Lernaeopoda tenuis n. sp. andPseudolernaeopoda caudocapta n. g., n. sp. (Copepoda,Lernaeopodidae) parasitic onTriakis maculata (Kner & Steindachner) from the Chilean coast,South Pacific

Summary One new genus and two new species of the Lernaeopodidae (Copepoda: Siphonostomatoida), parasitic onTriakis maculata off the coast of Chile, are described and illustrated. The new genus,Pseudolernaeopoda, parasitizes the cloaca of its host. Its type-species,P. caudocapta, can be distinguished fromLernaeopoda by the absence of modified uropods. A new species ofLernaeopoda, L. tenuis, was found on the gills of the same host. In absence of the male, the generic affinities of this species must be considered only tentative. ac]19850409     

Complete structure elucidation of a trisaccharide from a patient with mannosidosis by carbon-13 nuclear magnetic resonance spectrometry

Through the exclusive use of ¹³C NMR data a relatively complex oligosaccharide was completely characterized in terms of its sugar composition, ring forms, anomeric configurations, sequence, and interresidue linkage positions. Thus, the unique structure of a trisaccharide from a patient with α-mannosidosis was deduced to be Manpα1–3Manp β1–4N-acetylglucosamine. The study demonstrated that the empirical approach of matching chemical shifts between those of known and unknown structures and the use of chemical shift-predictive rules, obtained from a series of related compounds, can be successfully applied to the elucidation of carbohydrate structure in spite of the spectral complexities derived from the multiplicity of similar chemical shifts. This study also demonstrates that as more structure-chemical shift relationships are established with carbohydrates, the easier it will be to use ¹³C NMR for their characterization.     

Human hemoglobin expression in Escherichia coli: importance of optimal codon usage.

The overexpression of a nonfusion product of human beta-globin in Escherichia coli from its cDNA sequence has been accomplished for the first time. Expression of beta-globin from its native cDNA required the use of the strong bacteriophage T7 promoter. In this system, beta-globin accumulated to approximately 10% of total E. coli proteins. alpha-Globin was not expressed in the T7 system using the native cDNA. For the expression of alpha-globin, synthetic genes containing optimal E. coli codons were constructed. Neither synthetic alpha- nor beta-globin gene alone was expressed from the lac or tac promoter. Globin expression was achieved when the two synthetic alpha- and beta-globin genes were combined as an operon downstream of the lac promoter. The two proteins combined intracellularly with endogenous heme, which was concomitantly overproduced to yield tetrameric hemoglobin as roughly 5-10% of total E. coli protein. Cloning the alpha- and beta-globin cDNAs in a construct identical with the lac promoter did not yield globin production, establishing the requirement for optimal codon usage. The recombinant beta-globin from the T7 expression system was purified and reconstituted in vitro with heme and native alpha chains. N-terminal analyses showed that the beta-globin produced in the T7 system and the tetrameric hemoglobin produced from the synthetic genes contained an additional beta 1 methionine residue. Two additional mutants, beta 1 Val----Met and beta 1 Val----Ala were produced using the T7 system. Functional and structural properties of the purified hemoglobins will be discussed in the following papers.     

Storage in water ofRhizobium phaseoli cells harvested from continuous culture and its effect on biological nitrogen fixation

Summary Taken cells from cultures ofRhizobium phaseoli grown at different dilution rates were tested for plant growth promotion in a bioassay withPhaseolus vulgaris, before and after storage in water for one month. Cells grown at higher dilution rates showed a greater loss of effectiveness     

Formation of closed vesicles from a simple phosphate diester. Preparation and some properties of vesicles of dihexadecyl phosphate

Upon sonication in water above 55°, dihexadecyl phosphate forms aqueous dispersions. Gel filtration, substrate entrapment and electron microscopic investigations indicate that these dispersions consist of closed vesicles possessing the characteristics of single bilayer liposomes. These dispersions are quite sensitive to the presence of salts. These wholly synthetic phosphate diester vesicles provide one of the simplest models for the mimicry of membrane and transport functions.     

Simple and rapid determination of carboplatin in plasma by high-performance liquid chromatography. Error pattern and application to clinical pharmacokinetic studies

Carboplatin is an antitumor agent widely employed in cancer chemotherapy. A specific and selective method for the determination of carboplatin in human plasma and its applications to pharmacokinetic investigations is described. One ultrafiltration step, through a Centrifree micropartition system (Amicon) at 2000 g for 10 min, is the only requirement as sample treatment. The resulting solution is injected into an Inertsil ODS-2 (5 μm, 25 cm×4.6 mm I.D.) analytical column. The mobile phase consisted of 0.1 M potassium dihydrogenphosphate with 1 mM dipotassium edetate adjusted to a pH between 3 and 3.5. The limit of quantitation was 1 mg/l. The method showed good recovery (100.68±5.49%) and precision: the within-day relative standard deviation (RSD) for carboplatin (3–350 mg/l) was 2.07% and the between-day RSD for carboplatin, in the previously described range, was 1.31%. We determined the assay error pattern for proper weighting of serum level data in pharmacokinetic models. The selectivity (discrimination between the parent drug and platinum-containing species such as carboplatin metabolites), simplicity and speed of this assay for free carboplatin quantitation should facilitate pharmacokinetic investigations and therapeutic drug monitoring.     

