High throughput and quantitative enzymology in the genomic era.

Accurate predictions from models based on physical principles are the ultimate metric of our biophysical understanding. Although there has been stunning progress toward structure prediction, quantitative prediction of enzyme function has remained challenging. Realizing this goal will require large numbers of quantitative measurements of rate and binding constants and the use of these ground-truth data sets to guide the development and testing of these quantitative models. Ground truth data more closely linked to the underlying physical forces are also desired. Here, we describe technological advances that enable both types of ground truth measurements. These advances allow classic models to be tested, provide novel mechanistic insights, and place us on the path toward a predictive understanding of enzyme structure and function.

Revealing enzyme functional architecture via high-throughput microfluidic enzyme kinetics.

Systematic and extensive investigation of enzymes is needed to understand their extraordinary efficiency and meet current challenges in medicine and engineering. We present HT-MEK (High-Throughput Microfluidic Enzyme Kinetics), a microfluidic platform for high-throughput expression, purification, and characterization of more than 1500 enzyme variants per experiment. For 1036 mutants of the alkaline phosphatase PafA (phosphate-irrepressible alkaline phosphatase of Flavobacterium), we performed more than 670,000 reactions and determined more than 5000 kinetic and physical constants for multiple substrates and inhibitors. We uncovered extensive kinetic partitioning to a misfolded state and isolated catalytic effects, revealing spatially contiguous regions of residues linked to particular aspects of function. Regions included active-site proximal residues but extended to the enzyme surface, providing a map of underlying architecture not possible to derive from existing approaches. HT-MEK has applications that range from understanding molecular mechanisms to medicine, engineering, and design.

Sub-chronic (13-week) oral toxicity study in rats with recombinant human lactoferrin produced in the milk of transgenic cows.

The oral toxicity of recombinant human lactoferrin (rhLF) produced in the milk of transgenic cows was investigated in Wistar rats by daily administration via oral gavage for 13 consecutive weeks, 7 days per week. The study used four groups of 20 rats/sex/dose. The control group received physiological saline and the three test groups received daily doses of 200, 600 and 2000 mg of rhLF per kg body weight. Clinical observations, growth, food consumption, food conversion efficiency, water consumption, neurobehavioural testing, ophthalmoscopy, haematology, clinical chemistry, renal concentration test, urinalysis, organ weights and gross examination at necropsy and microscopic examination of various organs and tissues were used as criteria for detecting the effects of treatment. Overall, no treatment-related, toxicologically significant changes were observed. The few findings that may be related to the treatment (lower cholesterol in high-dose females, lower urinary pH in high-dose males and females and very slightly higher kidney weight in high-dose females) were considered of no toxicological significance. Based on the absence of treatment-related, toxicologically relevant changes, the no-observed-adverse-effect level (NOAEL) was considered to be at least 2000 mg/kg body weight/day.

Effects of dietary linoleic acid on pancreatic carcinogenesis in rats and hamsters.

It has been suggested that linoleic acid (LA) is responsible for the promoting effect of dietary polyunsaturated fat on pancreatic carcinogenesis via an accelerated prostaglandin synthesis, caused by metabolism of LA-derived arachidonic acid in (pre)neoplastic tissue. The purpose of the present study was to investigate whether dietary LA is the cause of pancreatic tumor promotion by a high fat diet. Five groups of 30 azaserine-treated rats and 5 groups of 30 N-nitrosobis(2-oxopropyl)amine-treated hamsters were maintained for 6 months (rats) and 12 months (hamsters) on high fat (25 weight %) AIN diets containing 2, 4, 6, 10, or 15 weight % LA. The results indicated that the strongest enhancing effect on the growth of pancreatic (pre)neoplastic lesions in rats and hamsters was obtained with 4 and 2 weight % of dietary LA, respectively. At higher LA levels the tumor response seemed to decrease rather than increase. In both rats and hamsters the fatty acid profiles of blood plasma and pancreas showed an accurate reflection of the dietary fatty acid profiles: a proportional increase in LA levels was observed in plasma and pancreas with increasing dietary LA. In both species plasma and pancreatic AA levels remained constant, except for arachidonic acid levels in rat plasma, which significantly increased with increasing dietary LA levels. Fatty acid profiles in hamster pancreatic tumors did not differ from fatty acid profiles in nontumorous pancreatic tissue from hamsters fed the same diet. Prostaglandin (PG) E2, 6-keto-PGF1 alpha, PGF2 alpha, and thromboxane B2-concentrations in nontumorous pancreatic tissue were similar among the diet groups. Ductular adenocarcinomas from hamster pancreas showed significantly higher levels of 6-keto-PGF1 alpha, PGF2 alpha, and thromboxane B2, but not of PGE2 in comparison with nontumorous pancreas. It is concluded that the strongest pancreatic tumor promotion by dietary LA is 4 weight % in rats and 2 weight % or less in hamsters, and that PGs may be involved in the development of ductular adenocarcinomas induced in hamster pancreas by N-nitrosobis(2-oxopropyl)amine.

