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Objective: To evaluate the role of simulation models and previous surgical experience on subjective and objective stress levels of students performing their 1st elective surgery within the veterinary curriculum., Sample: 141 third-year veterinary students., Methods: Using a pre-post experimental design, salivary alpha-amylase, and cortisol were evaluated as markers of physiologic stress response before students' first elective surgery. Student self-reported State-Trait Anxiety Inventory (STAI) scores and quantitative measures of experience were correlated to biomarker results., Results: No association was found for change in salivary biomarkers of stress, alpha-amylase, and cortisol, between baseline and presurgical samples accounting for gender, age, type of elective surgery performed, previous surgical experience, or simulation model use. Salivary cortisol levels were markedly elevated falling between the 66th and 99th percentile compared to an age and gender-matched population. Salivary alpha-amylase levels were also 2 to 3 times higher than those recorded by other health professionals. Veterinary student STAI scores were high falling between the 65th and 73rd percentile compared to working adults in the general population., Clinical Relevance: Veterinary students' salivary cortisol, alpha-amylase, and STAI scores fell into the upper 2/3rds of the general population, demonstrating a high level of stress. Simulation models and previous surgical experience were not associated with decreased stress. Further evaluation of the implementation of high-fidelity simulation models and the role of stress on performance is indicated.

Currently, intraoperative tumour margin imaging is not routinely utilized in veterinary medicine. Optical coherence tomography (OCT) allows for real-time assessment of tissue morphology of 1-2 mm depth. The aims of this study were (1) to compare the histologic and OCT features of excised canine skin and subcutaneous specimens, and (2) to determine the diagnostic accuracy of OCT for surgical margin evaluation. The authors hypothesized that OCT imaging would correlate well with histopathology and that OCT would be sensitive for detection of incomplete margins. Eighty dogs were prospectively enrolled. Tumours were excised, and the surgical margins were imaged using a spectral domain OCT system. The tumour type and completeness of excision were determined by histopathology. Nine blinded observers received training in OCT image interpretation and were then given a set of OCT images and videos. The observers assigned each image/video a grade from 1 (no tumour) to 4 (tumour) and the results were compared to histopathology. The overall median sensitivity and specificity of OCT imaging for detection of incomplete margins were 86.7% and 84.6%, respectively. A potential limitation is that observers had varied experience with OCT image interpretation, ranging from no prior experience to participating in a previous OCT project. OCT is sensitive for detection of incomplete margins and could be a promising real-time surgical margin imaging modality. Further study is needed to evaluate intraoperative applications of OCT and its impact on tumour recurrence and long-term outcome., (© 2022 The Authors. Veterinary and Comparative Oncology published by John Wiley & Sons Ltd.)

Objective: To evaluate outcomes of dogs with parathyroid carcinoma (PTC) treated by surgical excision and to describe the incidence of postoperative hypocalcemia, degree of hypocalcemia, duration of hospitalization, duration of calcium supplementation, and survival time., Animals: 100 client-owned dogs with PTC admitted to academic, referral veterinary institutions., Procedures: In a retrospective multi-institutional study, medical records of dogs undergoing surgical excision of PTC between 2010 to 2019 were reviewed. Signalment, relevant medical history, clinical signs, clinicopathologic testing, imaging, surgical findings, intraoperative complications, histologic examination, and survival time were recorded., Results: 100 dogs with PTC were included, and 96 dogs had clinical or incidental hypercalcemia. Common clinical signs included polyuria (44%), polydipsia (43%), hind limb paresis (22%), lethargy (21%), and hyporexia (20%). Cervical ultrasonography detected a parathyroid nodule in 91 of 91 dogs, with a single nodule in 70.3% (64/91), 2 nodules in 25.3% (23/91), and ≥ 3 nodules in 4 (4/91)% of dogs. Hypercalcemia resolved in 89 of 96 dogs within 7 days after surgery. Thirty-four percent of dogs developed hypocalcemia, on the basis of individual analyzer ranges, within 1 week after surgery. One dog had metastatic PTC to the prescapular lymph node, and 3 dogs were euthanized for refractory postoperative hypocalcemia. Estimated 1-, 2-, and 3-year survival rates were 84%, 65%, and 51% respectively, with a median survival time of 2 years., Conclusions and Clinical Relevance: Excision of PTC results in resolution of hypercalcemia and excellent long-term tumor control. Surgical excision of PTC is recommended because of resolution of hypercalcemia and a good long-term prognosis. Future prospective studies and long-term follow-up are needed to further assess primary tumor recurrence, metastasis, and incidence of postoperative hypocalcemia.

Background: A strategy for reducing adverse pregnancy outcomes is the expanded implementation of maternity waiting homes (MWHs). We assessed factors influencing MWH use, as well as the association between MWH stay and obstetric outcomes in a hospital in rural Ethiopia., Methods: Data from medical records of the Glenn C. Olson Memorial Primary Hospital obstetric ward were cross matched with records from the affiliated MWH between 1 and 2011 to 31 March 2014. Poisson regression with robust variance was conducted to estimate the relative risk (RR) of childbirth complications associated with MWH use vs. non-use. Five key informant interviews of a convenience sample of three MWH staff and two users were conducted and a thematic analysis performed of social, cultural, and economic factors underlying MWH use., Results: During the study period, 489 women gave birth at the hospital, 93 of whom were MWH users. Common reasons for using the MWH were post-term status, previous caesarean section/myomectomy, malposition/malpresentation, and low-lying placenta, placenta previa, or antepartum hemorrhage, and hypertension or preeclampsia. MWH users were more likely than non-users to have had a previous caesarean Sec. (15.1 % vs. 5.3 %, p < 0.001) and to be post-term (21.5 % vs. 3.8 %, p < 0.001). MWH users were also more likely to undergo a caesarean Sec. (51.0 % vs. 35.4 %, p < 0.05) and less likely (p < 0.05) to have a spontaneous vaginal delivery (49.0 % vs. 63.6 %), obstructed labor (6.5 % vs. 14.4 %) or stillbirth (1.1 % vs. 8.6 %). MWH use (N = 93) was associated with a 77 % (adjusted RR = 0.23, 95 % Confidence Interval (CI) 0.12-0.46, p < 0.001) lower risk of childbirth complications, a 94 % (adjusted RR = 0.06, 95 % CI 0.01-0.43, p = 0.005) lower risk of fetal and newborn complications, and a 73 % (adjusted RR = 0.27, 95 % CI 0.13-0.56, p < 0.001) lower risk of maternal complications compared to MWH non-users (N = 396). Birth weight [median 3.5 kg (interquartile range 3.0-3.8) vs. 3.2 kg (2.8-3.5), p < 0.001] and 5-min Apgar scores (adjusted difference = 0.25, 95 % CI 0.06-0.44, p < 0.001) were also higher in offspring of MWH users. Opportunity costs due to missed work and need to arrange for care of children at home, long travel times, and lack of entertainment were suggested as key barriers to MWH utilization., Conclusions: This observational, non-randomized study suggests that MWH usage was associated with significantly improved childbirth outcomes. Increasing facility quality, expanding services, and providing educational opportunities should be considered to increase MWH use.

