Expression of a group II phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus in Escherichia coli: Recovery and renaturation from bacterial inclusion bodies

A synthetic gene encoding the Group 11 phospholipase A₂ (PLA₂) from the venom of Agkistrodon piscivorus piscivorus has been constructed and expressed with high efficiency in Escherichia coli. No enzymatic activity was recovered when the polypeptide contained the initiator Met residue. Replacement of an Asn residue penultimate to the initiator Met with Ser or Gly permitted removal of the initiator Met by the endogenous methionine aminopeptidase. The amino-terminal serine (N-Ser) and amino-terminal glycine PLA₂'s were isolated from intracellular inclusion bodies and were renatured with 25% recovery. Automated Edman degradation confirmed the removal of the initiator Met and confirmed the sequence of the first 40 residues of N-Ser PLA₂. The recombinant proteins were purified to apparent homogeneity and showed the same specific activity as the wild-type protein. N-Ser PLA₂ demonstrated the same kinetics of activation as the wild type enzyme on large vesicles of zwitterionic lipid.     

Occurrence,localization, and possible significance of an ornithine-containing lipid in Paracoccus denitrificans

A ninhydrin-positive, phosphorus-negative lipid from Paracoccus denitrificans ATCC 13543 has been isolated and purified by mild alkaline methanolysis followed by silicic acid column chromatography and preparative thin-layer chromatography. The lipid was identified as an ornithine-containing lipid. The major ester-linked fatty acid was cis vaccenic acid. Major amide-linked fatty acids were 3-OH-20:1 and 3-OH-18:0. Ornithine-containing lipid was a major lipid component of P. denitrificans. Phospholipids made up about 57% and ornithine-containing lipid about 14% of the weight of the total lipid of the organism. The ratios of lipid ornithine: lipid phosphorus were 0.23, 0.65 and 0.58 in cytoplasmic membrane, outer membrane, and an NaCl extract, which is thought to represent chiefly outer membrane, respectively. Thus ornithine-containing lipid appears to be present in larger amounts in outer membrane than cytoplasmic membrane. No substantial variations in lipid ornithine levels were noted in stationary phase versus exposnential phase organisms, organisms grown in complex medium versus organisms grown in minimal medium with and without amino acid supplements, or in organisms grown in low phosphate-containing medium.Non standard abbreviations TLC thin-layer chromatography - Tris-HCl tris(hydroxymethyl)aminomethane hydrochloride - TMS trimethylsilyl - TFA triluoroacetyl - NPPN ninhydrin-positive, phosphorus-negative - ECL equivalent chain length     

The rates of sediment-water exchange of oxygen and sediment bioturbation in Lough Neagh,Northern Ireland

Oxygen is transported 30 mm into the sediment at an 8 m depth site in eutrophic Lough Neagh by the irrigational activities of the benthic fauna. Faunal activity also mixes the upper 20 mm of sediment. Sediment oxygen uptake rate, redox potential-depth profile and the chlorophylla concentration were measured in the upper sediment layers from February to November 1979. Chlorophylla input to the sediment, following the Spring phytoplankton maximum, remained in the 0–1 cm sediment layer but did cause the redox potential profile to change from one with potentials around 400 mV in the upper 50 mm to one with a strong gradient over the 0–30 mm region. The start of benthic faunal activity in May caused the chlorophylla to be mixed into the 1–2 cm layer and also caused oxygen to be transported into the sediment at a rate sufficient to change the redox potential back to its initial state. The biodiffusion coefficient for solids in the upper 20 mm was estimated to be 6 × 10⁻⁸ cm² s⁻¹. Oxygen transport in the pore, waters of the upper sediment layers was considered to be best described as advection, caused by the irrigational activities of the benthic fauna.     

Developmental patterns of ornithine decarboxylase activity in organogenesis phase rat embryos in culture and in utero

Summary Ornithine decarboxylase activity was measured during organogenesis in rat embryos grown in utero and whole rat conceptuses maintained in an in vitro culture system. Ornithine decarboxylase levels in vivo showed a distinct peak at embryonic age 10.5 d. Despite identical morphology, protein content, crown rump length and numbers of somites cultured embryos displayed a different developmental pattern and possessed less than half the ODC activity of that in vivo. The data suggest that the normal embryonic programming of ODC activity is significantly altered by the culture environment and that further biochemical comparisons of embryos growing in utero and in vitro may be required to evaluate properly the applicability of this technique to detailed studies of teratogenesis and developmental biology. This work was supported by NIH-5-507-RR5359-17 and a 1980 Research Starter Grant from the Pharmaceutical Manufacturers Association Foundation.     

Nucleotide sequences of the trpG regions of Escherichia coli,Shigella dysenteriae,Salmonella typhimurium and Serratia marcescens

The nucleotide sequences of Serratia marcescens trpG and the corresponding regions of Escherichia coli, Shigella dysenteriae and Salmonella typhimurium trpD have been determined. Analysis of the nucleotide sequence divergence suggests the following evolutionary relationships: Serratia-[Salmonella, (Escherichia, Shigella)]. Partial reconstruction of ancestral nucleotide sequences and subsequent analysis of nucleotide substitutions show that the majority of nucleotide substitutions in the evolution of trp(G)D are transitions that result in a reduction of G + C content. Since most of the nucleotide substitutions are in the third position of codons, bias in synonymous codon usage also reflects G + C content. The trpE-trp(G)D junction in the four organisms is characterized by overlapping translation termination and initiation codons. The relative positions of trpE and trp(G)D thus became fixed in evolution before the fusion of trpG and trpD. Nucleotide sequences representing the fusion of trpG and trpD in Escherichia, Shigella and Salmonella are not more nor less divergent than other portions of the trp(G)D coding sequences.     

Some electrophysiological consequences of electrogenic sodium and potassium transport in cardiac muscle: a theoretical study

A previous paper described a kinetic model for electrogenic sodium-potassium transport in cardiac muscle, combining a thermodynamically-constrained transport model with simple passive permeabilities for sodium and potassium to generate a cardiac action potential (Chapman, Kootsey & Johnson, 1979). The present paper explores the extent to which this simplest of active-passive transport models can account (without further modification) for the electrophysiological behavior of cardiac muscle. The long term (several minutes) changes in the duration of the action potential observed following a change in stimulation rate are predicted by the model through a shift in the steady-state current-voltage relationship caused by small changes in inside ion concentrations. The diastolic hyperpolarization observed following an increase in rate is also predicted, including the linear relationship between the maximum diastolic depolarization and the rate of stimulation. Varying the outside potassium concentration in the model produces changes in the rest potential and current-voltage relationship similar to published data. Deviations from ideal potassium electrode behavior occur at both high and low concentrations because of effects on the pump. The model not only predicts the observed shift of the current-voltage curve in the depolarizing direction with increasing [K⁺]₀, but also the crossing of the curve in normal [K ⁺]₀ without having to assume a variation in g_K. Anoxia was introduced into the model by changing the concentrations of ATP and ADP, thereby enabling the model to account for the rapid diastolic depolarization observed in myocardial ischemia.     

Morphological and functional specializations of the shell, musculature and pallial glands in the Lithophaginae (Mollusca: Bivalvia)

The structure of the pallial glands in limestone and coral boring species of Lithophaga (Bivalvia: Lithophaginae) is examined and related to evolutionary trends in habitat specialization. Boring glands of all species occur in the middle mantle fold but display a progressive degree of complexity from simple epithelial to ducted sub-epithelial structures coinciding with increasing specialization of habitat from natural limestone to species specific live coral dwellers. The size of this gland is relatively reduced anteriorly and along the entire length of the mantle in live coral borers.