Nursing and midwifery revalidation.

This article details the Nursing and Midwifery Council revalidation requirements essential for all registered nurses and midwives in the United Kingdom. Nursing revalidation is effective from April 2016 and is built on the pre-existing post-registration education and practice. Unlike the previous process, revalidation provides a more robust system which is clearly linked to the code and should assist towards the delivery of quality and safe effective care.

Organic neem compounds inhibit soft-rot fungal growth and improve the strength of anthracite bricks bound with collagen and lignin for use in iron foundry cupolas.

AIMS: To examine organic neem compounds for their effective growth inhibition of saprotrophic soft-rot fungi on anthracite bricks bound with collagen and lignin for use in iron foundry cupolas as an alternative fuel source. METHODS AND RESULTS: Azadirachtin, crude neem oil (NO), and clarified neem oil extract (CNO) were combined with copper to inhibit the growth of the soft-rot fungus, Chaetomium globosum. A synergistic interaction was observed between CNO and a low dose of copper on nutrient media (two-factor anova with triplicate replication: P < 0·05). Interaction was confirmed on lab-scale collagen-lignin-anthracite briquettes by measuring their unconfined compressive (UC) strength. The effective collagen strength of the briquettes was enhanced by applying CNO to their surface prior to inoculation: the room temperature UC strength of the briquettes was 28 ± 4·6% greater when CNO (0·4 mg cm(-2) ) was surface-applied, and was 43 ± 3·0% greater when CNO plus copper (0·14 µg cm(-2) ) were surface-applied. CONCLUSION: Surface application of CNO and copper synergistically prevents fungal growth on bindered anthracite briquettes and increases their room temperature strength. SIGNIFICANCE AND IMPACT OF THE STUDY: This novel organic fungicidal treatment may increase the storage and performance of anthracite bricks in iron foundries, thereby saving 15-20% of the energy used in conventional coke production.

Improved removal of arsenic from groundwater using pre-corroded steel and iron tailored granular activated carbon.

The authors have combined corrosion of steel fittings or perforated sheets with granular activated carbon (GAC) that had been pre-treated with Fe(III)-citrate, to produce an innovative and low-maintenance technique for removing arsenic from groundwater. Removal of arsenic was measured using two GAC column configurations: rapid small scale column tests (RSSCT's) and mini-column tests. Independent variables included pH, pre-corrosion procedure, and idling of the column (i.e. intentionally stopping flow for defined times in order to create reducing conditions). Use of corroded steel plus pre-treated GAC removed arsenic to below 10 microg/L for up to 248,000 bed volumes (BV) at pH 6, compared to 7,000 BVs for pre-treated GAC without pre-corroded steel. Performance was not as good at pH 6.5 or 7.5. Idling the system recovered the iron corrosion ability by reducing the passive Fe(III) layer on pre-corroded steel surface, as a result the BVs to arsenic breakthrough was doubled. But idling also caused brief periods of arsenic and iron release after restart, due to reductive dissolution of arsenic-containing ferric oxides. GAC was also effective as filtration media for removal of iron (hydr)oxide particles (and associated arsenic) that was released from the pre-corroded iron.

Alfalfa yield response to inoculation with recombinant strains of Rhizobium meliloti with an extra copy of dctABD and/or modified nifA expression.

The construction of rhizobial strains which increase plant biomass under controlled conditions has been previously reported. However, there is no evidence that these newly constructed strains increase legume yield under agricultural conditions. This work tested the hypothesis that carefully manipulating expression of additional copies of nifA and dctABD in strains of Rhizobium meliloti would increase alfalfa yield in the field. The rationale for this hypothesis is based on the positive regulatory role that nifA plays in the expression of the nif regulon and the fact that a supply of dicarboxylic acids from the plant is required as a carbon and energy source for nitrogen fixation by the Rhizobium bacteroids in the nodule. These recombinant strains, as well as the wild-type strains from which they were derived, are ideal tools to examine the effects of modifying or increasing the expression of these genes on alfalfa biomass. The experimental design comprised seven recombinant strains, two wild-type strains, and an uninoculated control. Each treatment was replicated eight times and was conducted at four field sites in Wisconsin. Recombinant strain RMBPC-2, which has an additional copy of both nifA and dctABD, increased alfalfa biomass by 12.9% compared with the yield with the wild-type strain RMBPC and 17.9% over that in the uninoculated control plot at the site where soil nitrogen and organic matter content was lowest. These increases were statistically significant at the 5% confidence interval for each of the three harvests made during the growing season. Strain RMBPC-2 did increase alfalfa biomass at the Hancock site; however, no other significant increases or decreases in alfalfa biomass were observed with the seven other recombinant strains at that site. At three sites where this experiment was conducted, either native rhizobial populations or soil nitrogen concentrations were high. At these sites, none of the recombinant strains affected yield. We conclude that RMBPC -2 can increase alfalfa yields under field conditions of nitrogen limitation, low endogenous rhizobial competitors, and sufficient moisture.

