Structural and functional characterization of second-coordination sphere mutants of soybean lipoxygenase-1.

Lipoxygenases are an important class of non-heme iron enzymes that catalyze the hydroperoxidation of unsaturated fatty acids. The details of the enzymatic mechanism of lipoxygenases are still not well understood. This study utilizes a combination of kinetic and structural probes to relate the lipoxygenase mechanism of action with structural modifications of the iron's second coordination sphere. The second coordination sphere consists of Gln(495) and Gln(697), which form a hydrogen bond network between the substrate cavity and the first coordination sphere (Asn(694)). In this investigation, we compared the kinetic and structural properties of four mutants (Q495E, Q495A, Q697N, and Q697E) with those of wild-type soybean lipoxygenase-1 and determined that changes in the second coordination sphere affected the enzymatic activity by hydrogen bond rearrangement and substrate positioning through interaction with Gln(495). The nature of the C-H bond cleavage event remained unchanged, which demonstrates that the mutations have not affected the mechanism of hydrogen atom tunneling. The unusual and dramatic inverse solvent isotope effect (SIE) observed for the Q697E mutant indicated that an Fe(III)-OH(-) is the active site base. A new transition state model for hydrogen atom abstraction is proposed.     

Defects in the processing of procollagen to collagen are demonstrable in cultured fibroblasts from patients with the Ehlers-Danlos and osteogenesis imperfecta syndromes

This is a study of the processing of procollagen to collagen in cultures of skin and tendon fibroblasts. Processing was markedly increased by growing cells for 2-4 days postconfluence and then adding ascorbate to the medium for 2 days prior to labeling with [3H] proline. With this system, more than two-thirds of the pro-alpha chains of type I procollagen in the culture medium, and more than 90% of those in the cell layer, were rapidly processed to pC-alpha, pN-alpha, or alpha chains. Purified, exogenous procollagen was also rapidly processed in cell-free culture medium. The results showed for the first time that exogenous procollagen can be processed in conditioned cell-free medium. The system was then used to compare the processing of procollagen in the medium of normal fibroblasts, cells from one bovine and four human variants of osteogenesis imperfecta, and those from eight human variants of the Ehlers-Danlos syndrome. The cells could be divided into three groups, based on their ability to process type I procollagen: normal, consistently slow, and very slow. The cause of the decreased processing was shown to be associated with either a mutation causing a shortening of an alpha chain or decreased activity of procollagen N-proteinase in cell-free culture medium. Decreased processing of procollagen to collagen occurred with cultured fibroblasts from patients with different forms of both osteogenesis imperfecta and Ehlers-Danlos syndrome. Both of these disease syndromes are associated with abnormalities in the structure or metabolism of procollagen in fibrous connective tissues, bones, and teeth. The results show that defects in the structure, synthesis, or processing of procollagen are readily demonstrated with cultured fibroblasts.     

P Nutrition during Seed Development : Leaf Senescence, Pod Retention, and Seed Weight of Soybean

Soybean (Glycine max [L.] Merrill) leaf senescence, which may partially result from mineral redistribution, appears to limit grain yield. Two experiments were designed to test the effects of supplemental inorganic phosphate (Pi), K, malate, and methionine (Met) infusions on senescence and yield. A novel stem infusion technique using pediatric intravenous kits was developed to supply these nutrients throughout seed growth. An average of 48.4 milliliters per plant was successfully infused into lower stem internodes during a 4 to 6 week period. Senescence was unaffected by K or malate infusions, but was delayed by Pi infusions (up to 8 days) and by increased nutrient solution Pi levels (up to 21 days) in separate experiments. Treatments which delayed senescence also improved yield as much as 3-fold, due primarily to increased pod retention and secondarily to increased seed size. Met infusions further increased pod retention at the lower, infused nodes, and thus increased total plant yield also. The influence of higher Pi levels during reproductive growth on soybean pod retention and yield may have been the result of sustained sucrose export due to altered C partitioning in leaves. The role of Met in improving yield was not clear. However, these results clearly demonstrate the importance of adequate Pi for delaying senescence and improving pod retention and yield.     

Poliovirus type 3: molecular cloning of the genome and nucleotide sequence of the region encoding the protease and polymerase proteins.

Overlapping cDNA clones representing the entire genome of poliovirus type 3 have been prepared in E. coli by two separate methods. Cloning of RNA . cDNA hybrids produced a more comprehensive set of clones with generally larger cDNA inserts than cloning of double - stranded cDNA. A restriction map of the entire genome and the nucleotide sequence of 2003 bases from the 3' terminus, comprising the region encoding the protease and polymerase proteins, are presented.     

Search for rhythmicity during hibernation in the European hamster

Temporal patterns of hibernation were studied by continuous monitoring of body temperature by radiotelemetry over 6 months in European hamsters, Cricetus cricetus, at constant temperature and photoperiod. Entrances into hibernation occurred mostly at the end of the night (0000–0800 hours), while arousals were randomly distributed between day and night. This is at variance with a control of bout duration by a clock with a period of 24 h. Consequently, the timing of entrances implies a phase-resetting of the circadian clock on each arousal. Persistence of circadian rhythmicity with a period different from 24 h during deep hibernation was investigated examining whether the durations of torpor bouts were integer multiples of a constant period. A non-parametric version of the classical contingency test of periodicity was developed for this purpose. Periods ranging from 21 to 29 h were tested. Nine animals out of ten showed at least one significant period in this range (P<0.01), either below 24 h (21.8±0.5 h, n=4) or above (27.3±0.5 h, n=7). However, we have found a theoretical model of bout durations for which the contingency test of periodicity sometimes gives false significant results. This indicates that the power of the test is weak. With this reservation our results suggest that a circadian oscillator controls the duration of a bout of hibernation, which would occur after an integer, but variable and possibly temperature-dependent number of cycles.Abbreviations _b a contingency test (see Appendix) - SCN suprachiasmatic nuclei - period - T _b body temperature     

Decay-accelerating factor CD55 is identified as the receptor for echovirus 7 using CELICS, a rapid immuno-focal cloning method.

Using an anti-receptor mAb that blocks the attachment of echovirus 7 and related viruses (echoviruses 13, 21, 29 and 33), we have isolated a complementary DNA clone that encodes the human decay-accelerating factor (CD55). Mouse cells transfected with the CD55 clone bind echovirus 7, and this binding is blocked by the anti-receptor mAb. The method used (CELICS) allows rapid and direct cloning of genes encoding cell surface receptors. It is based on episomal replication and high efficiency expression of complementary DNA clones in the vector pCDM8 in COS or WOP cells, in conjunction with a sensitive immuno-focal screen that uses antibody probes linked to beta-galactosidase. Receptor positive cells were identified by a colour change and isolated individually using a micromanipulator. DNA extracted from a small number of cells was then cloned directly in Escherichia coli.