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1.  In vitro exposure of bovine morulae to Ureaplasma diversum. 
Ureaplasma diversum has been associated with infertility in the cow experimentally and in naturally occurring cases. However, the pathogenic mechanism is undetermined. The purpose of this study was to determine whether ureaplasmas are pathogenic for bovine morulae in vitro. Twenty-one morulae were recovered from three superovulated, mature, Holstein cows six or seven days postestrus. The embryos were divided into three groups (A,B,C) and incubated for 16 hours at 37 degrees C in humidified air with 10% CO2. Group A was incubated in embryo culture medium alone, Group B was incubated in culture medium with sterile ureaplasma broth added and Group C was incubated in culture medium containing 1.7 X 10(6) colony forming units Ureaplasma diversum strain 2312. After incubation, the morulae were examined using an electron microscope. Structures morphologically identical to U. diversum were present on the outer surface of the zonae pellucidae of all the morulae exposed to the organism and none were present on the unexposed control embryos. No other morphological differences were observed in either the ureaplasma-exposed embryos or the two groups of control embryos. Ureaplasma diversum was isolated from three of the five embryos incubated in culture medium with sterile ureaplasma broth added. These three embryos were recovered from one donor cow which cultured positive for U. diversum from the vulva and flush fluid. This finding suggests that the contaminating organisms entered the embryo culture wells either in the embryo collection medium or attached to the embryos.(ABSTRACT TRUNCATED AT 250 WORDS)
PMCID: PMC1255303  PMID: 3607652
2.  Ureaplasma diversum infection in vitro alters prostaglandin E2 and prostaglandin F2a production by bovine endometrial cells without affecting cell viability. 
Infection and Immunity  1994;62(5):1528-1533.
Bovine epithelial and stromal cells of the endometrium were inoculated with Ureaplasma diversum, pathogenic strain 2312, at 10(6) or 10(3) color-changing units (ccu)/ml in the presence of 1% fetal bovine serum (depleted of steroids by dextran-charcoal treatment) to assess the effect of infection on prostaglandin biosynthesis. When the inoculum of U. diversum was 10(6) ccu/ml, the concentration of U. diversum in the culture medium decreased with time. U. diversum was found on the epithelial and stromal cell monolayers, increasing in titer 100-fold, indicating that attachment and eventually growth occurred. When the inoculum was 10(3) ccu/ml, the titer of U. diversum remained the same or increased in the supernatant and increased on epithelial and stromal cells. The effect of infection was evaluated by measurement of the primary prostaglandin produced by each cell type, prostaglandin F2a for epithelial cells and prostaglandin E2 for stromal cells. Infection with U. diversum significantly decreased prostaglandin F2a accumulation, by 44.7% +/- 6.0% at 10(6) ccu/ml (P < or = 0.005) and 15.8% +/- 5.3% at 10(3) ccu/ml (P < or = 0.05) in epithelial cells. Prostaglandin E2 accumulation by stromal cells was decreased by 34.0% +/- 4.0% at 10(6) ccu/ml (P < or = 0.001) and by 13.5% +/- 2.7% at 10(3) ccu/ml (P < or = 0.005). Infection with 10(6) ccu/ml did not alter endometrial cell viability, as shown by protein measurement, trypan blue dye exclusion, and cell plating efficiency tests. Thus, alterations in prostaglandin production were not due to cell deterioration. These observations suggest that U. diversum can alter prostaglandin E2 and prostaglandin F2a patterns in primary cultures of bovine endometrial cells without affecting cell viability.
PMCID: PMC186347  PMID: 8168914
3.  Protein A gold identification of ureaplasmas on the bovine zona pellucida. 
The object of this study was to develop a prefixation protein A gold labelling technique for Ureaplasma diversum and to apply this to bovine embryos. Sixteen hour cultures of Ureaplasma diversum strain 2312 were incubated with either specific antiserum or nonimmune serum, followed by exposure to protein A gold and negative staining. The ureaplasmas which were incubated with specific antiserum were labelled with gold particles while those ureaplasmas which were incubated with nonimmune serum were not labelled. Twenty-three unhatched, day 7 bovine embryos were then incubated in either embryo culture medium (ECM) alone, ECM with sterile ureaplasma broth added or ECM with 1.7 X 10(6) colony forming units of Ureaplasma diversum strain 2312 per embryo. After 16 hours, the embryos were washed twice and incubated with either specific antiserum or nonimmune serum. The embryos were then incubated with medium containing protein A gold and examined by electron microscopy. No ureaplasmas were identified on the zona pellucida of the control embryos. Ureaplasmas were identified on the outer surface of the zona pellucida of 13 of the 17 embryos which had been exposed to the organism. Of these, the embryos which were incubated with specific antiserum had labelled ureaplasmas while the embryos which were incubated with nonimmune serum had unlabelled ureaplasmas on the zona pellucida. It was concluded that the protein A gold method was a suitable technique for the identification of ureaplasmas in EM preparations. The presence of ureaplasmas on the outer surface of the bovine zona pellucida following in vitro exposure to the organism was confirmed.(ABSTRACT TRUNCATED AT 250 WORDS)
