The effect of pyridostigmine pretreatment on oxime efficacy against intoxication by soman or VX in rats. This study was done to assess the effects of pyridostigmine (PYR) on a) the accumulation of labelled VX and soman within the brain, b) the therapeutic efficacy of atropine and oxime (2-PAM or HI-6) against intoxication by VX and soman and c) oxime-induced reactivation of inhibited acetylcholinesterase (AChE). In all experiments, rats were given PYR (131 micrograms/kg, im; I70 dose for whole blood AChE) or vehicle 30 min prior to nerve agent. In estimating 3H-agent the accumulation in the brain or estimating blood AChE activity, sufficient soman (47 micrograms/kg, iv) or VX (21.3 micrograms/kg, iv) was given to inhibit 50% of brain AChE activity. In assessing therapeutic efficacy and oxime-induced reactivation of blood AChE, rats were pretreated with PYR, challenged with agent and treated with atropine (16 mg/kg, im) and HI-6 or 2-PAM (100 umoles/kg, im) 30 sec post agent. Whole blood was collected by tail bleeding to monitor peripheral AChE activity at various time points before and after PYR and challenge. Pyridostigmine failed to alter covalent binding of labelled VX or soman in the brain. The 24-hr survival data showed that PYR reduced the therapeutic benefit of atropine and oxime against VX intoxication (but not soman). Protective ratios in VX-challenged rats given vehicle or PYR and treated with atropine + 2-PAM decreased slightly from 2.5 to 2.1 (p > .05), whereas with atropine + HI-6 they decreased significantly from 3.8 to 2.4. Also, AChE reactivation by HI-6 in VX-challenged rats was greater (p < .05) in vehicle- than in PYR-pretreated rats. HI-6 significantly reactivated AChE activity in both pretreatment groups (PYR or vehicle) given soman. The data suggest that PYR decreases the overall recovery of inhibited AChE in VX-challenged rats given HI-6; under the conditions used, this adverse effect decreases atropine+oxime efficacy against VX-induced lethality.


The effect of tetrabutylammonium ion on chicken brain acetylcholinesterase activity in vitro. The effect of tetrabutylammonium ion on chicken brain acetylcholinesterase activity was investigated. Tetrabutylammonium ion (1-4mM) reversibly inhibited acetylcholinesterase activity of chicken brain (34-73%) in a concentration dependent manner, the IC50 being about 1.1mM. A Lineweaver-Burk plot, as well as a Dixon plot, indicated that the nature of inhibition is noncompetitive with a Km value of 0.134mM and a Ki value of 1.58mM at 37 degrees C. An Arrhenius plot showed that the transition temperature is significantly reduced in the presence of tetrabutylammonium ion.


Relationship between activities of cytochrome P-450 monooxygenases in human placental microsomes and binding of benzo(a)pyrene metabolites to calf thymus DNA. Benzo(a)pyrene metabolism in human placental microsomes from smokers was studied. Benzo(a)pyrene metabolites were separated using high pressure liquid chromatographic technique. Reaction of benzo(a)pyrene with a microsomal fraction of placenta from individuals who smoke cigarettes during pregnancy yields 7,8 dihydroxy benzo(a)pyrene as a major metabolite, while 3'-hydroxy benzo(a)pyrene, 4,5 dihydroxy benzo(a)pyrene and quinones constitute minor metabolites. The activities of arylhydrocarbon hydroxylase and 7-ethoxycoumarin deethylase exhibited much higher activities in smokers than in nonsmokers. Examination of specific binding of monoclonal antibodies to cytochrome P-450 isozymes in placental microsomes revealed that cigarette smoking specifically enhanced the level of cytochrome P-450 c and d isozymes in human placental microsomes. Coincubation of 3H-benzo(a)pyrene and calf thymus DNA with placental microsomes yielded acid insoluble 3H-B(a)P from smokers, suggesting that cigarette smoking may induce placental enzymes which convert benzo(a)pyrene into ultimate metabolites to form carcinogen-DNA adducts.


Evaluation of acetaminophen-induced developmental toxicity using FETAX. Potential mechanisms of acetaminophen-induced developmental toxicity were evaluated using FETAX (Frog Embryo Teratogenesis Assay-Xenopus). Early Xenopus laevis embryos were exposed to acetaminophen for 96-h in two definitive concentrations-response assays with and without an exogenous metabolic activation system (MAS). Two static renewal tests of acetaminophen and the MAS treated with carbon monoxide, cimetidine, ellipticine, diethyl maleate, and supplemented with glutathione were also performed. Addition of the MAS decreased the 96-h LC50 and EC50 (malformation) values of unactivated acetaminophen 3.9-fold and 7.1-fold, respectively. Addition of the carbon monoxide- and ellipticine-inhibited MAS, as well as the glutathione-supplemented MAS decreased the developmental toxicity of activated acetaminophen to levels near that of the unactivated parent compound. Cimetidine-inhibited MAS also reduced the developmental toxicity of acetaminophen, but not to the extent observed with the carbon monoxide- and ellipticine-inhibited, or glutathione-supplemented MAS. Addition of the diethyl maleate-treated MAS substantially increased the developmental toxicity of acetaminophen. Results indicate that a highly reactive intermediate formed as the result of MFO-mediated metabolism (possibly P-448) significantly increased the developmental toxicity of acetaminophen. Glutathione was also found to play a major role in intermediate detoxification in vitro.


Influence of rumen fermentation on response to endophyte-infected tall fescue seed measured by a rat bioassay. Possible alteration of toxicity of endophyte-infected tall fescue by ruminal fermentation was studied using 28 Harlan Sprague-Dawley rats (avg. initial wt., 141 g). These were assigned randomly to one of four treatments in a 2*2 factorial consisting of a 14-day growth period with weights and feed consumption data determined on days 0, 5, 10 and 14. Treatments were: endophyte-infected Kentucky-31 tall fescue seed (E+) or endophyte free Johnstone tall fescue seed (E-) that was incubated for 0 (NON) or 24 hours (INC) with rumen fluid collected from a 290-kg cannulated steer fed a diet containing 37% endophyte-infected tall fescue hay. Diets consisted of 50% lab chow, 39% fescue seed and 11% rumen contents (air-dry basis). Alkaloid content (N-acetyl plus N-formyl loline) for the four diets were 2540, 2680, 0, and 0 micrograms/g for E+NON, E+INC, E-NON, and E-INC diets, respectively. E- treatments gained faster, consumed more feed and converted feed more efficiently (P < .05) than did E+ fed groups. No difference in feed intake was observed within E+ treatments, however, the E+INC diet gained faster (P < .05) and converted feed more efficiently (P < .05) than did E+NON fed rats (2.56 vs 1.96 g/d and 5.94 vs 7.51 g of feed/g of gain for gain and feed conversion, respectively). Including endophyte-infected seed in rat diets depressed performance (ie. intake, gain and feed efficiency). This depression was partially alleviated by a 24-hour incubation with rumen fluid contents suggesting that toxicity of endophyte-infected tall fescue is lessened due to rumen microbial action.


Clinical counterpoint: gonadotropin-releasing hormone deficiency: perspectives from clinical investigation. Advances in our understanding of the pathophysiology of Kallmann syndrome have come from an interdisciplinary approach involving developmental biology, clinical investigation, and molecular biology. It is equally clear that progress to date is but the first chapter of what will be a fascinating biological story. It now seems likely that the full expression of reproductive potential from the neuroendocrine perspective is likely to be as complicated as other aspects of reproduction, such as the multigene control of external genital differentiation. An analogous story may well emerge for the neuroendocrine control of reproduction in which the GnRH gene is encoded on the eighth chromosome, the protein guiding the embryonic journey of the GnRH-producing neuron to the hypothalamus lies on the X chromosome, and many, as yet to be determined, other genetic loci collaborate in the full expression of reproductive potential. Such a detailed study is warranted not only because of the clinical and genetic implications for an individual patient with this disorder, but also from an organizational theme for the evolution of the species (and its potential regulation). Given the pressing nature of world population growth, obtaining such understanding and its applications to fertility and contraception is crucial. These advances will only come from enlightened interactions of clinical investigators, molecular geneticists, and developmental biologists in which interdisciplinary approaches should be fostered. This should be an exciting story to follow given the remarkable nature of the tools at hand to study these clinical conditions.


Arthroscopic surgery for osteochondritis dissecans of the femoropatellar joint of the horse. Arthroscopic surgery for the treatment of osteochondritis dissecans was undertaken on 252 femoropatellar joints in 161 horses (82 Thoroughbreds, 39 Quarter Horses, 16 Arabians, 9 Warmbloods and 15 others of various breeds). There were 53 females and 108 males. Twenty-two horses were 1 year of age at the time of surgery, 68 were yearlings, 36 were 2-year-olds, 21 were 3-year-olds, and 14 were > or = 4 years old. Ninety-one had bilateral involvement and 70 had unilateral disease. Follow-up information was obtained on 134 horses, including 79 racehorses and 55 non-racehorses: 86 (64%) of these 134 horses returned to their intended use, 9 (7%) were in training, 21 (16%) were unsuccessful and 18 (13%) were unsuccessful due to other defined reasons. Horses with Grade I lesions (< 2 cm in length) had a significantly higher success rate (78%) than did horses with Grade 2 (2-4 cm) or Grade 3 (> 4 cm) lesions (63% and 54% success rates respectively). A significantly higher success rate was also noted for horses operated on as 3-year-olds compared with the remainder of the study population. A significantly lower success rate was noted for yearlings than for the remainder of the population. There was no significant difference in outcome as related to sex of animal involved, racehorse versus non-racehorse, lesion location, unilateral versus bilateral involvement, presence or absence of patellar or trochlear groove lesions, or presence or absence of loose bodies.


Fractures of the lateral malleolus of the tibia in 16 horses. Clinical and radiological features of 16 horses with fractures of the lateral malleolus of the tibia are reported. The paper describes surgical techniques used, results obtained and discusses justification for removal. Fourteen fractures were unilateral and two bilateral. There was no left:right disparity. The history included a known traumatic incident in 14 cases. All animals had a tarsocrural joint effusion and 10 had palpable thickening of the lateral collateral ligaments. Crepitus was also palpable in 10 horses. The fracture was identified in all dorsoplantar and 14 of 18 dorsomedial-plantarolateral oblique radiographic projections. Nine fractures were simple and 9 comminuted. All fractures were removed via a tarsocrural arthrotomy. Approaches were dorsolateral in 14 joints, plantarolateral in 3 and dorsolateral and plantarolateral in 1. After an ascending exercise programme horses returned to work 6 months after surgery: 15 horses were free of lameness after 17-62 months, with 13 animals performing at a level similar to pre-injury standard.


