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We tested the hypothesis that brown-headed cowbirds (Molothrus ater) harbor Sarcocystis neurona, the agent of equine protozoal myeloencephalitis (EPM), and act as intermediate hosts for this parasite. In summer 1999, wild caught brown-headed cowbirds were collected and necropsied to determine infection rate with Sarcocystis spp. by macroscopic inspection. Seven of 381 (1.8%) birds had grossly visible sarcocysts in leg muscles with none in breast muscles. Histopathology revealed two classes of sarcocysts in leg muscles, thin-walled and thick-walled suggesting two species. Electron microscopy showed that thick-walled cysts had characteristics of S. falcatula and thin-walled cysts had characteristics of S. neurona. Thereafter, several experiments were conducted to confirm that cowbirds had viable S. neurona that could be transmitted to an intermediate host and cause disease. Specific-pathogen-free opossums fed cowbird leg muscle that was enriched for muscle either with or without visible sarcocysts all shed high numbers of sporocysts by 4 weeks after infection, while the control opossum fed cowbird breast muscle was negative. These sporocysts were apparently of two size classes, 11.4+/-0.7 microm by 7.6+/-0.4 microm (n=25) and 12.6+/-0.6 microm by 8.0+/-0 microm (n=25). When these sporocysts were excysted and introduced into equine dermal cell tissue culture, schizogony occurred, most merozoites survived and replicated long term and merozoites sampled from the cultures with long-term growth were indistinguishable from known S. neurona isolates. A cowbird Sarcocystis isolate, Michigan Cowbird 1 (MICB1), derived from thin-walled sarcocysts from cowbirds that was passaged in SPF opossums and tissue culture went on to produce neurological disease in IFNgamma knockout mice indistinguishable from that of the positive control inoculated with S. neurona. This, together with the knowledge that S. falcatula does not cause lesions in IFNgamma knockout mice, showed that cowbird leg muscles had a Sarcocystis that fulfills the first aim of Koch's postulates to produce disease similar to S. neurona. Two molecular assays provided further support that both S. neurona and S. falcatula were present in cowbird leg muscles. In a blinded study, PCR-RFLP of RAPD-derived DNA designed to discriminate between S. neurona and S. falcatula showed that fresh sporocysts from the opossum feeding trial had both Sarcocystis species. Visible, thick-walled sarcocysts from cowbird leg muscle were positive for S. falcatula but not S. neurona; thin-walled sarcocysts typed as S. neurona. In 1999, DNA was extracted from leg muscles of 100 wild caught cowbirds and subjected to a PCR targeting an S. neurona specific sequence of the small subunit ribosomal RNA (SSU rRNA) gene. In control spiking experiments, this assay detected DNA from 10 S. neurona merozoites in 0.5g of muscle. In the 1999 experiment, 23 of 79 (29.1%) individual cowbird leg muscle samples were positive by this S. neurona-specific PCR. Finally, in June of 2000, 265 cowbird leg muscle samples were tested by histopathology for the presence of thick- and thin-walled sarcocysts. Seven percent (18/265) had only thick-walled sarcocysts, 0.8% (2/265) had only thin-walled sarcocysts and 1.9% (5/265) had both. The other half of these leg muscles when tested by PCR-RFLP of RAPD-derived DNA and SSU rRNA PCR showed a good correlation with histopathological results and the two molecular typing methods concurred; 9.8% (26/265) of cowbirds had sarcocysts in muscle, 7.9% (21/265) had S. falcatula sarcocysts, 1.1% (3/265) had S. neurona sarcocysts, and 0.8% (2/265) had both. These results show that some cowbirds have S. neurona as well as S. falcatula in their leg muscles and can act as intermediate hosts for both parasites.

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Sarcocysts were dissected from the tongue of a nine-banded armadillo (Dasypus novemcinctus). DNA was extracted and characterised by PCR amplification followed by restriction fragment length polymorphism analysis and nucleotide sequencing. A total of 1879 nucleotides were compared; the sarcocyst DNA sequence was identical to that reported for Sarcocystis neurona. DNA was extracted from the sarcocysts of five more nine-banded armadillos. A 254-nucleotide sequence was determined for each and found to be identical to S. neurona. Western blot techniques for detection of anti-S. neurona antibody were developed for use with armadillo plasma and samples from 19 wild-caught and 17 captive-raised armadillos were examined. Whereas all of the 19 wild-caught armadillos had antibodies to S. neurona, only one of 17 captive-raised armadillos did. These results suggest that the nine-banded armadillo are naturally infected with S. neurona.

