Subunit interaction in extracellular superoxide dismutase: effects of mutations in the N-terminal domain.

Human extracellular superoxide dismutase (hEC-SOD) is a secreted tetrameric protein involved in protection against oxygen free radicals. Because EC-SOD is too large a protein for structural determination by multidimensional NMR, and attempts to crystallize the protein for X-ray structural determination have failed, the three-dimensional structure of hEC-SOD is unknown. This means that alternative strategies for structural studies are needed. The N-terminal domain of EC-SOD has already been studied using the fusion protein FusNN, comprised of the 49 N-terminal amino acids from hEC-SOD fused to human carbonic anhydrase (HCAII). The N-terminal domain in this fusion protein forms a well-defined three-dimensional structure, which probably contains alpha-helical elements and is responsible for the tetramerization of the protein. In this work, we have extended the studies, using site-directed mutagenesis in combination with size-exclusion chromatography, CD, and fluorescence spectroscopy, to investigate the nature of the tetrameric interaction. Our results show that the hydrophobic side of a predicted amphiphatic alpha-helix (formed by residues 14-32) in the N-terminal domain is essential for the subunit interaction.     

Carbon starvation of Salmonella typhimurium does not cause a general increase of mutation rates.

Mutation rates in bacteria can vary depending on the genetic target studied and the specific growth conditions of the cells. Here, two different methods were used to determine how rates of mutation to antibiotic resistance, auxotrophy, and prototrophy were influenced by carbon starvation on agar plates. The rate of mutation to rifampin resistance was increased by starvation as measured by fluctuation tests, similar to what has been reported previously for Escherichia coli. In contrast, the rates of mutation to various types of auxotrophy were unaffected or decreased as measured by both fluctuation tests and a repeated-streaking procedure. Similarly, the rates of reversion to prototrophy of his and lac nonsense and missense mutations were unaffected by starvation. Thus, mutation rates of different genetic targets can be affected differently by starvation and we conclude that carbon starvation is not generally mutagenic in Salmonella typhimurium.     

Evolutionary relationships among members of the genus Chlamydia based on 16S ribosomal DNA analysis.

Nucleotide sequences from strains of the four species currently in the genus Chlamydia, C. pecorum, C. pneumoniae, C. psittaci, and C. trachomatis were investigated. In vitro-amplified RNA genes of the ribosomal small subunit from 30 strains of C. pneumoniae and C. pecorum were subjected to solid-phase DNA sequencing of both strands. The human isolates of C. pneumoniae differed in only one position in the 16S rRNA gene, indicating genetic homogeneity among these strains. Interestingly, horse isolate N16 of C. pneumoniae was found to be closely related to the human isolates of this species, with a 98.9% nucleotide similarity between their 16S rRNA sequences. The type strain and koala isolates of C. pecorum were also found to be very similar to each other, possessing two different 16S rRNA sequences with only one-nucleotide difference. Furthermore, the C. pecorum strains truncated the 16S rRNA molecule by one nucleotide compared to the molecules of the other chlamydial species. This truncation was found to result in loss of a unilaterally bulged nucleotide, an attribute present in all other eubacteria. The phylogenetic structure of the genus Chlamydia was determined by analysis of 16S rRNA sequences. All phylogenetic trees revealed a distinct line of descent of the family Chlamydiaceae built of two main clusters which we denote the C. pneumoniae cluster and the C. psittaci cluster. The clusters were verified by bootstrap analysis of the trees and signature nucleotide analysis. The former cluster contained the human isolates of C. pneumoniae and equine strain N16. The latter cluster consisted of C. psittaci, C. pecorum, and C. trachomatis. The members of the C. pneumoniae cluster showed tight clustering and strain N16 is likely to be a subspecies of C. pneumoniae since these strains also share some antigenic cross-reactivity and clustering of major outer membrane protein gene sequences. C. psittaci and strain N16 branched early out of the respective cluster, and interestingly, their inclusion bodies do not stain with iodine. Furthermore, they also share less reliable features like normal elementary body morphology and plasmid content. Therefore, the branching order presented here is very likely a true reflection of evolution, with strain N16 of the species C. pneumoniae and C. psittaci forming early branches of their respective cluster and with C. trachomatis being the more recently evolved species within the genus Chlamydia.     