Objective: To compare in vitro knot holding strength of the laparoscopic Miller's knot (LMK), open Miller's knot (MK), open surgeon's throw (Sx), and laparoscopic surgeon's throw (LSx) in a vascular pedicle model when used as the first throw for vascular ligation., Study Design: Experimental study., Sample Population: Ten constructs each of the Miller's knot and surgeon's throw performed openly and laparoscopically with 2-0 polyglyconate suture., Methods: Knot holding strengths of the LMK, MK, LSx, and Sx knots were evaluated on balloon dilation catheters used as vascular pedicle models. Laparoscopic knots were tied in a laparoscopic box trainer. Knot constructs were pressure tested to failure. Results were compared by Kruskal-Wallis and Steel-Dwass comparisons., Results: Both MK and LMK had mean leakage pressures above 300 mm Hg. The MK leaked at higher pressure than all other knots, including the LMK (P < .001). The LMK leaked at greater pressures compared with the Sx and the LSx (P < .001). No difference was detected between leaking pressures of the Sx and the LSx (P = .226), with both leaking at pressures below 40 mm Hg., Conclusion: The LMK created a more secure first throw compared with the Sx and leaked at supraphysiologic pressures., Clinical Significance: The LMK has excellent knot holding strength on a vascular pedicle model and may be further evaluated for clinical application., (© 2020 The American College of Veterinary Surgeons.)

Coxsackievirus typically infects humans via the gastrointestinal tract, which has a large number of microorganisms collectively referred to as the microbiota. To study how the intestinal microbiota influences enteric virus infection, several groups have used an antibiotic regimen in mice to deplete bacteria. These studies have shown that bacteria promote infection with several enteric viruses. However, very little is known about whether antibiotics influence viruses in a microbiota-independent manner. In this study, we sought to determine the effects of antibiotics on coxsackievirus B3 (CVB3) using an in vitro cell culture model in the absence of bacteria. We determined that an aminoglycoside antibiotic, neomycin, enhanced the plaque size of CVB3 strain Nancy. Neomycin treatment did not alter viral attachment, translation, or replication. However, we found that the positive charge of neomycin and other positively charged compounds enhanced viral diffusion by overcoming the negative inhibitory effect of sulfated polysaccharides present in agar overlays. Neomycin and the positively charged compound protamine also enhanced plaque formation of reovirus. Overall, these data provide further evidence that antibiotics can play noncanonical roles in viral infections and that this should be considered when studying enteric virus-microbiota interactions. IMPORTANCE Coxsackieviruses primarily infect the gastrointestinal tract of humans, but they can disseminate systemically and cause severe disease. Using antibiotic treatment regimens to deplete intestinal microbes in mice, several groups have shown the bacteria promote infection with a variety of enteric viruses. However, it is possible that antibiotics have microbiota-independent effects on viruses. Here we show that an aminoglycoside antibiotic, neomycin, can influence quantification of coxsackievirus in cultured cells in the absence of bacteria., (Copyright © 2019 Woods Acevedo et al.)

Salmonella is one of the most common foodborne pathogens found in retail fresh meat products. The purpose of this study was to characterize the Salmonella that is found in common types of fresh ground meats available to consumers in grocery stores in the Brookings, South Dakota, area. Salmonella serotypes were detected in 50 (19%) of 261 retail fresh ground meat samples, with 2 (2%) of 115 ground turkey samples, 6 (14%) of 42 chicken samples, and 42 (40%) of 104 ground pork samples testing positive for Salmonella. The Salmonella isolates were sequenced using an Illumina MiSeq genome sequencer. The resulting genomic sequences were analyzed to determine the serotypes of the isolates and to detect the presence of virulence and antibiotic resistance genes. The Salmonella isolated from the ground meats belonged to 23 different serotypes. The predominant serotype isolated from ground chicken was Enteriditis (5 of 6, 83%). Among the ground pork isolates, the most common serotypes were the potential monophasic variant of Typhimurium (5 of 42, 12%), Uganda (5 of 42, 12%), Anatum (4 of 42, 10%), Derby (3 of 42, 7%), Infantis (3 of 42, 7%), and London (3 of 42, 7%). Among the 45 Salmonella isolates tested to determine their resistance to common veterinary antibiotics, 25 (56%) were found to be susceptible to all 14 antibiotics tested, 11 (24%) were resistant to 1 antibiotic, 4 (9%) were resistant to 2 antibiotics, 1 (2%) was resistant to 3 antibiotics, 2 (4%) were resistant to 4 antibiotics, 1 (2%) was resistant to 8 antibiotics, and 1 (2%) was resistant to 10 antibiotics. The most common antibiotic resistances observed in this study were to streptomycin (15 of 45, 33%), tetracycline (11 of 45, 24%), and sulfisoxazole (7 of 45, 16%). The results of phenotypic evaluation of antibiotic resistance profiles of Salmonella isolates correlated well with the antibiotic resistance genes detected in the genomic sequences of the isolates.

RNA viruses exist in genetically diverse populations due to high levels of mutations, many of which reduce viral fitness. Interestingly, intestinal bacteria can promote infection of several mammalian enteric RNA viruses, but the mechanisms and consequences are unclear. We screened a panel of 41 bacterial strains as a platform to determine how different bacteria impact infection of poliovirus, a model enteric virus. Most bacterial strains, including those extracted from cecal contents of mice, bound poliovirus, with each bacterium binding multiple virions. Certain bacterial strains increased viral co-infection of mammalian cells even at a low virus-to-host cell ratio. Bacteria-mediated viral co-infection correlated with bacterial adherence to cells. Importantly, bacterial strains that induced viral co-infection facilitated genetic recombination between two different viruses, thereby removing deleterious mutations and restoring viral fitness. Thus, bacteria-virus interactions may increase viral fitness through viral recombination at initial sites of infection, potentially limiting abortive infections., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Objective: To quantify uptake of long-acting reversible contraceptives (LARC)-intrauterine devices (IUDs) and hormonal implants-among U.S. Army active-duty female soldiers and identify characteristics associated with uptake., Methods: This retrospective cohort study used the Stanford Military Data Repository, which includes all digitally recorded health encounters for active-duty U.S. Army soldiers from 2011 to 2014. We analyzed data from women aged 18-44 years to assess rates of LARC initiation using medical billing codes. We then evaluated predictors of LARC initiation using multivariable regression., Results: Among 114,661 servicewomen, 14.5% received a LARC method; among those, 60% received an IUD. Intrauterine device insertions decreased over the study period (38.7-35.9 insertions per 1,000 women per year, β=0.14, 95% confidence interval [CI] -0.23 to -0.05, P<.05), whereas LARC uptake increased, driven by an increase in implant insertions (20.3-35.4/1,000 women per year, β=0.41, CI 0.33-0.48, P<.001). Younger age was a positive predictor of LARC uptake: 32.4% of IUD users and 62.6% of implant users were in the youngest age category (18-22 years) compared with 9.6% and 2.0% in the oldest (36-44 years). The likelihood of uptake among the youngest women (compared with oldest) was most marked for implants (adjusted relative risk 7.12, CI 5.92-8.55; P<.001). A total of 26.2% of IUD users had one child compared with 13.2% among non-LARC users (adjusted relative risk 1.94, CI 1.85-2.04, P<.001). The majority (52.2%) of those initiating IUDs were married, which was predictive of uptake over never-married women (adjusted relative risk 1.52, CI 1.44-1.59, P<.001)., Conclusion: Among servicewomen, we observed low but rising rates of LARC insertion, driven by increasing implant use. Unmarried and childless soldiers were less likely to initiate LARC. These findings are consistent with potential underutilization and a need for education about LARC safety and reversibility in a population facing unique consequences for unintended pregnancies.