A plant selectable marker gene based on the detoxification of the herbicide dalapon.

A gene from Pseudomonas putida coding for a dehalogenase capable of degrading 2,2 dichloropropionic acid (2,2DCPA), the active ingredient of the herbicide dalapon, has been isolated and characterised. In plant transformation experiments the gene was shown to confer resistance to 2,2DCPA at a tissue culture level where 2,2DCPA could be used to select for transformants. At the whole plant level, transformed plants showed resistance to 2,2DCPA at concentrations up to 5 times the recommended dose rate of dalapon when it was sprayed on their leaves. At lower concentrations, the herbicide caused a non-lethal yellowing of sensitive plants which clearly distinguished them from resistant plants. The mode of action of chlorinated aliphatic acids is not known but they probably affect many enzyme pathways. The results described here are the first example of engineering a plant resistant to a herbicide that does not have one specific enzyme as its target site. This gene has several advantages as a marker in plant breeding and genetic studies. For example, the herbicide is readily available and has low toxicity, transformants can be selected at both the tissue culture and the whole plant level, a large number of transformed plants can easily be screened even in the field, and there is a very low probability of selecting spontaneous mutants.

Identification of a 110-kDa nonintegrin cell surface laminin-binding protein which recognizes an A chain neurite-promoting peptide.

Laminin is a potent promoter of neurite outgrowth, and a synthetic peptide of 19 amino acids, PA22-2, from the A chain has been found to promote process formation. Using peptide affinity chromatography, we have identified a 110-kDa, cell surface ligand from both neural cells and brain which binds this sequence. This binding protein does not share immunological identity with the B1 chain of integrin, and reduction does not alter its mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibody to the 110-kDa protein stained cellular processes in vivo. Sequence analysis of the first 18 amino acids from the amino terminus yielded almost exact sequence identity with nucleolin, a major 110-kDa nucleolar phosphoprotein. Antibody to nucleolin, however, does not interact with the neural-derived, laminin-peptide-binding 110-kDa protein. The 110-kDa protein appears to be a ligand for a specific site on laminin.

Adult and fetal human mesangial cells interact with specific laminin domains.

Mesangial cells are centrally located pericytes in the renal glomerulus. They are surrounded by an extracellular matrix and directly contact the glomerular basement membrane in vivo. Because these interactions are critical for renal development and function, we have studied human mesangial cell interactions with laminin, a major adhesive component of basement membranes present in the extracellular matrix of the mesangium. Human fetal and adult mesangial cell attachment was stimulated by both laminin and the laminin-derived synthetic peptides YIGSR-NH2, CQAGTFALRGDNPQG-NH2, and CIKVAVS-NH2. Furthermore, mesangial cells spread on laminin as well as on both the RGD-containing and CIKVAVS peptides. When added in solution, all three peptides inhibited mesangial cell attachment to laminin, and the latter two peptides inhibited mesangial cell spreading on laminin. Laminin affinity column chromatography demonstrated several low-molecular-mass laminin-binding proteins ranging from between 35 and 42 kDa, which predominated in fetal mesangial cells, whereas a higher molecular mass laminin-binding protein of 65 kDa was predominant in adult mesangial cells. Western blot analysis with an anti-32-kDa laminin-binding protein antibody showed increased expression of both 31- and 42-kDa proteins in fetal mesangial cells when compared with the adult. The antisera to the 32-kDa laminin-binding protein also inhibited fetal mesangial spreading on the CIKVAVS peptide. Western blot analysis with an anti-67-kDa laminin-binding protein antibody revealed a 110-kDa protein in adult mesangial cells that was not present in fetal mesangial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Degradation of the Herbicide Glyphosate by Members of the Family Rhizobiaceae.