PMCID: PMC1255543  PMID: 2469532
4.  The effect of intrauterine inoculation with Ureaplasma diversum on bovine fertility. 
To determine the influence of Ureaplasma diversum on bovine fertility 11 uninfected virgin heifers with normal ovarian cyclic activity were randomly allocated to test or control groups. At a synchronized estrus, five test heifers were given an intrauterine broth inoculum containing 1.09 x 10(8) to 1.4 x 10(9) colony forming units of U. diversum and six control animals were infused with sterile ureaplasma broth medium. All animals were artificially inseminated within one hour of infusion. Pregnancy was diagnosed in one of five test heifers and all of six controls by serum progesterone concentrations measured to 25 days postinsemination. The difference in pregnancy rates between the two groups was statistically significant (p = 0.0152). It was concluded that under the conditions of this experiment U. diversum is capable of causing infertility in cattle.
PMCID: PMC1255361  PMID: 3453263
5.  Invasion of Ureaplasma diversum in bovine spermatozoids 
BMC Research Notes  2011;4:455.
Ureaplasma diversum has been associated with infertility in cows. In bulls, this mollicute colonizes the prepuce and distal portion of the urethra and may infect sperm cells. The aim of this study is to analyze in vitro interaction of U. diversum isolates and ATCC strains with bovine spermatozoids. The interactions were observed by confocal microscopy and the gentamycin internalization assay.
U. diversum were able to adhere to and invade spermatozoids after 30 min of infection. The gentamicin resistance assay confirmed the intracellularity and survival of U. diversum in bovine spermatozoids.
The intracellular nature of bovine ureaplasma identifies a new difficulty to control the reproductive of these animals.
PMCID: PMC3219583  PMID: 22032232
Ureaplasma diversum; bovine spermatozoid; invasion; confocal microscopy
6.  Isolation of Ureaplasma diversum and mycoplasmas from genital tracts of beef and dairy cattle in Saskatchewan 
We report herein a survey in which cultures of bovine reproductive tracts for Ureaplasma diversum and mycoplasmas were carried out in order to better understand the role of these organisms in granular vulvitis (GV). Samples cultured were vulvar swabs from clinically normal cows or ones with GV, preputial swabs or raw semen from bulls, and abomasal contents of aborted fetuses.
Ureaplasma diversum was isolated from 104 (43.3%) of 240 dairy cows, 32 (27.1%) of 118 beef cows, 43 (47.2%) of 91 beef heifers, 23 (67.6%) of 34 beef bulls, and three (60%) of five dairy bulls. Mycoplasmas were isolated from 18 (7.5%) dairy cows, two (1.6%) beef cows, three (8.8%) beef bulls, and one dairy bull. No isolation was made from 97 aborted fetuses. For 65 dairy cows and 30 beef heifers with vulvar lesions, the isolation rates for ureaplasmas of 62.5% and 69.7%, respectively, were significantly higher (X2) than those for normal animals (37.5% and 30.3%). On immunofluorescent serotyping of 137 of the 205 isolates, there were 66 in serogroup C (strain T44), 18 in serogroup B (strain D48), eight in serogroup A (strain A417 or strain 2312), 14 cross-reacting, and 31 that were not identified. It was concluded that U. diversum is commonly present in the lower reproductive tract of beef/dairy cattle in Saskatchewan and is associated with granular vulvitis.
PMCID: PMC1481172  PMID: 17423929
7.  Measurement of the cytotoxic effects of different strains of Mycoplasma equigenitalium on the equine uterine tube using a calmodulin assay. 
The cytopathic effects induced by five strains of Mycoplasma equigenitalium for cells of equine uterine tube explants were tested by measuring changes in cellular and extracellular concentrations of calmodulin (CaM). Calmodulin concentrations in samples of total homogenate (TH) and total homogenate supernates (THS) of the infected equine uterine tube explants were significantly lower than respective measurements on noninfected controls. In tissue culture medium fractions (TCM) of some infected explants, CaM concentrations were significantly higher than noninfected controls (p > 0.95). The results suggest that M. equigenitalium colonization on ciliated cells of the equine uterine tube can affect the permeability of the cell membrane leading to leakage or release of CaM during cell breakdown. Measurement of CaM concentrations in samples of TH revealed significant differences in the cytotoxic effects induced by different strains of M. equigenitalium on the equine uterine tube (EUT). The data suggests that some strains of M. equigenitalium may have a role in reproductive failure in the mare. In addition comparisons of the means of the concentrations of CaM in samples of TH or THS in EUT explants from four mares in the follicular and four in the luteal phase of the estrous cycle were found to be not significantly different.