Histopathological studies of experimental lyme disease in the dog.

Experimental borrelia infection was induced in 62 specific--pathogen-free beagle dogs by exposure to Ixodes scapularis ticks harbouring the spirochaete Borrelia burgdorferi. Clinical signs of Lyme disease occurred in 39/62 dogs, the remaining 23 being subclinically infected. Clinical signs consisted of one to six episodes of transitory lameness with joint swelling and pain, most commonly affecting the elbow or shoulder joints. The polymerase chain reaction and culture demonstrated that the dogs remained infected for up to 581 days. At necropsy, gross findings consisted of lymphadenopathy in the area of tick attachment. Microscopical changes consisted of effusive fibrinosuppurative inflammation or nonsuppurative inflammation, or both, affecting synovial membranes, joint capsules and associated tendon sheaths. Plasma cells dominated areas of chronic inflammation, with CD3(+) T cells being present in lesser numbers. Microscopical signs of arthritis were polyarticular and more widespread than indicated by clinical signs, and most of the subclinically affected animals also had synovitis. In areas of tick attachment to the skin, hyperkeratosis and a mixture of suppurative and nonsuppurative dermatitis were encountered. Lymphadenopathy in superficial lymph nodes resulted from follicular and parafollicular hyperplasia. In 14/62 dogs, lymphoplasmacytic periarteritis and perineuritis were noted, resembling lesions found in human Lyme disease and syphilis, in which an underlying microangiopathy has been proposed.

Canine viruses and multiple sclerosis.

Serum and cerebrospinal fluid from multiple sclerosis (MS) patients and control subjects were tested and compared for presence and titer of neutralizing antibody against the most common canine viruses. Canine viruses included canine distemper virus (CDV), canine adenovirus 1 (CAV-1), canine parainfluenza virus (CPIV), canine herpesvirus (CHV), canine coronavirus (CCV), and canine parvovirus (CPV). Neutralizing titers against measles virus (MV) and human adenovirus 8 (HA8) were also tested. Significantly elevated (p less than 0.05) antibody levels in sera from MS patients were found only against MV and CDV, but this depended upon the study population and the method of evaluation. The CDV-neutralizing component in serum could be absorbed on MV-infected cells. Results of this study failed to establish a link between canine viruses and MS.

Demyelinating canine distemper encephalomyelitis: measurement of myelin basic protein in cerebrospinal fluid.

Beagle dogs were experimentally infected with the Cornell A75-17 strain of canine distemper virus. At three time points post-infection (PI), immunoreactive myelin basic protein (MBP) was measured in cerebrospinal fluid (CSF). Levels were correlated with neuropathological findings, interferon in CSF and virus isolation from the brain. CSF from animals inoculated with Cornell A75-17 strain often showed detectable immunoreactive MBP late in the disease course. As anticipated from earlier morphological studies, CSF drawn around day 20 PI lacked MBP while subsequent samples were positive. Dogs with severe demyelination had elevated values of immunoreactive MBP while dogs with only mild inflammation had little or none. Release of MBP or MBP peptides into CSF of dogs with canine distemper may be a valuable laboratory test in studies of the natural history of this disease and in assessing the response to treatment. Whether an immune response to MBP plays an immunopathogenic role in the chronic, demyelinating phase of canine distemper encephalitis remains to be determined.

Effects of dietary fish oil (MaxEPA) on N-nitrosobis(2-oxopropyl)amine (BOP)-induced pancreatic carcinogenesis in hamsters.