Several strains of the family Rhizobiaceae were tested for their ability to degrade the phosphonate herbicide glyphosate (isopropylamine salt of N-phosphonomethylglycine). All organisms tested (seven Rhizobium meliloti strains, Rhizobium leguminosarum, Rhizobium galega, Rhizobium trifolii, Agrobacterium rhizogenes, and Agrobacterium tumefaciens) were able to grow on glyphosate as the sole source of phosphorus in the presence of the aromatic amino acids, although growth on glyphosate was not as fast as on P(i). These results suggest that glyphosate degradation ability is widespread in the family Rhizobiaceae. Uptake and metabolism of glyphosate were studied by using R. meliloti 1021. Sarcosine was found to be the immediate breakdown product, indicating that the initial cleavage of glyphosate was at the C-P bond. Therefore, glyphosate breakdown in R. meliloti 1021 is achieved by a C-P lyase activity.

Organ-specific modulation of gene expression in transgenic plants using antisense RNA.

We have shown leaf-specific inhibition GUS gene expression in transgenic Nicotiana plants using an antisense RNA with a 41-base homology spanning the translation start codon of the gene. GUS was expressed from the nominally constitutive 35S promoter and the antisense RNA was expressed from the light-regulated ca/b promoter of Arabidopsis thaliana. A range of GUS inhibition from 0 to 100% was obtained by screening a small population of transgenic plants and the specific levels of inhibition observed were stably inherited in two generations. An antiGUS 'gene' dosage effect was observed in plants which were homozygous for antiGUS. RNA detection results suggest that duplex formation with the 41 base pair antiGUS RNA destabilized the GUS mRNA and that an excess of antisense RNA was not required. Our results demonstrate the potential of antisense RNA as a strategy for obtaining plant mutants, especially 'down mutations' in essential genes where only a short 5' sequence of the mRNA is required. They also suggest that the 'position effect' on gene expression could be used in conjunction with an antisense RNA strategy to provide a versatile approach for crop improvement.

The effect of presumptive polyadenylation signals on the expression of the CAT gene in transgenic tobacco.

Promoter::gene fusions which differed only in the presumed polyadenylation signals attached to the 3' end of the gene, have been used to examine the effect of these signals on expression of the gene in protoplasts and in transgenic plants. The gene constructs were comprised of the 35S promoter, the chloramphenicol acetyl transferase (CAT) gene and DNA sequences carrying the presumed polyadenylation signals of the nopaline synthase (nos) gene or of transcript 7 (T-7) from the T-DNA ofAgrobacterium. Our results show that levels of gene expression were not significantly affected by the orientation or absence of these sequences. We therefore suggest that the practice of cloning presumptive polyadenylation sequences at the 3' end of a gene for expression in plants may be unnecessary.

Nucleotide sequence and mutagenesis of the nifA gene from Azotobacter vinelandii.

The nucleotide sequence of the nifA gene from Azotobacter vinelandii was determined. This gene encodes an Mr = 58,100 polypeptide that shares significant sequence identity when compared to nifA-encoded products from other organisms. Interspecies comparisons of nifA-encoded products reveal that they all have a consensus ATP binding site and a consensus DNA binding site in highly conserved regions of the respective polypeptides. The nifA gene immediately precedes the nifB-nifQ gene region but is unlinked to the major nif gene cluster from A. vinelandii. A potential regulatory gene precedes and is apparently cotranscribed with nifA. Mutant strains that have a deletion or a deletion plus an insertion within nifA are incapable of diazotrophic growth and they fail to accumulate nitrogenase structural gene products.

Laminin receptors for neurite formation.

Laminin, a basement membrane glycoprotein promotes both cell attachment and neurite outgrowth. Separate domains on laminin elicit these responses, suggesting that distinct receptors occur on the surface of cells. NG108-15 neuroblastoma-glioma cells rapidly extend long processes in the presence of laminin. We report here that 125I-labeled laminin specifically binds to these cells and to three membrane proteins of 67, 110, and 180 kDa. These proteins were isolated by affinity chromatography on laminin-Sepharose. The 67-kDa protein reacted with antibody to the previously characterized receptor for cell attachment to laminin. Antibodies to the 110-kDa and 180-kDa bands demonstrated that the 110-kDa protein was found in a variety of epithelial cell lines and in brain, whereas the 180-kDa protein was neural specific. Antibodies prepared against the 110-kDa and 180-kDa proteins inhibited neurite outgrowth induced by the neurite-promoting domain of laminin, whereas antibodies to the 67-kDa laminin receptor had no effect on neurite outgrowth. We conclude that neuronal cells have multiple cell-surface laminin receptors and that the 110-kDa and 180-kDa proteins are involved in neurite formation.

Expression and functional analysis of the Rhizobium meliloti nifA gene.

