The effect of levodopa-carbidopa intestinal gel infusion long-term therapy on motor complications in advanced Parkinson's disease: a multicenter Romanian experience.

Chronic treatment with oral levodopa is associated with an increased frequency of motor complications in the late stages of Parkinson's disease (PD). Continuous administration of levodopa-carbidopa intestinal gel (LCIG-Duodopa(®), Abbott Laboratories), which has been available in Romania since 2009, represents an option for treating patients with advanced PD. Our primary objective was to report changes in motor complications after initiation of LCIG therapy. The secondary objectives were as follows: to determine the impact of LCIG therapy on the daily levodopa dose variation before/and after LCIG, to collect patient self-assessments of quality of life (QoL), and to study the overall tolerability and safety of LCIG administration. A retrospective analysis (2009-2013) of LCIG therapy and the experience in nine neurology centers in Romania was performed. The impact of LCIG therapy was evaluated by analyzing changes in motor fluctuations, dyskinesia and the patients' QoL after initiating therapy. The safety of LCIG therapy was estimated by noting agent-related adverse events (AEs) and medical device-related AEs. In the 113 patients included, we observed a significant improvement in PD symptoms after initiation of LCIG therapy. The "on" period increased, with a mean value of 6.14 h, and the dyskinesia period was reduced, with a mean value of 29.4 %. The quantified non-motor symptoms subsided. The patients exhibited significant improvements in QoL scores. There were few AEs and few cases of LCIG therapy discontinuation. LCIG is an important and available therapeutic option for managing patients with advanced PD.

Randomized trial of safinamide add-on to levodopa in Parkinson's disease with motor fluctuations.

Levodopa is effective for the motor symptoms of Parkinson's disease (PD), but is associated with motor fluctuations and dyskinesia. Many patients require add-on therapy to improve motor fluctuations without exacerbating dyskinesia. The objective of this Phase III, multicenter, double-blind, placebo-controlled, parallel-group study was to evaluate the efficacy and safety of safinamide, an α-aminoamide with dopaminergic and nondopaminergic mechanisms, as add-on to l-dopa in the treatment of patients with PD and motor fluctuations. Patients were randomized to oral safinamide 100 mg/day (n = 224), 50 mg/day (n = 223), or placebo (n = 222) for 24 weeks. The primary endpoint was total on time with no or nontroublesome dyskinesia (assessed using the Hauser patient diaries). Secondary endpoints included off time, Unified Parkinson's Disease Rating Scale (UPDRS) Part III (motor) scores, and Clinical Global Impression-Change (CGI-C). At week 24, mean ± SD increases in total on time with no or nontroublesome dyskinesia were 1.36 ± 2.625 hours for safinamide 100 mg/day, 1.37 ± 2.745 hours for safinamide 50 mg/day, and 0.97 ± 2.375 hours for placebo. Least squares means differences in both safinamide groups were significantly higher versus placebo. Improvements in off time, UPDRS Part III, and CGI-C were significantly greater in both safinamide groups versus placebo. There were no significant between-group differences for incidences of treatment-emergent adverse events (TEAEs) or TEAEs leading to discontinuation. The addition of safinamide 50 mg/day or 100 mg/day to l-dopa in patients with PD and motor fluctuations significantly increased total on time with no or nontroublesome dyskinesia, decreased off time, and improved parkinsonism, indicating that safinamide improves motor symptoms and parkinsonism without worsening dyskinesia.

Magnetoelectric confinement and stabilization of Z pinch in a soft-x-ray Ar(+8) laser.

Magnetoelectric confinement and stabilization of the plasma column in a soft-x-ray Ar(+8) laser, which is excited by a capillary Z pinch, via the combined magnetic and electric fields of the gliding surface discharge is experimentally demonstrated. Unlike soft-x-ray lasers excited by the conventional capillary Z pinches, the magnetoelectric confinement and stabilization of plasma do provide the laser operation without using any external preionization circuit.

Manipulating grain processing method and roughage level to improve feed efficiency in feedlot cattle.