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The nine-banded armadillo (Dasypus novemcinctus) is an intermediate host of at least three species of Sarcocystis, Sarcocystis dasypi, Sarcocystis diminuta, and an unidentified species; however, life cycles of these species have not been determined. Following feeding of armadillo muscles containing sarcocysts to the Virginia opossum (Didelphis virginiana), the opossums shed sporulated Sarcocystis sporocysts in their faeces. Mean dimensions for sporocysts were 11.0x7.5 microm and each contained four sporozoites and a residual body. Sporocysts were identified as Sarcocystis neurona using PCR and DNA sequencing. A 2-month-old foal that was negative for S. neurona antibodies in the CSF was orally inoculated with 5x10(5) sporocysts. At 4 weeks post-infection, the foal had a 'low positive' result by immunoblot for CSF antibodies to S. neurona and by week 6 had a 'strong positive' CSF result and developed an abnormal gait with proprioceptive deficits and ataxia in all four limbs. Based on the results of this study, the nine-banded armadillo is an intermediate host of S. neurona.

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Sarcocystis neurona is a protozoan parasite that can cause neurological deficits in infected horses. The route of transmission is by fecal-oral transfer of sporocysts from opossums. However, the species identity and the lifecycle are not completely known. In this study, Sarcocystis merozoites from eight isolates obtained from Michigan horses were compared to S. neurona from a California horse (UCD1), Sarcocystis from a grackle (Cornell), and five Sarcocystis isolates from feral opossums from Michigan. Comparisons were made using several techniques. SDS-PAGE analysis with silver staining showed that Sarcocystis spp. from the eight horses appeared the same, but different from the grackle isolate. One Michigan horse isolate (MIH6) had two bands at 72 and 25kDa that were more prominent than the UCD1 isolate and other Michigan horse isolates. Western blot analysis showed that merozoites of eight of eight equine-derived isolates, and the UCD1 S. neurona isolate had similar bands when developed with serum or CSF of an infected horse. Major bands were seen at 60, 44, 30, and 16kDa. In the grackle (Cornell) isolate, bands were seen at 60, 44, 29, and 16kDa. DNA from merozoites of each of the eight equine-derived isolates and the grackle-derived isolate produced a 334bp PCR product (Tanhauser et al., 1999). Restriction fragment length polymorphism (RFLP) analysis of these horse isolates showed banding patterns characteristic for S. neurona. The grackle (Cornell) isolate had an RFLP banding pattern characteristic of other S. falcatula species. Finally, electron microscopy examining multiple merozoites of each of these eight horse isolates showed similar morphology, which differed from the grackle (Cornell) isolate. We conclude that the eight Michigan horse isolates are S. neurona species and the grackle isolate is an S. falcatula species.

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Equine protozoal myeloencephalitis is a common neurologic disease of horses in the Americas usually caused by Sarcocystis neurona. To date, the disease has not been induced in horses using characterized sporocysts from Didelphis virginiana, the definitive host. S. neurona sporocysts from 15 naturally infected opossums were fed to horses seronegative for antibodies against S. neurona. Eight horses were given 5x10(5) sporocysts daily for 7 days. Horses were examined for abnormal clinical signs, and blood and cerebrospinal fluid were harvested at intervals for 90 days after the first day of challenge and analyzed both qualitatively (western blot) and quantitatively (anti-17kDa) for anti-S. neurona IgG. Four of the challenged horses were given dexamethasone (0.1mg/kg orally once daily) for the duration of the experiment. All challenged horses immunoconverted against S. neurona in blood within 32 days of challenge and in CSF within 61 days. There was a trend (P = 0.057) for horses given dexamethasone to immunoconvert earlier than horses that were not immunosuppressed. Anti-17kDa was detected in the CSF of all challenged horses by day 61. This response was statistically greater at day 32 in horses given dexamethasone. Control horses remained seronegative throughout the period in which all challenged horses converted. One control horse immunoconverted in blood at day 75 and in CSF at day 89. Signs of neurologic disease were mild to equivocal in challenged horses. Horses given dexamethasone had more severe signs of limb weakness than did horses not given dexamethasone; however, we could not determine whether these signs were due to spinal cord disease or to effects of systemic illness. At necropsy, mild-moderate multifocal gliosis and neurophagia were found histologically in the spinal cords of 7/8 challenged horses. No organisms were seen either in routinely processed sections or by immunohistochemistry. Although neurologic disease comparable to naturally occurring equine protozoal myeloencephalitis (EPM) was not produced, we had clear evidence of an immune response to challenge both systemically and in the CNS. Broad immunosuppression with dexamethasone did not increase the severity of histologic changes in the CNS of challenged horses. Future work must focus on defining the factors that govern progression of inapparent S. neurona infection to EPM.