PMCID: PMC1263565  PMID: 1477802
8.  The effects of two Ureaplasma diversum strains on early pregnancy in heifers. 
Two field isolates of Ureaplasma diversum spp. were used to infect heifers at the time of insemination in a preliminary study to observe the effect of infection on early pregnancy. M84-14c-1 was a field isolate from a bull's prepuce typed by immunofluorescence to be similar to U. diversum strain T-44 (Group C). M84-477c-4 was a field isolate from bovine semen typed by immunofluorescence to be similar to U. diversum strain T-288 (Group A). All three heifers infected with M84-477c-4 had a mild granular vulvitis at some time during the trial. None was pregnant when slaughtered 27 days after infection. The result of infection with M84-14c-1, a preputial isolate, was not consistent. One heifer had no infection and a normal pregnancy, one heifer was infected with an abnormal pregnancy, and one heifer was open but ureaplasmas were not detected until day 17 of the trial.
PMCID: PMC1255382  PMID: 3453276
9.  Ureaplasma infection of cell cultures. 
Infection and Immunity  1986;52(2):437-444.
Studies were performed to characterize the effects of ureaplasmas in HeLa, 3T6, and CV-1 cell cultures. The ureaplasmas studied were human Ureaplasma urealyticum T960 (serotype VIII), bovine U. diversum T95, simian strain T167-2, ovine strain 1202, canine strain D1M-C, and feline strains 382 and FT2-B. FT2-B was the only ureaplasma to grow in the cell free culture medium, Dulbecco modified Eagle-Earle medium containing 10% fetal bovine serum. The growth pattern of the ureaplasmas varied in the different cell cultures, but each strain grew in at least two of the cell cultures, suggesting a requirement for a product of the cell culture and for low concentrations of urea. When growth occurred, organisms grew to concentrations that approached, but did not equal, those observed in 10B broth. Most, but not all, ureaplasmas grew quickly, reaching peak titers 2 days after infection. Canine strain D1M-C did not grow in 3T6, but showed rapid growth in HeLa and CV-1 cells, killing both cultures, In some systems, e.g., U. urealyticum T960 and simian strain T167-2, the infection persisted, and ureaplasmas could be recovered from cell cultures four passages after infection, when studies were terminated. The cell culture ureaplasmas grew on T agar, but not on mycoplasma agar medium.
PMCID: PMC261018  PMID: 3699891
10.  Susceptibilities of Mycoplasma bovis, Mycoplasma dispar, and Ureaplasma diversum strains to antimicrobial agents in vitro. 
The purpose of this study was to determine the susceptibility of various strains of Mycoplasma bovis, Mycoplasma dispar, and Ureaplasma diversum, which are prevalent causes of pneumonia in calves, to 16 antimicrobial agents in vitro. The MICs of the antimicrobial agents were determined by a serial broth dilution method for 16 field strains and the type strain of M. bovis, for 19 field strains and the type strain of M. dispar, and for 17 field strains of U. diversum. Final MICs for M. bovis and M. dispar were read after 7 days and final MICs for U. diversum after 1 to 2 days. All strains tested were susceptible to tylosin, kitasamycin, and tiamulin but were resistant to nifuroquine and streptomycin. Most strains of U. diversum were intermediately susceptible to oxytetracycline but fully susceptible to chlortetracycline; most strains of M. bovis and M. dispar, however, were resistant to both agents. Strains of M. dispar and U. diversum were susceptible to doxycycline and minocycline, but strains of M. bovis were only intermediately susceptible. Susceptibility or resistance to chloramphenicol, spiramycin, spectinomycin, lincomycin, or enrofloxacin depended on the species but was not equal for the three species. The type strains of M. bovis and M. dispar were more susceptible to various antimicrobial agents, including tetracyclines, than the field strains. This finding might indicate that M. bovis and M. dispar strains are becoming resistant to these agents. Antimicrobial agents that are effective in vitro against all three mycoplasma species can be considered for treating mycoplasma infections in pneumonic calves. Therefore, tylosin, kitasamycin, and tiamulin may be preferred over oxytetracycline and chlortetracycline.
PMCID: PMC187660  PMID: 8452363
11.  Immune response of heifers to vaginal submucosal or subcutaneous vaccination and intravaginal challenge with Ureaplasma diversum. 