The plaque assay is a common technique used to measure virus concentrations and is based upon the principle that each plaque represents a single infectious unit. As such, the number of plaques is expected to correlate linearly with the virus dilution plated, and each plaque should be formed by a single founder virus. Here, we examined whether more than one virus can contribute to plaque formation. By using genetic and phenotypic assays with genetically marked polioviruses, we found that multiple parental viruses are present in 5 to 7% of plaques, even at an extremely low multiplicity of infection. We demonstrated through visual and biophysical assays that, like many viral stocks, our viral stocks contain both single particles and aggregates. These data suggest that aggregated virions are capable of inducing coinfection and chimeric plaque formation. In fact, inducing virion aggregation via exposure to low pH increased coinfection in a flow cytometry-based assay. We hypothesized that plaques generated by viruses with high mutation loads may have higher coinfection frequencies due to processes restoring fitness, such as complementation and recombination. Indeed, we found that coinfection frequency correlated with mutation load, with 17% chimeric plaque formation for heavily mutagenized viruses. Importantly, the frequency of chimeric plaques may be underestimated by up to threefold, since coinfection with the same parental virus cannot be scored in our assay. This work indicates that more than one virus can contribute to plaque formation and that coinfection may assist plaque formation in situations where the amount of genome damage is high. IMPORTANCE One of the most common methods to quantify viruses is the plaque assay, where it is generally presumed that each plaque represents a single infectious virus. Using genetically marked polioviruses, we demonstrate that a plaque can contain more than one parental virus, likely due to aggregates within virus stocks that induce coinfection of a cell. A relatively small number of plaques are the products of coinfection for our standard virus stocks. However, mutagenized virus stocks with increased genome damage give rise to a higher amount of plaques that are chimeric. These results suggest that coinfection may aid plaque formation of viruses with genome damage, possibly due to processes such as complementation and recombination. Overall, our results suggest that the relationship between viral dilution and plaque number may not be linear, particularly for mutagenized viral populations., (Copyright © 2017 Aguilera et al.)

Neurotropic viruses initiate infection in peripheral tissues prior to entry into the central nervous system (CNS). However, mechanisms of dissemination are not completely understood. We used genetically marked viruses to compare dissemination of poliovirus, yellow fever virus 17D (YFV-17D), and reovirus type 3 Dearing in mice from a hind limb intramuscular inoculation site to the sciatic nerve, spinal cord, and brain. While YFV-17D likely entered the CNS via blood, poliovirus and reovirus likely entered the CNS by transport through the sciatic nerve to the spinal cord. We found that dissemination was inefficient in adult immune-competent mice for all three viruses, particularly reovirus. Dissemination of all viruses was more efficient in immune-deficient mice. Although poliovirus and reovirus both accessed the CNS by transit through the sciatic nerve, stimulation of neuronal transport by muscle damage enhanced dissemination only of poliovirus. Our results suggest that these viruses access the CNS using different pathways., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The host and viral factors that influence disease outcome during flavivirus infections are not fully understood. Using the live attenuated yellow fever virus (YFV) vaccine strain 17D as a model system we evaluated how viral dose, inoculation route and immunopathogenesis contributed to disease outcome in mice deficient in the type I IFN response. We found that YFV-17D infection of IFN-α/β receptor knockout mice resulted in three distinct disease outcomes: no clinical signs of disease, fatal viscerotropic disease or fatal neurotropic disease. Interestingly, viral load at disease onset did not correlate with disease outcome. However, we found increased immune infiltrates in the brain tissues of mice that developed neurotropic disease. Additionally, mice that developed viscerotropic disease, as characterized by liver and spleen pathology and/or intestinal haemorrhage, had significantly elevated levels of alanine aminotransferase, monocyte chemotactic protein and IFN-inducible protein (IP)-10 as compared with mice with no clinical signs of disease or neurotropic disease. Furthermore, mice treated with recombinant IP-10 throughout YFV-17D infection showed increased mortality and an increased percentage of mice with viscerotropic disease. Our results demonstrated that viral load did not correlate with pathogenesis, and the host immune response played a pivotal role in disease outcome and contributed to YFV-17D pathogenesis in mice., (© 2015 The Authors.)

Arboviruses such as yellow fever virus (YFV) are transmitted between arthropod vectors and vertebrate hosts. While barriers limiting arbovirus population diversity have been observed in mosquitoes, whether barriers exist in vertebrate hosts is unclear. To investigate whether arboviruses encounter bottlenecks during dissemination in the vertebrate host, we infected immunocompetent mice and immune-deficient mice lacking alpha/beta interferon (IFN-α/β) receptors (IFNAR⁻/⁻ mice) with a pool of genetically marked viruses to evaluate dissemination and host barriers. We used the live attenuated vaccine strain YFV-17D, which contains many mutations compared with virulent YFV. We found that intramuscularly injected immunocompetent mice did not develop disease and that viral dissemination was restricted. Conversely, 32% of intramuscularly injected IFNAR⁻/⁻ mice developed disease. By following the genetically marked viruses over time, we found broad dissemination in IFNAR⁻/⁻ mice followed by clearance. The patterns of viral dissemination were similar in mice that developed disease and mice that did not develop disease. Unlike our previous results with poliovirus, these results suggest that YFV-17D encounters no major barriers during dissemination within a vertebrate host in the absence of the type I IFN response.

