Engineering multiple properties of a protein by combinatorial mutagenesis

A method for simultaneously engineering multiple properties of a protein, based on the observed additivity of effects of individual mutations, is presented. We show that, for the gene V protein of bacteriophage f1, effects of double mutations on both protein stability and DNA binding affinity are approximately equal to the sums of the effects of the constituent single mutations. This additivity of effects implies that it is possible to deliberately construct mutant proteins optimized for multiple properties by combination of appropriate single mutations chosen from a characterized library.     

Differences in muscle endurance and recovery between fallers and nonfallers, and between young and older women

BACKGROUND: Significant morbidity and mortality are associated with falls in older adults. We tested the hypothesis that older women with a history of falls demonstrate decreased muscle endurance and longer recovery times following fatiguing exercise. METHODS: We evaluated dynamic endurance and recoverability of the quadriceps femoris of 29 young women (YW) (M age = 21.7), 26 older women with a history of falls (FA) (M age = 73.3), and 27 older women with no history of falls (NF) (M age = 71.2) using an isokinetic dynamometer. Subjects performed repeated maximal concentric knee extensions until the force output of two consecutive repetitions fell below 50% of their maximal voluntary contraction (MVC). Recovery was defined as the time required for the return of force output > or = 80% MVC for 2 consecutive repetitions, within a set consisting of 3 maximal contractions. One minute rest was allowed between sets. We collected electromyographic (EMG) data from the quadriceps during all testing to evaluate spectral shifts. RESULTS: ANOVA with a post-hoc Bonferroni-Dunn test revealed time to fatigue was significantly faster in FA than YW (p < .02) and in FA than NF (p < .05), but not different between YW and NF. Time to recovery was significantly slower in FA than YW (p = .01), but not different between YW and NF, or between FA and NF, EMG median frequency power shift (from the beginning to the end of the test) was significantly less in FA (p < .001) than either YW (p < .002) or NF (p < .05). CONCLUSIONS: Older women with a history of falls demonstrate decreased muscular endurance compared to YW and NF, and increased time to recover from fatiguing exercise when compared to young women.     

Evaluation of humoral immune responses in cattle grazing endophyte-infected or endophyte-free fescue

Anecdotal reports suggest cattle with fescue toxicosis may not respond to vaccination and thus, experience increased incidence of Bovine Respiratory Disease Complex (BRDC) when shipped to feedlots. Fescue toxicosis causes hypoprolactemia in cattle. Hypoprolactemia decreases humoral immune responses in mice. Therefore, a study was conducted to compare the magnitude of primary and secondary humoral immune responses against specific antigens in cattle grazing endophyte-infected or endophyte-free fescue. Angus steers were blocked by weight and allocated into four groups. Two groups grazed endophyte-infected (EI) fescue and the other two groups grazed endophyte-free (EF) fescue. All steers were injected IM on d 0 and 21 with lysozyme without adjuvant and concanavalin. A (Con A) with sheep red blood cells (SRBC) in incomplete adjuvant of Freund. Steers were bled on days 0, 21 and 35 post-vaccination. Average daily gains (ADG), alkaline phosphatase (ALP) activity, cholesterol concentrations, rectal temperatures, and serum prolactin concentrations were measured to confirm fescue toxicosis in steers grazing EI fescue. Antibodies to Con A and SRBC were determined by ELISA and hemagglutination assay, respectively. The ADG were decreased for the EI group during the first month. Rectal temperature were elevated and serum prolactin concentrations were decreased in the EI group. Cholesterol and ALP concentrations also were decreased in the EI group. Primary and secondary immune responses against Con A tended to be increased and were increased against SRBC in the EI group. Antibodies against lysozyme were not induced in either group. In conclusion, cattle grazing EI fescue mounted similar humoral immune responses to vaccination, despite hypoprolactemia, as cattle grazing EF fescue. Increases in bovine respiratory disease in cattle maintained on EI fescue probably is not associated with lack of humoral immune response to vaccination protocols as a result of fescue toxicosis.     

Restoration of squamous mucosa after ablation of Barrett's esophageal epithelium

BACKGROUND: Antireflux therapy has generally failed to induce regression of Barrett's epithelium. It was hypothesized that squamous epithelium could be restored if the columnar tissue was ablated while gastric acid secretion was suppressed. METHODS: Ten white men with Barrett's esophagus received 40 mg of omeprazole daily. Thereafter, every 2-5 weeks they underwent videotaped endoscopies to argon laser photoablate columnar tissue, obtain biopsy specimens, and assess results. Squamous re-epithelialization was assessed by correlation of videotapes and directed biopsies. RESULTS: Patients had one to eight areas ablated, totaling 0.5-12.0 cm2. Videotape assessments were corroborated by biopsy in all but one instance. Thirty-eight of 40 treatment locations partially or completely re-epithelialized with squamous tissue. Squamous regrowth appeared to occur by spread from contiguous squamous borders and de novo from glandular tissue. Regrowth was influenced by the extent of squamous borders and completeness of ablations. Nonablated glandular tissue persisted beneath squamous epithelium. CONCLUSIONS: Ablation of Barrett's epithelium and suppression of acid secretion facilitated squamous re-epithelialization. A progenitor cell within the metaplastic tissue has the potential to differentiate normally.     