Twenty beef heifers were randomly assigned to five equal groups and vaccinated: Group 1--in vaginal submucosa (VM) with Ureaplasma diversum ultrasonicated whole cells (WC) in complete Freund's adjuvant (CFA); Group 2--in VM with U. diversum cell membranes (CM) in CFA; Group 3--subcutaneously (SC) with CM in CFA; Group 4--in VM with CM alone; and Group 5--in VM with phosphate buffered saline (PBS) in CFA. A second vaccination with the same antigens in incomplete Freund's adjuvant was given after four weeks, and three weeks later, all heifers were challenged intravaginally with 3.6 x 10(7) colony-forming units (CFU) of U. diversum strain 2312. Immunoglobulins that reacted with U. diversum were measured in serum and cervicovaginal mucus (CVM) by an enzyme-linked-immunosorbent assay. In groups 1 and 2, vaccination by the VM route with WC or CM antigens, stimulated high levels of U. diversum-reactive IgG1 and IgG2 antibodies in serum as well as CVM, but a low IgA response only in CVM. In group 4, VM vaccination with CM (no adjuvant) elicited a minimal IgG1 and IgG2 response in serum and CVM. In group 3, SC vaccination with CM antigen stimulated high IgG1 and IgG2 reactivity in both serum and CVM, but no IgA reactivity. Very little IgM reactivity was detected in the four vaccinated groups. Intravaginal challenge resulted in characteristic granular vulvitis in all vaccinated and control heifers, with all animals remaining culture-positive for the 35 day observation period. The infection stimulated a marked increase in the specific IgA response in CVM of the three groups vaccinated with either, adjuvanted antigen.(ABSTRACT TRUNCATED AT 250 WORDS)
PMCID: PMC1263675  PMID: 8004535
12.  Activation of murine macrophages and lymphocytes by Ureaplasma diversum. 
Ureaplasma diversum is a pathogen in the bovine reproductive tract. The objective of the research was to study interactions with macrophages and lymphocytes which might elucidate aspects of pathogenetic mechanisms of this organism. We studied the activation of murine macrophages of C3H/HeN (LPS-responder) and C3H/HeJ (LPS-low-responder) genotype for TNF-alpha, IL-6, IL-1 and nitric oxide production and blastogenic response of C3H/HeJ splenocytes after Ureaplasma diversum stimulation. Live and heat-killed U. diversum induced TNF-alpha, IL-6 and IL-1 in peritoneal macrophage cultures of both C3H/HeN and C3H/HeJ mice in a dose dependent manner. Interferon-gamma modulated the cytokine production, by increasing the production of TNF-alpha, IL-6 and nitric oxide, but IL-1 secretion was only enhanced in C3H/HeJ macrophages stimulated by live ureaplasmas. Supernatant of U. diversum sonicate was mitogenic for murine spleen lymphocytes. The blastogenic response was dose dependent, and stimulation with both U. diversum and Concanavalin A seemed to have an additive effect. These results suggest that U. diversum, similar to other mycoplasmas, activates murine macrophages and lymphoid cells. The studies should be repeated with bovine cells in order to elucidate pathogenetic aspects of inflammation in cattle caused by U. diversum.
PMCID: PMC1263712  PMID: 7889459
13.  The Distribution of Mycoplasmas and Ureaplasmas in the Genital Tract of Normal Artificial Insemination Bulls 
Bull semen is commonly contaminated with mycoplasmas. To determine the source of contamination, semen and the genital tracts of 45 artificial insemination bulls were cultured for these organisms. The results indicate that mycoplasmas colonize the prepuce and the distal part of the urethra. Only rarely were they found in the ampullae or seminal vesicles. In 92% of the bulls with contaminated semen the same Mycoplasma species or Ureaplasma diversum was isolated from the prepuce and urethral orifice as was found in the semen. This suggests that the prepuce and distal urethra is the source of contamination. Colonization of the genital tracts with Mycoplasmas or U. diversum was not associated with histological changes.
PMCID: PMC1680053  PMID: 17422489
Mycoplasma; ureaplasma; genital tract; semen; artificial insemination; microbial colonization
14.  Invasion of Ureaplasma diversum in Hep-2 cells 
BMC Microbiology  2010;10:83.
Understanding mollicutes is challenging due to their variety and relationship with host cells. Invasion has explained issues related to their opportunistic role. Few studies have been done on the Ureaplasma diversum mollicute, which is detected in healthy or diseased bovine. The invasion in Hep-2 cells of four clinical isolates and two reference strains of their ureaplasma was studied by Confocal Laser Scanning Microscopy and gentamicin invasion assay.
The isolates and strains used were detected inside the cells after infection of one minute without difference in the arrangement for adhesion and invasion. The adhesion was scattered throughout the cells, and after three hours, the invasion of the ureaplasmas surrounded the nuclear region but were not observed inside the nuclei. The gentamicin invasion assay detected that 1% of the ATCC strains were inside the infected Hep-2 cells in contrast to 10% to the clinical isolates. A high level of phospholipase C activity was also detected in all studied ureaplasma.