Enterotoxigenic Escherichia coli (ETEC) is an important diarrheal agent of young domestic animals. Currently, there are no commercially available non-living vaccines to protect weaned pigs from the disease and no major veterinary biologics company markets a postweaning ETEC vaccine of any kind. While efforts have been made to develop a non-living postweaning ETEC vaccine for pigs, studies have been limited to the assessment of immune responses to experimental immunogens. In the present study, we describe a reproducible gnotobiotic piglet model of post-weaning ETEC diarrhea and efficacy tests in that model of subunit vaccines consisting of K88 (F4) fimbriae and/or heat labile enterotoxin (LT) delivered by the intranasal route. We also report antibody responses to the vaccine antigens. Piglets vaccinated with both antigens mounted a substantial immune response with serum and cecal antibody titers to K88 antigen significantly greater than those of controls. Serum anti-LT antibody titers were also significantly greater than those of controls. Piglets vaccinated with both antigens remained healthy following challenge with ETEC. At least some pigs vaccinated with either antigen alone, and most of the control piglets developed dehydrating diarrhea and suffered significant weight loss. The results of this study suggest that an intranasal vaccine consisting of both antigens is highly protective against a vigorous experimental challenge of pigs with K88+ ETEC, while that against either antigen alone is not. The current study provides a system whereby various ETEC antigens and/or combinations of antigens can be tested in exploring strategies for the development of vaccines for ETEC., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Objective: To assess the level of interest of university students enrolled in veterinary science courses toward becoming a veterinarian, reasons supporting or discouraging their interest, when those attitudes were formed, and future plans for those pursuing veterinary medicine as a career., Design: Cross-sectional study., Sample: 585 university students in South Dakota enrolled in 2 veterinary science courses over a 6-year period., Procedures: Each year, students enrolled in the 2 courses answered survey questions pertaining to their interest in becoming a veterinarian, background, and future plans., Results: Most students enrolled in these courses desired to become a veterinarian at some time in their lives. Females were more likely than males to indicate veterinary medicine as their current career choice. Most students developed their interest during grades 10 to 12. Females developed an interest in veterinary medicine earlier than did males. Enjoyment of animals, intellectual stimulation, and the opportunity to actively work outdoors were cited frequently as reasons for interest in veterinary medicine. Increased duration of education, high educational costs, and preveterinary coursework difficulty were major reasons for disinterest in becoming a veterinarian. Of students pursuing the profession, desired practice type correlated strongly with previous animal experience., Conclusions and Clinical Relevance: Considering that most students, especially males, developed their interest in veterinary medicine during grades 10 to 12, elementary school may be the best starting point for exposing students to veterinary medicine. To increase interest in large animal practice among students entering veterinary school, livestock experiences should be provided to children with no farm experience during their elementary, middle, and high school years. In our survey population, cost and duration of veterinary education had a significant negative influence on student interest in the profession.

Background: Current treatments for chronic hepatitis C virus (HCV) employing pegylated interferon (PEG-IFN) plus ribavirin are successful in approximately 50% of patients. Consensus IFN (CIFN) is a recombinant type I IFN that has demonstrated efficacy where conventional therapy has failed. We evaluated the host cell antiviral response and anti-HCV actions induced by IFN-alpha2a, PEG-IFN-alpha2b or CIFN on cultured immortalized human hepatocytes, Huh7 human hepatoma cells and Huh7 cells that harboured genetically distinct HCV RNA replicons or were infected with HCV 2a., Methods: Cultured cells were treated with each IFN at relevant dosing based upon the pharmacological attainable in vivo serum maximum IFN concentrations. Gene expression and antiviral properties were measured using protein, RNA and virus quantification assays., Results: CIFN treatment maximally triggered Janus kinase signal transducer and activator of transcription signalling in association with enhanced IFN-stimulated gene (ISG) expression. Increased antiviral potency of CIFN was associated with enhancement of IFN-induced blockade upon viral protein synthesis, protection of the cellular IFN promoter stimulator-1 (IPS-1) protein from HCV proteolysis and reduced replication of an IFN-resistant HCV replicon variant. Microarray analyses revealed that CIFN treatment induced a distinct pattern of ISG expression in cultured hepatocytes compared with other IFNs., Conclusions: CIFN exhibits increased anti-HCV potency over IFN-alpha2a and PEG-IFN through maximal and distinct induction of ISG expression and enhanced intracellular innate antiviral response, while protecting IPS-1 from HCV proteolysis. CIFN might offer a treatment regimen imparting translational control programmes and restoration of the retinoic acid-inducible gene-1/IPS-1 pathway and could be considered for previous treatment failures.

Antiviral immunity in mammals involves several levels of surveillance and effector actions by host factors to detect viral pathogens, trigger alpha/beta interferon production, and to mediate innate defenses within infected cells. Our studies have focused on understanding how these processes are regulated during infection by hepatitis C virus (HCV) and West Nile virus (WNV). Both viruses are members of the Flaviviridae and are human pathogens, but they each mediate a very different disease and course of infection. Our results demonstrate common and unique innate immune interactions of each virus that govern antiviral immunity and demonstrate the central role of alpha/beta interferon immune defenses in controlling the outcome of infection.

Galectins (Gal) are an evolutionarily conserved family of 15 carbohydrate-binding proteins (lectins) that are widely distributed in normal and neoplastic cells in a wide range of organisms. They have roles in inflammation, cell adhesion, tumor progression, and metastasis. The function and distribution of Gal-3 and Gal-1 are well characterized, but less information is available about Gal-4. Recent studies have localized Gal-4 in the enterochromaffin cells of the porcine and murine small intestine. We examined the expression of Gal-4 in primary and metastatic human ileal carcinoid tumors as well as in carcinoid tumors of the stomach, lung, and rectum. A total of 44 primary and 42 ileal metastatic carcinoid tumors were examined by immunohistochemistry using tissue microarrays (TMA) with monoclonal antibodies to Gal-4, Gal-3, and Gal-1. Pulmonary (n = 7), rectal (n = 6), and gastric (n = 6) carcinoids were examined with larger tissue sections. A total of 18 pancreatic neuroendocrine tumors were also examined with larger tissue sections. Western blots of three ileal carcinoids were also done. Gal-4 was most highly expressed in the ileal carcinoids and the levels of expression tended to be higher in primary ileal carcinoids compared to the metastatic tumors (p = 0.069). All 18 pancreatic neuroendocrine tumors were negative for Gal-1, Gal-3, and Gal-4. Western blot showed a 32 kDa band for Gal-4 in the ileal carcinoids. Gal-3 and Gal-1 were not detected in the metastatic ileal carcinoids by Western blotting. Gastric carcinoids also expressed Gal-4, but very few pulmonary or rectal carcinoids were positive for Gal-4 (p = 0.002). Lower levels of Gal-1 and Gal-3 expression were present in ileal carcinoids compared to primary pulmonary and rectal tumors. These results show a differential distribution of Gal-4 in carcinoid tumors in different locations of the gastrointestinal tract and the lungs.

Viral signaling through retinoic acid-inducible gene-I (RIG-I) and its adaptor protein, IFN promoter-stimulator 1 (IPS-1), activates IFN regulatory factor-3 (IRF-3) and the host IFN-alpha/beta response that limits virus infection. The hepatitis C virus (HCV) NS3/4A protease cleaves IPS-1 to block RIG-I signaling, but how this regulation controls the host response to HCV is not known. Moreover, endogenous IPS-1 cleavage has not been demonstrated in the context of HCV infection in vitro or in vivo. Here, we show that HCV infection transiently induces RIG-I- and IPS-1-dependent IRF-3 activation. This host response limits HCV production and constrains cellular permissiveness to infection. However, HCV disrupts this response early in infection by NS3/4A cleavage of IPS-1 at C508, releasing IPS-1 from the mitochondrial membrane. Cleavage results in subcellular redistribution of IPS-1 and loss of interaction with RIG-I, thereby preventing downstream activation of IRF-3 and IFN-beta induction. Liver tissues from chronically infected patients similarly demonstrate subcellular redistribution of IPS-1 in infected hepatocytes and IPS-1 cleavage associated with a lack of ISG15 expression and conjugation of target proteins in vivo. Importantly, small-molecule inhibitors of NS3/4A prevent cleavage and restore RIG-I signaling of IFN-beta induction. Our results suggest a dynamic model in which early activation of IRF-3 and induction of antiviral genes are reversed by IPS-1 proteolysis and abrogation of RIG-I signaling as NS3/4A accumulates in newly infected cells. HCV protease inhibitors effectively prevent IPS-1 proteolysis, suggesting they may be capable of restoring this innate host response in clinical practice.