Septic arthritis in 15 standardbred racehorses after intra-articular injection. Case histories, results of synovial fluid analyses, treatment regimens and outcome are described for 15 adult Standardbred horses with confirmed post-injection septic arthritis. Joint sepsis followed injection of corticosteroids, hyaluronic acid, polysulphated glycosaminoglycan, or local anaesthetic. The median interval from injection to appearance of clinical signs was 2.5 days, and median interval from injection to referral was 9 days. The median initial synovial leucocyte count on admission was 57 x 10(9)/litre, but there was a wide range of values (18-258 x 10(9)/litre). The median synovial neutrophil percentage was 95% (77-99%). All bacterial isolates were Gram-positive cocci, 86% of which were staphylococci. All treated horses (12/15) initially received broad-spectrum parenteral antibiotic therapy, and the articulations of all horses except one were lavaged, either with non-surgical through-and-through techniques only (N = 3), or surgically with arthrotomy (N = 1) or arthroscopy (N = 7). The owners of all treated horses were contacted and racing records were consulted. Eleven of 12 horses returned to racing. Outcome was judged as either satisfactory (3/12) if the horse had returned to racing levels similar to or better than before treatment, or unsatisfactory (9/12) if the horse had poorer performance or could not return to racing. The 3 horses with satisfactory follow-up had been treated with arthroscopy and post-surgical closed suction drainage. The results of bacterial cultures suggest that the initial antimicrobial agents used should be effective against penicillin-resistant staphylococci.


A retrospective study of 192 horses affected with septic arthritis/tenosynovitis. The medical records of 192 horses with septic arthritis/tenosynovitis 1979-1989 were reviewed. Forty-three horses developed infection after an intra-articular injection, 46 following a penetrating wound, 25 following surgery, 66 were foals less than 6 months old, and 12 were adult horses without a known aetiology. Haematogenous infection of a joint occurs in adult horses and should be considered as a differential diagnosis in horses with an acute onset of severe lameness. The aetiology of the infection had a significant effect on the type of bacteria identified by culture. Staphylococcus was cultured from most of the horses that developed infection following a joint injection or surgery, 69% of the horses from which an organism was identified. Horses that developed infection secondary to a penetrating wound frequently provided cultures of more than one organism; Enterobacteriaceae and anaerobes were more frequently isolated in this group. The most common organisms isolated from foals were Enterobacteriaceae; E. coli was identified in more than 27% of the foals. The hock was the most frequently involved joint. Multiple treatments were used over the 10-year period of study. Survival rates were lowest in foals; only 45% survived to be released from the hospital. Survival was greater in adult horses; 85% of the horses that were treated were released from the hospital. Survival was significantly greater in horses with septic tenosynovitis; all 14 of the horses that were treated survived. Survival was not significantly affected by the joint involved or by the type of bacteria cultured from the synovial fluid.(ABSTRACT TRUNCATED AT 250 WORDS)


Open drainage, intra-articular and systemic antibiotics in the treatment of septic arthritis/tenosynovitis in horses. Open drainage was used to treat 26 horses with persistent or severe septic arthritis/tenosynovitis. Infected synovial structures were drained through a small (3 cm) arthrotomy incision that was left open and protected by a sterile bandage. Joint lavage was performed in all 26 horses. In addition to systemic antibiotics, 23 of these horses were also treated with intra-articular antibiotics; amikacin (17 horses), gentamycin (2 horses), cefazolin (2 horses), and 2 horses were injected at different times with gentamycin and amikacin. The infection was eliminated from the involved synovial structures in 25 of 26 horses; 24 survived and were released from the hospital. The arthrotomy incisions healed by granulation in 16 horses; in 9 horses the arthrotomy incision was sutured closed once the infection was eliminated. Seventeen horses returned to soundness and resumed athletic function. Open drainage was an effective method of achieving chronic drainage from a joint or tendon sheath. It is indicated in horses that have established intra-synovial infections or in horses that do not respond to joint lavage through needles.


Antimicrobial susceptibility of bacterial isolates from 233 horses with musculoskeletal infection during 1979-1989. Bacterial culture and susceptibility results were analysed from 233 horses with septic arthritis/tenosynovitis or osteomyelitis that developed after fracture repair. Antibiotics were deemed highly effective, effective or ineffective if > or = 85%, 70-84.9% or < 70% of the isolates were susceptible respectively. In total, 424 bacterial types were isolated; 386 were aerobic or facultative and 38 were anaerobic. Enterobacteriaceae (28.8%) were the most common bacterial group isolated, followed by non-beta-haemolytic streptococci (13.0%), coagulase-positive staphylococci (11.8%), beta-haemolytic streptococci (9.4%), and coagulase-negative staphylococci (7.3%). The remainder of the organisms were other Gram-negative (15.8%), other Gram-positive (2.3%) and miscellaneous (2.6%) bacteria. Penicillin and ampicillin were highly effective against beta-haemolytic streptococci, but were ineffective against other bacteria. Ampicillin was no more effective than penicillin against most bacteria. Amikacin was the most effective antibiotic against the wide range of bacteria isolated in this study. Amikacin was highly effective against coagulase-positive staphylococci, Enterobacteriaceae and Pseudomonas and was effective against coagulase-negative staphylococci and Actinobacillus. Gentamycin was not highly effective against any bacterial group; but was effective against coagulase-positive and negative staphylococci, Pseudomonas, Salmonella and Actinobacillus. Kanamycin was ineffective against all bacteria with the exception of Actinobacillus. Cephalothin was highly effective against beta-haemolytic streptococci, coagulase-positive staphylococci and Actinobacillus and was effective against coagulase-negative staphylococci.(ABSTRACT TRUNCATED AT 250 WORDS)


Evaluation of progesterone treatment to create a model for equine endometritis. To investigate a model for equine endometritis, 12 mares with normal reproductive tracts were divided into 2 groups. All mares received progesterone in oil, 250 mg im, daily. At 5 days after initiation of progesterone administration, the uteri were inoculated with 10(6) colony forming units of Pseudomonas aeruginosa. The day of inoculation was designated Day 0. On Day 6, endometrial swab samples yielded P. aeruginosa in 5 mares; samples from the other 7 mares yielded heavy growth of Escherichia coli, Streptococcus zooepidemicus, Klebsiella pneumoniae, Enterobacter spp., Citrobacter diversus, Staphylococcus epidermidis and Streptococcus morbillorum. On Days 6, 7 and 8, Group A mares received intrauterine infusions of 6 g ticarcillin disodium and 0.2 g clavulanate potassium in 100 ml sterile saline. Group B mares received infusions of saline only. The incidence of swab specimens yielding no bacterial growth was significantly higher in Group A than Group B mares on Days 8 and 13 (4/6 vs 0/6). Swab samples from 5 of the 6 mares in Group A yielded growth of fungi on Days 13 and 19. Mares in Group B were then similarly treated with ticarcillin/clavulanate infusions, on Days 19, 20 and 21. The incidence of swab specimens yielding no bacterial growth was 2/6 and 1/6 on Days 21 and 26, respectively; fungi were not recovered from these mares at any time. The incidence of no-growth swabs after antibiotic treatment tended to be higher in Group A and incidence of fungal recovery after antibiotic treatment was significantly higher in Group A.(ABSTRACT TRUNCATED AT 250 WORDS)


Effects of high-intensity exercise on plasma catecholamines in the thoroughbred horse. In Study 1, a single speed test of 6 to 12 m/sec was performed for 2 mins at an incline of 5 degrees on a high-speed treadmill (single-step test). Only one speed was performed per session and blood samples were taken before and after the test. In Study 2 horses cantered for 1 min at increasing speeds of 6 to 13 m/sec on an incline of 3 degrees (multiple-step test). Blood samples were taken before exercise, throughout the test and during recovery. In the single-step test plasma concentrations of adrenaline and noradrenaline both increased at speeds of 9 m/sec, as did blood lactate. Mean concentrations of adrenaline and noradrenaline at the end of the 12 m/sec test were 153 and 148 nmol/litre, respectively. Plasma concentrations were similar over all speeds although there was a tendency for the increase in noradrenaline to be greater than that of adrenaline at the lower speeds. The multiple-step test resulted in smaller increases in both adrenaline and noradrenaline. Although again closely correlated, increases in adrenaline were 20-30% greater than those for noradrenaline. In both exercise models, changes in plasma adrenaline and noradrenaline values with exercise showed an exponential relationship to plasma lactate. A plasma half-life of less than 30 secs was indicated during recovery from the multiple-step test. Changes in adrenaline and noradrenaline were much greater than previously recorded in man and emphasise the importance of catecholamines in mediating the physiological response of the horse to exercise.


Outcome of treatment in 23 horses with progressive ethmoidal haematoma. This paper describes the outcome of treatment in 23 horses with an ethmoidal haematoma. In 22 cases a diagnosis could be made by endoscopic means alone but in 1 horse the lesion was confined to the maxillary sinus and a diagnosis was made only at surgery. One horse was destroyed at the owner's request but the other 22 underwent radical excision of the lesion via a facial flap approach under general anaesthesia. Post-operative haemorrhage was controlled by nasal packing with a gauze bandage and this was removed between the 2nd and 4th post-operative day. One horse died from encephalitis the day after surgery. Other complications included facial wound dehiscence, sequestration and suture periostitis. Of 21 horses followed up post-operatively there was definite recurrence of lesion in 2 cases and possibly a third. However, in 18 horses there was no evidence of recurrence (follow up times were 2 to 85 months). It is suggested that radical excision of the lesion provides an effective means of treatment.


Effect of dietary biotin supplement on equine hoof horn growth rate and hardness. Over a 10-month period, 24 randomly selected riding horses were fed various amounts of biotin. Statistically significant improvements in growth rates and hardness of hooves were produced by biotin supplementation. Greater growth rates and hardness were achieved at a daily dose of 15 mg than at 7.5 mg. Increased hoof hardness was greatest in the hoof quarters and toe. No ring formation occurred in hooves of horses fed biotin intermittently.


Effects of hypoxia and azotaemia on the pharmacokinetics of amikacin in neonatal foals. The effects of hypoxia and azotaemia on the pharmacokinetics of amikacin were evaluated in 20 full-term neonatal critically ill foals which required 24-h supportive care, antibiotics and dextrose-supplemented polyionic fluids given intravenously, nasal insufflation with oxygen and nutritional supplementation. There was no association between sepsis score or survival and pharmacokinetic parameters. Concurrent hypoxia and azotaemia were associated with significantly decreased clearance and increased peak and trough serum concentrations of amikacin; however, peaks or troughs did not exceed toxic values. Derangements in serum peak, trough and clearance values, which were present on admission, persisted over the 6-day duration of this study. Daily monitoring of serum amikacin concentration revealed a tendency to underdose (particularly in foals receiving aggressive fluid therapy), which necessitated increasing the dose/kg body weight (9-12 mg/kg) and increasing the dose interval (10-12 h) in 40% (8/20) of the cases, so that blood concentrations of amikacin could be maintained within the target range of 3-15 micrograms/ml. Amikacin-induced nephrotoxicity was not indicated by conventional laboratory testing, nor was it strongly suspected after examination of post mortem lesions.