The results presented herein will help better understand U. diversum infections, aswell as cellular attachment and virulence.
PMCID: PMC2907839  PMID: 20236540
15.  Humoral and secretory antibodies to Ureaplasma diversum in heifers following subcutaneous vaccination and vaginal infection. 
We measured antibody levels in serum and cervicovaginal mucus (CVM) of four heifers vaccinated with two inoculations of killed Ureaplasma diversum strain 2312 in incomplete Freund's adjuvant (IFA) two weeks apart, and six heifers given a placebo. Two weeks later, the vaccinates and four placebo heifers, were challenged by intravaginal inoculation with 6.4 x 10(8) colony-forming units of the homologous U. diversum strain. The remaining two placebo heifers served as unvaccinated, unchallenged controls. Antibody levels in serum and CVM of all heifers were determined by an enzyme-linked immunosorbent assay (ELISA). Vaccination stimulated specific IgG1 and IgG2 responses in serum and CVM but only a slight IgM and no IgA response. In both vaccinate and placebo heifers, subsequent intravaginal challenge resulted in a granular vulvitis (GV) with a predominant IgA response in the CVM. The GV gradually subsided during the 35 day observation period but ureaplasmas were consistently demonstrated by culture. We concluded that subcutaneous vaccination stimulated a specific, albeit nonprotective, IgG response in serum and CVM. In contrast, vaginal infection primarily induced a mucosal IgA response.
PMCID: PMC1263674  PMID: 8004534
16.  Rapid identification of mycoplasmas by indirect immunoperoxidase test using small square filter paper. 
The indirect immunoperoxidase test using small, square filter paper was used for rapid identification of mycoplasmas. Colonies of type strains of 22 mycoplasma species, 3 acholeplasma species, and three Ureaplasma diversum serogroups were stained by this test with high sensitivity and specificity. All of 49 isolates from bovine materials and cell cultures were easily identified by this test, and the results agreed with those obtained by growth inhibition test. Use of filter paper made it possible to add different kinds of antisera or conjugates to the same agar plate simultaneously and also to save antiserum and conjugate. This test proved to be a simple and useful technique for rapid identification of many mycoplasma species grown on agar medium.
PMCID: PMC265805  PMID: 3539989
17.  "Aggrecanase" activity is implicated in tumour necrosis factor alpha mediated cartilage aggrecan breakdown but is not detected by an in vitro assay. 
Molecular Pathology  1997;50(3):153-159.
AIMS: To develop an in vitro assay for the putative glutamyl endopeptidase, "aggrecanase", which is thought to degrade cartilage aggrecan, and to examine the role of the enzyme in tumour necrosis factor stimulated aggrecan cleavage. METHODS: Aggrecan fragments released by bovine nasal cartilage explants, with and without exposure to tumour necrosis factor alpha, were purified and analysed by western blotting and N-terminal sequencing. Intact bovine aggrecan was incubated with extracts of cartilage, lysed chondrocytes, or cartilage explant conditioned culture medium under a variety of conditions. Deglycosylated aggrecan was incubated with nasal cartilage explants. Proteoglycan breakdown was assessed by metachromatic assay of fragments in culture media, and cleavage of the substrate at the aggrecanase cleavage site was detected and measured using the antibody BC3, which recognises a neoepitope produced by aggrecanase cleavage of aggrecan. RESULTS: Aggrecan fragments generated from explants treated with tumour necrosis factor had N-terminal sequences consistent with cleavage of aggrecan at a restricted number of glutamyl bonds. Aggrecanase generated fragments were found in cartilage explant culture medium and chondrocyte monolayers. However, no aggrecanase activity could be detected in extracts of cartilage, or chondrocytes from which endogenous aggrecan fragments had been removed, under a variety of assay conditions. Deglycosylated aggrecan, added to explant cultures, efficiently inhibited endogenous aggrecan breakdown. CONCLUSIONS: Aggrecanase is active in cartilage and in chondrocyte monolayers, and its action is stimulated by tumour necrosis factor alpha. However, activity due to this enzyme could not be detected in vitro under our assay conditions, although a deglycosylated version of the substrate inhibited aggrecan breakdown in explant cultures.
PMCID: PMC379611  PMID: 9292151
To examine the relationships between biochemical composition and mechanical properties of articular cartilage explants during interleukin-1 (IL-1)-induced degradation and post-exposure recovery.
Bovine articular cartilage explants were cultured for up to 32 days with or without 20 ng/mL interleukin-1. The dynamic shear modulus |G*dyn| and equilibrium and dynamic unconfined compression moduli (Eequil and |E*dyn|) were measured at intervals throughout the culture period. In a subsequent recovery study, explants were cultured for 4 days with or without 20ng/mL IL-1 and for an additional 16 days in control media. The dynamic moduli |E*dyn| and |G*dyn| were measured at intervals during degeneration and recovery. Conditioned media and explant digests were assayed for sulfated glycosaminoglycans (sGAG) and collagen content.
