A new mutation inEscherichia coli K12,isfA,which is responsible for inhibition of SOS functions

A new mutation inEscherichia coli K12,isfA, is described, which causes inhibition of SOS functions. The mutation, discovered in a ΔpolA ⁺ mutant, is responsible for inhibition of several phenomena related to the SOS response inpolA ⁺ strains: UV- and methyl methanesulfonate-induced mutagenesis, resumption of DNA replication in UV-irradiated cells, cell filamentation, prophage induction and increase in UV sensitivity. TheisfA mutation also significantly reduces UV-induced expression of β-galactosidase fromrecA::lacZ andumuC′::lacZ fusions. The results suggest that theisfA gene product may affect RecA^* coprotease activity and may be involved in the regulation of the termination of the SOS response after completion of DNA repair. TheisfA mutation was localized at 85 min on theE. coli chromosome, and preliminary experiments suggest that it may be dominant to the wild-type allele.     

Nuclear and chloroplast transformation inChlamydomonas reinhardtii: strategies for genetic manipulation and gene expression

Transformation of the nuclear, chloroplast, and mitochondrial genomes can now be accomplished inChlamydomonas reinhardtii. Many biosynthetic pathways are carried out in the chloroplast, and efforts to manipulate these pathways will require that gene products be directed to this compartment. Chloroplast proteins are encoded in either the chloroplast or nuclear genome. In the latter case they are synthesized in the cytoplasm and imported post-translationally into the chloroplast. Thus, strategies for expressing foreign genes or overexpressing endogenous genes whose products reside in the chloroplast could involve either genome. This paper reviews the present status of transformation methodology for the nuclear and chloroplast genomes inChlamydomonas. Considerations for expressing gene products in the chloroplast are discussed. Experimental evidence for homologous recombination during transformation of the nuclear genome is presented.     

Insulin-like growth factor I enhances the formation of type I collagen in hydrocortisone-treated human osteoblasts

We have studied the effect of insulin-like growth factor I (IGF-I) on the formation of osteocalcin and type I collagen in isolated human osteoblasts. IGF-I at and above 0.1 nM stimulated the formation of type I collagen as measured by the type I procollagen carboxyterminal peptide (PICP), in human osteoblasts, incubated for 72 hrs in serumfree conditions. The secretion of osteocalcin was not affected by IGF-I while 1,25(OH)₂ vitamin D₃ significantly enhanced the formation of osteocalcin. When human osteoblast-like cells were incubated with hydrocortisone (1 M), a significant decrease in the release of both PICP and osteocalcin was seen. Addition of IGF-I to human osteoblasts also treated with hydrocortisone normalized the PICP-formation but did not affect the suppressed osteocalcin-formation. These data indicate that IGF-I reverses selective effects of hydrocortisone on bone.     

Site-directed mutagenesis of the yeast PRP20/1SRM1 gene reveals distinct activity domains in the protein product

Prp20/Srm1, a homolog of the mammalian protein RCC1 in Saccharomyces cerevisiae, binds to double-stranded DNA (dsDNA) through a multicomponent complex in vitro. This dsDNA-binding capability of the Prp20 complex has been shown to be cell-cycle dependent; affinity for dsDNA is lost during DNA replication. By analyzing a number of temperature sensitive (ts) prp20 alleles produced in vivo and in vitro, as well as site-directed mutations in highly conserved positions in the imperfect repeats that make up the protein, we have determined a relationship between the residues at these positions, cell viability, and the dsDNA-binding abilities of the Prp20 complex. These data reveal that the essential residues for Prp20 function are located mainly in the second and the third repeats at the amino-terminus and the last two repeats, the seventh and eighth, at the carboxyl-terminus of Prp20. Carboxyl-terminal mutations in Prp20 differ from amino-terminal mutations in showing loss of dsDNA binding: their conditional lethal phenotype and the loss of dsDNA binding affinity are both suppressible by overproduction of Gsp1, a GTP-binding constituent of the Prp20 complex, homologous to the mammalian protein TC4/Ran. Although wild-type Prp20 does not bind to dsDNA on its own, two mutations in conserved residues were found that caused the isolated protein to bind dsDNA. These data imply that, in situ, the other components of the Prp20 complex regulate the conformation of Prp20 and thus its affinity for dsDNA. Gsp1 not only influences the dsDNA-binding ability of Prp20 but it also regulates other essential function(s) of the Prp20 complex. Overproduction of Gsp1 also suppresses the lethality of two conditional mutations in the penultimate carboxyl-terminal repeat of Prp20, even though these mutations do not eliminate the dsDNA binding activity of the Prp20 complex. Other site-directed mutants reveal that internal and carboxyl-terminal regions of Prp20 that lack homology to RCC1 are dispensable for dsDNA binding and growth.     