'Laterally aggregated' polyacrylamide gels for electrophoresis. A new method is described for producing highly porous polyacrylamide matrices: polymerization in presence of a preformed hydrophilic polymer. If a standard mixture of monomers (e.g., 5%T, 4%C) is polymerized in presence of, e.g., polyethylene glycol (PEG) 10 kDa, lateral chain aggregation occurs, with formation of large pore sizes. In PEG 10 kDa, the transition from a small- to a large-pore gel is clearly apparent at 0.5% PEG addition and reaches a plateau already at 2.5% PEG. Even with shorter PEG fragments (6.2 and 1 kDa) this transition occurs, but with progressively larger amounts of PEG in solution (up to 25% for the 1 kDa species). Other polymers such as hydroxymethyl cellulose (1000 kDa) and polyvinyl-pyrrolidone (360 kDa and 25 kDa) are also able to elicit this phenomenon. It appears that lateral chain aggregation (before the cross-linking event) is induced via intra-chain hydrogen bonding, since urea and temperature strongly inhibit it, whereas tetramethylurea (an agent quenching hydrophobic interactions) does not hamper it. By scanning electron microscope, it is found that the maximum pore size obtained in a 5%T, 4%C gel in presence of 2.5% PEG 10 kDA is of the order of 0.5 micron, whereas the same 5%T, 4%C control gel would have an average pore diameter of 5 nm. Thus, an increment of pore size of about 2 orders of magnitude is obtained: in these new matrices, a 21000 bp DNA fragment exhibits a much greater migration than in a control gel in which the sample is entrapped at the application site.


The effect of gel structure on matrix orientation. Four polymeric gels of different structure, low electroendosmosis (LE) agarose, highest electroendosmosis (HEEO) agarose, beta-carrageenan, and polyacrylamide, were studied by transient electric birefringence to determine the importance of various structural features on the orientation of the gels in an electric field. The two types of agarose, but not the polyacrylamide or beta-carrageenan, exhibited anomalous orientation effects. Both agarose and beta-carrageenan exhibited large birefringence signals, suggesting that the noncovalent hydrogen bonds joining the agarose fibers within the matrix allow the high degree of orientation of the gel. The spatial arrangement of the sugars of the agarose backbone is necessary for the anomalous orientation effects in reversing electric fields.


Information on DNA conformation derived from transverse pore gradient gel electrophoresis in conjunction with an advanced data analysis applied to capillary electrophoresis in polymer media. Abnormally slow migration of DNA is conventionally viewed as being due to an abnormal conformation relative to "linear" standards. The evidence for this rests on a few instances where nonlinear DNA structures have been established by independent methods and yield low mobilities relative to standards. Transverse pore gradient gel electrophoresis of authentically bent kinetoplast DNA and of an upstream activator sequence (UAS) of an E. coli operon promoter shows in addition that curves of migration distance vs. gel concentration ("Ferguson curves") of such abnormally conformed DNA differ from those of "linear" standards. Since Ferguson curves are interpretable with regard to molecular size in concordance with a mathematical model (Ogston model), transverse pore gradient gel electrophoresis provides a simple means of correlating abnormally slow migration of DNA with molecular size. In addition, transverse pore gradient gel electrophoresis is able to distinguish between DNA banding which exhibits a steeper dependence on gel concentration than "linear" standards from one which shows the same dependence. The former appears characteristic of circularly bent DNA and gives rise to a substantial retardation, the latter of bending across a knot or kink in the DNA chain associated with a relatively minor retardation relative to standards. Circularly bent restriction fragments formed from kinetoplast DNA retain the characteristic intersecting Ferguson curves on the transverse pore gradient gel. Another authentically "abnormal" DNA structure recognizable on transverse pore gradient gels is supercoiled DNA derived from the reaction of topoisomerase with a plasmid. Different lengths of supercoiled sequences give rise to parallel Ferguson curves clearly intersecting with those of linear standards.(ABSTRACT TRUNCATED AT 250 WORDS)


Molecular sieving of lambda phage DNA in polyacrylamide solutions as a function of the molecular weight of the polymer. Electrophoresis of lambda phage DNA was carried out in solutions at various concentrations of uncrosslinked polyacrylamide of 0.6, 1, 5 and 9 x 10(6) molecular weight (Mw) with narrow Mw distribution. By inspection of mobilities in the various concentration ranges, it appears that mobilities decrease, and retardation increases, with increasing Mw. The relation between electrophoretic retardation and the Mw of the polymer was also interpreted (i) in the manner previously applied to nonlinear Ferguson plots and compatible with the Ogston model; and (ii) empirically, on the basis of the first derivatives of the functions describing the Ferguson plots at the polymer concentrations used. Interpretation (i) shows that the retardation increases linearly in the order of 0.6, 1, 5 and 9 x 10(6) Mw of polyacrylamide. Interpretation (ii) shows a nonlinear increase of retardation in the Mw range 5 to 9 x 10(6), and a decrease in retardation as Mw is raised from 0.6 to 5.0 x 10(6). Hypothetically, interpretation (ii) can be explained mechanistically by a progressive change, as the polymer size is increased, from a collision with the surface of the polymer fiber to one occurring after permeation in the interior of a random-coiled fiber. Interpretation (i) may fail to detect that change due to the large difference between DNA mobility in solutions of the smallest polymer and the free mobility. DNA peak detection in all of the four size classes of polyacrylamide in solution is limited to relatively narrow ranges of polymer concentration.(ABSTRACT TRUNCATED AT 250 WORDS)


Advances in DNA electrophoresis in polymer solutions. DNA electrophoresis in gels and solutions of agarose and polyacrylamide was objectively evaluated with regard to separation efficiency at optimal polymer concentrations. In application to DNA fragments, polyacrylamide gels were superior for separating fragments of less than 7800 bp, and agarose gels are the best choice for larger fragments. Agarose solutions are nearly as good as polyacrylamide gels for small DNA (< 300 bp). Agarose solutions have a higher efficiency than polyacrylamide solutions for DNA of less than 1200 bp. Separation efficiency sharply decreases with increasing length of DNA. Retardation in polyacrylamide solutions was found to depend on polymer length in a biphasic fashion. The choice of resolving polymer concentrations depends on the progressive stretching of DNA in proportion to polymer concentration. The rate of that stretching appears higher in polyacrylmide solution than in gels or in liquid or gelled agarose. Application of polymer solutions to capillary electrophoresis raises further problems concerning agarose plugs, DNA interactions with the polymers, operation at low field strength and long durations as well as detection sensitivity.


High-throughput automated DNA sequencing facility with fluorescent labels at the European Molecular Biology Laboratory. One of the aims of the facility is to develop and push the automated on-line DNA sequencing gel technology to its limit in sequence throughput, which may be somewhere around 100 kilobases of sequence per device per day. Key new developments were initiated and applied in operation on the European Molecular Biology Laboratory (EMBL) automated sequencer and its commercial version A.L.F. (Pharmacia). Sequencing speed was increased by a factor of 10-20, up to 1500 bases per hour per clone on ultrathin (about 100 microns) gels, while the resolution and reading length were extended to 1000 bases on gels with 50 cm separation length, using fluorescein-15-*dATP as the internal label. With our sequencing strategy, closing about 40% of the sequence with "walking" primers and F-15-*dATP as internal label, we sequenced both strands of a cosmid insert of 38.5 kb in length, each strand twice, in only 430 sequencing reactions and with average reading of 380 bases per reaction.


Manual sequencing using pulsed field.

Manual sequencing using pulsed field. The use of pulsed fields in manual sequencing opens up the compression zone found with a DC field and extends the range of resolution from a few hundred bases to several thousand bases. The band inversion problem is overcome with the proper pulsing conditions, and the bands are sharper than for the DC field case. Accurate visual reading is possible up to about 800-900 bases. The method is compatible with automation techniques, since the band spectrum is stretched continuously during migration, and the smaller fragments are run off the gel.


Physical genome analysis of bacteria.

Physical genome analysis of bacteria. Pulsed-field gel electrophoresis (PFGE) is a general analytical tool to separate large DNA molecules and may therefore be applied to problems from all areas of bacteriology. The genome size of bacteria covers the range of 0.6 to 10 megabase pairs. For genome fingerprinting, the bacterial chromosome is cleaved with a restriction endonuclease that gives a resolvable and informative number of five to one hundred fragments on the PFGE gel. Restriction enzymes are chosen according to GC content, degree of methylation, and codon usage of the respective bacterial genus. Macrorestriction fingerprinting allows the identification of bacterial strains and the distinction between related and unrelated strains. If fragment patterns of several restriction digestions are quantitatively evaluated, strains can be classified according to genetic relatedness at the level of genus, species, and biovar. In particular, members of a clonal lineage can be uncovered. Hence, any problem from applied, environmental, and clinical microbiology may be addressed by PFGE restriction analysis where the spatiotemporal spread of a bacterial clone is of interest. In bacterial genomics, PFGE is employed for the top-down construction of macrorestriction maps of the chromosome which yields data about genome organization, mobile genetic elements, and the arrangement of gene loci and gene families. The genomic diversity of a bacterial species is elucidated by comparative chromosome mapping. Map positions of restriction sites and gene loci of interest serve as landmarks to assess the extent of gross chromosomal modification, namely insertions, deletions and inversions. Intra- and interspecies comparisons of genome organization provide insights into the structure and diversity of bacterial populations and the phylogeny of bacterial taxa.


PCR amplification products are of limited use for the study of DNA/protein interaction. Conventional methods for labeling double-stranded DNA lead to high specific activity. Yet they often alter the target DNA sequence to such an extent as to prevent a meaningful protein/DNA interaction analysis. Therefore we tried to establish a polymerase chain reaction (PCR)-based method which allows radiolabeling to high specific activity and should maintain the protein binding capability of small double stranded DNA fragments. By using PCR it is possible to label double stranded DNA to high specificity, but the protein binding capability of such DNA is drastically reduced.


Pulsed-field gel electrophoresis analysis of a Pseudomonas aeruginosa pathovar. This paper presents the genome organization and mobility of Pseudomonas aeruginosa strains that had been isolated in half-year intervals from 30 patients with cystic fibrosis since the onset of colonization over a 2- to 8-year period. The chromosomes were digested with DraI or SpeI, separated by pulsed-field gel electrophoresis, blotted and hybridized with probes encoding housekeeping or virulence genes. Strains were differentiated by relatedness of macrorestriction fingerprints. After some turnover of strains during the first two years of colonization, each patient had acquired a set of strains that diversified during the course of the disease. In the majority of patients, two clonal lineages were found to account for colonization in the air passages but each lung habitat was characterized by some specific signature of bands in the macrorestriction fragment pattern.