The Bacillus subtilis endospore crust: protein interaction network,architecture and glycosylation state of a potential glycoprotein layer

The endospore of Bacillus subtilis is formed intracellularly upon nutrient starvation and is encased by proteinaceous shells. The outermost layer, the crust, is a postulated glycoprotein layer that is composed of six proteins: CotV, W, X, Y, Z and CgeA. Despite some insight into protein interactions and the identification of players in glycosylation, a clear picture of its architecture is still missing. Here, we report a comprehensive mutational analysis that confirms CotZ as the anchor of the crust, while the crust structure is provided by CotV, CotX and CotY. CotY seems to be the major structural component, while CotV and CotX are polar and co‐depend on each other and partially on CotW. CotW is independent of other crust proteins, instead depending on outer coat proteins, indicating a role at the interface of crust and coat. CgeA is co‐expressed with putative glycosyltransferases (CgeB and CgeD) and implicated in crust glycosylation. In accordance, we provide evidence that CgeB, CgeCDE, SpsA‐L, SpsM and SpsNOPQR (formerly YfnHGFED) contribute to the glycosylation state of the spore. The crust polysaccharide layer consists of functionally linked rhamnose‐ and galactose‐related variants and could contain rare sugars. It may therefore protect the crust against biological degradation and scavenging.     

Estimation of the percolation thresholds in lobenzarit disodium native dextran matrix tablets

The objective of the present work is to estimate for the first time the percolation threshold of a new series of dextran (native dextran of high molecular weight [B110-1-2, Mw = 2 x 10(6)]), in matrices of lobenzarit disodium (LBD) and to apply the obtained result to the design of hydrophilic matrices for the controlled delivery of this drug. The formulations studied were prepared with different amounts of excipient in the range of 20% to 70% wt/wt. Dissolution studies were performed using the paddle method (100 rpm) and one face water uptake measurements were performed using a modified Enslin apparatus. The Higuchi, zero-order, and Hixson-Crowell models as well as the nonlinear regression model were employed as empiric methods to study the release data. Values of diffusion exponent 0.563 < n < 0.786 (Korsmeyer equation) for dissolution profile and water uptake mechanism 0.715 < n < 1 (Davidson and Peppas equation) suggested anomalous or complex mechanisms. On the other hand, the contribution of the relaxation or erosion and of the diffusive mechanism in Peppas-Sahlin equation indicated that the main mechanism for drug delivery from tablets is swelling controlled delivery (K(r)/K(d) < 1). The critical points observed in kinetic parameters above 58.63% vol/vol of native dextran B110-1-2 plus initial porosity in the LBD-dextran matrices with a relative polymer/drug particle size of 4.17 were attributed to the existence of an excipient percolation threshold.     

Differential expression of the FAK family kinases in rheumatoid arthritis and osteoarthritis synovial tissues

The focal adhesion kinase (FAK) family kinases, including FAK and proline-rich kinase 2 (Pyk)2, are the predominant mediators of integrin αvβ3 signaling events that play an important role in cell adhesion, osteoclast pathology, and angiogenesis, all processes important in rheumatoid arthritis (RA). Using immunohistochemical and western blot analysis, we studied the distribution of phospho (p)FAK, pPyk2, pSrc, pPaxillin and pPLCγ in the synovial tissue (ST) from patients with RA, osteoarthritis (OA) and normal donors (NDs) as well as in RA ST fibroblasts and peripheral blood differentiated macrophages (PB MΦs) treated with tumor necrosis factor-α (TNFα) or interleukin-1β (IL1β). RA and OA STs showed a greater percentage of pFAK on lining cells and MΦs compared with ND ST. RA ST fibroblasts expressed pFAK at baseline, which increased with TNFα or IL1β stimulation. Pyk2 and Src were phosphorylated more on RA versus OA and ND lining cells and MΦs. pPyk2 was expressed on RA ST fibrobasts but not in MΦs at baseline, however it was upregulated upon TNFα or IL1β activation in both cell types. pSrc was expressed in RA ST fibroblasts and MΦs at baseline and was further increased by TNFα or IL1β stimulation. pPaxillin and pPLCγ were upregulated in RA versus OA and ND lining cells and sublining MΦs. Activation of the FAK family signaling cascade on RA and OA lining cells may be responsible for cell adhesion and migration into the diseased STs. Therapies targeting this novel signaling pathway may be beneficial in RA.