The translational initiation point for Rhizobium meliloti nifA, the specific activator for nitrogen fixation (nif) genes, was determined. When expressed in an Escherichia coli linked transcriptional-translational system the DNA coding for the R.meliloti nifA gene produced four polypeptide bands of 71 000, 67 000, 62 000 and 59 000 daltons. There are three in-frame ATG codons at the N-terminus of the gene; by replacing the poor ribosome binding sites of the native DNA with a synthetic consensus ribosome binding site prior to each of ATG codons the polypeptides were produced at enhanced levels and related to each of the initiation codons. The ability of these specifically expressed polypeptides to activate nif promoters fused to lacZ was determined. Only the fulllength polypeptide activated the Klebsiella pneumoniae nifH, R.meliloti nifH and fixA and Bradyrhizobium japonicum nifH and nifD promoters. The R.meliloti fixA promoter, contrary to previous evidence, could be activated in E.coli. Deletion of the putative N-terminal domain of the R.meliloti nifA gene product greatly increased the ability of the protein to activate nif promoters. However, deletions retaining part of this domain were not functional. This shows that the N-terminal domain is not essential for activity and that its presence decreases the full potential function of the protein. Our results are consistent with the suggesting that this domain has a regulatory role. In contrast to K.pneumoniae nifA protein, the function of the full length and domain deleted forms of R.meliloti nifA gene product was sensitive to oxygen in E.coli.

Parameters affecting the frequency of kanamycin resistant alfalfa obtained by Agrobacterium tumefaciens mediated transformation.

Kanamycin resistant plants of Medicago varia A2 were obtained by an optimized procedure for high frequency transformation using Agrobacterium tumefaciens infection of leaf and petiole tissue. Parameters which affected the frequency were explant type, the Agrobacterium strain used and the time allowed for cocultivation. Under optimum conditions, i.e., using the Agrobacterium strain A281 and a 4 day cocultivation period, the frequency of transformed leaflets obtained was greater than 70%.

Use of extracellular matrix components for cell culture.

Extracellular matrix components when used as a substratum in vitro can greatly influence cell behavior. The response observed is dependent on the type of cell and matrix used. Cells in vitro usually respond best to the matrix components with which they are normally in contact in vivo. More differentiated phenotypes are observed and cells generally survive longer on such matrices. In some cases, the presence of such matrices allows cells to be cultured in the absence of serum and growth factors. As more investigators try the matrices and matrix components described, as well as new components and combinations of them, it is anticipated that improvement in the culture of many cells can be expected.

Comparative organization of nitrogen fixation-specific genes from Azotobacter vinelandii and Klebsiella pneumoniae: DNA sequence of the nifUSV genes.

In the facultative anaerobe Klebsiella pneumoniae 17 nitrogen fixation-specific genes (nif genes) have been identified. Homologs to 12 of these genes have now been isolated from the aerobic diazotroph Azotobacter vinelandii. Comparative studies have indicated that these diverse microorganisms share striking similarities in the genetic organization of their nif genes and in the primary structure of their individual nif gene products. In this study the complete nucleotide sequence of the nifUSV gene clusters from both K. pneumoniae and A. vinelandii were determined. These genes are identically organized on their respective genomes, and the individual genes and their products exhibit a high degree of interspecies sequence homology.

Genes for basement membrane proteins are coordinately expressed in differentiating F9 cells but not in normal adult murine tissues.

We have obtained cDNA clones coding for the A, B1, and B2 chains of laminin by screening a cDNA library prepared from mouse EHS tumor poly(A)RNA in the lambda gt11 expression vector with polyclonal antibody against denatured laminin. These cDNA clones were used in combination with a cDNA clone coding for the alpha 1 type IV collagen chain to study the regulation of genes for these basement membrane proteins in retinoic acid-induced differentiating mouse F9 teratocarcinoma cells and in various adult murine tissues. The levels of mRNA for the laminin A, B1, and B2 chains and for the alpha 1 type IV collagen chain were increased simultaneously and reached a maximum at almost the same time during the differentiation of F9 cells, suggesting coordinate expression in these cells. The tissue levels of mRNA encoding for the basement membrane components, however, varied considerably. The highest level of the B1 chain mRNA was observed in kidney, whereas, the levels of mRNA for A and B2 chains were highest in heart. Almost the same levels of expression of the alpha 1(IV) collagen mRNA were found in kidney, lung, and heart. The results indicate that the expression of genes for the basement membrane proteins is not coordinately regulated in these tissues. It is thus possible that different subunit structures of the laminin molecule may exist in tissues.