A PacI/SwaI map of the Pseudomonas aeruginosa PAO chromosome. The PacI/SwaI macrorestriction map of the 5.9 Mb Pseudomonas aeruginosa PAO (DSM 1707) genome was constructed by fragment size comparisons of complete single and double digests and hybridization with probes of known genomic localization. P. aeruginosa PAO contains five recognition sites for PacI and six sites for SwaI. An integrated DpnI/PacI/SpeI/SwaI map was obtained by fragment pattern and Southern blot hybridization analyses of all combinations of double digestions and by two-dimensional pulsed-field gel electrophoresis mapping protocols.


Chromosomal localization of the HYP2-gene in Saccharomyces cerevisiae and use of pulsed-field gel electrophoresis for detection of irregular recombination events in gene disruption experiments. In the hypusine-containing protein (HP), a specific lysine residue is modified by spermidine to form the unusual amino acid hypusine (4-amino-2-hydroxybutyllysine). The HP has been designated as an eucaryotic translation initiation factor--eIF-5A--because of its stimulating effect in the methionyl-puromycin in vitro assay. Nevertheless, the precise function of this protein remains to be elucidated. In the yeast Saccharomyces cerevisiae two genes, HYP1 and HYP2, coding for two different forms of the HP, are present. The HYP1-gene is identical to the ANB1-gene and has already been localized on chromosome X. However, the chromosomal localization of the HYP2-gene has not been elucidated. By using pulsed-field gel electrophoresis (PFGE) and subsequent Southern blotting, we determined the localization of the HYP2-gene to chromosome V. Furthermore, PFGE was used for the detection of irregular recombination events, such as misintegration or integration into a duplicated gene, and in gene disruption experiments using haploid and diploid yeast cells. The obtained data support the critical role of the HP for cell viability.


Genetic heterogeneity of prostatic carcinoma-derived cell lines as emphasized by DNA fingerprinting. To investigate genetic heterogeneity during in vitro cultivation of human prostatic carcinomas following radical prostatectomy we performed DNA fingerprinting using the digoxygenin-labeled probes (GACA)4 and (GTG)5. DNA was isolated from fresh material stemming from different areas within one tumor and from cell cultures of the same material. The patterns which were obtained by the nucleolar organizer region (NOR)-specific probe (GACA)4 exhibit only a few prominent low molecular mass bands and no differences were observed between any of the tumors analyzed so far. Changes in the fingerprint pattern occurred between cell cultures derived from different areas within one tumor when the DNA was cleaved by HaeIII and signals detected with the (GTG)5 probe. The "area-specific" pattern was stable during several subcultivations of these cell lines, indicating genetic stability of these prostatic carcinoma cells in vitro. Thus individual cell lines derived from radical prostatectomy seem to represent a biological system very close to the situation in vivo.


Evaluation of isoelectric focusing runs as specific resistance/volthour plots. Isoelectric focusing (IEF) runs, e.g. on ultrathin gels, are characterized by an extensive change of gel electric parameters, caused by the rearrangement of carrier ampholyte components from a uniform distribution to a highly ordered pH gradient. A particularly important parameter is the specific resistance rho [Ohm*cm] which has been determined in polymerization mixtures (with and without carrier ampholytes) and in 125 x 0.15 mm ultrathin gels, pH 3-10 with 5% T, 3% C, 5% Servalyt carrier ampholytes, pH 3-10. The starting specific resistance rho of ultra-thin IEF gels, calculated from the geometric gel dimensions and electric current values (V, mA), is in agreement with the data determined directly in 30 mL samples of polymerization mixtures by using a conductivity meter. Electric specific conductivity/Volthour (Vh) plots proved to be a valuable tool for the evaluation of gel systems with and without protein samples during IEP runs. These plots are usually S-shaped, indicating that the key part of pH gradient formation takes places in a relatively short time. A "good" ultrahin gel, after a short lag phase, shows a rapid increase in specific resistance due to a rapid pH gradient formation and a slope of about 18 Ohm*cm/Vh. IEF is finished in about 3000 Vh. After prolonged gel storage, and especially in partially dried gels, the electrical parameters approach equilibrium only slowly, as indicated by the relatively shallow slope (8.9 Ohm*cm/Vh). Accordingly, separations need more than 4000 Vh.(ABSTRACT TRUNCATED AT 250 WORDS)


Analysis of members of the Ig-gene superfamily by thermal gradient polyacrylamide gel electrophoresis. A solid aluminum block, connected with a warm and cold thermostated waterbath, provided for a linear transversal temperature gradient (TG) during polyacrylamide gel electrophoresis (PAGE). Noncovalently bound heavy chain dimers as well as heavy-light chain dimers, derived from human monoclonal IgG, could be melted into monomers using a 40-75 degrees C TG under conditions of sodium dodecyl sulfate-PAGE. Using native PAGE, major histocompatibility complex (MHC) class I molecules, preloaded with the iodinated peptide FAPGNYPAL could be melted in a 4-40 degrees C TG to release the peptide. The method is in general applicable to thermal stability analysis of noncovalently bound hetero-oligomers if the product after melting possess different electrophoretic mobilities.


Mass spectrometric analysis of blotted proteins after gel electrophoretic separation by matrix-assisted laser desorption/ionization. The molecular masses of electroblotted proteins were determined with a time-of-flight mass spectrometer by matrix-assisted laser desorption/ionization directly from blot membranes. Therefore standard proteins, separated by polyacrylamide gel electrophoresis, were electroblotted onto polyvinylidene difluoride or polyamide membranes by standard procedures. Pieces of membrane containing the protein of interest were soaked in matrix solution and analyzed in the mass spectrometer.


Thin-layer electrophoresis with PhastSystem facilitates analysis of phosphoamino acids from proteins bound to Immobilon. A modification of thin-layer electrophoresis of phosphoamino acids on miniplates, using the PhastSystem (Pharmacia) is described. This method not only reduces the amount of radioactivity required but also the radioactive waste. In addition, it also decreases the handling of hazardous substances and takes only a fraction of the time required for the standard procedure.


Purification to single isoforms of a secreted epidermal growth factor receptor in a multicompartment electrolyzer with isoelectric membranes. A purified, size-homogeneous (100 kDa), desialylated form of a truncated, soluble form of the epidermal growth factor receptor secreted by A431 human tumor cells has been found, by isoelectric focusing in immobilized pH gradients, to consist of two major isoforms (with pIs of 6.96 and 6.71), one intermediate form (pI 6.45) and a number (> 10) of minor components. The two major components have been purified to charge homogeneity by isoelectric focusing in a multicompartment electrolyzer with buffering isoelectric membranes having the following pI values: 5.90, 6.63, 6.76, 6.92, 7.05 and 7.35. Such single pI species are presently used for attempts at crystal growing.


Preparative continuous separation of biological particles by means of free-flow magnetophoresis in a free-flow electrophoresis chamber. For sorting, cells or cellular components can specifically be labeled by antibody-coated magnetic beads. We have developed a device for continuous magnetic sorting based on the flow-chamber of a free-flow electrophoresis system. Magnetically labeled particles are injected into a given continuously flowing chamber buffer and pass an inhomogeneous magnetic field, configurated perpendicular to the flow direction. According to its magnetic moment, the magnetic material is deviated into the direction of the magnetic forces, while nonmagnetic material passes the field without interaction. The magnetic forces can be changed with the electrical current of the solenoids producing the magnetic field. As in the free-flow electrophoresis system, the particle fractions are collected in different vials. On-line control of the experiments can be performed by an optical scanning system. Experiments with model particles achieved a sorting purity of more than 99% at a rate of up to 5 X 10(8) particles per hour. In experiments with blood cells, a high enrichment of either B-or-T-lymphocytes was obtained. In contrast to free-flow electrophoresis, there is no limitation, in principle, regarding the type of chamber buffer to be used. This allows an optimal adaptation of the buffer conditions to the requirements of vital sorting. The preliminary results so far confirm this conclusion.


Identification of peaks in capillary zone electrophoresis based on actual mobilities. A procedure is proposed for the calculation of the actual effective mobility of a zone from its migration time. It is based on the use of internal standards with known mobilities; the use of two internal standards provides reliable mobility data even if the magnitudes of the effects of sample composition, capillary temperature, capillary length, migration distance, used voltage, as well as the tube length occupied by the injected sample are unknown. Formulas have been derived for the calculation of the actual mobilities, and their experimental verification has been carried out by using a model set of anionic solutes with mobilities ranging from -56 to -20 x 10(-9) m2V-1s-1 and chloride as the ion modelling the effect of the sample matrix.


Application of capillary electrophoresis in peptide research. Comparison of high performance liquid chromatograms (HPLC) with capillary electrophoresis shows the latter to be superior in many cases owing to rapid separation, high resolution and high sensitivity. This is demonstrated with two examples (i) the isolation of natural peptides from bovine tissue and (ii) characterization of a synthetic peptide mixture with the natural sequence (fragment) of the human immunodeficiency virus (HIV) transmembrane glycoprotein gp 41 env.


Enantiomers resolution in capillary zone electrophoresis by using cyclodextrins. Cyclodextrins added to the background electrolyte are shown to be useful for the resolution of racemic compounds in their enantiomers. Several parameters have to be controlled in order to achieve resolution, e.g., cyclodextrin type, concentration, analyte shape, as well as column temperature. The resolution of nor-epinephrine, epinephrine and isoproterenol in their enantiomers decreased by increasing the column temperature. Octopamine and ketamine have been resolved by supporting the background electrolyte with 2, 6-di-O-methyl-beta-cyclodextrin. In spite of the stronger inclusion-complex of ketamine than octopamine with the modified cyclodextrin its resolution was not satisfactory.


Capillary electrophoresis of macromolecules in 'syrupy' solutions: facts and misfacts. 'Syrupy' solutions of liquid linear polyacrylamide (> or = 10%T, 0%C) appear to be excellent for fractionation of oligonucleotides and, potentially, for DNA sequencing. For such analyses, the silica wall must be coated by covalently bound strings of polyacrylamide; otherwise, the electroosmotic flow will slowly pump out the viscous electrolyte solution. Due to the enormous viscosity (100 Pa s for an 8% T solution) the polymer strings must be prepared in situ, by filling the capillary with the appropriate monomer solution. The reaction, however, cannot be driven to better than 80-85% conversion: in 10%T, the concentration of unreacted monomers will thus be 200-300 mM. This will give a substantial background absorbance (even at 254 nm) and leave a huge amount of potentially harmful reacting species in the background electrolyte. A chemical scavenging method is proposed here: after polymerization, a 100 mM solution of cysteine is driven in from the cathode and allowed to react for up to 10 h. At the end of the reaction period, the excess cysteine and its acrylamido adduct are driven out electrophoretically and the column is reconstituted with its normal background electrolyte. Columns thus preconditioned have been found to perform extremely well and to last as long as the inner coating (and the linear polymer filling) will last. No 'carry over' from run to run was experienced.