The effects of feeding finishing diets containing whole corn with no roughage on performance and carcass characteristics of feedlot steers were evaluated in 6 trials conducted at commercial research facilities (Bos Technica Research Services Inc., Salina, KS) in the Southern Plains of the United States. One hundred and two feedlot pens containing 6,895 steers were represented. All trials were designed as randomized complete blocks with pen serving as the experimental unit. Steers were fed and managed similarly across all trials. Treatments consisted of a typical control finishing diet with various grain sources and processing methods that contained roughage and a finishing diet containing whole corn (8 to 23% of diet DM) but without added roughage. Final BW was greater (P < 0.1) for steers fed typical finishing diets than for steers fed whole corn diets without roughage in 5 of the 6 trials. Feeding finishing diets containing whole corn but without roughage resulted in decreased (P < 0.1) ADG and carcass ADG in 5 of the 6 trials. However, DMI also was less (P < 0.1) for steers fed whole corn finishing diets without roughage in all trials such that feeding whole corn diets without roughage improved (P < 0.05) G:F (BW basis) in 2 of the 6 trials, and improved (P < 0.1) G:F based on carcass weight in 5 of the 6 trials. Dry matter intake as a percentage of BW daily across trials was well predicted from percentage of dietary NDF from roughage, being 1.906 + 0.0199 (+/-0.0012) NDF (P < 0.05). Performance-based NE(g) content of the diet was greater (P < 0.07) for steers fed whole corn diets without roughage. Differences in USDA yield and quality grades were inconsistent. Results indicate that feeding diets containing whole corn with no added roughage tends to decrease DMI and ADG in finishing steers, but improves feed efficiency and performance-calculated dietary NE(g).

Effect of preslaughter feeding and ractopamine hydrochloride supplementation on growth performance, carcass characteristics, and end product quality in market dairy cows.

An experiment was conducted in market dairy cows to determine the effect of feeding time and ractopamine hydrochloride (RAC) on growth performance, carcass characteristics, and end product quality. In 3 replicates, 9 Holstein cows per replicate (n = 27; 659 +/- 25.3 kg initial BW) culled from 3 dairies were randomly assigned to 3 treatments: 1) slaughter immediately (control), 2) feed for 90 d (NoR), or 3) feed for 90 d with RAC (312 mg.cow(-1).d(-1)) for the final 32 d (RAC). On d 0, NoR and RAC cows were placed in individual pens and fed a high concentrate diet (86% concentrate, DM basis) for 90 d before slaughter. All cows were subjectively scored for BCS and locomotion score on d 0, and NoR and RAC cows were evaluated again after 90 d. Individual DMI was recorded daily throughout the trial, and BW was collected every 14 d. Age and age x treatment did not affect (P > 0.05) any of the traits evaluated in this study. When cows fed for 90 d (NoR and RAC combined) were compared with nonfed controls, fed cows had greater (P < 0.001) final BCS, BW and HCW, lower (P < 0.001) final locomotion score, and greater (P < 0.03) dressing percentage, external fat thickness, and marbling score. Fed cows also tended to have more desirable yield grade (P = 0.08), ribeye area (P = 0.11), fat color (P = 0.09), lean maturity (P = 0.06), and quality grade (P = 0.09) compared with control cows. Warner-Bratzler shear force was not affected (P = 0.23) by feeding. However, a 12-member trained sensory panel revealed that fed cow carcasses had more desirable (P < 0.04) tenderness, juiciness, and overall acceptability than control cow carcasses. Flavor intensity also tended (P = 0.10) to be more desirable for fed vs. control cows. No difference (P > 0.10) in off-flavor was detected among treatments. Finally, there was no effect (P > 0.10) of RAC on growth performance, carcass characteristics, or end product quality. In conclusion, feeding a high concentrate diet for 90 d improved important live animal, carcass, and end product characteristics related to the quality and palatability of beef from market dairy cows; however, no effect of RAC supplementation was observed.

Regulation of lipid accumulation in 3T3-L1 cells: insulin-independent and combined effects of fatty acids and insulin.