Continuous IL-1 stimulation triggered progressive decreases in Eequil, |E*dyn|, and |G*dyn| concomitant with the sequential release of sGAG and collagen from the explants. Brief IL-1 exposure resulted in a short release of sGAG but not collagen, followed by a gradual and incomplete repopulation of sGAG. The temporary sGAG depletion was associated with decreases in both |E*dyn| and |G*dyn| which also recovered after removal of IL-1. During IL-1-induced degradation and post-exposure recovery, explant mechanical properties correlated well with tissue sGAG concentration.
As previously shown for developing cartilages and engineered cartilage constructs, cytokine-induced changes in sGAG concentration (i.e., fixed charge density) are coincident with changes in compressive and shear properties of articular cartilage. Further, recovery of cartilage mechanical properties can be achieved by relief from proinflammatory stimuli and subsequent restoration of tissue sGAG concentration.
PMCID: PMC2745941  PMID: 19281879
interleukin-1; cartilage degradation; cartilage mechanics; composition-function relationships
19.  Mycoplasmas and bovine respiratory disease: studies related to pathogenicity and the immune response--a selective review. 
Three species of mycoplasma have been established as being of importance as causes of pneumonia in housed calves, based on pathogenicity studies and frequency of association with the disease. These three species are Mycoplasma bovis, M. dispar, and Ureaplasma diversum. M. bovis is the most pathogenic of these species but the disease outbreaks with which it is associated are sporadic. M. dispar is regularly isolated from pneumonic calves but is also found causing mild superficial and asymptomatic infections of the respiratory mucosa. The bovine ureaplasmas are serologically complex. They are distinct from ureaplasmas isolated from other non-ruminants by PAGE analysis, G + C content of DNA, and serology. A second species within the genus ureaplasma has been proposed to accommodate the bovine ureaplasmas, U. diversum. Control of mycoplasma respiratory infections of cattle based on immunization might be possible. Calves have been immunized against M. bovis and immunity has been related to antibody in the lung. M. dispar appears less immunogenic in calves than M. bovis and this may contribute to its pathogenicity.
PMCID: PMC2590553  PMID: 6382831
20.  Calmodulin stabilization of kinetochore microtubule structure to the effect of nocodazole 
The Journal of Cell Biology  1988;107(6):2243-2251.
To investigate the function of calmodulin (CaM) in the mitotic apparatus, the effect of microinjected CaM and chemically modified CaMs on nocodazole-induced depolymerization of spindle microtubules was examined. When metaphase PtK1 cells were microinjected with CaM or a CaM-TRITC conjugate, kinetochore microtubules (kMTs) were protected from the effect of nocodazole. The ability of microinjected CaM to subsequently protect kMTs from the depolymerizing effect of nocodazole was dose dependent, and was effective for approximately 45 min, with protection decreasing if nocodazole treatment was delayed for more than 60 min after injection of CaM. The CaM-TRITC conjugate, similar to native CaM, displayed the ability to activate bovine brain CaM- dependent adenylate cyclase in a Ca++-dependent manner and showed a Ca++-dependent mobility shift when subjected to PAGE. A heat-altered CaM-TRITC conjugate also protected kMTs from the effect of nocodazole. However, this modified CaM was not able to activate adenylate cyclase nor did it display a Ca++-dependent mobility shift when electrophoresed. In a permeabilized cell model system, both CaM analogs were observed to bind to the spindle in a Ca++-independent manner. In contrast, a performic acid-oxidized CaM did not have a protective effect on spindle structure when microinjected into metaphase cells before nocodazole treatment. The oxidized CaM did not activate adenylate cyclase and did not exhibit Ca++-dependent mobility on polyacrylamide gels. These results are interpreted as supporting the hypothesis that CaM binds to the mitotic spindle in a Ca++-independent manner and that CaM may serve in the spindle, at least in part, to stabilize kMTs.
PMCID: PMC2115647  PMID: 3198685
21.  Biosynthesis of the D2-cell adhesion molecule: post-translational modifications, intracellular transport, and developmental changes 
The Journal of Cell Biology  1984;99(6):2082-2091.