Atrazine and simazine determination in river water samples by micellar electrokinetic capillary chromatography. A rapid and sensitive micellar electrokinetic capillary chromatography analytical method was used for the determination of chlorotriazine herbicides in river water samples. Several electrolyte systems in the pH range 7-10 were tested in order to optimize the separation. The two compounds were separated in less than 10 min and the determination limit was about 0.4 ppb for each herbicide. Recovery values of the method were in the range 80%-117%.


Applications of an automated apparatus for two-dimensional electrophoresis, Model TEP-1, for microsequence analyses of proteins. Two-dimensional electrophoresis has become a rapid and powerful separation method for the preparation of samples for sequence analysis depending on the development of automated microsequencers accompanied by a blotting technique. We have constructed an automated apparatus for two-dimensional electrophoresis, Model TEP-1, which allows precise analytical runs under computer-control with a fully automated transfer from the first- to the second-dimensional gel. In the present paper we describe several applications of TEP-1 followed by blotting and sequencing to reveal the primary structure of proteins in a complex mixture. Several known and unknown proteins were analyzed. Enzymatic digests of proteins could be directly applied onto the TEP-1, in which a gel of lower molecular weight proteins was used as the second dimension. Subsequent blotting and sequencing provided internal sequence information. The use of TEP-1, followed by blotting, provides spots containing ca. 10-100 pmol protein. These figures permitted the identification of 10-40 amino acid residues from the N-terminus. The TEP-1 provides a simple and rapid separation of complex mixtures obtained from natural sources and chemical, biological or genetic products.


Immune complexes in blood: a new strategy for the analysis of their antigen portion. A method is described for the characterization of protein antigens from circulating immune complexes from plasma. Free immunoglobulins G were separated from larger immune complexes by gel filtration with a fast protein liquid chromatographic system. The collected immune complexes were dissociated with 4M urea into antigens and antibodies. With a second column run with 4M urea, antigens smaller than 120 kDa were separated from unloaded antibody fractions. After concentration, they were analyzed by two-dimensional gel electrophoresis.


Plasma protein map: an update by microsequencing. The reference plasma protein map, obtained with immobilized pH gradients in the first dimension of two-dimensional electrophoresis, is presented. By microsequencing, more than 40 polypeptide chains were identified. The new polypeptides and previously known proteins are listed in a table and labeled on the protein map, thus providing an update of the human plasma two-dimensional gel database.


Protein synthesis in murine organs during postimplantation development detected by two-dimensional gel electrophoresis. Mouse embryos were isolated from the uterus on days 10 to 11 of gestation and incubated in Dulbecco's modified Eagle's medium (DMEM) with [35S]methionine for 4 h. Subsequently, their hearts and the brains were dissected. The brain was divided into three parts, containing the telencephalon, mesencephalon, and myelencephalon. These tissues were then processed for two-dimensional (2-D) gel electrophoresis. Protein synthesis of the isolated tissues was analyzed for organ-and cell lineage-specific patterns. We studied proteins with isoelectric points (pI) ranging from 4 to 10 and relative molecular weights (M(r)) varying from 10000 to 200000 and found several significant quantitative and qualitative differences between the tissues and the developmental stages analyzed. In particular, we were able to distinguish between protein spots that we now attribute putatively to the corresponding embryonic organs. These differences may reflect some of the organ- and cell lineage-specific changes in protein synthesis and gene expression during early mammalian differentiation.


Examination of microheterogeneity in grass pollen allergens. The separation of timothy pollen extract by two-dimensional immunoblotting revealed microheterogeneity of the major allergens PhI p I and PhI p V. There was not only a diversity in size, 38 and 32 kDa for PhI p V and 37, 35, and 33 kDa for PhI p I, but also a separation into proteins of identical sizes but different pIs. Since former studies on the protein structure by amino acid analysis and N-terminal microsequencing did not reveal any differences, we examined other possibilities that might cause microheterogeneity. In allergens belonging to the PhI p I group, the variability in pI can be due to the carbohydrate structure and to the fact that charges are hidden in the interior of the protein, as shown by varying concentrations of urea. On the other hand we were not able to detect any such reasons for the existence of PhI p V isoallergens. Thus, we assume that they are stable conformational isomers of the proteins (allomorphism) or that there are only slight variations in the internal sequences of these proteins (polymorphism) causing distinct pIs.


A human myocardial two-dimensional electrophoresis database: protein characterisation by microsequencing and immunoblotting. This communication briefly describes how a human heart two-dimensional electrophoresis (2-DE) protein database is being established in our laboratory. The database contains more than 1500 polypeptides and approximately fifty proteins from 2-DE gels of human myocardial tissue have been characterised. Information about the proteins has been compiled including molecular weight (M(r)), isoelectric point (pI), sample spot (SSP) number, protein name, partial sequence, and antibody reacting with the protein. The first stage of this project involves the investigation of protein with pIs in the range pH 4-7. Future studies will employ immobilised pH gradient (IPG) gels as the first dimension of the 2-DE to examine basic proteins. The ultimate goal of this project is to establish a global picture of human heart protein expression in both normal and disease conditions.


Electrophoretic analysis of electrically trained skeletal muscle. Sheep latissimus dorsi muscle was electrically trained, thereby inducing fast-to-slow fibre-type transformation. Using a combination of one- and two-dimensional gel electrophoresis techniques with computer analysis, we have analysed altered expression of contractile protein isoforms at protein and mRNA level over a time course of electrical training extending to 5 months. Myosin heavy chain and regulatory myosin light chain analysis showed predominant expression of their slow isoforms (86% and 92%, respectively) after 3 months of training. At the same time point, however, tropomyosin analysis revealed that the slow isoform of the alpha-subunit accounted for 64% of the total alpha expression. Troponin T isoform switching proceeded more slowly over the same time course than tropomyosin and the thick filament proteins studied. Troponin T analysis revealed 5 fast and 2 slow isoforms in the sheep, of which the second slow isoform only became clearly visible after 5 months' training. At this time point the two slow isoforms were more predominant than their fast counterparts. This suggests that a wide heterogeneity of fast and slow isoform combinations are possible in the thin filament of skeletal muscle.


Effects of bile salt exposure on pancreatic duct barrier function and protein release determined by two-dimensional electrophoresis and lectin-affinoblotting. We analyzed effects of a bile salt on pancreatic duct barrier function and protein release into pancreatic juice in cats, using two-dimensional-electrophoresis and lectin-affinoblotting. Preanalyzed pancreatic juice was perfused along the pancreatic duct following duct exposure to a bile salt. In this case, pancreatic duct mucosa showed a partial loss of barrier function to the leakage of exocrine pancreatic proteins dependent upon molecular weight and charge. The release of a sialic acid possessing protein into pancreatic juice was observed.


Towards a two-dimensional database of common human proteins. A protein pattern of common human proteins was constructed by comparing the two-dimensional gel electrophoresis (2-DE) protein patterns from five cell lines of different germ layers. Total cell lysate and the isolated and purified nuclei of each cell line were separated by parallel electrophoresis runs in a multiple casting system of highest reproducibility. The computerized image analysis of the digitized 2-DE gels revealed a master protein pattern for each cell line. By comparison of all master protein patterns a 2-DE protein map of only common human proteins was constructed as a basis for a new 2-DE database. In a first step we have started characterizing a number of spots by microsequencing, amino acid composition analysis, and mass spectroscopy.


Using two-dimensional gel electrophoresis to study immune response against intracellular bacterial infection. Principal component analysis was applied to two-dimensional (2-D) gel electrophoresis patterns, obtained in various phases of infection. Untreated controls could be satisfactorily differentiated from patterns after infection on days 3 and 7 whereas day 10 of infection was grouped with the controls. Comparison of host cellular protein patterns could help to classify in vivo developing infection without requiring any so-called immune marker functions. Immunoaffinity separation of infected cells treated with detergent, followed by 2-D electrophoresis of negative as well as positive eluates, did not reveal radiolabeled bacterial protein antigens.


Application of high resolution two-dimensional polyacrylamide gel electrophoresis of polypeptides from cultured neonatal rat cardiomyocytes: regulation of protein synthesis by catecholamines. High resolution two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) was applied to cultured neonatal rat heart muscle cells, incubated for 72 h at 37 degrees C in serum-free medium, either in the absence or in the presence of 0.1 microM norepinephrine. After silver staining, about 340 and 550 protein spots could be seen in cardiomyocytes, cultured either in the absence or presence of norepinephrine. Of these spots, 141 could be further characterized according to isoelectric point and molecular weight, with 71 protein spots being present under both conditions. In cells cultivated in presence of norepinephrine, 58 new protein spots appeared, whereas 12 spots disappeared, and 22 spots increased (whereas 3 spots decreased) in intensity. In comparison with 2-D PAGE of rat cardiomyocytes, the protein pattern of the intact heart of neonatal rats is incongruent. 2-D PAGE of polypeptides of cultured neonatal rat cardiomyocytes may be a suitable tool to study the regulation of protein synthesis by various stimuli with relevance to cardiac growth adaptation, inotropy and heart failure.


Regulation of protein biosynthesis in neonatal rat cardiomyocytes by adrenoceptor-stimulation: investigations with high resolution two-dimensional polyacrylamide gel electrophoresis. The role of alpha- and beta-adrenoceptor-mediated regulation of protein patterns in cultured neonatal rat cardiomyocytes was studied with high resolution two-dimensional polyacrylamide gel electrophoresis. Spontaneously beating neonatal rat cardiomyocytes were cultured for 72 h at 37 degrees C in serum-free medium, supplemented with either the catecholamine norepinephrine (0.1 microM), or with norepinephrine (0.1 microM) plus the alpha 1-adrenoceptor blocker prazosin (1 microM), or with neither of these substances. In transmission light, 105 protein spots could be seen in the lysates from control cells, 244 spots were identified in lysates from norepinephrine-treated cells, and 114 protein species were counted in the case of lysates from cardiomyocytes which had been cultured in the presence of norepinephrine plus prazosin. This experimental approach allows a clear classification of alpha- and non-alpha (probably beta)-adrenoceptor-mediated catecholamine effects on protein synthesis in cardiomyocytes. In comparison with previous experiments (Chang et al., Electrophoresis, this issue, pp. 748-754), less protein species were identified in the untreated control cardiomyocytes, as well as in the norepinephrine treated cells. The only actual modification in the experimental setup in these two series was the addition of dimethyl sulfoxide--a substance known for its repressive effect on oncoprotein expression--to the culture medium, as solvent for prazosin.


Identification of human myocard proteins separated by two-dimensional electrophoresis. N-Terminal sequencing, internal sequencing and amino acid analysis were used to identify twelve proteins of the human myocard two-dimensional gel electrophoresis (2-DE) pattern. Amino acid analysis was shown to be a powerful tool in addition to sequencing. The identification of a disease-associated N-terminally blocked protein by internal sequencing was not successful. The twelve identified proteins are the basis of a human myocard 2-DE database.