Many strains of enterotoxigenic Escherichia coli (ETEC) that cause diarrhoea in young piglets secrete a heat-labile enterotoxin (LTp) that binds to specific glycoconjugates on porcine intestinal epithelial cells. Binding of LTp to an appropriate glycoconjugate facilitates the uptake and trafficking of the toxin into the cell, where it stimulates intracellular changes that promote fluid secretion and diarrhoea. The objective of the current study was to identify the LTp-binding glycoconjugates on porcine intestinal epithelial cells, the natural target cells for LTp. We found that LTp binds, in an age-correlated manner, to an acidic glycosphingolipid (GSL) that co-migrated with GM1 on thin-layer chromatography (TLC), a small acidic GSL that appears to be a sulphatide, a neutral GSL that co-migrated with neolactotetraglycosylceramide (nLc4) on TLC, and two glycoproteins (36 and 205 kDa). Of these potential LTp receptors, the GM1-co-migrating GSL was detected most intensely in young animals, while the other four LTp-binding glycoconjugates were detected most intensely in older pigs (> or= 4 weeks). Since ETEC primarily cause disease in young piglets, the GM1-co-migrating GSL is the most likely candidate for a functional LTp receptor.

High molecular weight glycoproteins, MUC1 and MUCX, originating from bovine milk, were compared with regard to their distribution in milk fat and skim milk fractions and for presence of carbohydrate structures. Polymorphic MUC1, which migrated into 6% resolving SDS-PAGE gels, was found in both milk fat globule membrane and skim milk phases of bovine milk. In contrast, MUCX, appearing as a non-polymorphic single band in 3% polyacrylamide stacking gels, was present only in the skim milk fraction. Peptide-N-glycosidase F digestion studies indicated that MUC1 and MUCX possessed N-glycans with MUC1 containing more N-glycans than MUCX. Exoglycosidase digestion studies revealed the existence of abundant terminal sialic acid residues in both MUC1 and MUCX. Lectin-binding studies showed that MUCX likely possessed more complex carbohydrate structures than MUC1. The complex carbohydrate structures carried by both MUC1 and MUCX suggest that they may have potential to bind a wide spectrum of pathogenic microorganisms. If that proves to be the case in vivo, such structures could have a role in preventing or reducing some infectious diseases.

Galectins are an evolutionarily conserved family of 15 different lectins found in various combinations in virtually every type of animal cell. One of the primary galectins expressed in intestinal epithelium is galectin-4, a tandem-repeat galectin with two carbohydrate-recognition domains in a single polypeptide chain. In the current study, we produced an anti-galectin-4 monoclonal antibody (MAb) for determining the distribution of galectin-4 in porcine small intestine to enhance our understanding of where galectin-4 performs its functions in the small intestine. In immunohistochemistry studies, this MAb detected galectin-4 primarily in the cytoplasm of absorptive epithelial cells lining intestinal villi. Mature epithelial cells at the villous tips stained the most intensely with this MAb, with progressively less intense staining observed along the sides of villi and into the crypts. In addition to its cytoplasmic localization, galectin-4 was also associated with nuclei in villous tip cells, indicating that some galectin-4 may migrate to the nucleus during terminal maturation of these cells. In intestinal crypts, a specific subset of cells, which may be enteroendocrine cells, expressed galectin-4 at a relatively high level. Galectin-4 distribution patterns were similar in all three regions (duodenum, jejunum, and ileum) of porcine small intestine.

Lactose-binding proteins with molecular masses of 14-, 17-, 18-, 28-, and 34-kDa were identified in extracts from porcine small intestinal mucosa. Amino acid sequence analysis of peptides generated by CNBr cleavage of the 34-kDa protein, the most abundant of these proteins, identified this protein as porcine galectin-4. To determine if a porcine homolog of murine galectin-6 is expressed in small intestine, primers for a reverse transcriptase-polymerase chain reaction (RT-PCR) were developed that amplified across the linker region of galectin-4, which is the region that differs between murine galectins-4 and -6. Using these primers, this RT-PCR approach identified two galectin-4 isoforms that differed in the length of their linker region. The larger isoform, galectin-4.1, is nine amino acids longer in its linker region than the smaller isoform, galectin-4.2. Based on nucleotide sequence similarities, the two isoforms are likely splice variants of galectin-4 pre-mRNA and not products of separate genes like murine galectins-4 and -6.

Diarrheal disease caused by enterotoxigenic Escherichia coli expressing the K88 (F4) fimbrial adhesin (K88 ETEC) is a significant source of mortality and morbidity among newborn and weaned piglets. K88 fimbrial adhesins are filamentous surface appendages whose lectin (carbohydrate-binding) activity allows K88 ETEC to attach to specific glycoconjugates (receptors) on porcine intestinal epithelial cells. There are three variants of K88 adhesin (K88ab, K88ac, and K88ad), which possess different, yet related, carbohydrate-binding specificities. We used porcine serum transferrin (pSTf) and purified glycosphingolipids (GSL) to begin to define the minimal recognition sequence for K88 adhesin variants. We found that K88ab adhesin binds with high affinity to pSTf (dissociation constant, 75 microM), while neither K88ac nor K88ad adhesin recognizes pSTf. Degradation of the N-glycan on pSTf by extensive metaperiodate treatment abolished its interaction with the K88ab adhesin, indicating that the K88ab adhesin binds to the single N-glycan found on pSTf. Using exoglycosidase digestion of the pSTf glycan, we demonstrated that K88ab adhesin recognizes N-acetylglucosamine (GlcNAc) residues in the core of the N-glycan on pSTf. All three K88 variants were found to bind preferentially to GSL containing a beta-linked N-acetylhexosamine (HexNAc), either GlcNAc or N-acetylgalactosamine, in the terminal position or, alternatively, in the penultimate position with galactose in the terminal position. Considering the results from pSTf and GSL binding studies together, we propose that the minimal recognition sequence for the K88 adhesin variants contains a beta-linked HexNAc. In addition, the presence of a terminal galactose beta-linked to this HexNAc residue enhances K88 adhesin binding.

Sulofenur is one of the diarylsulfonylureas developed as an anticancer agent. Sulofenur possesses a broad spectrum of activity in several solid tumor models and has undergone extensive clinical trials based on its impressive preclinical activity. However, the clinical response of sulofenur has been disappointing because of the side effect of anemia. Furthermore, the anticancer mechanism of sulofenur and its diarylsulfonylurea analogs still remains unknown. Elucidation of the metabolic fates of sulofenur may help to delineate the mechanism and provide information to guide the structural modification for more potent anticancer agents with less side effects. We have identified a glutathione conjugate and a mercapturic acid conjugate from sulofenur-dosed rats with the aid of liquid chromatography/mass spectrometry. The fraction of the dose of sulofenur as the glutathione conjugate in the dosed-rat bile over 5 h was 0.12 +/- 0.03%, and the mercapturic acid conjugate in urine over 24 h was 1.4 +/- 0.7%. Protein binding of the glutathione conjugate and mercapturic acid conjugate was determined to be 20 +/- 3 and 84 +/- 2%, respectively, as opposed to >99% of sulofenur. The high protein binding of sulofenur requires a higher than in vitro dose, which is believed to cause the side effect of anemia. The significance of this metabolic pathway is that both conjugates were found to be glutathione reductase inhibitors and to possess anticancer activity comparable to sulofenur against human colon adenocarcinoma GC(3)/c1 cells, a sulofenur-sensitive cell line. These conjugates may serve as new leads for the development of novel anticancer agents.

