A model of the quaternary structure of the Escherichia coli F1 ATPase from X-ray solution scattering and evidence for structural changes in the delta subunit during ATP hydrolysis.

The shape and subunit arrangement of the Escherichia coli F1 ATPase (ECF1 ATPase) was investigated by synchrotron radiation x-ray solution scattering. The radius of gyration and the maximum dimension of the enzyme complex are 4.61 +/- 0.03 nm and 15.5 +/- 0.05 nm, respectively. The shape of the complex was determined ab initio from the scattering data at a resolution of 3 nm, which allowed unequivocal identification of the volume occupied by the alpha3beta3 subassembly and further positioning of the atomic models of the smaller subunits. The delta subunit was positioned near the bottom of the alpha3beta3 hexamer in a location consistent with a beta-delta disulfide formation in the mutant ECF1 ATPase, betaY331W:betaY381C:epsilonS108C, when MgADP is bound to the enzyme. The position and orientation of the epsilon subunit were found by interactively fitting the solution scattering data to maintain connection of the two-helix hairpin with the alpha3beta3 complex and binding of the beta-sandwich domain to the gamma subunit. Nucleotide-dependent changes of the delta subunit were investigated by stopped-flow fluorescence technique at 12 degrees C using N-[4-[7-(dimethylamino)-4-methyl]coumarin-3-yl]maleimide (CM) as a label. Fluorescence quenching monitored after addition of MgATP was rapid [k = 6.6 s-1] and then remained constant. Binding of MgADP and the noncleavable nucleotide analog AMP . PNP caused an initial fluorescent quenching followed by a slower decay back to the original level. This suggests that the delta subunit undergoes conformational changes and/or rearrangements in the ECF1 ATPase during ATP hydrolysis.     

Cloning: new breakthroughs leading to commercial opportunities

Research on cloning animals, again, came to the forefront of public attention in 1997. Most scientists involved in biomedical and agricultural research have emphasized the benefits, of which there are many, of cloning to the public. Basic studies on nuclear transfer have and will continue to contribute to our understanding of how genomic activation and cell cycle synchrony affect nuclear reprogramming and cloning efficiencies, specifically. Also, more basic information on actual mechanisms and specific factors in the oocyte causing nuclear reprogramming is forthcoming. As new molecular approaches in functional genomics are combined with nuclear transfer experiments, new genes involved in nuclear reprogramming will be found. The commercial potentials of products stemming from discoveries in cloning are vast. Cloning will be a more efficient, faster and more useful way of making transgenic fetuses for cell therapies, adult animals for protein production and organs for xenotransplantation. Clearly there are new opportunities in animal cloning technology that will produce many benefits to society.     

Effect of Carbohydrate Position on Lysosomal Transport of Procathepsin L

To study the role of carbohydrate in lysosomal protein transport, we engineered two novel glycosylation signals (Asn-X-Ser/Thr) into the cDNA of human procathepsin L, a lysosomal acid protease. We constructed six mutant cDNAs encoding glycosylation signals at mutant sites Asn-138, Asn-175, or both sites together, in the presence or absence of the wild-type Asn-204 site. We stably transfected wild-type and mutant cDNAs into NIH3T3 mouse fibroblasts and then used species-specific antibodies to determine the glycosylation status, phosphorylation, localization, and transport kinetics of recombinant human procathepsin L containing one, two, or three glycosylation sites. Both novel glycosylation sites were capable of being glycosylated, although Asn-175 was utilized only 30–50% of the time. Like the wild-type glycosylation at Asn-204, carbohydrates at Asn-138 and Asn-175 were completely sensitive to endoglycosidase H, and they were phosphorylated. Mutant proteins containing two carbohydrates were capable of being delivered to lysosomes, but there was not a consistent relationship between the efficiency of lysosomal delivery and carbohydrate content of the protein. Pulse-chase labeling revealed a unique biosynthetic pattern for proteins carrying the Asn-175 glycosylation sequence. Whereas wild-type procathepsin L and mutants bearing carbohydrate at Asn-138 appeared in lysosomes by about 60 min, proteins with carbohydrate at Asn-175 were processed to a lysosome-like polypeptide within 15 min. Temperature shift, brefeldin A, and NH₄Cl experiments suggested that the rapid processing did not occur in the endoplasmic reticulum and that Asn-175 mutants could interact with the mannose 6-phosphate receptor. Taken together, our results are consistent with the interpretation that Asn-175 carbohydrate confers rapid transport to lysosomes. We may have identified a recognition domain in procathepsin L that is important for its interactions with the cellular transport machinery.     

The Material Culture of Steamboat Passengers: Archaeological Evidence from the Missouri River

The Material Culture of Steamboat Passengers: Archaeological Evidence from the Missouri River. Annalies Corbin. New York: Kluwer Academic/Plenum Publishers, 2000. 237 pp.     

THE MITOTIC APPARATUS: ISOLATION BY CONTROLLED pH

The isolation of the mitotic apparatus (MA) from the echinoderm egg was studied in detail, with particular attention given to the factors governing its stability. Successful isolation depends mainly on the pH of the isolation solution, slightly acid values being required. The use of a 1 M solution of hexanediol, buffered at pH 6.0 to 6.4, gives high yields of stable MA, while MA of poorer quality can be isolated in water buffered at pH 5.5 to 5.8. Isolation is possible only over a very narrow range of pH, as the cells become more difficult to break at lower values and the MA becomes unstable at higher values. Within this range the fibrous structure of the MA varies with the pH. The isolated MA disintegrates slowly when transferred to water at pH 7 and dissolves rapidly in solutions of high ionic strength.     

