Stimulation of the hexose-monophosphate pentose pathway by delta 1-pyrroline-5-carboxylic acid in human fibroblasts.

L-pyrroline-5-carboxylic acid, an intermediate in the interconversions of glutamic acid, ornithine and proline, is a potent stimulator of the hexose-monophosphate pentose pathway in cultured human fibroblasts. These studies suggest that pyrroline-5-carboxylate reductase, which catalyzes the conversion of pyrroline-5-carboxylate to proline coupled with the oxidation of NADPH, provides the NADP for the observed activation of the hexose-monophosphate pentose pathway.     

Clinical implications of developments in in vitro fertilisation.

During February 1979 to December 1983, 831 infertile couples were treated by in vitro fertilisation and embryo transfer. The problems they faced included deciding on the number of oocytes to be collected at laparoscopy, the numbers to be donated or fertilised, the numbers of embryos to be transferred and frozen, and whether abnormal embryos should be used for research or discarded. The 831 patients received a total of 1530 treatment cycles. Of the 763 patients for whom complete data were available, 136 (17.8%) became pregnant. The rate of pregnancy, however, increased dramatically from 7.4% when only one embryo was transferred to 21.1% and 28.1% when two and three embryos were transferred, respectively. The chance of multiple pregnancy also increased with the number of embryos transferred, but the risk (2% for twins) was far outweighed by the relatively poor result after transferring a single embryo. Out of 40 embryos freeze-thawed, 23 survived thawing and were transferred; of these, 4 (17%) resulted in pregnancy. Thirty four transfers of donor oocyte embryos also resulted in four pregnancies (12%), but two of these ended in abortion. Neither microscopy nor any other available test can determine the potential of an oocyte to result in pregnancy, so that discarding oocytes that may look abnormal simply reduces the chances of conception--both for the patient and for any prospective recipient of donor oocyte embryos. In any case, abnormal embryos tend to die when growth is allowed to continue in vitro. Probably all oocytes harvested from a patient should be inseminated and the utilisation of the embryos decided once the number developed is known.     

Deuterium NMR investigation of polymorphism in stratum corneum lipids

The intercellular lipid lamellae of stratum corneum constitute the major barrier to percutaneous penetration. Deuterium magnetic resonance and freeze-fracture electron microscopic investigation of hydrated lipid mixtures consisting of ceramides, cholesterol, palmitic acid and cholesteryl sulfate and approximating the stratum corneum intercellular lipid composition, revealed thermally induced polymorphism. The transition temperature of bilayer to hexagonal transition decreased as the ratio of cholesterol to ceramides in these mixtures was lowered. Lipid mixtures in which the stratum corneum ceramides were replaced by synthetic dipalmitoylphosphatidylcholine did not show any polymorphism throughout the temperature range used in the present study. The ability of the ceramide-containing samples to form hexagonal structures establishes a plausible mechanism for the assembly of the stratum corneum intercellular lamellae during the final stages of epidermal differentiation. Also, the bilayer to hexagonal phase transition of these nonpolar lipid mixtures could be used to enhance the penetration of drugs through skin.     

What spectroscopy can still tell us about the secondary structure of bacteriorhodopsin.

The recently published model of the structure of bacteriorhodopsin (bR), developed by fitting the peptide chain to a high-resolution, three-dimensional density map, rules out the existence of transmembrane beta-sheet and provides an accurate estimate of the helix content. The precise geometry of the dihedral angles in the helical regions of the polypeptide cannot yet be specified from the diffraction data, however. Published data on the circular dichroism (CD) spectrum between 190 and 240 nm, and the infrared (IR) spectrum in the amide I band suggest that the helical conformation in bR may be, for the most part, a rather unusual one. The precise structural model, which specifies the number of residues in transmembrane helices, can now be used as an additional constraint in seeking models of the helical conformation that are in quantitative agreement with the CD and IR spectroscopic data. Further spectroscopic measurements can also be used to determine whether there are changes in the unusual dihedral-angle conformation within the helices during the photocycle.     