Strains of enterotoxigenic Escherichia coli that express K88 fimbriae are among the most common causes of diarrhea in young pigs. Adhesion of bacteria to receptors on intestinal epithelial cells, mediated by K88 fimbriae, is the initial step in the establishment of infection. Three antigenic variants of K88 fimbriae exist in nature: K88ab, K88ac, and K88ad. K88ac is the most prevalent and may be the only variant of significance in swine disease. Each K88 fimbrial variant is composed of multiple antigenic determinants. Some of these determinants are shared among the three variants and may be referred to as conserved epitopes, whereas others are unique to a specific variant and may be referred to as variable epitopes. In this study, monoclonal antibodies (MAbs) specific to either variable or conserved epitopes of K88ac fimbriae were produced. The specificity of each MAb was tested by enzyme-linked immunosorbent and immunoblot assays. Fab fragments were prepared from these MAbs and were tested for their ability to block the binding of K88-positive bacteria and purified fimbriae to porcine enterocyte brush border vesicles and purified K88 receptors, respectively. The purified receptors were intestinal mucin-type sialoglycoproteins (IMTGP) isolated from porcine enterocytes (A. K. Erickson, D. R. Baker, B. T. Bosworth, T. A. Casey, D. A. Benfield, and D. H. Francis, Infect. Immun. 62:5404-5410, 1994). Fab fragments prepared from MAbs specific for variable epitopes blocked the binding of bacteria to brush borders and of fimbriae to IMTGP. However, those from MAbs specific for a conserved epitope did not. These observations indicate that the receptor-binding domain of a K88ac fimbria is contained, at least in part, within the antigenically variable epitopes of that fimbria. Epitope mapping for one of the MAbs, which recognizes a linear epitope on K88ac fimbriae, indicated that this MAb binds to the region from amino acid no. 64 to no. 107 on the major subunit of K88ac fimbriae.

The three antigenic variants of the K88 fimbrial adhesin (K88ab, K88ac, and K88ad) of enterotoxigenic Escherichia coli (ETEC) each exhibit unique specificity with regard to their hemagglutination characteristics. The variants are also unique in the specificity of their binding to the brush borders of enterocytes isolated from pigs with different genetic backgrounds. Diversity in enterocyte binding specificity suggests the existence of several K88 receptors, expressed individually or in various combinations on porcine enterocytes. Three candidate receptors have been identified that may explain the adhesion of K88 fimbrial variants to various porcine enterocytes. These receptors are an intestinal mucin-type sialoglycoprotein (IMTGP), an intestinal transferrin (GP74), and an intestinal neutral glycosphingolipid (IGLad). The IMTGP binds K88ab and K88ac, but not K88ad. The GP74 binds K88ab, but not K88ac or K88ad, and the IGLad binds K88ad, but not K88ab or K88ac. Each of the candidate receptors has been found in brush borders that are adhesive for the fimbriae that bind the respective receptor. They have not been found in brush borders that are not adhesive for those same fimbriae. The presence of IMTGP was highly correlated with susceptibility of neonatal gnotobiotic pigs to ETEC expressing K88ab or K88ac.

In this study, we identified a receptor for the K88ad fimbrial adhesin of Escherichia coli in neutral glycosphingolipid preparations from intestinal epithelial cells of K88ad-adhesive pigs, which was absent in preparations from K88ad-nonadhesive pigs. Neither K88ab nor K88ac adhesin variants bound to this neutral glycosphingolipid. Because this receptor is an intestinal glycosphingolipid that binds K88ad adhesin, it has been designated IGLad. Carbohydrate compositional analysis of a partially purified preparation of IGLad identified galactose, glucose, and N-acetylglucosamine in a ratio of 1.5:1.0:0.5 as the major monosaccharides. Preliminary characterization experiments using lectins showed that IGLad contains the terminal glycanic structure Galbeta1-4GlcNAc. Removal of terminal beta-linked galactose residues from IGLad decreased the recognition of IGLad by the K88ad adhesin, indicating that terminal beta-linked galactose is an essential component of the K88ad adhesin recognition site on IGLad. Studies with purified glycosphingolipid standards demonstrated that K88ad adhesin binds to neolactotetraosylceramide (nLc4Cer) (Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glcbeta1-1Cer) , lactotriosylceramide (GlcNAcbeta1-3Galbeta1-4Glcbeta1-1Cer) and lactotetraosylceramide (Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glcbeta1-1Cer) . Based on these studies, IGLad appears to be nLc4Cer.

Three antigenic variants of the K88 fimbrial adhesin exist in nature, K88ab, K88ac, and K88ad. Enterotoxigenic Escherichia coli (ETEC) strains that produce these fimbriae cause life-threatening diarrhea in some but not all young pigs. The susceptibility of pigs to these organisms has been correlated with the adherence of bacteria to isolated enterocyte brush borders. Whether that correlation holds for multiple K88 variants and over a broad genetic base of pigs is unknown and was the impetus for this study. We also desired to examine the correlation of the expression of a porcine intestinal brush border mucin-type glycoprotein (IMTGP) which binds K88ab and K88ac with the susceptibility of piglets to K88(+) ETEC. Of 31 neonatal gnotobiotic pigs inoculated with K88ab+ or K88ac+ ETEC, 13 developed severe diarrhea, became dehydrated, and died or became moribund. Another pig became severely lethargic but not dehydrated. In vitro brush border adherence analysis was not possible for 10 of the severely ill pigs due to colonization by challenge strains. However, of the 17 pigs that did not become severely ill, 8 (47%) had brush borders that supported the adherence of K88ab+ and K88ac+ bacteria in vitro, suggesting a poor correlation between in vitro brush border adherence and piglet susceptibility to K88(+) ETEC. By contrast, the expression of IMTGP was highly correlated with susceptibility to K88(+) ETEC. Of the 12 pigs that produced IMTGP, 11 developed severe diarrhea. The other pig that produced IMTGP became lethargic but not severely diarrheic. Only 2 of 18 pigs that did not produce IMTGP became severely diarrheic. Colonizing bacteria were observed in histologic sections of intestines from all pigs that expressed IMTGP except for the one that did not develop severe diarrhea. However, colonizing bacteria were observed in histologic sections from only one pig that did not produce IMTGP. The bacterial concentration in the jejuna and ilea of pigs expressing IMTGP was significantly greater (P < 0.005) than that in pigs not expressing IMTGP. These observations suggest the IMTGP is a biologically relevant receptor for K88ab+ and K88ac+ E. coli or a correlate for expression for such a receptor.