The insulin-independent and combined effects of fatty acids (FA; linoleic and oleic acids) and insulin in modulating lipid accumulation and adipogenesis in 3T3-L1 cells was investigated using a novel protocol avoiding the effects of a complex hormone 'induction' mixture. 3T3-L1 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) plus serum (control) or in DMEM plus either 0.3 mmol/l linoleic or oleic acids with 0.3 mmol/l FA-free bovine serum albumin in the presence or absence of insulin. Cells were cultured for 4 to 8 days and cell number, lipid accumulation, peroxisome proliferator-activated receptor-gamma (PPAR-γ) and glucose transporter 4 (GLUT-4) protein expression were determined. Cell number appeared to be decreased in comparison with control cultures. In both oleic acid and linoleic acid-treated cells, notably in the absence (and presence) of insulin, oil-red O stain-positive cells showed abundant lipid. The percentage of cells showing lipid accumulation was greater in FA-treated cultures compared with control cells grown in DMEM plus serum (P < 0.001). Treatment with both linoleic and oleic acid-containing media evoked higher levels of PPAR-γ than observed in control cultures (P < 0.05). GLUT-4 protein also increased in response to treatment with both linoleic and oleic acid-containing media (P < 0.001). Lipid accumulation in 3T3-L1 cells occurs in response to either oleic or linoleic acids independently of the presence of insulin. Both PPAR-γ and GLUT-4 protein expression were stimulated. Both proteins are considered markers of adipogenesis, and these observations suggest that these cells had entered the physiological state broadly accepted as differentiated. Furthermore, 3T3-L1 cells can be induced to accumulate lipid in a serum-free medium supplemented with FA, without the use of induction protocols using complex hormone mixtures. We have demonstrated a novel model for the study of lipid accumulation that will improve the understanding of adipogenesis in adipocyte lineage cells.

Influence of endosperm vitreousness and kernel moisture at harvest on site and extent of digestion of high-moisture corn by feedlot steers.

Six ruminally and duodenally cannulated Angus-Jersey crossbred steers (450 kg of BW) were used in a 6 x 6 Latin square to evaluate the effect of kernel vitreousness and moisture on intake and digestibility of high-moisture corn. Arranged in a 2 x 3 factorial, diets included a floury (FLO) or a vitreous (VIT) endosperm corn hybrid harvested at 28.1% (DRY), 31.2% (MID), or 35.7% (WET) kernel moisture content. Diet DM consisted of 88.25% high-moisture corn, 6% chopped alfalfa hay, 2% corn gluten meal, 0.75% urea, and 3% supplement. Supplement was included to ensure that the diets contained a minimum (DM basis) of 0.6% Ca, 0.6% K, 0.2% S, 33 mg/kg of monensin, and 11 mg/kg of tylosin. Geometric mean diameter of lyophilized high-moisture corn tended to be less (P = 0.06) for VIT than for FLO, and the calculated particle surface area was 15.8% greater (P = 0.03). An interaction of vitreousness with the quadratic effect of moisture was noted (P < 0.001), such that fraction a and effective degradation for starch tended to be greater for the vitreous hybrid at the least and greatest moisture content but lower for the vitreous hybrid at the intermediate moisture content. Intake and ruminal disappearance of DM, OM, and starch were not influenced by vitreousness or moisture, with ruminal starch disappearance averaging 90.9%. Intestinal starch digestion measured as a percentage of starch entering the intestines averaged 91% and was greater (P < 0.05) for VIT than FLO corn. Averaged across moisture levels, total tract starch digestibility was greater (P < 0.003) for VIT than FLO. Compared with FLO kernels, VIT kernels appeared to be more brittle and therefore shattered more readily when rolled, particularly at the driest kernel moisture level. Furthermore, increased surface area of smaller particles may have been responsible for the greater starch utilization from VIT corn. In contrast with the results from other in situ and in vivo trials with dry-rolled corn grain, in which the starch from vitreous hybrids was less rapidly or completely digested, hybrids with more vitreous starch, when fed as high-moisture corn, had greater total tract starch digestibility, primarily due to greater postruminal starch digestion.

Effect of varying levels of fatty acids from palm oil on feed intake and milk production in Holstein cows.

To determine the optimum feeding level of fatty acids of palm oil (PALM; Energizer RP10; 86.6% palmitic acid) on milk production, lactating cows (n = 18) were randomly assigned to a treatment sequence in replicated 4 x 4 Latin squares. Animals were assigned to squares by parity (3 multiparous and 1 primiparous squares with primiparous in the incomplete square). The 4 diets were designed to provide 0, 500, 1,000, and 1,500 g of PALM per day. Cows were fed individually with feed intake measured daily. Each period lasted 16 d with milk production and composition determined the final 2 d. Milk production, milk composition and feed intake data were analyzed using the MIXED procedure of SAS. Milk yields were 30.9, 34.0, 34.2, and 34.2 kg/ d (SEM = 1.9) for the 0, 500, 1,000, and 1,500 g levels, respectively. Milk yield was increased by the addition of PALM; however, there were no differences among the levels of PALM. Milk fat percentage was also increased from 3.44% for 0 g to 3.95% (SEM = 0.17) across all levels of PALM but there were no differences among the PALM treatments. Dry matter intakes were 23.3, 26.4, 24.7, and 23.8 kg/d (SEM = 1.4) for the 0, 500, 1,000 and 1,500 g levels, respectively. The addition of PALM increased milk yield and milk fat percentage, and no adverse effects on dry matter intake were observed.