Posttranslational modifications and intracellular transport of the D2- cell adhesion molecule (D2-CAM) were examined in cultured fetal rat neuronal cells. Developmental changes in biosynthesis were studied in rat forebrain explant cultures. Two D2-CAM polypeptides with Mr of 187,000-210,000 (A) and 131,000-158,000 (B) were synthesized using radiolabeled precursors in cultured neurons. A and B were found to contain only N-linked complex oligosaccharides, and both polypeptides appeared to be polysialated as determined by [14C]mannosamine incorporation and precipitation with anti-polysialic acid antibody. The two polypeptides were sulfated in the trans-Golgi compartment and phosphorylated at the plasma membrane. D2-CAM underwent rapid intracellular transport, appearing at the cell surface within 35 min of synthesis. A and B were shown to be integral membrane proteins as seen by radioiodination by photoactivation employing a hydrophobic labeling reagent. In rat forebrain explant cultures, D2-CAM was synthesized as four polypeptides: A (195,000 Mr), B (137,000 Mr), C (115,000 Mr), and a group of polypeptides in the high molecular weight region (HMr) between 250,000 and 350,000. Peptide maps of the four polypeptides yielded similar patterns. Biosynthesis of C and HMr increased with age, relative to A and B. A and B were sulfated in embryonic brain, however, sulfation was not noticeable at postnatal ages. Phosphorylation, on the other hand, of A and B was observed at all ages examined. We suggest that D2-CAM function may be modified during development by changes in the relative synthesis of the different polypeptides, as well as by changes in their glycosylation and sulfation.
PMCID: PMC2113562  PMID: 6501413
22.  Aggrecanolysis and in vitro matrix degradation in the immature bovine meniscus: mechanisms and functional implications 
Arthritis Research & Therapy  2009;11(6):R173.
Little is known about endogenous or cytokine-stimulated aggrecan catabolism in the meniscal fibrocartilage of the knee. The objectives of this study were to characterize the structure, distribution, and processing of aggrecan in menisci from immature bovines, and to identify mechanisms of extracellular matrix degradation that lead to changes in the mechanical properties of meniscal fibrocartilage.
Aggrecanase activity in the native immature bovine meniscus was examined by immunolocalization of the aggrecan NITEGE neoepitope. To investigate mechanisms of cytokine-induced aggrecan catabolism in this tissue, explants were treated with interleukin-1α (IL-1) in the absence or presence of selective or broad spectrum metalloproteinase inhibitors. The sulfated glycosaminoglycan (sGAG) and collagen contents of explants and culture media were quantified by biochemical methods, and aggrecan catabolism was examined by Western analysis of aggrecan fragments. The mechanical properties of explants were determined by dynamic compression and shear tests.
The aggrecanase-generated NITEGE neoepitope was preferentially localized in the middle and outer regions of freshly isolated immature bovine menisci, where sGAG density was lowest and blood vessels were present. In vitro treatment of explants with IL-1 triggered the accumulation of NITEGE in the inner and middle regions. Middle region explants stimulated with IL-1 exhibited substantial decreases in sGAG content, collagen content, and mechanical properties. A broad spectrum metalloproteinase inhibitor significantly reduced sGAG loss, abrogated collagen degradation, and preserved tissue mechanical properties. In contrast, an inhibitor selective for ADAMTS-4 and ADAMTS-5 was least effective at blocking IL-1-induced matrix catabolism and loss of mechanical properties.
Aggrecanase-mediated aggrecanolysis, typical of degenerative articular cartilage, may play a physiologic role in the development of the immature bovine meniscus. IL-1-induced release of sGAG and loss of mechanical properties can be ascribed primarily to the activity of MMPs or aggrecanases other than ADAMTS-4 and ADAMTS-5. These results may have implications for the clinical management of osteoarthritis.
PMCID: PMC3003508  PMID: 19919704
23.  Production of monoclonal antibodies against calmodulin by in vitro immunization of spleen cells 
The Journal of Cell Biology  1983;96(4):1149-1154.
Monoclonal antibodies against the highly conserved ubiquitous calcium- binding protein, calmodulin (CaM), were produced by immunization of mouse primary spleen cell cultures. Dissociated spleen cells were cultured for 5 d in the presence of mixed thymocyte culture conditioned media (TCM) and purified bovine testes CaM (50 ng-1 mg). Following immunization, cells were fused with mouse myeloma cells (SP2/0, Ag 8.653) and cultured for 2-3 wk before initial screening for antibody. In five independent immunizations there was a range of 25-44% of the initial polyclonal cultures which produced antibodies reacting with purified CaM as determined by immunoassay. 80% of the cloned hybridoma produced IgM immunoglobulins while the remaining clones were IgG producers. This ratio was changed to 50% IgM and 50% IgG by subsequent extension of the in vitro immunization periods and reduced amounts of antigen and extended in vitro culturing. In vitro immunization introduces a new dimension to monoclonal antibody production where limited antigen or poorly antigenic proteins are of interest. The monoclonal antibodies produced in this study have enabled us to to selectively localize CaM in association with distinct subcellular structures, mitochondria, stress fibers, centrioles, and the mitotic spindle.
PMCID: PMC2112333  PMID: 6833395
24.  Conversion of embryonic form to adult forms of N-CAM in vitro: results from de novo synthesis of adult forms 
The Journal of Cell Biology  1985;101(2):412-419.