We have previously identified two mucin-type sialoglycoproteins from porcine intestinal epithelial cells with approximate molecular masses of 210 (intestinal mucin-type glycoprotein IMTGP-1) and 240 kDa (IMTGP-2) as receptors for the K88ab and K88ac fimbrial adhesins of Escherichia coli. These receptors are detected in intestinal brush border membrane preparations from pigs with adhesive phenotypes but not from pigs with nonadhesive phenotypes and are postulated to be important determinants of the susceptibility of pigs to K88ab+ and K88ac+ enterotoxigenic E. coli infections. Using exoglycosidase digestion studies, we have now determined that beta-linked galactose is an important component in the recognition of IMTGP-1 and IMTGP-2 by the K88ac adhesin. In addition, we observed a differential distribution of the K88ac adhesin binding activity of IMTGP-1 and IMTGP-2 along the crypt-villus axis, suggesting that receptor activity is dependent on the maturation state of the intestinal epithelial cells. Brush borders from immature intestinal epithelial cells possessed the highest concentrations of IMTGP-1 and IMTGP-2 receptor activity, with a progressive decrease in receptor activity as the cells mature. To characterize the differences in the carbohydrate moieties of IMTGP-1 and IMTGP-2, we developed a procedure for purifying the receptors, using phenol extraction followed by serial lectin affinity chromatography. Carbohydrate compositional analysis of the purified receptors indicated that the carbohydrate moieties of IMTGP-1 and IMTGP-2 consist of both N- and O-glycans containing galactose, glucose, sialic acid, mannose, N-acetylgalactosamine, N-acetylglucosamine, and fucose. The major difference between the two receptors is that IMTGP-2 contains a higher percentage of monosaccharides (mannose and glucose) commonly found in N-glycans.

We evaluated intestinal epithelial membrane preparations from five phenotypes of pigs, distinguished by the variant of K88 fimbrial adhesin (K88ab, K88ac, K88ad) which bind to their intestinal epithelial cells (A-all three variants, B-K88ab and K88ac, C-K88ab and K88ad, D-K88ad, and E-none of the variants), for the presence of K88 adhesin receptors. Intestinal brush border membranes were prepared from 20 animals (four from each phenotype). Brush border proteins, that had been separated using SDS-PAGE and transferred to nitrocellulose membranes, were overlaid with biotinylated K88 adhesin, 35S-labelled K88+ Escherichia coli, or biotinylated K88+ E. coli. Biotinylated K88ab and K88ac fimbrial adhesins and labelled E. coli expressing K88ab or K88ac adhesin bound to 210- and 240-kDa receptors in phenotype A and B, but not phenotype C, D, or E animals. In contrast, no phenotype-specific receptors were identified for the K88ad adhesin. Previously, purified K88ab and K88ac fimbriae were shown to block K88ad binding, but purified K88ad fimbriae were unable to block K88ab or K88ac binding in phenotype A animals. These results point to the existence of three K88 adhesin receptors to account for the observed phenotypes: (1) Receptor bcd binds all three variants and is found in phenotype A pigs, (2) Receptor bc (210- and 240-kDa receptors) binds K88ab and K88ac and is found in phenotype A and B pigs, and (3) Receptor d binds K88ad and is found in phenotype C and D pigs.

This study examined whether the provision of norepinephrine, as would be encountered within the highly innervated gastrointestinal system, affected the growth rate of enterotoxigenic Escherichia coli (ETEC) and the expression of the K99 pilus adhesin virulence-related factor. The addition of norepinephrine to serum-containing medium resulted in a 3- to 7-fold increase in the growth rate of the K99+ ETEC strain B44 as compared to growth in vehicle supplemented medium or medium supplemented with normetanephrine, a norepinephrine metabolite that contains one more methyl group than norepinephrine. ELISA analysis revealed that K99 pilus adhesin expression was increased in norepinephrine supplemented culture as compared to normetanephrine and vehicle supplemented controls. This increase occurred from 9 to 15 hours of incubation which represented the exponential growth phase for the norepinephrine supplemented culture. These results indicate that addition of norepinephrine affects both ETEC growth and expression of a specific virulence factor.

Four phenotypes of pigs distinguished by the variant(s) of K88 fimbrial adhesin (K88ab, K88ac, K88ad) that bind to their intestinal epithelial cells (I-none of the variants, II-K88ad, III-K88ab and K88ac, and IV-all three variants) have been identified. We hypothesize that the differences between the phenotypes are defined by the presence or absence of K88 adhesin receptors. We propose a three-receptor model to account for the observed phenotypes: 1) Receptor bed which binds all three variants and is found in phenotype IV, 2) Receptor be which binds K88ab and K88ac and is found in phenotype III and IV, and 3) Receptor d which binds K88ad and is found in phenotype II. We have identified the be receptor activity as a pair of mucin-type sialoglycoproteins (210 and 240 kDa). Although neither the bcd nor d receptor has been identified biochemically, their presence has been established using both blocking and receptor localization studies. Blocking studies using phenotype IV brush borders demonstrated that K88ab and K88ac fimbriae block the binding of E. coli expressing any of the K88 variants, but K88ad fimbriae block only K88ad E. coli binding. These results indicate that two receptors (bcd and bc) exist in the phenotype IV animals. Receptor localization studies on intestinal sections from phenotype IV animals showed that K88ab and K88ac adhesin binding is continuous from the crypt to the tip of the villus. The binding of the K88ad adhesin binding is multifocal in phenotype IV pigs, but continuous from crypt to tip of the villus in sections of phenotype II pigs. These studies verify the presence of two receptors (bcd and bc) in phenotype IV animals, and indicate that the K88ad receptor in phenotype IV animals (bcd) is different than in phenotype II animals (d).

The effects of protamine on the phosphorylase phosphatase activity of porcine cardiac protein phosphatase 2A1 (PP2A1) were complex and ionic strength dependent. Under ionic strength conditions that protamine activation was optimal, activation of PP2A1 by either dilution or heparin was prevented. A time-dependent deactivation of the protamine-stimulated phosphatase activity was observed when PP2A1 was preincubated with protamine. Protamine forms a very tight association with phosphorylase a, which is optimal at a 1:1 protamine:phosphorylase a monomer molar ratio. Protamine activation of PP2A1 activity, however, is not substrate-directed since the basic polypeptide did not stimulate either the activity of the catalytic subunit or trypsinolysis of [32P]phosphorylase a. The interaction of protamine with phosphorylase a does not apparently involve the phosphorylation site in the protein substrate (ser 14). The activation of PP2A1 by protamine is proposed to involve part of the basic polypeptide, not associated with phosphorylase a monomer, interacting with the regulatory and/or the catalytic subunit(s) of the phosphatase. A minimal model for the activation of PP2A1 by protamine was tested kinetically. In this model, free PP2A1 binds with decreasing affinities to the protamine:phosphorylase a complex, free phosphorylase a, and free protamine. Protamine decreases the K(m) of PP2A1 for the phosphorylase a monomer 5-fold and increases the Vmax 17-fold. Interaction of free protamine with PP2A1 inhibits the phosphatase activity.