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Gamma-interferon knockout mice have become the model animal used for studies on Sarcocystis neurona. In order to determine the viability of S. neurona sporocysts and to evaluate the course of the disease in these mice, sporocysts were collected from opossums (Didelphis virginiana), processed, and stored for varying periods of time. Gamma-interferon knockout mice were then inoculated orally with different isolates at different doses. These animals were observed daily for clinical signs until they died or it appeared necessary to humanely euthanize them. 15 of 17 (88%) mice died or showed clinical signs consistent with neurologic disease. The clinical neurologic symptoms observed in these mice appeared to be similar to those observed in horses. 15 of 17 (88%) mice were euthanized or dead by day 35 and organisms were observed in the brains of 13 of 17 (77%) mice. Dose appeared not to effect clinical signs, but did effect the amount of time in which the course of disease was completed with some isolates. The minimum effective dose in this study was 500 orally inoculated sporocysts. Efforts to titrate to smaller doses were not attempted. Direct correlation can be made between molecularly characterized S. neurona sporocysts and their ability to cause neurologic disease in gamma-interferon knockout mice.

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Studies designed to investigate the causative agent of equine protozoal myeloencephalitis and its life cycle have been hampered by the marked similarity of Sarcocystis neurona to other Sarcocystis spp. present in the same definitive host. Random-amplified polymorphic DNA techniques were used to amplify DNA from isolates of S. neurona and Sarcocystis falcatula. DNA sequence analysis of polymerase chain reaction (PCR) products was then used to design PCR primers to amplify specific Sarcocystis spp. DNA products. The ribosomal RNA internal transcribed spacer was also amplified and compared between S. neurona and S. falcatula. Useful sequence heterogeneity between the 2 organisms was identified, creating potential markers to distinguish these Sarcocystis spp. These markers were used to characterize Sarcocystis isolates from opossum (Didelphis virginiana) feces. Our data suggest that S. neurona and S. falcatula can be differentiated with these markers and that multiple Sarcocystis spp., including S. neurona and S. falcatula, are shed by opossums.

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Mutational damage to human mitochondrial DNA (mtDNA) can cause disorders in oxidative phosphorylation; speculation that such damage is involved in degenerative diseases and aging is common. We have detected deletions in mouse mtDNA which resemble those found in elderly humans or patients with certain mtDNA disorders. Five different mtDNA deletions, predicted from the positions of short, direct DNA repeats, were present in aged, but not young, mice. Deleted regions were surrounded by either exact or inexact repeats and occurred in both the major and minor regions of the mtDNA genome. The abundance of a particular deletion was generally related to the thermodynamic stability of the bounding repeat sequence. Deletions in aged mice were present at low levels (less than 0.01% of total mtDNA). However, in contrast to results from aged humans, deletions were more abundant in liver than in brain, heart, or skeletal muscle. These results make it possible to predict the location and relative abundance of deletions in any sequenced mtDNA, including inbred mouse strains differing in inherent natural lifespan. The inbred mouse model will allow a critical examination of the relationship between the presence and abundance of mtDNA deletions and the aging process.