Genetic sex determination,sex chromosome size and sex-specific lifespans across tetrapods

Sex differences in lifespan are ubiquitous across the tree of life and exhibit broad taxonomic patterns that remain a puzzle, such as males living longer than females in birds and vice versa in mammals. The prevailing unguarded X hypothesis explains sex differences in lifespan by differential expression of recessive mutations on the X or Z chromosome of the heterogametic sex, but has only received indirect support to date. An alternative hypothesis is that the accumulation of deleterious mutations and repetitive elements on the Y or W chromosome might lower the survival of the heterogametic sex (‘toxic Y’ hypothesis). Here, we use a new database to report lower survival of the heterogametic relative to the homogametic sex across 136 species of birds, mammals, reptiles and amphibians, as expected if sex chromosomes shape sex-specific lifespans, and consistent with previous findings. We also found that the relative sizes of both the X and the Y chromosomes in mammals (but not the Z or the W chromosomes in birds) are associated with sex differences in lifespan, as predicted by the unguarded X and the ‘toxic Y’. Furthermore, we report that the relative size of the Y is negatively associated with male lifespan in mammals, so that small Y size correlates with increased male lifespan. In theory, toxic Y effects are expected to be particularly strong in mammals, and we did not find similar effects in birds. Our results confirm the role of sex chromosomes in explaining sex differences in lifespan across tetrapods and further suggest that, at least in mammals, ‘toxic Y’ effects may play an important part in this role.     

Biocontrol of the Sugarcane Borer Eldana saccharina by Expression of the Bacillus thuringiensis cry1Ac7 and Serratia marcescens chiA Genes in Sugarcane-Associated Bacteria

The cry1Ac7 gene of Bacillus thuringiensis strain 234, showing activity against the sugarcane borer Eldana saccharina, was cloned under the control of the tac promoter. The fusion was introduced into the broad-host-range plasmid pKT240 and the integration vector pJFF350 and without the tac promoter into the broad-host-range plasmids pML122 and pKmM0. These plasmids were introduced into a Pseudomonas fluorescens strain isolated from the phylloplane of sugarcane and the endophytic bacterium Herbaspirillum seropedicae found in sugarcane. The ptac-cry1Ac7 construct was introduced into the chromosome of P. fluorescens using the integration vector pJFF350 carrying the artificial interposon Omegon-Km. Western blot analysis showed that the expression levels of the integrated cry1Ac7 gene were much higher under the control of the tac promoter than under the control of its endogenous promoter. It was also determined that multicopy expression in P. fluorescens and H. seropedicae of ptac-cry1Ac7 carried on pKT240 caused plasmid instability with no detectable protein expression. In H. seropedicae, more Cry1Ac7 toxin was produced when the gene was cloned under the control of the Nm^r promoter on pML122 than in the opposite orientation and bioassays showed that the former resulted in higher mortality of E. saccharina larvae than the latter. P. fluorescens 14::ptac-tox resulted in higher mortality of larvae than did P. fluorescens 14::tox. An increased toxic effect was observed when P. fluorescens 14::ptac-tox was combined with P. fluorescens carrying the Serratia marcescens chitinase gene chiA, under the control of the tac promoter, integrated into the chromosome.     

EGF-like domain calcium affinity modulated by N-terminal domain linkage in human fibrillin-1

Calcium binding epidermal growth factor-like domains (cbEGFs) are present in many extracellular proteins, including fibrillin-1, Notch-3, protein S, factor IX and the low density lipoprotein (LDL) receptor, which perform a diverse range of functions. Genetic mutations that cause amino acid changes within these proteins have been linked to the Marfan syndrome (MFS), CADASIL, protein S deficiency, haemophilia B and familial hypercholesterolaemia, respectively. A number of these mutations disrupt calcium binding to cbEGFs, emphasising the critical functional role of calcium in these proteins.We have determined the calcium binding affinity of two sites within a cbEGF pair (cbEGF12-13) from human fibrillin-1 using two-dimensional nuclear magnetic resonance (NMR) and fluorescence techniques. Fibrillin-1 is a mosaic protein containing 43 cbEGF domains, mainly arranged as tandem repeats. Our results show that the cbEGF13 site in the cbEGF12-13 pair possesses the highest calcium affinity of any cbEGF investigated from fibrillin-1. A comparative analysis of these and previously reported calcium binding data from fibrillin-1 demonstrate that the affinity of cbEGF13 is enhanced more than 70-fold by the linkage of an N-terminal cbEGF domain. In contrast, comparison of calcium binding by cbEGF32 in isolation relative to when linked to a transforming growth factor beta-binding protein-like domain (TB6-cbEGF32) reveals that the same enhancement is not observed for this heterologous domain pair. Taken together, these results indicate that fibrillin-1 cbEGF Ca2+ affinity can be significantly modulated by the type of domain which is linked to its N terminus. The cbEGF12-13 pair is located within the longest contiguous section of cbEGFs in fibrillin-1, and a number of mutations in this region are associated with the most severe neonatal form of MFS. The affinities of cbEGF domains 13 and 14 in this region are substantially higher than in the C-terminal region of fibrillin-1. This increased affinity may be important for fibrillin assembly into 10-12 nm connective tissue microfibrils and/or may contribute to the biomechanical properties of the microfibrillar network.