Characterization of Nine Novel Mutations in the CD40 Ligand Gene in Patients with X-Linked Hyper IgM Syndrome of Various Ancestry

X-linked immunodeficiency with hyper-IgM (HIGMX-1) is a rare disorder caused by defective expression of the CD40 ligand (CD40L) by activated T lymphocytes, resulting in inefficient T-B cell cooperation and failure of B cells to undergo immunoglobulin isotype switch. In the present work, we describe nine patients of various ancestry who bear different mutations in the X chromosome–specific CD40L gene. Two of the mutations were nonsense mutations, one each resulting in premature stop codons at amino acid residues 39 and 140. Three patients had single point missense mutations, one each at codons 126, 140, and 144. Another patient had a 4-bp genomic deletion in exon 2, resulting in a frameshift and premature termination. Three patients showed insertions, one each of 1, 2, and 4 nt, probably because of polymerase slippage, resulting in frameshift mutation and premature termination. Overall, these observations confirm the heterogeneity of mutations in HIGMX-1. However, the identification of two patients whose mutation involves codon 140 (previously shown to be altered in two other unrelated subjects) suggests that this may be a hotspot of mutation in HIGMX-1. In two additional patients with clinical and immunological features indistinguishable from canonical HIGMX-1, no mutation was detected in the coding sequence, in the 5' flanking region, or in the 3' UTR.     

Chromatographic resolution of some cyclic sulfoximides derived from prochiral and chiral sulfoxides

Three endocyclic sulfoximides of the 1-aryl- and 1-alkyl-3-oxo-benzo[d]-isothia (IV)-azole 1-oxide type (1-substituent = 2′-carboxyphenyl, 2′-carbethoxyphenyl, and octyl, respectively) were found to be well resolved on a chiral phase derived from bovine serum albumin (BSA). Selectivities (α) of 1.74, 1.12, and 1.44, respectively, were obtained. The retention behaviour of 1-octyl-3-oxo-benzo[d]isothia(IV)-azole 1-oxide was further investigated in some detail as a function of the mobile phase composition and the elution order was established from optically active material obtained from the enantiopure sulfoxide precursor. An enantiomeric excess of 85.4% was obtained in the cyclocondensation reaction of the octyl-substituted sulfoxide precursor with hydrazoic acid to the corresponding endocyclic sulfoximide. © 1995 Wiley-Liss, Inc.     

The morphological background to imbibition in seeds ofPinus sylvestris L. of different provenances

An examination was made of the structure of the coats of Scots pine (Pinus sylvestris L.) seeds of different provenance and the contribution of this factor to differences in imbibition. The seed coat layers derived from the integument, the sarcotesta, sclerotesta and endotesta did little to restrict imbibition, even though the sclerotesta of the northern provenance seeds was composed of a double multicellular layer and the sarcotesta contained large numbers of pigmented, phenol-bearing cells. In addition to the micropyle, the sclerotesta was found to possess structural openings at the chalazal end and at the ridge joining the two halves of the seed, but being covered by the pigmented cells of the sarcotesta, these did not allow water to enter any more than did the micropyle itself. Imbibition was chiefly regulated by the lipophilic covers surrounding the endosperm, which are mainly of nucellar origin, especially by the megaspore membranes nearest to the endosperm, the outer and inner exine. The nucellar cap covering the micropylar end of the endosperm proved to be impermeable to water, and its edge extended between the exine layers, which further enhanced the importance of the endosperm covers as regulators of imbibition.     

The PiGMaP consortium linkage map of the pig (Sus scrofa)

A linkage map of the porcine genome has been developed by segregation analysis of 239 genetic markers. Eighty-one of these markers correspond to known genes. Linkage groups have been assigned to all 18 autosomes plus the X Chromosome (Chr). As 69 of the markers on the linkage map have also been mapped physically (by others), there is significant integration of linkage and physical map data. Six informative markers failed to show linkage to these maps. As in other species, the genetic map of the heterogametic sex (male) was significantly shorter (16.5 Morgans) than the genetic map of the homogametic sex (female) (21.5 Morgans). The sex-averaged genetic map of the pig was estimated to be 18 Morgans in length. Mapping information for 61 Type I loci (genes) enhances the contribution of the pig gene map to comparative gene mapping. Because the linkage map incorporates both highly polymorphic Type II loci, predominantly microsatellites, and Type I loci, it will be useful both for large experiments to map quantitative trait loci and for the subsequent isolation of trait genes following a comparative and candidate gene approach.     

