Repositioning of a domain in a modular polyketide synthase to promote specific chain cleavage.

Macrocyclic polyketides exhibit an impressive range of medically useful activities, and there is great interest in manipulating the genes that govern their synthesis. The 6-deoxyerythronolide B synthase (DEBS) of Saccharopolyspora erythraea, which synthesizes the aglycone core of the antibiotic erythromycin A, has been modified by repositioning of a chain-terminating cyclase domain to the carboxyl-terminus of DEBS1, the multienzyme that catalyzes the first two rounds of polyketide chain extension. The resulting mutant markedly accelerates formation of the predicted triketide lactone, compared to a control in which the repositioned domain is inactive. Repositioning of the cyclase should be generally useful for redirecting polyketide synthesis to obtain polyketides of specified chain lengths.

Oxaliplatin-DNA adduct formation in white blood cells of cancer patients.

In this study, we investigated the kinetics of oxaliplatin-DNA adduct formation in white blood cells of cancer patients in relation to efficacy as well as oxaliplatin-associated neurotoxicity. Thirty-seven patients with various solid tumours received 130 mg m(-2) oxaliplatin as a 2-h infusion. Oxaliplatin-DNA adduct levels were measured in the first cycle using adsorptive stripping voltammetry. Platinum concentrations were measured in ultrafiltrate and plasma using a validated flameless atomic absorption spectrometry method. DNA adduct levels showed a characteristic time course, but were not correlated to platinum pharmacokinetics and varied considerably among individuals. In patients showing tumour response, adduct levels after 24 and 48 h were significantly higher than in nonresponders. Oxaliplatin-induced neurotoxicity was more pronounced but was not significantly different in patients with high adduct levels. The potential of oxaliplatin-DNA adduct measurements as pharmacodynamic end point should be further investigated in future trials.

Polyketide synthesis in vitro on a modular polyketide synthase.

BACKGROUND: The 6-deoxyerythronolide B synthase (DEBS) of Saccharopolyspora erythraea, which synthesizes the aglycone core of the antibiotic erythromycin A, contains some 30 active sites distributed between three multienzyme polypeptides (designated DEBS1-3). This complexity has hitherto frustrated mechanistic analysis of such enzymes. We previously produced a mutant strain of S. erythraea in which the chain-terminating cyclase domain (TE) is fused to the carboxyl-terminus of DEBS1, the multienzyme that catalyzes the first two rounds of polyketide chain extension in S. erythraea. This mutant strain produces triketide lactone in vivo. We set out to purify the chimaeric enzyme and to determine its activity in vitro. RESULTS: The purified DEBS1-TE multienzyme catalyzes synthesis of triketide lactones in vitro. The synthase specifically uses the (2S)-isomer of methylmalonyl-CoA, as previously proposed, but has a more relaxed specificity for the starter unit than in vivo. CONCLUSIONS: We have obtained a purified polyketide synthase system, derived from DEBS, which retains catalytic activity. This approach opens the way for mechanistic and structural analyses of active multienzymes derived from any modular polyketide synthase.

Translocation of beta crystallin in neural cells in response to stress.

While extralenticular expression of proteins in the alpha crystallin (small heat shock protein) family is well documented, that for proteins in the beta/gamma-superfamily is less well established. Here we show, using SDS-PAGE, Western blotting and confocal microscopy, that there is a constitutive level of beta crystallin expression in mouse N1E-115 neural cells. Furthermore, upon heat shock at 43 degrees C or 55 degrees C, or cold shock at 30 degrees C, beta crystallin immunoreactivity translocated predominantly from the nuclear region into the cytoplasmic region of the cells. In conditions of stress, it may be important for beta crystallin to be recruited into the cytoplasm to stabilise other proteins via its high beta-sheet content, and/or to ensure that storage levels of cytoplasmic Ca2+ are maintained.

Effect of mutations of murine lens alphaB crystallin on transfected neural cell viability and cellular translocation in response to stress.