Factors influencing hemorrhagic transformation in ischemic stroke: a clinicopathological comparison.

As hemorrhagic transformation (HTr) is a frequent complication and can worsen the outcome of acute ischemic stroke, our aim was to assess the risk factors of HTr. Using the database of our neuropathological laboratory, 245 consecutive acute ischemic stroke patients were analyzed. An exploratory logistic regression procedure was carried out to find the best multiple model identifying the factors associated with HTr. The autopsy revealed ischemic infarct in 175 (71%) and ischemic infarct with HTr in 70 (29%) patients. Mean age was 71.5 +/- 11.4 years (mean +/- SD) and 74.8 +/- 10.2 years (mean +/- SD), respectively. The multiple model confirmed age in case of embolic stroke, and diabetes mellitus and infarct size as independent risk factors of HTr. It seems that not serum glucose level but diabetes mellitus in the case history is an independent predictor of HTr.

Residual feed intake of purebred Angus steers: effects on meat quality and palatability.

Relationships between residual feed intake (RFI) and other performance variables were determined using 54 purebred Angus steers. Individual feed intake and BW gain were recorded during a 70-d post-weaning period to calculate RFI. After the 70-d post-weaning test, steers were fed a finishing ration to a similar fat thickness (FT), transported to a commercial facility, and slaughtered. A subsample of carcasses (n = 32) was selected to examine the relationships among RFI, meat quality, and palatability. Steers were categorized into high (> 0.5 SD above the mean; n = 16), medium (mid; +/- 0.5 SD from the mean; n = 21), and low (< 0.5 SD below the mean; n = 17) RFI groups. No differences were detected in ADG, initial BW, and d 71 BW among the high, mid, and low RFI steers. Steers from the high RFI group had a greater DMI (P = 0.004) and feed conversion ratio (FCR; DMI:ADG; P = 0.002) compared with the low RFI steers. Residual feed intake was positively correlated with DMI (r = 0.54; P = 0.003) and FCR (r = 0.42; P = 0.002), but not with initial BW, d 71 BW, d 71 ultrasound FT, initial ultrasound LM area, d 71 ultrasound LM area, or ADG. The FCR was positively correlated with initial BW (r = 0.46; P = 0.0005), d 71 BW (r = 0.34; P = 0.01), and DMI (r = 0.40; P = 0.003) and was negatively correlated with ADG (r = -0.65; P = 0.001). There were no differences among RFI groups for HCW, LM area, FT, KPH, USDA yield grade, marbling score, or quality grade. Reflectance color b* scores of steaks from high RFI steers were greater (P = 0.02) than those from low RFI steers. There was no difference between high and low RFI groups for LM calpastatin activity. Warner-Bratzler shear force and sensory panel tenderness and flavor scores of steaks were similar across RFI groups. Steaks from high RFI steers had lower (P = 0.04) off-flavor scores than those from low RFI steers. Cook loss percentages were greater (P = 0.005) for steaks from low RFI steers than for those from mid RFI steers. These data support current views that RFI is independent of ADG, but is correlated with DMI and FCR. Importantly, the data also support the hypothesis that there is no relationship between RFI and beef quality in purebred Angus steers.

Effects of pasteurization of potato slurry by-product fed in corn-or barley-based beef finishing diets.