In the present study the chemopreventive potential of 25% fat (HF) diets containing 2 wt% linoleic acid (LA) and including 0.0, 1.2, 2.4, 4.7, 7.1 or 9.4 wt% dietary fish oil (MaxEPA) has been investigated using the N-nitrosobis(2-oxopropyl)amine (BOP)-hamster model for pancreatic cancer. The number of pancreatic borderline lesions (BLL) was significantly higher (P < 0.05) in the HF groups containing 1.2, 2.4 or 9.4 wt% MaxEPA in comparison with the HF group without MaxEPA. MaxEPA inhibited the metabolism of LA to arachidonic acid (AA) and of AA to prostaglandins (PGs) in both blood plasma and pancreatic microsomes. The pancreatic levels of PGE2 (P < 0.05), 6-keto-PGF1 alpha (P < 0.01) and PGF2 alpha (P < 0.05) decreased significantly with increasing dietary MaxEPA. The levels of PGE2 (P < 0.001), 6-keto-PGF1 alpha (P < 0.05), PGF2 alpha (P < 0.001) and thromboxane (TX) B2 (P < 0.001) in pancreatic adenocarcinomas were higher than in non-tumorous pancreas. The MaxEPA had no significant effect on the BrdU labeling index (LI) in acinar, ductular or centroacinar cells, nor on the LI in BOP-induced pancreatic lesions. It is concluded that (i) dietary fish oil has a slight enhancing effect on BOP-induced pancreatic carcinogenesis in hamsters and (ii) dietary fish oil dose-dependently inhibits the conversion of LA to AA and of AA to certain PGs and (iii) dietary fish oil does not influence the cell proliferation in hamster pancreas.

Azaserine-induced pancreatic carcinogenesis in rats: promotion by a diet rich in saturated fat and inhibition by a standard laboratory chow.

Dietary fat has been shown to enhance pancreatic carcinogenesis. Uncertainty still exists whether the amount of linoleic acid or the amount of fat is the main determining factor. In the present study the effects of a high lard, a low lard, a linoleic acid supplemented low lard and a laboratory chow diet were investigated on the development of (pre)neoplastic pancreatic lesions in rats treated with azaserine. The rats were killed 15 months after carcinogen treatment and the pancreata were examined for the number and size of putative preneoplastic lesions and for the occurrence of neoplasms. The linoleic acid supplemented low lard group showed a significantly increased number of basophilic foci as compared to the low lard group. Rats maintained on the linoleic acid supplemented diet or the laboratory chow developed significantly less atypical acinar cell nodules larger than 1.0 mm in diameter and adenocarcinomas as compared to the high lard group. Animals maintained on the low lard diet developed significantly less adenocarcinomas than rats on the high lard diet did. Overall, the number of benign and malignant pancreatic tumours was consistently higher in the high lard group and consistently lower in the linoleic acid supplemented low lard group than the number of these types of tumours in the low lard group, with the exception of the number of carcinomas in situ, which was lower in the high lard group. The laboratory chow group showed a significant lower number of atypical acinar cell nodules with a diameter over 1.0 mm and a lower number of adenocarcinomas as compared to both the high lard and the low lard group. It is concluded that a diet high in saturated fat has a promoting and that laboratory chow has an inhibitory effect on pancreatic carcinogenesis in azaserine-treated rats.

Lack of inhibitory effects of beta-carotene, vitamin C, vitamin E and selenium on development of ductular adenocarcinomas in exocrine pancreas of hamsters.

The effects of vitamins E and E, beta-carotene and selenium on development of N-nitrosobis(2-oxopropyl)amine (BOP)-induced pancreatic tumours in hamsters were investigated. Dietary supplementation of vitamin C, alone as well as in combination with beta-carotene resulted in consistently lower numbers of advanced ductular lesions. The differences with the controls, however, did not reach the level of statistical significance. Beta-Carotene alone demonstrated no inhibitory effect on the development of (pre)neoplastic lesions in the pancreas. Vitamin E or Se, either alone or in combination, had no effect on the development of advanced ductular lesions in BOP-treated hamsters.

Expression and secretion of outer surface protein (OSP-A) of Borrelia burgdorferi from Escherichia coli.

Outer surface protein A (OspA) is encoded by the ospA gene from Borrelia burgdorferi. This protein induces immunity against infection in mice. The cloning and expression of OspA in Escherichia coli have been previously described, but the secretion of OspA into culture media in E. coli has not yet been reported. In this report we demonstrate that a chimeric OspA protein was secreted into culture media by E. coli when it also harbors the hemolysin secretion genes hlyBD. The OspA fusion protein was also overexpressed from a T7 promoter and purified by immobilized metal ion chromatography. This was possible because the fusion protein contains six histidyl residues in its N-terminus.

Identification of negative strand and positive strand RNA of canine distemper virus in animal tissues using single stranded RNA probes.