Environmental assessment of recombinant DNA fermentations

Summary This issue of theJournal of Industrial Microbiology contains a compilation of papers presented at the 1992 National Meeting of the Society for Industrial Microbiology in two symposia entitled Environmental Assessment of Recombinant DNA Fermentations. It focuses on three areas of particular interest to industry usingEscherichia coli K-12 strains to make recombinant proteins: (i) the current regulatory environment; (ii) plant design; (iii) results from five different companies all of whom are using or planning to use recombinantE. coli in commercial fermentations. The results from all five companies pursuing the questions of environmental fate and the potential for gene transfer in different studies reached the same conclusions. That is, recombinantE. coli K-12 strains and their plasmidless hosts were unable to survive in any environmental microcosm tested. Additionally, there was absolutely no evidence of gene transfer despite the use of highly sensitive techniques to measure such an event. It seems reasonable to conclude thatE. coli K-12 strains with recombinant, non-conjugating, poorly mobilizable plasmids do not represent environmental hazards in the event of an accidental release of such microorganisms into the environment.     

Isolation of Escherichia coli synthesized recombinant eukaryotic proteins that contain epsilon-N-acetyllysine.

Recombinant porcine (rpST) and bovine somatotropins (rbST) synthesized in Escherichia coli contain the amino acid, epsilon-N-acetyllysine. This amino acid was initially discovered in place of the normal lysine144 in a modified reversed-phase HPLC (RP-HPLC) species of rpST. Mass spectrometry and amino acid sequencing of a tryptic peptide isolated from this RP-HPLC purified protein were used to identify this altered residue as epsilon-N-acetyllysine. Ion-exchange chromatography was utilized to prepare low isoelectric point (pI) forms of rpST and rbST, which are enriched in epsilon-N-acetyllysine. Electrospray mass spectrometry demonstrated that the majority of the protein in these low pI fractions contained species 42 Da larger than normal. Immobilized pH gradient electrophoresis (IPG) of the ion-exchange purified low pI proteins was used to isolate several monoacetylated species of rpST and rbST. The location of the acetylated lysine in each IPG-purified protein was determined by tryptic peptide mapping and amino acid sequencing of the altered tryptic peptides. Amino acid analyses of enzymatic digests of rpST and rbST were also used to confirm the presence of epsilon-N-acetyllysine in these recombinant proteins. These data demonstrate that a significant portion of rpST and rbST produced in E. coli contain this unusual amino acid.     

Thrombospondin expression in traumatized skeletal muscle

Summary Biochemical and immuno-microscopic techniques were used to study temporal involvement of thrombospondin in relation to fibrinogen in muscle regeneration using a rat skeletal muscle-wound model. In undamaged control muscle, no fibrinogen and minimal thrombospondin antigen was found. Following crushing injury, fibrin networks appear immediately, followed by a gradual ordered accumulation of thrombospondin (within a few hours) in the vicinity of the vascular bed and adjacent endomysial connective tissue. Later, thrombospondin becomes associated with connective tissue and basal laminae around muscle fibers throughout the damaged muscle, maximal labelling occurring 3–6 days post-injury. Thrombospondin immunoreactivity decreased thereafter to near normal levels after 7 days post-injury, coincident with the appearance of regenerating muscle fibers. In contrast, little fibrin material remained by five days after injury. Quantitative radioimmunoassay of soluble thrombospondin antigen and radioimmune labelling of thick frozen sections reinforced the qualitative immuno-microscopic observations, with levels peaking at 3–4 days post-trauma, 10-fold over control levels. SDS-PAGE immunoblotting of non-reduced muscle extracts three days after a crush assault shows that the bulk of the thrombospondin incorporated into the injury site exists in a polymerized state (1000 kD). These results demonstrate that the temporal appearance and disappearance of thrombospondin in the healing of a crushing lesion in muscle is related more closely to the regeneration phase of muscle than to the coagulation phase.     

Degradation of T-cell receptor chains in the endoplasmic reticulum is inhibited by inhibitors of cysteine proteases.

The endoplasmic reticulum, or an organelle closely associated with it, contains proteases that can be used to remove partially assembled or improperly folded proteins. Very little is known at present about the types of protease that degrade these proteins. The beta chain and cluster of differentiation (CD)3 delta subunit of the human T-cell antigen receptor (TCR) are degraded shortly after synthesis. In this study Chinese hamster ovary (CHO) cells transfected with either beta or delta were incubated with a panel of protease inhibitors, and the rates of degradation of the transfected proteins were followed using chain-specific enzyme-linked immunosorbent assays (ELISAs). Of the protease inhibitors tested, degradation of both chains was highly sensitive to sulfhydryl reagents and peptidyl inhibitors of cysteine proteases. Concentrations of inhibitors that produced near complete inhibition of degradation in the endoplasmic reticulum did not cause gross changes in cellular ATP levels nor did they significantly slow constitutive secretion from CHO cells. The inhibitors did not affect the ability of CHO cells to synthesize and assemble disulphide-linked TCR zeta dimers. We conclude that the protease inhibitors were not toxic to cells and did not affect the biosynthetic activity of the endoplasmic reticulum. Furthermore, they did not alter the ability of the endoplasmic reticulum to deliver its content to the Golgi apparatus. Taken together, these results suggest that the cysteine protease inhibitors slow degradation in the endoplasmic reticulum through an action on cysteine proteases. The results imply that the endoplasmic reticulum contains cysteine proteases that can be used to remove retained proteins.