Pasteurization of vegetable by-products such as potato slurry (PS) before feeding may be necessary to prevent the spread of pathogens and beef carcass blemishes. We hypothesized that pasteurization would increase ruminal fermentability of PS starch. Four ruminally cannulated crossbred beef steers (initial BW = 432) were used in a 4 x 4 Latin square experiment with a 2 x 2 factorial arrangement of treatments to examine the main effects and interactions of pasteurization (54.4 degrees C for 2 h) of PS and grain type (GT; dry-rolled corn and barley) on ruminal and total tract digestion of beef finishing diets. Diets contained 7% alfalfa hay and 14% PS (DM basis) and were fed ad libitum three times daily. Corn-based diets had 71.7% corn, whereas barley-based diets had 60% barley and 11.7% corn. Pasteurization resulted in greater (P = 0.004) soluble, rapidly degradable starch (34.3 vs. 26.7% for pasteurized and nonpasteurized PS, respectively). Ruminal fluid pH was more acidic (P < 0.07) for corn-based diets than for barley-based diets (P = 0.07) at 0200 and 2100 (sample time x GT; P < 0.05). Ruminal fluid pH was more acidic (P = 0.06) at 1400 for corn-based diets containing pasteurized PS compared with other dietary treatments (sample time x pasteurization x GT; P = 0.04). Minimum and maximum ruminal pH were greater (P < 0.10) for barley-based diets than for corn-based diets. Ruminal fluid pH was < 6.0 for a greater (P = 0.04) proportion of the day for corn-based compared with barley-based diets. In vitro incubation measurements revealed that pasteurization of PS resulted in lower (P = 0.06) ruminal fluid ammonia N concentration. Ruminal fluid ammonia N concentration was lower (P = 0.11) for barley-based diets than for corn-based diets. Steers fed barley-based diets had greater (P = 0.02) DMI and lesser (P < 0.05) total tract digestibility of DM and ADF compared with steers fed corn diets. Pasteurization increased (P = 0.10) total tract starch digestibility. Results indicate pasteurization increased rapidly degradable starch, ruminal starch fermentability, and total tract starch digestibility of PS. Grain type interacted with pasteurization such that feeding corn-based diets containing pasteurized PS resulted in periodic reductions in ruminal pH. Feeding management may be more critical when feeding pasteurized PS in beef finishing diets.

Effect of cutting height and genetics on composition, intake, and digestibility of corn silage by beef heifers.

This research was conducted to determine the effect of corn genetics and cutting height on the composition and nutritive characteristics of corn silage. An in situ study involving eight commercially available corn hybrids indicated main effects and interactions (P < 0.01) of hybrid and cutting height on NDF, ADF, and starch content and on in situ DM and NDF degradablility. Four ruminally cannulated Angus heifers (initial BW = 378 +/- 3 kg) were used in a 4 x 4 Latin square digestion experiment with a 2 x 2 factorial treatment arrangement. Main effects and interactions of hybrid (Pioneer Hi-Bred Int., Inc., hybrids 3335 and 3223) and cutting height (LO = 20.3 cm, and HI = 61 cm) were evaluated. Dietary treatment consisted of 40% chopped alfalfa hay and 60% corn silage. Although corn silage hybrids used were of equivalent maturity at harvest (60% milkline), 3335 treatments had 37.8% starch and 34.8% NDF, whereas 3223 treatments had 33.7% starch and 38.6% NDF. The LO treatments averaged 3.1 percentage units greater in NDF and 3.45 percentage units less in starch content than the HI treatments. Intake of DM was greater for heifers fed 3335-HI than 3335-LO; however, DMI was greater by heifers fed 3223-LO than 3223-HI (hybrid x cutting height interaction, P < 0.05). Starch intake was greater (P < 0.05) and NDF intake was less (P < 0.05) by heifers fed HI vs. LO and fed 3335 vs. 3223 dietary treatments. Digestibility of DM, starch, and NDF was greater (P < 0.05) by heifers fed 3223 than 3335 dietary treatments, but digestibility differences were not observed (P > 0.10) between cutting heights. Rate of in situ DM and starch degradability was not affected (P > 0.10) by hybrid or cutting height; however DM degradability was greater (P < 0.05) for HI than LO corn silage substrates at 8, 16, and 24 h of incubation. Rate of NDF degradability tended (P = 0.08) to be greater for 3223 than for 3335, and for LO compared with HI corn silage. Degradability of NDF was greater (P < 0.05) for 3223 compared with 3335 substrates at 24, 36, and 48 h of incubation. These data suggest fiber may not be an accurate measure of corn silage quality. Whereas cutting height affected chemical composition, we observed genetics to have a greater effect on corn silage quality.

The Mures-Uzhgorod-Debrecen study: a comparison of hospital stroke services in Central-Eastern Europe.