In this report, we describe a technique for identifying negative strand (genome) and positive strand (messenger) RNA of canine distemper virus (CDV) in dog tissues by using single stranded RNA probes. Plasmids (pSP64-P and pSP65-P) which contain insert DNA corresponding to the P gene of CDV were transcribed by SP6 polymerase in the presence of radioisotope to produce radiolabeled single stranded RNA probes. RNA transcribed from pSP65-P is complementary to the negative strand (genome) and RNA produced from pSP64-P is complementary to the positive strand (message) of CDV. The binding specificity of the single stranded RNA probes was determined on Northern-blots. The use of these RNA probes in hybridization assays resulted in greater sensitivity and specificity than that obtained from double stranded DNA probes (either whole plasmids or purified insert DNA) which were labeled by the nick translation reaction. We also describe the making of single stranded DNA probes by reverse transcription labeling of complementary RNA. The complementary RNA was produced by the transcription of cloned DNA (pSP64-P and pSP65P). Single stranded RNA probes and single stranded DNA probes were similar in sensitivity. The single stranded RNA and DNA probes were applied to ethanolacetic acid fixed tissue sections from dogs infected with CDV-A75/17. We used 32P-labeled probes in tissue hybridizations and 35S-labeled probes in in situ hybridizations to identify negative and positive stranded CDV RNA. In this report we demonstrate that single stranded RNA and DNA probes can be used successfully in tissue hybridization and in situ hybridization assays to study viral expression in this virus-host system.

Pathogenicity of morbilliviruses for terrestrial carnivores.

Many different species of the order Carnivora are susceptible to canine distemper and the mortality rate varies greatly between species. Ailuridae, Canidae, Hyaenidae, Mustelidae, Procyonidae, Ursidae, Viverridae and now Felidae have been reported to be susceptible to canine distemper virus infection. Although distemper outbreaks in dogs, fur farms and in zoo carnivores have been greatly reduced in recent years due to vaccination, there are still regular outbreaks in free-living carnivores. Unexpected outbreaks of canine distemper have occurred in exotic felids in a California wildlife park and in the Serengeti in Tanzania as well as in javelinas (collared peccaries, Tayassu tajacu) in Arizona. Although safe and efficacious in dogs, modified live canine distemper virus vaccines may be dangerous for a variety of zoo and wildlife carnivores, especially red pandas (Ailurus fulgens) and black footed ferrets (Mustela nigripes).

Cross-reactivity between B. burgdorferi and other spirochetes affects specificity of serotests for detection of antibodies to the Lyme disease agent in dogs.

Western immunoblots, the kinetics-based enzyme-linked immunosorbent assay (KELA), and the microagglutination test were used to evaluate cross-reactivity among antibodies to serovars of Leptospira interrogans (leptospiral serovars), and B. burgdorferi from naturally infected dogs, and to Serpulina (Treponema) hyodysenteriae from vaccinated rabbits. Whole-cell lysates from Borrelia spp., leptospiral serovars, and Serpulina spp. were used for SDS-PAGE, western blots, and KELA. Crossreactivity occurred between the antibodies to B. burgdorferi and leptospiral serovars when tested on the heterologous antigens. Antibodies to leptospiral serovars tended to cross-react more strongly with antigens of B. burgdorferi spp. than did antibodies to B. burgdorferi when tested against antigens of leptospiral serovars. The antibodies against B. burgdorferi showed a lesser degree of cross-reactivity to the antigens of S. hyodysenteriae and S. innocens than they did to leptospiral serovars. We conclude that cross-reactivity occurs between B. burgdorferi and leptospiral serovars. Validation and interpretation of ELISA tests for detection of antibody activity to whole cell lysates of the Lyme agent must take this cross-reactivity into consideration. Conversely, dogs infected with the Lyme agent do not show significant cross-reactivity in the microagglutination test for antibody to the leptospiral serovars.

Dietary fat and carcinogenesis.

Epidemiologic investigations have suggested a relationship between dietary fat intake and various types of cancer incidences. Furthermore, epidemiologic studies as well as studies with animal models have demonstrated that not only the amount but also the type of fat consumed is important. At present, the mechanism by which dietary fat modulates carcinogenesis has not been elucidated. The effects of dietary fat on the development of tumours have been summarized in the present review with emphasis on colorectal, pancreas, breast and prostate cancer. It is concluded that influence on synthesis of prostaglandins and leukotrienes may be the universal mechanism by which dietary fats modulate carcinogenesis.

Up-regulation of inducible nitric oxide synthase mRNA in dogs experimentally infected with Borrelia burgdorferi.

