A streptomycin-resistant Escherichia coli mutant with ribosomes temperature-sensitive in the suppression of a nonsense codon.

Summary Cell free extracts from a streptomycin-resistant E. coli mutant which is also temperature-sensitive for Q phage were studied for suppression of a nonsense mutation at various temperatures. The streptomycin-resistant ribosomes of the mutant were found to be temperature-sensitive in suppression of an amber mutation in f2 phage coat protein while retaining the ability to synthesize proteins at an elevated temperature (42° C). The restriction of amber suppression at 42° C is assumed to be related to an alteration in the ribosomal protein S12 of the streptomycin-resistant mutant which also causes a change in its electrophoretic mobility.     

Outer membrane protein a and other polypeptides regulate capsular polysaccharide synthesis in E. coli K-12.

Summary capR (lon) mutants of Escherichia coli K-12 are mucoid on minimal agar because they produce large quantities of capsular polysaccharide. When such mutants are transformed to tetracycline resistance by plasmid pMC44, a hybrid plasmid that contains a 2 megadalton (Mdal) endonuclease EcoR1 fragment of E. coli K-12 DNA joined to the cloning vehicle-pSC101, capsular polysaccharide synthesis is inhibited and the transformed colonies exhibit a nonmucoid phenotype. Re-cloning of the 2 Mdal EcoR1 fragment onto plasmid pHA105, a min-colE1 plasmid, yielded plasmid pFM100 which also inhibited capsular polysaccharide synthesis in the capR mutants. A comparison of the polypeptides specified by both plasmids pFM100 and pMC44 in minicells demonstrated that seven polypeptide bands were specified by the 2 Mdal DNA, one of which was previously demonstrated to be outer membrane protein a; also known as 3b or M2 (40 kilodaltons, Kdal). Plasmid mutants no longer repressing capsular polysaccharide synthesis were either unable to specify the 40 K dal outer membrane protein a or were deficient in synthesis of 25 K dal and 14.5 K dal polypeptides specified by the 2 Mdal DNA fragment. Studies with a minicell-producing strain that also contained a capR mutation indicated that the capR gene product regulated processing of at least one normal protein, the precursor of outer membrane protein a.     

Large scale, analytical method for isolating basal lateral plasma membranes from rat duodenum.

A procedure is described for obtaining large amounts of basal lateral plasma membranes from the rat duodenal epithelium. The yield is approximately 50%, and the purification factor is 18; preparations from 25 rats routinely contain 100 mg of protein. The procedure depends on mild homogenization with a nitrogen cavitation bomb, followed by removal of brush borders by sedimentation in a weak centrifugal field. Basal lateral membranes in the resulting supernatant are partially purified by differential centrifugation in a medium which approximates their equilibrium density, and then further purified by equilibrium density gradient centrifugation in a high capacity zonal rotor. Brush border membranes may be isolated from the 450 x g pellet. Since both brush border and basal lateral membranes may be isolated from the same homogenate, this preparative procedure is suitable for such analytical purposes as determinations of distribution of enzyme activities between the two surfaces of the epithelium. The large scale of the isolation procedure makes it an appropriate starting point for purification of specific basal lateral membrane components.     

Functional disturbances in brain following injury

It was shown previously that local cerebral glucose utilization is less than 50% of normal in all cortical areas of rat brain 3 days following a focal freeze-lesion and that this effect of trauma is significantly diminished by dexamethasone (0.25 mg/Kg/day), and by indomethacin (7.5 mg/Kg single dose). To elucidate the mechanism of action of steroids and non-steroidal antiinflammatory drugs in traumatized brain, the effects of dexamethasone and indomethacin on arachidonic acid release, malondialdehyde production and prostaglandin synthesis in the lesion area were investigated. Five seconds after a freezing lesion arachidonic acid was significantly increased in the lesion area of untreated animals. Neither dexamethasone nor indomethacin had any effect on this release. The thiobarbituric acid reaction, as an estimate of malondialdehyde and non-enzymatic free radical lipoperoxide formation from unsaturated free fatty acids showed no change in the control and lesion areas of untreated and both dexamethasone and indomethacin treated groups. There was a marked increase in PGF2 alpha, PGE2, PGD2 in the lesion area of untreated animals. Indomethacin prevented the formation of prostaglandins by more than 90% while dexamethasone had no effect. These results suggest that some components of the arachidonic acid metabolism must be involved in functional disturbances resulting from trauma while steroid action is mediated in injured brain independently from the prostaglandin cascade.     

Bacteriorhodopsin mutants containing single substitutions of serine or threonine residues are all active in proton translocation.