Genetic variability of pepper (Capsicum annuum L.) seed proteins studied by 2-D electrophoresis with immobilized pH gradients. Ten pepper (Capsicum annuum L.) inbred lines were successfully differentiated by two-dimensional electrophoresis with immobilized pH gradients. Qualitative polymorphism of water-soluble and urea/detergent-soluble seed proteins, respectively, was investigated by computer analysis and used for establishing a dendrogram derived from maximum-parsimony analysis. The dendrogram calculated from urea/detergent-soluble proteins shows four types of distance indices, whereas water-soluble proteins show two sets of inbred lines with similar intraset distance indices. The validity of the dendrograms with respect to quantitative inherited traits, such as cold tolerance and earliness, will be tested by field trials.


Isoelectric focusing and immunoblotting of the platelet membrane glycoprotein complex IIb. IIIA following urea solubilization. Effective solubilization of the major platelet membrane component, the glycoprotein IIb.IIIa complex, can be achieved with 8 M urea. By avoiding nonionic detergents in the separation medium it is possible to obtain clear immunoblot patterns without interference from the isoelectric focusing matrix. Upon running on a pH 4.25-5.25 immobilized pH gradient, immunoreactive bands corresponding to the nonreduced IIb.IIIa complex stain between pH 4.5 and 5.0. The method appears of significant potential utility in evaluating glycoprotein IIb.IIIa polymorphisms under different clinical conditions.


Qualitative and quantitative changes in barley seed protein patterns during the malting process analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with respect to malting quality. Seeds of two barley cultivars, similar in total protein content and malt extract yield but different in their final attenuation values, were malted. Samples taken at daily intervals during the malting process were extracted sequentially with Tris-HCl buffer, aqueous 2-propanol, aqueous 2-propanol containing 0.5% dithiothreitol, and 4 M urea, containing 0.5% dithiothreitol and 1% Nonidet P-40. The protein composition of these extracts was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and computer densitometry to determine whether differences observed in the rate or extent of protein modification are related to the malting quality character final attenuation. It was found that, common to both cultivars, the albumin and globulin proteins were relatively resistant to proteolysis, whereas the hordeins suffered a dramatic breakdown during malting, with the D hordein being degraded most rapidly, followed by the B and C hordeins. Besides these similarities, differences between both cultivars were observed in the relative rates of D hordein degradation, as this rate was considerably higher in the cultivar with high malting quality. Similar, but much less distinct kinetics were seen with certain B hordeins. Since a possible relationship might exist between the rate of proteolysis of the D hordeins and the character final attenuation, we analyzed a larger number of barley cultivars with different final attenuation values with a simplified technique. For the ten cultivars examined, differences during germination were again seen in the rates of modification of the D hordeins. However, significant correlations between the D hordein breakdown and final attenuation values were not obtained, so that we propose that there exists at best a loose correlation between the relative rate of proteolysis of these proteins and the malting quality character final attenuation.


Improved phenotyping of alpha 1-antichymotrypsin (ACT) by isoelectric focusing and immunoprinting: first demonstration of a deficient protein variant in the ACT system. Genetic variation of human alpha 1-antichymotrypsin (ACT) was investigated in sera using thin-layer polyacrylamide gel isoelectric focusing (pH range 4.0-6.5) followed by immunoprinting with a monospecific anti-human ACT antibody. Sialidase-treated samples showed a microheterogeneous banding pattern which consisted of two major and several additional minor components with isoelectric points between pH 5.0 and 5.3. A population study of 200 unrelated individuals from southern Germany revealed no genetic variation. In a clinical investigation, however, we found a unique banding pattern in a female patient suffering from chronic obstructive pulmonary disease. In comparison with the monomorphic normal type the detected variant phenotype shows two additional bands that have lower intensities and are located cathodically to their major bands. Inheritance of the deficient IEF variant "ACT Bochum" was confirmed by a family study. To our knowledge this is the first genetic ACT mutant to be observed at the protein level.


Genetic study of orosomucoid by isoelectric focusing and immunoprinting in patients with carcinoma. The carbohydrate moiety of the orosomucoid (ORM) molecule shows microheterogeneity [1] and the pteridine-containing variant seems to be tumor-specific [2-4]. However, there also exists a genetic (protein-related) polymorphism coded by the ORM1 and ORM2 loci on chromosome 9 [5, 6]. To investigate the relationship between ORM1 gene products and the development of carcinoma, we analyzed the ORM1 phenotypes of desialated sera from 125 patients with carcinoma. The allele frequencies were estimated for ORM1*F1 0.556, ORM1*F2 0.012 and ORM1*S 0.432. In comparison to healthy individuals from the same geographical area [6] the ORM1 S phenotypes are significantly more frequent in carcinoma patients. The patients' sera frequently showed additional ORM-positive proteins which focused slightly cathodically to the ORM 2 A band. These proteins may represent posttranslational modifications of the ORM1*S allele product. Whether these modifications are tumor-specific and related to the carbohydrate moiety of the molecule must be confirmed in further studies.


An outbreak of gentamicin-resistant Klebsiella bacteraemia at a children's hospital. An outbreak of Klebsiella bacteraemia at the Ethio-Swedish Childrens' Hospital, Addis Abeba, Ethiopia is described. The epidemic occurred between February 1988 and February 1990 and appeared to originate in the neonatal nursery and subsequently spread to involve other wards in the hospital. Resistance to several antibiotics, including gentamicin, was common. The importance of effective surveillance and the early institution of control measures for the prevention of nosocomial infection are discussed.


Relapsing fever in children--demographic, social and clinical features. Louse-borne relapsing fever (LBRF) is an acute febrile illness endemic Ethiopia. To date reports of childhood LBRF are few. The demographic, social and clinical features of eighty children with LBRF admitted to Ethio-Swedish Children's Hospital, Addis Abeba between 1989 and 1991 is presented. The mean age of patients was 8.8 years (range 4 months to 15 years). The male to female ratio was 1.2:1. Seventy-seven (97%) patients came from Addis Abeba. They came from poor families living in overcrowded homes. Fever, headache, right upper quadrant pain, chills and rigors were common symptoms. Fever and hepatosplenomegaly were common signs. Three drug regimens were used in the treatment of patients. A combination of penicillin and tetracycline, chloramphenicol alone and erythromycin alone, all given for 3 days. There was only one death. The literature on LBRF in adults is reviewed and the results are compared (1).


Stress effects on hormonal growth factors in tobacco tissues indicated by special changes in the isoelectric peroxidase patterns. The isoelectric peroxidase patterns of tobacco tissue cultures allow us to draw inferences to cell elongation and cell division because certain zones in acid pH ranges respond to the influence of auxines and gibberellins (promoting cell elongation) and others respond to the influence of cytokinins (promoting cell division). Stress, due to the absence of phosphate and presence of lead in the medium, causes characteristic changes in the intensity of these sensitive zones in peroxidase patterns. It may be deduced that the increase and decrease of these zones correspond to stimulation and inhibition of cell elongation and cell division, respectively. Cell elongation remains almost unaltered by lack of phosphate but is markedly inhibited by lead, while cell division is enhanced. However, stress brings about a reduction of dry weight. Reactions to stress can be observed earlier in patterns of peroxidase than in growth.


Discontinuous electrophoresis of beta-caseins for the determination of bovine caseins in milk and dairy products. Discontinuous acidic anodic polyacrylamide gel electrophoresis enables the separation of bovine beta-caseins from those of ovine and caprine. Interfering protein bands as a consequence of ripening or processing have not been detected. After evaluation of the stained gel by laser densitometry, quantification was performed with calibration standards on the same gel by the ratio of the peak areas from bovine to ovine and bovine to caprine, respectively. Thus, independence from the extractability of proteins affected by denaturation and ripening (which might in some cases raise the limit of detection) is achieved. The range of quantification extends from 5 to approximately 70% bovine casein in relation to total casein.


National tuberculin test survey in Ethiopia. To plan and implement an effective National Tuberculosis Control Programme, it is essential to know the incidence of tuberculosis. Thus a tuberculin survey was conducted in Ethiopia between December 1987 and April 1990. A sample of 55 children, aged 6 to 10 years was selected from each of 480 representative clusters from 16 weredas (districts), which were chosen randomly. A total of 26,529 children were given intradermal tuberculin test and the results read in 26,269. There were 23,595 children who had no BCG vaccination of whom 2,503 (10.6%) were positive; this gives a "risk of infection" of 1.4% which is lower than the 3.0% reported for the same age group during the tuberculin survey in 1953-1955, suggesting that there has been a reduction in the tuberculin test positivity rate.


Fish species identification by peptide mapping of the myosin heavy chain. Fish species of seafood made of washed fish flesh (e.g. imitation crab meat from surimi) were identified by peptide mapping of the myosin heavy chain (MHC). In the first step the MHC was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in 7.5% T gel, followed by a second SDS-PAGE in a 15% T gel with proteolytic digestion of the MHC in the gel. The resulting peptides gave species specific patterns.


Bronchial asthma in Jima: a prospective analysis of 204 patients. Two hundred and four (204) consecutive adult asthmatic patients who visited the outpatient clinic of Jima Institute of Medical Sciences from May 1 to August 30, 1989 were prospectively studied. Data on age, sex, ethnic distribution and the associated etiological factors were documented. The age range was from 17-75 (mean 33.4) years and the male to female ratio was 1.4:1. The majority (80%) were not members of the ethnic group indigenous to the area. Of the total, 175 (85%) were not natives of Jima town and its surroundings. One hundred and fifty (85.7%) of the latter developed the disease in Jima. Ninety-six (64%) of the 150 patients contracted the disease in less than 5 years of their stay in the area. Infections, emotional stress, dust, and weather changes were the main precipitating factors. The majority of the attacks occurred from June to October. Allergic rhinitis was present in 88% of the patients and atopic dermatitis in 3%. Forty-one (20%) patients had a family history of asthma. Forty-eight percent of the patients claimed that change of climate relieved their symptoms. Eosinophilia was documented in 51.5% and parasitic infestations were observed in 39% of patients. The study shows that a variety of environmental factors such as pollen dust, fungus and climatic changes play a major role and may account for the high performance of asthma in Jima town. It is recommended that further studies be conducted to find out the causative factors so that appropriate preventive measures can be implemented.