Stroke mortality is extremely high in Central-Eastern European countries. The high rate of risk factors and differences in health care services might be among the factors resulting in high stroke morbidity and mortality in this region. As only few prospectively collected information are available from this region, we decided to evaluate some characteristics of stroke services in neurological departments of a Romanian, a Ukrainian and a Hungarian city in the framework of the Mures-Uzhgorod-Debrecen comparative epidemiological study. We registered demographic data, the absence or presence of the most important risk factors, and clinical signs on admission and at discharge. We recorded the application of various diagnostic methods, stroke treatment and recommendations for secondary prevention. Follow-up is planned after 30 days and after 1 year. The paper summarizes the methodology of this prospective epidemiological study of stroke patients hospitalized in neurological departments in Târgu Mures, Uzhgorod and Debrecen, three Central-Eastern European cities in Romania, Ukraine and Hungary, respectively.

Thermo Sequenase DNA polymerase and T. acidophilum pyrophosphatase: new thermostable enzymes for DNA sequencing.

A combination of thermostable enzymes has been developed that produces higher quality cycle sequences. Thermo Sequenase DNA polymerase is a thermostable enzyme engineered to catalyze the incorporation of ddNTPs with an efficiency several thousandfold better than other thermostable DNA polymerases. Since the enzyme also catalyzes pyrophosphorolysis at dideoxy termini, a thermostable inorganic pyrophosphatase is needed to remove the pyrophosphate produced during sequencing reactions. Thermoplasma acidophilum inorganic pyrophosphatase (TAP) is thermostable and effective for converting pyrophosphate to orthophosphate. The use of the combination of Thermo Sequenase polymerase and TAP for cycle sequencing yields sequence data with uniform band intensities, allowing the determination of longer, more accurate sequence reads. Uniform band intensities also facilitate interpretation of sequence anomalies and the presence of mixed templates. Sequencing PCR products of DNA amplified from heterozygous diploid individuals results in signals of equal intensity from each allele.

Site-directed mutagenesis of alpha-tubulin. Reductive methylation studies of the Lys 394 region.

Previous studies have implicated at least two regions in alpha-tubulin that are important for the regulation of microtubule assembly. These regions include a cluster of basic residues consisting of Arg 390, His 393, and Lys 394 and the highly acidic carboxyl terminus. Lys 394 is highly reactive to HCHO and NaCNBH3. The reductive methylation of Lys 394 by these reagents is thought to be responsible for the profound inhibitory effects of low concentrations of HCHO on microtubule assembly (cf. Szasz J., M. B. Yaffe, M. Elzinga, G. S. Blank, and H. Sternlicht. 1986. Biochemistry. 25:4572-4582). In this study we reexamined the basis for this inhibition. Lys 394 in a human keratinocyte alpha-tubulin (k alpha 1) was replaced by a glutamic acid residue using site-directed mutagenesis. The mutant K394E was synthesized in vitro using rabbit reticulocyte lysates, and its ability to coassemble with bovine brain microtubule protein (MTP) before and after reaction with HCHO and NaCNBH3 was compared with that of wild-type. No differences in the coassemblies of the unmethylated proteins were detected suggesting that Lys 394 is not essential for microtubule assembly. However, methylated K394E prepared at low HCHO concentrations (< 1 mM) incorporated into microtubules to a greater extent (approximately 30-40%) than methylated wild-type. This result is consistent with the hypothesis that methylation of Lys 394 interferes with microtubule assembly. However, the extent of protection afforded by the replacement of Lys 394 with Glu 394 was less than half as large as that predicted from the earlier studies. We tentatively conclude that another residue(s) besides Lys 394 contributes significantly to the assembly-inhibition observed with low concentrations of HCHO. Since this residue(s) is less reactive than Lys 394, it would have to inhibit assembly substoichiometrically when methylated. Potential candidates for this residue include bulk lysyl residue(s), a lysyl residue(s) with intermediate reactivity toward HCHO, and the NH2-termini. The NH2-termini are especially attractive candidates since they appear to have a structural role in microtubule assembly.

Ethoxyformylation of tubulin with [3H]diethyl pyrocarbonate: a reexamination of the mechanism of assembly inhibition.