We examined the influence of over-expressed native and mutant murine lens alphaB crystallin on the response of a murine neural cell line to heat and ionic strength shock. Native and mutant (F27R and KK174/175LL) murine alphaB crystallin amplicons were subcloned into a Lac-Switch IPTG-inducible RSV promoter eukaryotic vector, and transfected into N1E-115 cells using lipofectin. Expression was induced maximally 8 h after addition of IPTG (optimal final concentration 1 mM) to the medium. Cells grew normally after transfection with native and mutant murine alphaB crystallin. We demonstrated expression of the protein using specific anti-alpha crystallin antibodies. Viability under severe heat and ionic strength stress increased when cells had been transfected with native alphaB crystallin, but not with mutants F27R or KK174/175LL. Heat shock caused translocation of both native and mutant alphaB crystallins into the central region of the cells. These results show that mutations in alphaB crystallin that effect its chaperone-like activity may also influence viability of N1E-115 neural cells under stress, while not influencing the distribution of the protein within the cell.

Increased transplantation of kidneys with multiple renal arteries in the laparoscopic live donor nephrectomy era: surgical technique and surgical and nonsurgical donor and recipient outcomes.

BACKGROUND: For anatomical and technical reasons, many transplant centers restrict laparoscopic live donor nephrectomy (in contrast with open live donor nephrectomy) to left kidneys. HYPOTHESIS: This change in surgical practice increases procurement and transplantation rates of live donor kidneys with multiple renal arteries (RAs), without affecting donor and recipient outcomes. DESIGN AND SETTING: Retrospective review at an academic tertiary care referral center comparing laparoscopically procured single vs multiple-RA kidney grafts (April 1997 to October 2000). PATIENTS: Seventy-nine consecutive left laparoscopic live kidney donors and 78 transplant recipients. MAIN OUTCOME MEASURES: Donor and recipient complications and postoperative length of stay; cold and warm ischemia time; operating time; short-term and long-term graft function; and survival. RESULTS: We noted multiple RAs in 21 (27%) of all kidneys. The proportion of donors with 1 or more perioperative complications was 19% in the single-RA group vs 10% in the multiple-RA group (P was not significant). For the recipients, we noted no significant differences between groups with respect to surgical complications, quality of early and late graft function, rejection rates, graft losses (all immunologic), and graft survival. Cold and warm ischemia time and length of stay were similar for donors and recipients in both groups. Median operating times were significantly longer for the multiple-RA vs single-RA group (difference, 41 minutes for donors and 45 minutes for recipients; P<.02). CONCLUSIONS: While the introduction of laparoscopic live donor nephrectomy has significantly increased the number of grafts with multiple RAs (compared with historical open controls), this change in practice is safe for both donors and recipients from a patient outcome-based perspective. However, from an economic perspective, the longer operating time associated with multiple-RA grafts provides strong added rationale for optimization of surgical instruments and techniques to make right-sided laparoscopic nephrectomy a routine intervention.

Phosphatidylinositol 3-kinases regulate ERK and p38 MAP kinases in canine colonic smooth muscle.

In canine colon, M2/M3 muscarinic receptors are coupled to extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinases. We tested the hypothesis that this coupling is mediated by enzymes of the phosphatidylinositol (PI) 3-kinase family. RT-PCR and Western blotting demonstrated expression of two isoforms, PI 3-kinase-alpha and PI 3-kinase-gamma. Muscarinic stimulation of intact muscle strips (10 microM ACh) activated PI 3-kinase-gamma, ERK and p38 MAP kinases, and MAP kinase-activated protein kinase-2, whereas PI 3-kinase-alpha activation was not detected. Wortmannin (25 microM) abolished the activation of PI 3-kinase-gamma, ERK, and p38 MAP kinases. MAP kinase inhibition was a PI 3-kinase-gamma-specific effect, since wortmannin did not inhibit recombinant activated murine ERK2 MAP kinase, protein kinase C, Raf-1, or MAP kinase kinase. In cultured muscle cells, newborn calf serum (3%) activated PI 3-kinase-alpha and PI 3-kinase-gamma isoforms, ERK and p38 MAP kinases, and stimulated chemotactic cell migration. Using wortmannin and LY-294002 to inhibit PI 3-kinase activity and PD-098059 and SB-203580 to inhibit ERK and p38 MAP kinases, we established that these enzymes are functionally important for regulation of chemotactic migration of colonic myocytes.