Wheat gliadin: digital imaging and database construction using a 4-band reference system of agarose isoelectric focusing patterns. An isoelectric focusing method using thin-layer agarose gel has been developed for wheat gliadin. Using flat-bed units with a third electrode, up to 72 samples per gel may be analyzed. Advantages over traditional acid polyacrylamide gel electrophoresis methodology include: faster run times, nontoxic media, and greater sample capacity. The method is suitable for fingerprinting or purity testing of wheat varieties. Using digital images captured by a flat-bed scanner, a 4-band reference system using isoelectric points was devised. Software enables separated bands to be assigned pI values based upon reference tracks. Precision of assigned isoelectric points is shown to be on the order of 0.02 pH units. Captured images may be stored in a computer database and compared to unknown patterns to enable an identification. Parameters for a match with a stored pattern may be adjusted for pI interval required for a match, and number of best matches.


Chylous mesenteric and retroperitoneal cysts of developmental origin amongst Ethiopians: report of four cases. Chylous mesenteric and retroperitoneal cysts of developmental origin are unusual and rarely diagnosed with accuracy before laparotomy. They may produce compression of viscera and may rupture with haemorrhage. This is the first report of four cases, with varied presentations, from an African country.


Characterization of endopolygalacturonase (EC 3.2.1.15) from Aspergillus niger as glycoprotein by electrophoretic methods and lectin affino-blotting. Chromatographically purified endopolygalacturonase (PG) from Aspergillus niger was deglycosylated with N-glycosidase F (PNGase F) and characterized by means of sodium dodecyl sulfate (SDS)-electrophoresis, polyacrylamide gel electrophoresis (PAGE) without denaturing agents, isoelectric focusing (IEF) and lectin affino-blotting. The results show that PG, which is apparently homogeneous in SDS-PAGE but heterogeneous in IEF and PAGE, consists of at least two polypeptide chains with different glycosylation patterns. The component with the higher electrophoretic mobility is deglycosylated with PNGase F and reacts with concanavalin A (Con A) and Galanthus nivalis agglutinin (GNA), indicating a "high mannose" or "hybrid"-type of glycoprotein (GP). The other component may contain O-glycosidically linked mannose, N-acetylglucosamine or glucose.


The effect of driving force on intramolecular electron transfer in proteins. Studies on single-site mutated azurins. An intramolecular electron-transfer process has previously been shown to take place between the Cys3--Cys26 radical-ion (RSSR-) produced pulse radiolytically and the Cu(II) ion in the blue single-copper protein, azurin [Farver, O. & Pecht, I. (1989) Proc. Natl Acad. Sci. USA 86, 6868-6972]. To further investigate the nature of this long-range electron transfer (LRET) proceeding within the protein matrix, we have now investigated it in two azurins where amino acids have been substituted by single-site mutation of the wild-type Pseudomonas aeruginosa azurin. In one mutated protein, a methionine residue (Met44) that is proximal to the copper coordination sphere has been replaced by a positively charged lysyl residue ([M44K]azurin), while in the second mutant, another residue neighbouring the Cu-coordination site (His35) has been replaced by a glutamine ([H35Q]azurin). Though both these substitutions are not in the microenvironment separating the electron donor and acceptor, they were expected to affect the LRET rate because of their effect on the redox potential of the copper site and thus on the driving force of the reaction, as well as on the reorganization energies of the copper site. The rate of intramolecular electron transfer from RSSR- to Cu(II) in the wild-type P. aeruginosa azurin (delta G degrees = -68.9 kJ/mol) has previously been determined to be 44 +/- 7 s-1 at 298 K, pH 7.0. The [M44K]azurin mutant (delta G degrees = -75.3 kJ/mol) was now found to react considerably faster (k = 134 +/- 12 s-1 at 298 K, pH 7.0) while the [H35Q]azurin mutant (delta G degrees = -65.4 kJ/mol) exhibits, within experimental error, the same specific rate (k = 52 +/- 11 s-1, 298 K, pH 7.0) as that of the wild-type azurin. From the temperature dependence of these LRET rates the following activation parameters were calculated: delta H++ = 37.9 +/- 1.3 kJ/mol and 47.2 +/- 0.7 kJ/mol and delta S++ = -86.5 +/- 5.8 J/mol.K and -46.4 +/- 4.4 J/mol.K for [H35Q]azurin and [M44K]azurin, respectively. Using the Marcus relation for intramolecular electron transfer and the above parameters we have determined the reorganization energy, lambda and electronic coupling factor, beta. The calculated values fit very well with a through-bond LRET mechanism.


Characterization of peptides related to neuropeptide tyrosine and peptide tyrosine-tyrosine from the brain and gastrointestinal tract of teleost fish. Neuropeptide Y was isolated from the brain of the Atlantic cod, Gadus morhua and its primary structure established as Tyr-Pro-Ile*-Lys-Pro-Glu*-Asn-Pro-Gly-Glu10-Asp-Ala-Pro-Ala-Asp*-G lu*-Leu*-Ala- Lys*-Tyr20-Tyr-Ser-Ala-Leu-Arg-His-Tyr-Ile-Asn-Leu30-Ile-Thr -Arg-Gln-Arg-Tyr- CONH2. Residues denoted by an asterisk are different from the corresponding sequence of human neuropeptide Y. A structurally similar peptide was isolated from the brain of the trout, Oncorhynchus mykiss. Trout neuropeptide Y contains four substitutions (Ile3-->Val, Ala14-->Thr, Asp15-->Glu and Ser22-->Thr) compared with cod neuropeptide Y. A second peptide of the neuropeptide Y family was identified in the trout brain and this component was structurally similar to peptide tyrosine-tyrosine previously isolated from frog intestine (six amino acid substitutions) and identical to a peptide isolated from the pancreas of the closely related species, Oncorhynchus kisutch (Coho salmon). Peptide tyrosine-tyrosine, with the same primary structure as the brain peptide, was also isolated from an extract of the trout stomach. The data indicate that a peptide analogous to mammalian neuropeptide Y is present in the brain of teleost fish and a peptide analogous to mammalian peptide tyrosine-tyrosine is present in brain, gastrointestinal tissue and pancreas. We speculate, therefore, that the putative gene duplication that led to pancreatic polypeptide in the higher vertebrates took place after the time of divergence of fish and tetrapods.


Substitution of Arg214 at the substrate-binding site of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens. The gene encoding p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens was cloned in Escherichia coli to provide DNA for mutagenesis studies on the protein product. A plasmid containing a 1.65-kbp insert of P. fluorescens chromosomal DNA was obtained and its nucleotide sequence determined. The DNA-derived amino acid sequence agrees completely with the chemically determined amino acid sequence of the isolated protein. The enzyme is strongly expressed under influence of the vector-encoded lac promotor and is purified to homogeneity in a simple three-step procedure. The relation between substrate binding, the effector role of substrate and hydroxylation efficiency was studied by use of site-directed mutagenesis. Arg214, in ion-pair interaction with the carboxy moiety of p-hydroxybenzoate, was replaced with Lys, Gln and Ala, respectively. The affinity of the free enzymes for NADPH is unchanged, whereas the affinity for the aromatic substrate is strongly decreased. For enzymes Arg214-->Ala and Arg214-->Gln, the effector role of substrate is lost. For enzyme Arg214-->Lys, binding of p-hydroxybenzoate highly stimulates the rate of flavin reduction. In the presence of substrate or substrate analogues, the reduced enzyme Arg214-->Lys fails to stabilize the 4 alpha-hydroperoxyflavin intermediate, essential for efficient hydroxylation. Like the wild-type, enzyme Arg214-->Lys is susceptible to substrate inhibition. From spectral and kinetic results it is suggested that secondary binding of the substrate occurs at the re side of the flavin, where the nicotinamide moiety of NADPH is supposed to bind.


Purification, properties and primary structure of thioredoxin from Aspergillus nidulans. This paper reports the purification and the properties of a thioredoxin from the fungus Aspergillus nidulans. This thioredoxin is an acidic protein which exhibits an unusual fluorescence emission spectrum, characterized by a high contribution of tyrosine residues. Thioredoxin from A. nidulans cannot serve as a substrate for Escherichia coli thioredoxin reductase. Corn NADP-malate dehydrogenase is activated by this thioredoxin in the presence of dithiothreitol, while fructose-1,6-bisphosphatase is not. The amino acid sequence of Aspergillus thioredoxin was determined by automated Edman degradation after cleavage with trypsin, SV8 protease, chymotrypsin and cyanogen bromide. The masses of tryptic peptides were verified by plasma-desorption mass spectrometry. The mass of the protein was determined by electrospray mass spectrometry and shown to be in agreement with the calculated mass derived from the sequence (M(r) = 11,564). Compared to thioredoxins from other sources, the protein from A. nidulans displays a maximal sequence similarity with that from yeast (45%).


Subtilisin removes the surface layer of the phage fd coat. The major coat protein of native filamentous phage fd is vulnerable to digestion by subtilisin, but not by any of a number of other proteolytic enzymes. Degradation by the non-specific protease subtilisin occurs at specific sites in the N-terminal portion of g8p. The N-terminal part of the protein is considered to be the outer layer of a two-layered coat. Thus, subtilisin treatment results in a monolayered phage particle. These particles possess the morphology and stability of native phage fd. Furthermore, subtilisin proteolysis proved to be an efficient instrument in detecting variations in the topology of the g8p of related filamentous phages.


Analysis of the isoleucyl-tRNA synthetase reaction by total rate equations. Magnesium and spermidine in the tRNA kinetics. Derivation of a steady-state rate equation for the aminoacyl-tRNA synthetases is described, and its suitability for the analysis of various details of the reaction is tested. The equation is applied to the magnesium and spermidine dependences of the isoleucyl-tRNA synthetase reaction. Earlier work [Airas, R.K. (1990) Eur. J. Biochem. 192, 401-409] is expanded by experiments and calculations of the tRNA kinetics. The analysis suggests the following new details in addition to the earlier results: (a) The binding of tRNA to the enzyme (and not only the rate of the aminoacylation reaction) is affected by the presence of the Mg2+ and spermidine in the tRNA molecule. At least two bound Mg2+ or spermidines are required. (b) tRNA and PPi partly inhibit the binding of each other to the enzyme. (c) The transfer reaction is rather slow, and, at least under some conditions, it participates in rate limitation. (d) A Mg(2+)-induced reduction in the aminoacylation rate seems to be directed to the dissociation of the aminoacyl-tRNA from the enzyme. This dissociation rate is enhanced if a Mg2+ is first dissociated from the enzyme or tRNA. An increase in the Mg2+ concentration shifts the rate limitation from the transfer reaction towards dissociation of the product.


Effect of inorganic pyrophosphate on the pretransfer proofreading in the isoleucyl-tRNA synthetase from Escherichia coli. A total rate equation was used to calculate the discrimination of valine by the isoleucyl-tRNA synthetase from Escherichia coli. The PPi present in the cell makes the backward reaction or the pyrophosphorolysis of the E.aa-AMP possible. If the E.Ile-AMP has been corrected for wrong aminoacyl adenylation by the pretransfer proofreading, the pyrophosphorolysis rapidly equilibrates the corrected E.Ile-AMP with E.Ile and thus spoils the effect of the proofreading. The loss of the corrected species is avoided if there is a barrier (perhaps conformational) formed by a slow reaction step between the noncorrected E.Ile-AMP and the corrected (*E)tRNA(Ile-AMP). If such a slow conformational change exists, the increase in accuracy from the pretransfer proofreading would be beneficial, and, in addition, the PPi increases the accuracy by optimizing the initial discrimination of the wrong amino acid.