We have previously identified two K88ac adhesion receptors (210 and 240 kDa) which are present in membrane preparations from adhesive but not nonadhesive porcine intestinal brush border cells; these adhesin receptors are postulated to be important determinants of the susceptibility of pigs to K88ac+ enterotoxigenic Escherichia coli infections (A.K. Erickson, J.A. Willgohs, S.Y. McFarland, D.A. Benfield, and D.F. Francis, Infect. Immun. 60:983-988, 1992). We now describe a procedure for the purification of these two receptors. Receptors were solubilized from adhesive intestinal brush border vesicles using deoxycholate and were purified by gel filtration chromatography on Sepharose CL-4B and then by hydroxyapatite chromatography. Amino acid compositional analyses indicated that the two receptors have similar amino acid compositions. The most distinguishing characteristic of both receptors is a high percentage of threonine and proline residues. Neuraminidase treatment caused the K88ac adhesin receptors to migrate with a slower mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, indicating that these receptors are sialoglycoproteins. Results from lectin-binding studies indicated that the receptors contain O-linked oligosaccharides composed of galactosyl (beta-1,3)N-acetylgalactosamine, alpha-linked fucose, galactosyl(beta-1,4)N-acetylglucosamine, sialic acid, galactose, and N-acetylgalactosamine. Collectively, these characteristics indicate that the K88ac adhesin receptors are mucin-type sialoglycoproteins.

Protein phosphatases 2A1 and 2A2 were isolated from porcine heart tissue extracts by precipitation at pH 5.0 and separated by chromatography on DEAE-Sephacel. Phosphatase 2A1 was then purified to apparent homogeneity by chromatography on phenyl-Sepharose, aminohexyl-Sepharose, Sephacryl S-300, and L-tyrosine-agarose. Phosphatase 2A2 was purified to apparent homogeneity by chromatography on phenyl-Sepharose, DEAE-Sephacel, aminohexyl-Sepharose and L-tyrosine-agarose. Purified phosphatases 2A1 and 2A2 had specific activities of 2200 and 2710 nanomoles of phosphate released from phosphorylase a/mg protein, respectively. The apparent molecular weights of phosphatases 2A1 and 2A2 on gel filtration were 155 and 105 kDa, respectively. Both enzymes contain 70 and 37 kDa subunits and 2A1 also contains a 57 kDa subunit. The 37 kDa catalytic subunit (2Ac) was obtained from the purified phosphatases by treatment with room temperature ethanol followed by sucrose density gradient centrifugation or gel filtration chromatography.

Fructose 2,6-bisphosphate and glucose 1,6-bisphosphate are apparent noncompetitive inhibitors of porcine protein phosphatase 2A2 having Ki values of 0.38 and 0.56 mM, respectively. The inhibitory effects were on the catalytic subunit and were not substrate directed. In addition, fructose 2,6-bisphosphate caused a time-dependent inactivation of phosphatase activity toward phosphorylase a. This inactivation was antagonized by MnCl2. The fructose 2,6-bisphosphate-inactivated enzyme had increased p-nitrophenyl phosphate phosphatase activity. These effects are similar to the known effects of ATP on type 2A phosphatases.

In this study, we identified two brush border glycoproteins (210 and 240 kDa) that bind both K88ac+ Escherichia coli and purified K88ac adhesin. The specificity of these binding glycoproteins for the K88ac adhesin was demonstrated in studies in which the binding of 35S-labeled K88ac+ E. coli and biotinylated K88ac adhesin to these glycoproteins was blocked in the presence of a 100-fold molar excess of unlabeled K88ac adhesin but not in the presence of the K99 adhesin. Pretreatment of adhesive brush borders with sodium metaperiodate destroyed both binding activities, indicating that the interaction between the K88ac adhesin and the binding glycoproteins requires the glycoprotein carbohydrate moiety. It was demonstrated previously that K88ac+ E. coli binds to adhesive brush borders but not to nonadhesive brush borders (R. Sellwood, R. A. Gibbons, G. W. Jones, and J. M. Rutter, J. Med. Microbiol. 8:405-411, 1975). In the present study, brush borders isolated from 10 different pigs were tested first for brush border adhesiveness and then for the presence of the binding glycoproteins. In all cases, the binding glycoproteins were detected only in the adhesive brush border preparations. These two binding glycoproteins may be the receptors used by K88ac+ ETEC to adhere to intestinal brush border cells. Their presence on adhesive brush borders and absence on nonadhesive brush borders may be the basis for resistance and susceptibility of pigs to K88ac+ ETEC infections.

Mitogen-activated protein kinase (MAP kinase) is a 42 kd serine/threonine protein kinase whose enzymatic activity requires phosphorylation of both tyrosyl and threonyl residues. As a step in elucidating the mechanism(s) for activation of this enzyme, we have determined the sites of regulatory phosphorylation. Following proteolytic digestion of 32P-labeled pp42/MAP kinase with trypsin, only a single phosphopeptide was detected by two-dimensional peptide mapping, and this peptide contained both phosphotyrosine and phosphothreonine. The amino acid sequence of the peptide, including the phosphorylation sites, was determined using a combination of Fourier transform mass spectrometry and collision-activated dissociation tandem mass spectrometry with electrospray ionization. The sequence for the pp42/MAP kinase tryptic phosphopeptide is similar (but not identical) to a sequence present in the ERK1- and KSS1-encoded kinases. The two phosphorylation sites are separated by only a single residue. The regulation of activity by dual phosphorylations at closely spaced threonyl and tyrosyl residues has a functional correlate in p34cdc2, and may be characteristic of a family of protein kinases regulating cell cycle transitions.

Bovine myelin basic protein (MBP) was found to be an excellent in vitro substrate (apparent Km = 50 microM) for MAP (mitogen-activated protein) kinase and can be used in lieu of microtubule-associated protein 2 for purification and functional studies of the enzyme. MBP phosphotransferase activity co-purified with MAP kinase during sequential DE52, phenyl-Superose, and gel filtration chromatography, and kinase activities for the two substrates were co-regulated by mitogen stimulation. MAP kinase phosphorylated MBP exclusively on threonine, and only one major phosphopeptide was generated by digestion with trypsin or endoproteinase Lys-C. Using mass spectrometry, we determined that the phosphorylation site is threonine 97, present in the conserved triproline loop of MBP, with (partial) sequence -Thr-Pro-Arg-Thr97-Pro-Pro-Pro-. Thr97 is a known in vivo phosphorylation site in MBP although enzymes capable of phosphorylating this site have not been identified previously. MAP kinase phosphorylated peptide 88-109 from rabbit MBP and a synthetic peptide 91-109 from human MBP but did not phosphorylate either the histone H1 peptide, utilized by p34cdc2, or the peptide substrate for the recently described proline-directed kinase. Thus, the sequence surrounding threonine 97 in bovine MBP may contain essential features of a recognition sequence for MAP kinase.

A variety of antibodies to microtubule-associated protein (MAP) have been used to demonstrate that phosphorylation of a 350 kDa microtubule-associated protein is stimulated 2-3 fold by epidermal growth factor or serum in quiescent 3T3-L1 fibroblasts and by insulin in 3T3-L1 adipocytes. Phosphorylation occurs on serine residues, and is maximal by 15-20 min. The phosphoprotein has been identified as MAP1A by specific immunoprecipitation with a well-characterized monoclonal antibody.