To study their role in proton translocation by bacteriorhodopsin, 22 serine and threonine residues presumed to be located within and near the border of the transmembrane segments have been individually replaced by alanine or valine, respectively. Thr-89 was substituted by alanine, valine, and aspartic acid, and Ser-141 by alanine and cysteine. Most of the mutants showed essentially wild-type phenotype with regard to chromophore regeneration and absorption spectrum. However, replacement of Thr-89 by Val and of Ser-141 by Cys caused striking blue shifts of the chromophore by 100 and 80 nm, respectively. All substitutions of Thr-89 regenerated the chromophore at least 10-fold faster with 13-cis retinal than with all-trans retinal. The substitutions at positions 89, 90, and 141 also showed abnormal dark-light adaptation, suggesting interactions between these residues and the retinylidene chromophore. Proton pumping measurements revealed 60-75% activity for mutants of Thr-46, -89, -90, -205, and Ser-226, and about 20% for Ser-141----Cys, whereas the remaining mutants showed normal pumping. Kinetic studies of the photocycle and of proton release and uptake for mutants in which proton pumping was reduced revealed generally little alterations. The reduced activity in several of these mutants is most likely due to a lower percentage of all-trans retinal in the light-adapted state. In the mutants Thr-46----Val and Ser-226----Ala the decay of the photointer-mediate M was significantly accelerated, indicating an interaction between these residues and Asp-96 which reprotonates the Schiff base. Our results show that no single serine or threonine residue is obligatory for proton pumping.     

The retinylidene Schiff base counterion in bacteriorhodopsin

Previous studies of bacteriorhodopsin have indicated interactions between Asp-85, Asp-212, Arg-82, and the retinylidene Schiff base. The counterion environment of the Schiff base has now been further investigated by using single and double mutants of the above amino acids. Chromophore regeneration from bacterioopsin proceeds to a normal extent in the presence of a single aspartate or glutamate residue at position 85 or 212, whereas replacement of both charged amino acids in the mutant Asp-85----Asn/Asp-212----Asn abolishes the binding of retinal. This indicates that a carboxylate group at either residue 85 or 212 is required as counterion for formation and for stabilization of the protonated Schiff base. Measurements of the pKa of the Schiff base reveal reductions of greater than 3.5 units for neutral single mutants of Asp-85 but only decreases of less than 1.2 units for corresponding substitutions of Asp-212, relative to the wild type. Substitutions of Asp-85 show large red shifts in the absorption spectrum that are partially reversible upon addition of anions, whereas mutants of Asp-212 display minor red shifts or blue shifts. We conclude, therefore, that Asp-85 is the retinylidene Schiff base counterion in wild-type bacteriorhodopsin. In the mutant Asp-85----Asn/Asp-212----Asn formation of a protonated Schiff base chromophore is restored in the presence of salts. The spectral properties of the double mutant are similar to those of the acid-purple form of bacteriorhodopsin. Upon addition of salts the folded structure of wild-type and mutant proteins can be stabilized at low pH in lipid/detergent micelles. The data indicate that exogenous anions serve as surrogate counterions to the protonated Schiff base, when the intrinsic counterions have been neutralized by mutation or by protonation.     

The use of tryptophan mutants in identifying the 296 nm transient absorbing species during the photocycle of bacteriorhodopsin

The transient absorption at 296 nm was part of the spectroscopic evidence that initiated the proposal that tyrosinate (Tyr-) is formed during, and important to, the photocycle of bacteriorhodopsin (bR). Recent evidence against such a proposal comes from the results of NMR, UV Raman as well as electron cryo-microscopic structural studies. This makes it credible to assign this absorption to a charge perturbation of the lowest energy absorption of one of the tryptophan (Trp) residues in bR. The transient absorption at 296 nm is examined for each of 8 tryptophan mutants in which Trp is substituted by phenylalanine or cysteine, which absorb at shorter wavelength. It is shown that while all go through the photocycle, all but Trp-182 mutant show this transient absorption. This strongly suggests the assignment of this absorption to a charge perturbaton of the lowest energy absorption of Trp-182 during the photocycle. The chemical identity of the perturbing charge(s) is briefly discussed.     

Ultrastructural and histochemical observations on the tegument of Gastrodiscoides hominis (Paramphistoma: Digenea).

The tegument of the paramphistome, Gastrodiscoides hominis, is basically similar to that of other digeneans. It is folded into concentrically arranged furrows and ridges bearing numerous tightly packed tubercules, and extends into the oral cavity. An area of specialized tegument is present on the ventral surface, anterior to the disc region. Mitochondria are absent from the tegumental syncytium and underlying tegumental cells, suggesting that the tegument may serve principally as a protective layer rather than in active uptake phenomena. However, extensions of the lymph and parenchyma systems are closely associated with the base of the tegumental syncytium and may provide ATP for active processes. Ciliated and non-ciliated sensory papillae are present, particularly around the oral opening. Numerous lymph channels are present in the sub-tegument and may be involved in osmoregulation.