Mechanism of carboxypeptidase-Y-catalysed peptide semisynthesis. The initial rate steady-state kinetics of carboxypeptidase-Y-catalyzed hydrolysis and aminolysis reactions with some alpha-N-benzoyl-L-tyrosinyl compounds has been investigated using L-valinamide as the nucleophile in aminolysis. Hydrolysis of alpha-N-benzoyl-L-tyrosinyl ethyl ester, 4-nitroanilide, and -amide has been studied in the pH range 4-9. The results are interpreted in terms of the classical serine proteinase mechanism, which involves enzyme-substrate complex formation, followed by acylation and deacylation of the enzyme. The three reactions share the same deacylation step. It is rate-determining with the ester substrate, but with the 4-nitroaniline acylation is and this is even more pronounced with the amide. From the pH dependencies, no change of rate-determining step is apparent in the range pH 4-9. For the 4-nitroanilide and the amide substrates, the kinetic parameter, Kc/Km, is influenced by an ionizing group with a pK value of 6. Probably this is the active-site histidine residue, which thus is active in acylation in its deprotonated form. That group affects the deacylation reaction similarly as seen from the kinetics of the ester substrate. Aminolysis occurs in parallel to hydrolysis in the presence of reactive nucleophiles. Here L-valinamide was used as model nucleophile. The analysis of the observed kinetic effects of L-valinamide on the initial rate behaviour of carboxypeptidase-Y-catalyzed hydrolysis reactions suggests a reaction mechanism which involves (a) the binding of the free nucleophile to the free enzyme and (b) reaction of the free nucleophile with the acyl-enzyme complex forming an enzyme-aminolysis product complex, which dissociates into the free enzyme and the aminolysis product. The reactions are characterized by a number of kinetic parameters, the values of which are determined. The results of aminolysis progress reactions indicate that the formation of the product in high yields is strongly dependent on the leaving group of the substrate. The initial production of aminolysis product, however, is the same for the three substrates. But the fact that their Kc/Km values differ by several orders of magnitude leads to significantly different progresses of the aminolysis. The ester substrate is the only one that efficiently competes with and hinders the hydrolysis of the aminolysis product.


Purification and properties of a novel arylmalonate decarboxylase from Alcaligenes bronchisepticus KU 1201. A novel decarboxylase which catalyzes an enantioselective decarboxylation of alpha-aryl-alpha-methylmalonates to alpha-arylpropionates has been purified from a soil bacterium Alcaligenes bronchisepticus KU 1201. The enzyme was purified 300-fold to homogeneity, judged from the analysis of N-terminal amino acid sequence, and found to be a monomeric enzyme of apparent 24 kDa. The enzyme catalyzes a decarboxylation giving alpha-arylalkanoates from substituted malonates such as alpha-arylmalonate and alpha-alkyl-alpha-arylmalonates. The decarboxylase is not a biotin containing enzyme because avidin have no influence on the enzyme activity. In addition, the enzyme does not require known co-factors (ATP, ADP and coenzyme A) for maximum activity. The enzyme activity was inhibited by sulfhydryl agents. The electronic effect of the substituents on kcat for the enzymic decarboxylation of arylmalonates has been studied. The logarithm of relative value of kcat gave a linear correlation to Hammett's sigma with a rho value of +1.9, for substituted phenylmalonates. Comparing the relative activities, it is clear that the enzyme prefers alpha-arylmalonates to alpha-aryl-alpha-methylmalonates. Thus, the enzyme was tentatively named as arylmalonate decarboxylase.


Regulation of vimentin expression in cultured epithelial cells. Most cell types start expressing vimentin when brought into tissue culture. Using both vimentin-expressing (HeLa) and vimentin-negative (MCF-7) epithelial cell lines, we have identified the cis-regulatory DNA elements involved in this process. Sequences located 1.1-0.6 kb upstream of the vimentin transcription-initiation site strongly enhance expression in HeLa cells, but are silenced in MCF-7 cells. Other regulatory elements in the vimentin promoter (an enhancer 3.2-2.6 kb upstream and a minimal promoter region including the CAAT-box) are potentially active in both cell types, but are silenced by the 0.5-kb fragment in MCF-7 cells. Deletion of this fragment restores transcriptional activity of a transfected vimentin promoter. Our data indicate that a double AP 1/jun-binding site present in the 0.5-kb fragment mediates the induction of vimentin expression in cultured epithelial cells, while silencing sequences located within the same fragment are responsible for the absence of vimentin expression in MCF-7 cells. In contrast to MCF-7 cells, a transfected vimentin promoter and gene are transcriptionally active in the vimentin-negative epithelial cell line T24. Transfection studies show that type-III-intermediate-filament expression is not impaired at any level in these cells. Upon transfection and expression of a desmin construct in T24 cells not only desmin, but also vimentin was detected. Both proteins assembled into intermediate filaments. This induction of vimentin expression appeared to be regulated at the post-transcriptional level.


Regulatory phosphorylation of phosphoenolpyruvate carboxylase in protoplasts from Sorghum mesophyll cells and the role of pH and Ca2+ as possible components of the light-transduction pathway. The light-dependent phosphorylation of the photosynthetic phosphoenolpyruvate carboxylase (PyrPC) was shown to occur in protoplasts from Sorghum mesophyll cells. It was accompanied by an increase in PyrPC protein-serine-kinase activity and conferred the target-specific functional properties, i.e. an increase in Vmax and apparent Ki for L-malate, as previously found with the whole leaf. The light-dependent regulatory phosphorylation of PyrPC was (a) specifically promoted by the weak bases NH4Cl and methylamine (agents which increase cytosolic pH), but not by KNO3, (b) inhibited by the cytosolic protein-synthesis inhibitor, cycloheximide, thus confirming that protein turnover is a component of the signal-transduction cascade, as reported in [4], (c) found to moderately decrease in the presence of EGTA and to be strongly depressed when the Ca(2+)-selective ionophore A23187 was added to the incubation medium together with EGTA. Addition of Ca2+, but not of Mg2+, to the Ca(2+)-depleted protoplasts partially, but significantly, relieved the inhibition. Calcium deprivation apparently affected the in-situ light-activation of the PyrPC protein kinase. These data indicated that both Ca2+ and an increase in cytosolic pH are required for the induction of PyrPC protein kinase activity/PyrPC phosphorylation in illuminated protoplasts from Sorghum mesophyll cells.


Analysis of soluble human and mouse interferon-gamma receptors expressed in eukaryotic cells. The extracellular domains of the human and mouse interferon-gamma receptors were produced in insect Spodoptera frugiperda cells infected with recombinant baculoviruses and in mammalian Chinese-hamster-ovary cells. The receptors expressed in both systems are secreted into the culture medium. Their signal peptides are cleaved off and the proteins show heterogeneity in glycosylation which, however, does not affect the capacity to bind interferon gamma or specific antibodies. The soluble mouse receptors exhibit binding capacities similar to those of cell-surface-anchored receptors, whereas the human receptors exhibit a lower binding capacity. All soluble receptors inhibit the binding of interferon gamma to cellular receptors and neutralize the antiviral activity exerted by interferon gamma. These receptors could therefore be useful for structure/function analyses and in vivo studies.


Zinc-specific activation of a HeLa cell nuclear protein which interacts with a metal responsive element of the human metallothionein-IIA gene. Transcription of metallothionein genes is activated by heavy metals such as zinc and cadmium, and a DNA element called metal responsive element (MRE) is essential for this process. By mobility-shift assay, we identified a HeLa-cell nuclear protein which specifically binds to MREa of human metallothionein-IIA gene. This protein, named ZRF (zinc-regulatory factor), is present in the cells untreated with heavy metals. Zinc is essential for, and increases in a dose-dependent manner, the binding of ZRF to MREa. Other heavy metals which can also induce metallothioneins, including cadmium, copper and mercury, do not activate ZRF. A MREa-containing oligonucleotide that can bind ZRF confers heavy metal-inducibility to a heterologous promoter, suggesting that ZRF is a zinc-dependent transcriptional activator. In addition to the MRE core sequence, the surrounding sequences are also important for both ZRF binding in vitro, and zinc-dependent transcriptional activation in vivo. MREa by itself responds not only to zinc but also to other metallothionein-inducing heavy metals, indicating that the ZRF protein, not the MREa sequence, is responsible for the zinc specificity.


Allosteric characteristics of GTP cyclohydrolase I from Escherichia coli. The kinetic and regulatory properties of GTP cyclohydrolase I were investigated using an improved enzyme assay and direct determination of the product, dihydroneopterin triphosphate. The enzyme was purified from Escherichia coli to absolute homogeneity as demonstrated by N-terminal sequencing of up to 50 amino acid residues. A 30-residue internal fragment showed 42% similarity with rat liver GTP cyclohydrolase I. The enzyme did not obey Michaelis-Menten kinetics or show a sigmoid reaction curve. The substrate saturation kinetics were found to be slow with low response to minor changes in GTP concentrations. GTP cyclohydrolase I has a relatively high apparent Km. The values are slightly different for enzyme purified by GTP-agarose (100 microM) and UTP-agarose (110 microM). Low turnover numbers of 12/min and 19/min were calculated for the respective enzyme preparations. GTP-cyclohydrolase-I activity was modulated in Vmax by K+, divalent cations, UTP and tetrahydrobiopterin. Divalent cations, such as Mg2+, had an activating effect with an optimum at 8 mM Mg2+. A different catalytic function and formation of a new, unidentified product by GTP cyclohydrolase I was observed in the presence of Ca2+. In the presence of 1 mM EDTA and Mg2+, GTP-cyclohydrolase-I activity was strongly inhibited by chelate complexes. UTP proved not to be a competitive inhibitor, but a positive modulator. The inhibition by chelate complexes was totally abolished by UTP. Tetrahydrobiopterin showed an inhibitory effect, with 50% inhibition at 100 microM tetrahydrobiopterin. UTP was able to reduce the inhibition by tetrahydrobiopterin. Using monoclonal antibody 1F11 (related to the GTP-binding site), and monoclonal antibody NS7 (mimicking tetrahydrobiopterin), different binding sites were demonstrated for GTP and tetrahydrobiopterin on each enzyme subunit. Western-blot competition analysis revealed a UTP-binding site different from the binding sites of GTP and tetrahydrobiopterin. Based on the kinetic behaviour and the kind of modulations observed we defined GTP cyclohydrolase I as an M-class allosteric enzyme.