Methionine transamination—metabolic function and subcellular compartmentation

Enzymatic activities catalysing the inter-conversion of L-methionine and its oxy analogue 4-methylthio-2-oxobutyric acid (2,4-KMB) were detected in the liver, skeletal muscle and heart of the laboratory rat and of sheep. In both species the highest activity of methionine transamination was found in the liver and was located in the cytoplasm and mitochondria. We propose that physiological and nutritional role of the cytoplasmic methionine transamination is amination of 2,4 KMB and formation of L-methionine while in mitochondria the activity is responsible for disposal of excess methionine is oxidised through oxidative decarboxylation of 2,4 KMB.     

Inoculation with native soil improves seedling survival and reduces non-native reinvasion in a grassland restoration

Losses of grasslands have been largely attributed to widespread land-use changes, such as conversion to row-crop agriculture. The remaining tallgrass prairie faces further losses due to biological invasions by non-native plant species, often with resultant ecosystem degradation. Of critical concern for conservation, restoration of native grasslands has been met with little success following eradication of non-native plants. In addition to the direct and indirect effects of non-native invasive plants on beneficial soil microbes, management practices targeting invasive species may also negatively affect subsequent restoration efforts. To assess mechanisms limiting germination and survival of native species and to improve native species establishment, we established six replicate plots of each of the following four treatments: (1) inoculated with freshly collected prairie soil with native seeds; (2) inoculated with steam-pasteurized soil with native seeds; (3) noninoculated with native seeds; or (4) noninoculated/nonseeded control. Inoculation with whole soil did not improve seed germination; however, addition of whole soil significantly improved native species survival, compared to pasteurized soil or noninoculated treatments. Inoculation with whole soil significantly decreased reestablishment of non-native invasive Bothriochloa bladhii (Caucasian bluestem); at the end of the growing season, plots receiving whole soil consisted of approximately 30% B. bladhii cover, compared to approximately 80% in plots receiving no soil inoculum. Our results suggest invasion and eradication efforts negatively affect arbuscular mycorrhizal hyphal and spore abundances and soil aggregate stability, and inoculation with locally adapted soil microbial communities can improve metrics of restoration success, including plant species richness and diversity, while decreasing reinvasion by non-native species.     

Anti-Candida activity of four antifungal benzothiazoles

Abstract Anti- Candida activity of 6-amino-2- n -pentylthiobenzothiazole (I), benzylester of (6-amino-2-benzothiazolylthio)acetic acid (II) and of 3-butylthio-(1,2,4-triazolo)-2,3-benzothiazole (III) was followed and compared to that of 2-mercaptobenzothiazole (IV). I and II exhibited good activity against the C. albicans yeast form, similar to IV. They were inhibitorily active against other Candida strains, IC₅₀ values being of the order of 10⁻⁵ M, which means better activity than IV. Compound I also exhibited inhibitory activity on germ-tube formation and mycelial growth in the C. albicans strains, while II, III and IV were not active in these tests. III was the least active form of the compounds tested, IC₅₀ values being of the order of 10⁻⁴ M. All the compounds tested were highly active on a nystatin-resistant C. albicans mutant, with IC₅₀s of the order of 10⁻⁶ M−10⁻⁵ M.     

The thickness of the mucus layer in different segments of the rat intestine

Summary The thickness of the pre-epithelial mucus layer has been measured in different gut segments of rats kept under normal (ad libitum) feeding conditions, and after 48 h of fasting, using cryostat sections and celloidin stabilization from samples containing luminal contents. The mucus layer of the stomach, duodenum, jejunum, ileum, caecum, proximal colon, colon transversum, distal colon and rectum was studied in five groups of male rats (10, 40, 70 and 150 days of age, and older). Underad libitum feeding conditions, a distinct and continuous mucus layer, with a thickness of more than 3 μm, was only observed in the colon transversum, in the distal colon, in the rectum and in the stomach. No pre-epithelial mucus layer was observed in the duodenum and jejunum where the glycocalix from the apical membrane of the superficial cells appeared to be in a direct contact with the luminal ingesta. In the ileum, caecum and the proximal colon, the surface epithelium of the mucosa was only partly covered by a mucus layer of highly variable thickness. After 48 h of fasting, a mucus layer of 28.8 ± 25.6 μm and 93.3 ± 59.4 μm thickness, respectively, was found in the duodenum and jejunum of adult rats, but no increase in the thickness of the mucus layer was observed in the rat hind gut.