protein are required. To this aim, we successfully expressed the human vitamin C transporter-1 (hSVCT1; SLC23A1) in Xenopus laevis oocytes and isolated highly pure protein in microgram amounts. Recombinant hSVCT1 was functional when expressed in oocytes and glycosylated. Structural analysis of purified hSVCT1 by transmission electron microscopy and single particle analysis unveiled its shape, dimensions and low-resolution structure as well as the existence of a major monomeric and minor dimeric population. Chemical crosslinking of isolated oocyte membranes containing expressed hSVCT1 indicated similar oligomeric states of hSVCT1 in lipid bilayers. This work reports the first purification and structural analysis of a human SVCT protein and opens the way for future functional and structural studies using purified hSVCT1.     

The potential of AFLPs in biosystematics: A first application inSolanum taxonomy(Solanaceae)

Using the AFLP technique highly informative DNA fingerprints were generated from 19 taxa ofSolanum sect.Petota (potatoes) and three taxa ofSolanum sect.Lycopersicum (tomatoes). Both phenetic and cladistic analyses were conducted from the individual genotypic level to the species level. An AFLP fingerprint, using a combination of suitable AFLP primers, generated 12 to 71 scorable fragments per genotype which was sufficient for taxonomic interpretation. The classifications based on the molecular markers were generally in agreement with current taxonomic opinions. Unexpectedly,S. microdontum was associated with ser.Megistacroloba rather than with ser.Tuberosa, andS. demissum (ser.Demissa) and species of ser.Acaulia appeared closely affiliated. AFLP is an efficient and reliable technique to generate biosystematic data and therefore a promising tool for evolutionary studies.     

Demonstration of the ascorbate dependence of membrane-bound dopamine beta-monooxygenase in adrenal chromaffin granule ghosts

Chromaffin granule ghosts from bovine adrenal medullae have been used to examine the ability of membrane-bound dopamine beta-monooxygenase to interact directly with intravesicular ascorbate and to investigate vectorial electron transfer from external ascorbate across the ghost membrane. Ghosts prepared by a modification of published procedures were shown to be fully active in both dopamine uptake and norepinephrine production. Dopamine uptake is dependent on the presence of a magnesium and ATP ionic complex, is abolished by reserpine, and reaches a steady-state level in the presence of dopamine beta-monooxygenase, ascorbate, catalase, and fumarate. Omission of ascorbate either inside or outside the ghosts greatly enhances dopamine accumulation, which reaches levels of approximately 30 nmol/mg under these conditions. Correspondingly, in the presence of all components, norepinephrine production reached approximately 100 nmol/mg in 30 min of incubation. Norepinephrine production was strictly magnesium-ATP-dependent, inhibited by either reserpine or dopamine beta-monooxygenase inactivation, and was markedly reduced when ascorbate was omitted from either inside or outside the ghosts. In the presence of limiting amounts of internal ascorbate, rapid norepinephrine production occurred which corresponded to the amount of initial ascorbate present, followed by a much slower endogenous norepinephrine production observable after complete depletion of internal ascorbate. The endogenous rate of norepinephrine production likely represents epinephrine-supported dopamine beta-monooxygenase turnover. Taken together, the data demonstrate that facile norepinephrine production by membrane-bound dopamine beta-monooxygenase occurs only when internal ascorbate is present, terminates upon depletion of internal ascorbate, and can only be sustained at a significant rate when reducing equivalents from external ascorbate are available.