In this study we reexamined the basis for the profound inhibitory effects of low concentrations of diethyl pyrocarbonate (DEP) on tubulin's ability to assemble into microtubules [cf. Lee, Y. C., Houston, L. I., & Himes, R. H. (1976) Biochem. Biophys. Res. Commun. 70, 50-56]. Assembly inhibition at low DEP concentrations can be resolved into two components: a component reversible with hydroxylamine (attributed to monoethoxyformylation of histidyl residues) that contributes approximately 40% of the inhibition and a hydroxylamine-resistant component (attributed to ethoxyformylation of non-histidyl residues) that contributes approximately 60% of the inhibition. Comparisons between the extent of assembly inhibition associated with each component and the degree of residue modification argue for the involvement of a small number of highly reactive residues in the inhibition process. To identify these residues, tubulin was reacted with limiting concentrations of [3H]DEP and subjected to tryptic digestion and HPLC analysis. Only one moderately reactive histidyl residue was detected. This residue (approximately 2-3-fold more reactive than the bulk histidyl residues) eluted in an apparently large, hydrophobic fragment. We failed to detect any non-histidyl residues that were exceptionally reactive to [3H]DEP. However, we did observe that the N-terminal methionyl residues in native protein were ethoxyformylated at rates comparable to that of the bulk histidyl residues. In denatured protein these methionyl residues were ethoxyformylated to a much larger extent (approximately 3-4-fold) than the bulk histidyl residues. We suggest that the N-terminal methionyl residues in tubulin are partly buried or are in a salt-bridge interaction in native protein and that ethoxyformylation of these residues disrupts tubulin structure and interferes with microtubule assembly.

Expression of a human alpha-tubulin: properties of the isolated subunit.

We examined the in vitro expression and biochemical properties of the isolated alpha subunit of tubulin both in rabbit reticulocyte lysates and in Escherichia coli extracts. Both systems produce soluble, full-length human alpha-tubulin polypeptide. When alpha-tubulin mRNA is translated in rabbit reticulocyte lysates, the isolated alpha subunit is fully functional as assayed by coassembly with bovine brain tubulin using temperature-dependent or taxol/salt assembly procedures. The conformation of the isolated alpha subunit was probed by limited proteolytic digestion with chymotrypsin and by reductive methylation. Limited proteolysis studies indicated that the "monomeric" alpha subunit is highly susceptible to chymotrypsin digestion and becomes resistant to chymotrypsin cleavage following incorporation into the heterodimer. Reductive methylation indicated that the unassociated alpha subunit has a highly reactive lysyl residue essential for microtubule assembly similar to that observed in the heterodimer. In contrast, alpha-tubulin expressed in E. coli lysates was incapable of coassemblying with bovine brain tubulin. Differences in assembly competence of the two alpha-tubulin products appear to be related to formylation of the N-terminal methionine in the procaryotic synthesized subunit. These findings suggest that the amino-terminal methionine of alpha-tubulin plays an essential role in the isolated subunit and/or in the heterodimer, a hypothesis supported by chemical reactivity studies [Sherman, G., Rosenberry, T.L., & Sternlicht, H. (1983) J. Biol. Chem. 258, 2148-2156] which imply that this residue is in a salt-bridge interaction in the dimer.

Microtubule assembly is dependent on a cluster of basic residues in alpha-tubulin.

Previous studies have shown that tubulin, a major protein component of the microtubule, is rendered assembly incompetent when a highly reactive lysine residue (HRL) in the alpha polypeptide of tubulin dimer is reductively methylated [cf. Sherman, G., Rosenberry, T. L., & Sternlicht, H. (1983) J. Biol. Chem. 258, 2148-2156]. In this study we demonstrate that the HRL in bovine brain tubulin is Lys-394, a residue proximal in the alpha-tubulin sequence to the highly negatively charged carboxy-terminus region (residues 412-450) previously implicated in assembly. pH studies were undertaken to probe the local environment of Lys-394. These studies indicated that Lys-394 reactivity toward HCHO is sensitive to the titration of a pKa 6.3 group presumed to be a histidine residue. This assignment is supported by our finding that histidine modification via diethyl pyrocarbonate strongly affects Lys-394 reactivity toward HCHO as well as microtubule assembly. We propose on the basis of secondary structure considerations and published sequence data for a variety of tubulins that Lys-394 is part of an evolutionarily conserved cluster of basic residues (effective charge: 2+ to 2.5+ at neutral pH) composed of Lys-394, His-393, and Arg-390, which is important for tubulin function and which renders Lys-394 reactive as a nucleophile.