Results of treatment of inferior vena cava syndrome with expandable metallic stents

A 73-year-old male was admitted to our hospital because of detection of Shigella flexneri 2a from his stool. Antimicrobial treatment with levofloxacin (LVFX) was started, but could not eliminate the organism in the stool. In the examination of drug susceptibility, this strain was highly resistant to all new quinolones. The minimal inhibitory concentration of norfloxacin, ofloxacin and ciprofloxacin to this strain was 12.5 micrograms/ml, 6.25 micrograms/ml and 6.25 micrograms/ml, respectively. The dual mutations were detected in the codon 83 and 87 of the gyrA gene by sequencing the quinolone-resistance determining region (QRDR). There was, however, no significant difference between the intracellular uptake of ciprofloxacin in this strain and in the ciprofloxacin-sensitive strain. The amount of ciprofloxacin in this strain unchanged when carbonyl cyanide m-chlorophenyl hydrazone (CCCP) was added. These results suggest that the advanced resistance in Shigella flexneri against new quinolones could be acquired by only this dual mutations without the change of the active efflux mechanism.     

Extrahepatic metabolism of 4-methylumbelliferone and lidocaine in the anhepatic rabbit

OBJECTIVE: The initially well-fixed implants of total hip replacement (THR) are in the long-term subject to aseptic loosening. Many cytokines can contribute to osteolysis due to osteoclast recruitment and/or activation. However, in this respect tumor necrosis factor-alpha (TNF-alpha) plays a pivotal role, because it upregulates interleukin-1 and 6 and granulocyte-macrophage colony stimulating factor. The aim of this study was to assess the eventual presence, cellular localization and extent of expression of TNF-alpha in the synovial-like membrane at the implant or at the cement to bone interface compared to control synovial membrane. METHODS: Twenty samples from the synovial-like membrane of the periprosthetic tissues were compared to control samples. TNF-alpha containing cells were visualized using an avidin-biotin-peroxidase complex (ABC) method and analyzed by light microscopy, double labelling and image analysis. RESULTS: TNF-alpha was found in the periprosthetic tissues in fibroblasts and vascular endothelial cells, but mainly in the macrophages was it found to coincide with areas containing implant-derived debris. TNF-alpha containing cells were more numerous in the synovial-like membrane in the interface tissue from the proximal stem area (2816 +/- 318 cells) than in the control synovial membrane (565 +/- 93 cells, p < 0.01). Interestingly, similarly high TNF-alpha expression (3452 +/- 582 cells) was also seen in the synovial-like membrane of the pseudocapsule. CONCLUSION: These findings suggest that the foreign body-type host reaction caused by THR is characterized by the high expression of TNF-alpha. Because such expression occurred in the interface tissue between the implant and surrounding bone, TNF-alpha, due to its pivotal direct and indirect role in the activation and recruitment of osteoclasts, may contribute to periprosthetic osteolysis and to the loosening of THR.     

Regeneration of active receptor recognition domains on the B subunit of cholera toxin by formation of hybrids from chemically inactivated derivatives

In order to test the hypothesis that binding sites of cholera toxin for its receptor, the monosialoganglioside GM1, are shared between adjacent beta-polypeptide chains, two inactive chemical derivatives of the B subunit of cholera toxin (CTB) were prepared and were subsequently used for the construction of hybrid CTB pentamers. One inactive derivative consisted of CTB specifically modified in the single essential Trp-88 residue of each beta-chain. This residue was modified by formylation, a treatment preserving the structural integrity of CTB. The other inactive derivative consisted of CTB specifically succinylated in three amino groups located in or near the receptor binding site. Using [1,4-14C]succinic anhydride for the site-specific succinylation and analysis of radiolabeled tryptic fragments of S-carboxymethylated [14C]sssCTB revealed that the amino groups specifically modified were the alpha-amino group of Thr-1 and the epsilon-amino groups of respectively Lys-34 and Lys-91. Upon submitting equal amounts of formylated CTB and site-specific succinylated CTB to a denaturation-renaturation cycle, hybrid pentamers were formed which in contrast to the parental compounds were able to bind GM1. The affinity of hybrid CTB for GM1, as estimated by a competitive solid-phase radiobinding assay was unexpectedly high and only 2.5-fold lower than that of its native counterpart. The number of active binding sites on hybrid CTB was determined from: (i) titration with the oligosaccharide moiety of GM1 (oligo-GM1) and monitoring the reversal of the Trp fluorescence quenching by iodide ions and (ii) rapid gel filtration over a superdex HR column of a mixture of hybrid CTB and an excess of 3H-labeled oligo-GM1. The data are in agreement with the formation of one active binding per four reconstituted binding sites in hybrid CTB, which is consistent with a random association of CTB monomers during the denaturation-renaturation cycle.