The up-regulation of the inducible nitric oxide synthase (iNOS) mRNA was determined by RT-PCR in 25 tissues each from 22 specific pathogen-free (SPF) dogs experimentally infected with Borrelia burgdorferi by tick exposure and from five uninfected control dogs. Using primers specific for a homologous region of the human and canine iNOS sequence, and canine macrophage mRNA, we isolated and partially sequenced canine iNOS. A sequence of 1775 bases was obtained and primers specific for canine iNOS mRNA constructed to investigate the expression of iNOS in dog tissues in response to infection with B. burgdorferi. In 12 out of 22 dogs infected with B. burgdorferi, acute lameness occurred within 55-82 days after infection whereas the other 10 dogs showed no or only mild clinical signs despite persistent infection up to Day 175. The numbers of iNOS mRNA-positive tissues in dogs with acute lameness were significantly higher than in dogs without lameness, while uninfected dogs showed only negligible iNOS expression. Dogs with acute lameness also had higher numbers of borrelia-positive tissues as well as higher scores in histopathological evaluations than infected dogs without lameness. Our results show that the expression of iNOS mRNA is related to the number of B. burgdorferi-positive tissues and the severity of inflammation as assessed by histopathology. These results implicate an up-regulation of the iNOS mRNA as part of the host's immune response to borrelia infection and a possible role for NO in the pathogenesis of canine Lyme arthritis.

Genetic characterization of canine distemper virus in Serengeti carnivores.

The lion (Panthera leo) population in the Serengeti ecosystem was recently afflicted by a fatal epidemic involving neurological disease, encephalitis and pneumonia. The cause was identified as canine distemper virus (CDV). Several other species in the Serengeti were also affected. This report presents CDV H and P gene sequences isolated from Serengeti lions (Panthera leo), spotted hyenas (Crocuta crocuta), bat-eared fox (Otocyon megalotis) and domestic dog (Canis familiaris). Sequence analyses demonstrated that the four Serengeti species carry closely related CDV isolates which are genetically distinct from other CDV isolates from various species and locations. The results are consistent with the conclusions that: (1) a particularly virulent strain of CDV emerged among Serengeti carnivores within the last few years; (2) that strain has recognizable shared-derived (synapomorphic) genetic differences in both H and P genes when compared to CDV from other parts of the world; and (3) that the CDV strain has frequently crossed host species among Serengeti carnivores.

Disposition, accumulation and toxicity of iron fed as iron (II) sulfate or as sodium iron EDTA in rats.

A study was performed to provide data on the disposition, accumulation and toxicity of sodium iron EDTA in comparison with iron (II) sulfate in rats on administration via the diet for 31 and 61 days. Clinical signs, body weights, food consumption, food conversion efficiency, hematology, clinical chemistry and pathology of selected organs were used as criteria for disclosing possible harmful effects. Determination of iron and total iron binding capacity in blood plasma and non-heme iron analysis in liver, spleen and kidneys were used to assess the disposition and accumulation of iron originating from sodium iron EDTA or iron (II) sulfate. It was concluded that, under the conditions of the present study, iron is accumulated from the diet in liver, spleen and kidneys in a dose-dependent manner, and iron derived from FeEDTA is taken up and/or accumulated less efficiently in liver and spleen than iron from FeSO(4). Moreover, feeding iron up to 11.5 and 11.2 mg/kg body weight/day, derived from FeSO(4) and FeEDTA, respectively, did not result in tissue iron excess nor in any other toxicologically significant effects.

Modulation of pancreatic carcinogenesis by antioxidants.

Previously performed short-term (4-month) studies demonstrated that vitamins C and E, beta-carotene and selenium modulate growth of early putative preneoplastic acinar lesions induced in rat pancreas by azaserine. The present paper summarizes the results of long-term studies performed with azaserine-treated rats maintained on diets high in either beta-carotene, vitamins C and E or selenium. It appeared that rats given a diet high in beta-carotene, vitamin C or selenium, but not vitamin E, developed fewer pancreatic tumours than controls. The chemopreventive effects of these micronutrients were most pronounced when beta-carotene and/or selenium were given during the promotion phase of the carcinogenic process. Surprisingly, cell proliferation in azaserine-induced preneoplastic acinar lesions was higher in rats given beta-carotene and/or selenium via the diet in comparison to controls. It is considered unlikely that any antioxidant alone can be associated with protection against cancer. It is concluded that dietary supplementation of combinations of antioxidants may have practical application in chemoprevention of cancer.

Dog lymphocyte cultures facilitate the isolation and growth of virulent canine distemper virus.

Optimal conditions for the isolation and growth of virulent canine distemper virus (CDV) in canine thymic and peripheral blood lymphocyte cultures were determined. Peak virus titers were seen from 3 to 6 days postinoculation of lymphocytes and depended on the multiplicity of infection. Dog lymphocytes were at least as susceptible as canine macrophages to infection with virulent CDV. Virus replication in lymphocytes resulted in higher virus titers than in dog lung macrophages. Peripheral blood lymphocytes (PBL) from CDV-immune dogs were as susceptible to CDV as were PBL from susceptible dogs.