Identification of the coding region for the putidaredoxin reductase gene from the plasmid of Pseudomonas putida

The first 12 NH₂-terminal amino acids of the Pseudomonas putida putidaredoxin reductase were shown to be Met-Asn-Ala-Asn-Asp-Asn-Val-Val-Ile-Val-Gly-Thr. Comparison of these data with the DNA sequence of the BamHI-HindIII 197-base fragment derived from the PstI 2.2-kb fragment obtained from the P. putida plasmid showed that the putidaredoxin reductase gene was downstream from the cytochrome P-450 gene and the intergenic region had the 24-nucleotide sequence TAAACACATGGGAGTGCGTGCTAA. The Shine-Dalgarno sequence GGAG was detected in this region. The initiating triplet for the reductase gene was GTG, which normally codes for valine, but in the initiating codon position codes for methionine. From the amino acid sequence and X-ray data comparisons with other flavoproteins, what appears to be the AMP binding region of the FAD can be recognized in the NH₂-terminal portion of the reductase involving residues 5–35.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

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.     

Single channel kinetics of a glutamate receptor.

The glutamate receptor-channel of locust muscle membrane was studied using the patch-clamp technique. Muscles were pretreated with concanavalin A to block receptor-channel desensitization, thus facilitating analysis of receptor-channel gating kinetics. Single channel kinetics were analyzed to aid in identification of the molecular basis of channel gating. Channel dwell-time distributions and dwell-time autocorrelation functions were calculated from single channel data recorded in the precence of 10-4M glutamate. Analysis of the dwell time distributions in terms of mixtures of exponential functions revealed there to be at least three open states of the receptor-channel and at least four closed states. Autocorrelation function analysis showed there to be at least three pathways linking the open states with the closed. This results in a minimal scheme for gating of the glutamate receptor-channel, which is suggestive of allosteric models of receptor-channel gating.     

New and noteworthy South American species of Agalinis (Scrophulariaceae)

Agalinis bandeirensis is a Brazilian species distinguished by linear bracts, short pedicels, and villous stamens.Agalinis ramulifera, from southern Brazil, is distinguished by many, short, leafy branches and small flowers.Agalinis linarioides subsp.rojasi, from Paraguay, is distinguished by paniculate inflorescences, short calyx lobes, and broad corollas.Gerardia bangii, G. digitalis, andG. meyeniana are recognized as species ofAgalinis and appropriate transfers are made.     

New species of Aristolochia (Aristolochiaceae) from Peru

Aristolochia fosteri is a new species with unusual branched trichomes collected in Pasco near Oxapampa.Aristolochia hutchisonii is a prostrate species found in Amazonas and Cajamarca that is related toA. weberbaueri.Aristolochia barbouri is a narrow-leaved, herbaceous liana from Madre de Dios.     

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.     

Three new species of Elleanthus (Orchidaceae) from Central America

Elleanthus stolonifer andE. tillandsioides are new species in theE. poiformis complex of sectionChloidelyna.Elleanthus lentii is a new species in sectionStachydelyna. Elleanthus stolonifer is widespread in the mountains of Costa Rica and Panama;E. tillandsioides is found only in the lowland forests of southeastern Costa Rica;E. lentii is limited to the Cordillera de Guanacaste of Costa Rica.     

Two new species of Esterhazya (Scrophulariaceae) from Brazil

Barringer, Kerry (Department of Botany, Field Museum of Natural History, Chicago, IL 60605). Two new species of Esterhazya (Scrophulariaceae) from Brazil. Brittonia 37: 195–198. 1985.—Esterhazya petiolata is a distinctive species with ovate, petiolate leaves, spreading brances, and broadly tubular-campanulate flowers. It is known from the Serra Dourada of Goiás. Esterhazya eitenorum has narrow, lanceolate leaves, acute calyx lobes, and purple-pink flowers and is known from the planalto of Mt. Itatiaia.