Purification and characterization of a vanadate-sensitive nucleotide tri- and diphosphatase with acid pH optimum from rat bone

Rat bone was extracted with KCl and Triton X-100, and a tartrate-resistant acid phosphatase activity was purified by protamine sulfate precipitation, ion-exchange chromatography (CM-cellulose), and gel filtration on Sephadex G-200 according to previously described procedures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining demonstrated a major band with an apparent monomer molecular size of approximately 14,000 Da. The enzyme is active with p-nitrophenylphosphate (p-NPP) but exhibits a 5- to 10-fold higher affinity towards several nucleotides of which ATP and ADP are the most readily hydrolyzed substrates based on kinetic studies. Based on sensitivity towards proteolytic treatment and detergent removal, as well as pH-optimum studies, a single enzyme was found to be responsible for activity towards nucleotide phosphates as well as p-NPP. This nucleotide tri- and diphosphatase constitutes around 15% of the total acid phosphatase activity in rat bone. The activity with ATP as substrate in contrast to that with p-NPP was inhibited in a noncompetitive fashion by MgCl2, sodium metavanadate, and p-chloromercuribenzoate. Enzyme activity with p-NPP and ATP is dependent on the presence of KCl and detergent and is activated by Fe3+ and ascorbate. The reported characteristics of the enzyme suggest that it functions as a unique membrane acid ATPase.     

Selective radioactive labeling of cell surface sialoglycoproteins by periodate-tritiated borohydride.

Low concentrations of sodium metaperiodate induce specific oxidative cleavage of sialic acids between carbon 7 and carbon 8 or carbon 8 and carbon 9. The aldehydes formed can easily be reduced with NaB3H4 to tritiated 5-acetamido-3,5-dideoxy-L-arabino-2-heptulosonic acid or 5-acetamido-3,5-dideoxy-L-arabino-2-octulosonic acid. At 0 degrees, the periodate anion penetrates the cell plasma membrane very slowly and only externally exposed sialic acids are oxidized. This was shown by (a) limited labeling of the sialoglycoproteins in a preparation of inside-out erythrocyte vesicles; (b) trapping 14C-labeled fetuin within resealed erythrocyte ghosts; fetuin was then poorly labeled, whereas the erythrocyte sialoglycoproteins were highly labeled; (c) comparison of labeled glycoproteins of mouse lymphoid cells before and after treatment with neuraminidase. This simple method of specifically introducing a radioactive label into cell surface sialic acids is useful in the study of cell surface sialic acid-containing glycoproteins.     

Improvement of anther culture technique: Activated charcoal bound in agar medium in combination with liquid medium and elevated CO2 concentration

Anthers of different species of the genera Anemone, Clematis, Papaver and Nicotiana were cultured by floating on a liquid medium which overlay an agarified charcoal medium . This technique proved to be superior to conventional methods i.e. culture on either solid or liquid media. Cold treatment of Anemone anthers for 7 days after inoculation on the double layer medium gave about the same frequency of embryos per anther as corresponding cultures cold treated before inoculation. An elevation of the CO₂ concentration to 2% stimulated embryogenesis in anther cultures of Anemone canadensis, Anemone vitifolia, Papaver setigerum and Papaver radicatum . Cold treatment of cultures of Anemone canadensis inhibited embryogenesis if the ensuing culture was performed in 2% CO_2. On the other hand, cold treatment was stimulating, with an optimum of about 20 days, if the cultures were maintained in normal air. Chemical analysis of untreated anthers of Anemone canadensis showed the presence of abscisic acid (2.2 × 10⁻⁶ g/g anthers). Cold treatment reduced the concentration of abscisic acid to 0.6 × 10⁻⁶ g/g anthers. By use of assays with Lemna gibba as test organism, activated charcoal was shown to adsorb abscisic acid that was added to the medium. Medium treated with charcoal before inoculation of anthers of Anemone canadensis provided to inhibit embryo production.