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A cDNA encoding the mouse carbonic anhydrase V gene was isolated by reverse transcription and polymerase chain reaction from BALB/c mouse liver mRNA. Vectors containing the full coding sequence as well as two different NH2-terminal truncated genes expressed enzymatically active protein in Escherichia coli. The carbonic anhydrase V produced by a vector containing the full coding sequence, which includes a possible NH2-terminal mitochondrial targeting signal, was proteolytically processed by E. coli and contained several amino-terminal ends. The two NH2-terminal truncated vectors deleted, respectively, 1) the 29-amino acid putative targeting sequence and 2) 51 amino acids, yielding a protein equivalent to a carbonic anhydrase (CA) V isolated from mouse liver mitochondria; and both vectors produced homogeneous protein fractions. These latter two forms of CA V had identical steady-state constants for the hydration of CO2, with maximal values of kcat/Km at 3 x 10(7) M-1 s-1 and kcat at 3 x 10(5) s-1 with an apparent pKa for catalysis of 7.4 determined from kcat/Km. In catalytic properties, mouse CA V is closest to CA I; however, in inhibition by acetazolamide, ethoxzolamide, and cyanate, CA V is very similar to CA II. Mouse CA V has a tyrosine at position 64, where the highly active isozyme II has histidine serving as a proton shuttle in the catalytic pathway. Investigation of a site-specific mutant of CA V containing the replacement Tyr64-->His showed that the unique kinetic properties of CA V are not due to the presence of tyrosine at position 64.

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The osteocalcin (OC) gene was initially described as a single copy gene encoding the bone specific vitamin K dependent and vitamin D regulated protein. We report here the presence of multiple copies of the gene in mouse and rat. Southern blot analysis and restriction mapping of genomic DNA from several strains of mice indicated the presence of at least three copies of the OC coding sequence within a 19 kb fragment. Two closely linked OC genes contain the proximal promoter region with intact coding sequences. The third potential OC gene includes a 3.5 kb insert between an OC promoter-like region and a coding region that has several amino acid substitutions distributed among functional domains when compared with the normal gene. The 940 nucleotides upstream of the modified coding region lack the well defined 5' regulatory elements that support basal and hormone-responsive transcriptional control. In rats either one or more OC genes were observed in different strains or in Sprague Dawley rats obtained from different suppliers.

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Maximal turnover rates for the dehydration of HCO3- catalyzed by the zinc metalloenzyme carbonic anhydrase III are limited by a proton transfer to zinc-bound hydroxide in the active site. We have used site-directed mutagenesis to place a proton donor, histidine, at position 64 and used 18O exchange between CO2 and water measured by mass spectrometry to determine the rates of intramolecular proton transfer to the zinc-bound hydroxide. In a series of site-specific mutants, the values of pKa of the zinc-bound water ranged from approximately 5 to 9. The rate constants for proton transfer obeyed a Brønsted correlation and showed sharp curvature characteristic of facile proton transfers. Application of Marcus rate theory shows that this proton transfer has the small intrinsic energy barrier (near 1.5 kcal/mol) characteristic of rapid proton transfer between nitrogen and oxygen acids and bases, but has an observed overall energy barrier (near 10 kcal/mol), indicating the involvement of accompanying, energy requiring processes such as solvent reorganization or conformational change.

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Site-directed mutagenesis is widely used to examine structure/function relationships in proteins. We have designed a bacterial expression vector series which is optimized for efficient site-directed mutagenesis and subsequent protein synthesis without intervening subcloning steps. The vectors, derived from the T7 expression vectors of Studier and his collaborators [Studier et al., Methods Enzymol. 185 (1990) 60-89], are small and have a bacteriophage f1 origin of replication for production of single-stranded (ss) DNA. Both single-site mutants [using ssDNA and mutating oligodeoxyribonucleotides (oligos)] and cassette mutants (mutagenesis of a short region by inserting double-stranded oligos into unique restriction sites) are rapidly synthesized and expressed with these vectors. Vector construction and use are detailed with examples showing the expression of the sequences encoding human carbonic anhydrases II and III. Production levels of greater than 60 mg of protein per liter of culture have been obtained.