During normal development, the neural cell adhesion molecule N-CAM changes at the cell-surface from a sialic acid-rich embryonic, or E form, to several adult, or A forms that have less sialic acid (E-to-A conversion). To investigate the cellular and molecular mechanisms that underlie these changes, we have established conditions under which E-to- A conversion occurs in cultured explants of central nervous system tissues. Mouse cerebellum, chick spinal cord, and chick retina that express the E form of N-CAM were dissected and cultured on collagen gels. After 3-6 d in culture, increased proportions of A forms were synthesized, as revealed by specific immunoprecipitation and immunoblotting. The rate of E-to-A conversion and the proportions of the different A forms synthesized in vitro were similar to those observed for the tissues in vivo at comparable times. In addition, the explants incorporated radioactive precursors of amino sugars into N- CAM, and the electrophoretic mobilities of the E and A forms of N-CAM were altered by treatment with neuraminidase in a way comparable to that found for N-CAM obtained directly from tissue. These results suggest that the post translational processing in vitro was similar to that in vivo. Logistic studies on cell division and death in the explants suggested that E-to-A conversion resulted mainly from a specific increase in synthesis of A forms in individual cells rather than as a consequence of differential birth or death within distinct cell populations. The data were consistent with the possibility that the increase in synthesis of A forms occurred either in cells that had previously synthesized E forms or in a distinct population of cells that already synthesized A forms. Cells dissociated from embryonic central nervous system tissues and cultured in vitro were also found to undergo E-to-A conversion at the same rate as the explant cultures, which suggests that if intercellular signals were responsible for initiation of the change in synthetic pattern, they had already occurred in vivo before the time of culture. In pulse-chase experiments, the E form of N-CAM that was synthesized during the first day after explantation persisted as E form for several days, at times when newly synthesized N-CAM was predominantly in A forms. These results indicate that in cultured neural tissue, the E form of N-CAM is not processed into A forms but is gradually degraded and replaced by newly synthesized A forms.(ABSTRACT TRUNCATED AT 400 WORDS)
PMCID: PMC2113676  PMID: 4019580
25.  Differential contributions of Ng-CAM and N-CAM to cell adhesion in different neural regions 
The Journal of Cell Biology  1986;103(1):145-158.
Individual neurons can express both the neural cell adhesion molecule (N-CAM) and the neuron-glia cell adhesion molecule (Ng-CAM) at their cell surfaces. To determine how the functions of the two molecules may be differentially controlled, we have used specific antibodies to each cell adhesion molecule (CAM) to perturb its function, first in brain membrane vesicle aggregation and then in tissue culture assays testing the fasciculation of neurite outgrowths from cultured dorsal root ganglia, the migration of granule cells in cerebellar explants, and the formation of histological layers in the developing retina. Our strategy was initially to delineate further the binding mechanisms for each CAM. Antibodies to Ng-CAM and N-CAM each inhibited brain membrane vesicle aggregation but the binding mechanisms of the two CAMs differed. As expected from the known homophilic binding mechanism of N-CAM, anti-N- CAM-coated vesicles did not co-aggregate with uncoated vesicles. Anti- Ng-CAM-coated vesicles readily co-aggregated with uncoated vesicles in accord with a postulated heterophilic binding mechanism. It was also shown that N-CAM was not a ligand for Ng-CAM. In contrast to assays with brain membrane vesicles, cellular systems can reveal functional differences for each CAM reflecting its relative amount (prevalence modulation) and location (polarity modulation). Consistent with this, each of the three cellular processes examined in vitro was preferentially inhibited only by anti-N-CAM or by anti-Ng-CAM antibodies. Both neurite fasciculation and the migration of cerebellar granule cells were preferentially inhibited by anti-Ng-CAM antibodies. Anti-N-CAM antibodies inhibited the formation of histological layers in the retina. The data on perturbation by antibodies were correlated with the relative levels of expression of Ng-CAM and N-CAM in each of these different neural regions. Quantitative immunoblotting experiments indicated that the relative Ng-CAM/N-CAM ratios in comparable extracts of brain, dorsal root ganglia, and retina were respectively 0.32, 0.81, and 0.04. During culture of dorsal root ganglia in the presence of nerve growth factor, the Ng-CAM/N-CAM ratio rose to 4.95 in neurite outgrowths and 1.99 in the ganglion proper, reflecting both polarity and prevalence modulation. These results suggest that the relative ability of anti-Ng-CAM and anti-N-CAM antibodies to inhibit cell-cell interactions in different neural tissues is strongly correlated with the local Ng-CAM/N-CAM ratio.(ABSTRACT TRUNCATED AT 400 WORDS)
PMCID: PMC2113806  PMID: 3522601

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