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Among the seven known isozymes of carbonic anhydrase in higher vertebrates, isozyme III is the least efficient in catalytic hydration of CO2 and the least susceptible to inhibition by sulfonamides. We have investigated the role of two basic residues near the active site of human carbonic anhydrase III (HCA III), lysine 64 and arginine 67, to determine whether they can account for some of the unique properties of this isozyme. Site-directed mutagenesis was used to replace these residues with histidine 64 and asparagine 67, the amino acids present at the corresponding positions of HCA II, the most efficient of the carbonic anhydrase isozymes. Catalysis by wild-type HCA III and mutants was determined from the initial velocity of hydration of CO2 at steady state by stopped-flow spectrophotometry and from the exchange of 18O between CO2 and water at chemical equilibrium by mass spectrometry. We have shown that histidine 64 functions as a proton shuttle in carbonic anhydrase by substituting histidine for lysine 64 in HCA III. The enhanced CO2 hydration activity and pH profile of the resulting mutant support this role for histidine 64 in the catalytic mechanism and suggest an approach that may be useful in investigating the mechanistic roles of active-site residues in other isozyme groups. Replacing arginine 67 in HCA III by asparagine enhanced catalysis of CO2 hydration 3-fold compared with that of wild-type HCA III, and the pH profile of the resulting mutant was consistent with a proton transfer role for lysine 64. Neither replacement enhanced the weak inhibition of HCA III by acetazolamide or the catalytic hydrolysis of 4-nitrophenyl acetate.

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Among the isozymes of carbonic anhydrase, isozyme III is the least efficient in the catalysis of the hydration of CO2 and was previously thought to be unaffected by proton transfer from buffers to the active site. We report that buffers of small size, especially imidazole, increase the rate of catalysis by human carbonic anhydrase III (HCA III) of (1) 18O exchange between HCO3- and water measured by membrane-inlet mass spectrometry and (2) the dehydration of HCO3- measured by stopped-flow spectrophotometry. Imidazole enhanced the rate of release of 18O-labeled water from the active site of wild-type carbonic anhydrase III and caused a much greater enhancement, up to 20-fold, for the K64H, R67H, and R67N mutants of this isozyme. Imidazole had no effect on the rate of interconversion of CO2 and HCO3- at chemical equilibrium. Steady-state measurements showed that the addition of imidazole resulted in increases in the turnover number (kcat) for the hydration of CO2 catalyzed by HCA III and for the dehydration of HCO3- catalyzed by R67N HCA III. These results are consistent with the transfer of a proton from the imidazolium cation to the zinc-bound hydroxide at the active site, a step required to regenerate the active form of enzyme in the catalytic cycle. Like isozyme II of carbonic anhydrase, isozyme III can be enhanced in catalytic rate by the presence of small molecule buffers in solution.

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Sequence analysis of a mouse testicular alpha-tubulin partial cDNA, pRD alpha TT1, reveals an isotype that differs from both the somatic and the predominant testicular alpha tubulins at approximately 30% of the 212 amino acid residues determined. Although this mouse testicular cDNA retains the highly conserved sequence, Glu-Gly-Glu-Glu, found in the carboxyl termini of many alpha tubulins, the protein extends substantially beyond this sequence and does not terminate with a C-terminal tyrosine. Using rabbit antiserum prepared to a novel synthetic peptide predicted from this mouse testis alpha-tubulin cDNA, we have have detected by immunoblot and indirect immunofluorescence an antigenic epitope present in testicular alpha tubulin that is not detectable in brain alpha tubulins. We find that the antiserum specifically binds to the manchettes and meiotic spindles of the mouse testis but not with neural fibers or tubulin extracts of the adult mouse brain. These results demonstrate that at least one of the multiple alpha-tubulin isotypes of the mammalian testis is expressed and used in male germ cells but not in the brain.

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We have mapped the cleavage sites of four restriction enzymes which recognize six-base sequences within the nuclear ribosomal (rRNA) genes of twelve vertebrates, including several placental mammals (Homo sapiens, man; Bos taurus, cow; Equus caballus, horse; Sus scofra, pig; Ovis aries, sheep; Rattus rattus, rat), a marsupial (Didelphis marsupialis, opossum), a bird (Gallus domesticus, chicken), an amphibian (Xenopus laevis), a reptile (Alligator mississipiensis), a bony fish (Cynoscion nebulosus, sea trout), and a cartilagenous fish (Carcharhinus species, requiem shark). These animals represent a span of approx. 400 million years of evolutionary divergence. Our data identify restriction sites in the rRNA genes which are highly conserved among higher vertebrates and therefore are likely to be in functionally important regions. Additionally, the restriction enzyme sites identified will be useful in cloning and sequencing the rRNA genes in any vertebrate. Finally, the consistent size and conserved sequence homology suggests that these rRNA gene segments will be useful as internal controls in hybridization experiments involving other genomic regions in vertebrates.

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