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Population-based reference intervals (RIs) are vital tools used to characterize health and disease based on laboratory values. The science and statistical basis for RI generation have evolved over the past 50 yr. Current veterinary-specific guidelines by the American Society of Veterinary Clinical Pathology exist for establishing RIs from nondomestic and wild animals. A list of 35 items that should be included during generation and publication of reference data was distilled from the currently available RI guidelines. The archives of five peer-reviewed journals were searched and 106 articles presenting laboratory reference data from nondomestic or wildlife species were identified and each reviewed by two authors to determine compliance with the list of 35 items. A compliance score was calculated as the number of articles that fulfilled the item out of the number where it would have been appropriate to fulfill the item. Most articles reported the number of reference individuals (compliance score 0.98), their partitioning demographics (compliance score 0.95), and sample collection and handling practices (compliance scores 0.97 and 0.96, respectively). Common deficiencies included omitting discussion of the validation status of the analytical methods for the species being evaluated (compliance score 0.12), documentation of use of exclusion criteria (compliance score 0.51), outlier detection (compliance score 0.43), appropriate statistical methods for the reference population (compliance score 0.34), and calculation and presentation of confidence intervals around the reference limits (compliance score 0.35). Compliance scores were not statistically different when stratified on the number of individuals in the largest and smallest evaluated group or the format of the article (full vs short format). Articles that cited RI generation guidelines fulfilled more of the required steps and provided a more complete description of their data (compliance score 0.74) than those that did not cite guidelines (compliance score 0.58). Additional attention to the science of and recommendations for RI generation is recommended to strengthen the utility of published data.

Background: Measuring concentrations of metabolites of estradiol and progesterone in urine, instead of measuring serum concentrations, is common in research and also is used in patient care. The primary aim of this study was to demonstrate that analysis of urine samples dried on filter paper by gas chromatography with tandem mass spectrometry (GC-MS/MS) provides results similar to serum analyzed by radioimmunoassay (RIA). Secondary aims were to show that collection of four samples during the day (4-spot method) can be substituted for a 24-h collection, and that analysis of urine from dried samples is equivalent to liquid urine samples., Methods: This prospective observational study compared results of urine and serum analyses. Urine samples from women throughout the menstrual cycle and single samples from postmenopausal women were evaluated. Urine was collected onto filter paper and dried. Dried urine was extracted, hydrolyzed, and derivatized prior to analysis by GC-MS/MS. Hormone concentrations were normalized to creatinine. Single samples were used to compare results of 24-h urine collection to the 4-spot method from a separate population of women and men. A subset of these samples were used to compare results from dried urine to liquid urine., Results: The primary study showed good reliability in the comparisons between the dried urine and serum assays. During the menstrual cycles of a subset of four women, urine metabolite concentrations followed the same pattern as serum concentrations. Comparison of 4-spot to 24-h urine collections and of dried to liquid urine measurements had intraclass correlation coefficients (ICC) greater than 0.95, indicating excellent agreement., Conclusions: For estradiol and progesterone, the dried urine assay is a good surrogate for serum testing. The 4-spot method can be used instead of 24-h urine collections and dried urine results are comparable to liquid urine. The dried urine assay is useful for some clinical assessments of hormone disorders and may be useful in large epidemiologic studies due to ease of sample handling., Competing Interests: Precision Analytical, Inc. is a commercial laboratory offering hormone testing to medical practitioners and individuals. All statistics were calculated and interpreted by an independent agent.

Background: Leukocyte adhesion deficiency (LAD) or CD18 deficiency is an autosomal recessive immunodeficiency which has been described in people, cattle, dogs, and knockout mice., Objectives: The study goals were to characterize the clinicopathologic, immunologic, and molecular genetic features of feline LAD (FLAD) in a neutered male adult Domestic Longhair cat with severe leukocytosis and recurrent infections., Methods: Flow cytometry evaluated surface expression of CD18 on neutrophils. In vitro functional assays assessed CD18-dependent neutrophil adhesion and T-cell proliferation. Genomic DNA and cDNA were used to identify a causative mutation in the coding sequence of the integrin β 2 subunit (ITGB2) gene., Results: The affected cat developed periodontitis during the first months of life followed by recurrent infections poorly responsive to antibiotic therapy, accompanied by extreme neutrophilia. Neutrophils from the proband, compared to feline controls, did not express any CD18 on the cell surface. Adhesion of affected neutrophils was severely impaired with and without phorbol-myristate-acetate activation. The proband's T-cells proliferated weakly to 1 pg but normally to 100 pg staphylococcal enterotoxin A, suggesting a CD18-independent T-cell response at higher doses. Molecular genetic analysis of the ITGB2 gene revealed a 24 bp deletion at the exon 2 to intron 2 boundary (c.46_58 + 11del), predicting premature translational termination due to abnormal splicing of exon 1 to exon 3 or 4., Conclusions: Feline LAD exhibits features similar to LAD in other species. However, clinical episodes in FLAD appeared milder allowing for an extended life expectancy under long-term antimicrobial therapy, possibly due to an alternative, CD18-independent T-cell proliferation pathway., (© 2017 American Society for Veterinary Clinical Pathology.)

Through a series of investigations involving different levels of contextual fidelity we developed scales of perceived urgency for several dimensions of the auditory, visual, and tactile modalities. Psychophysical ratings of perceived urgency, annoyance, and acceptability as well as behavioral responses to signals in each modality were obtained and analyzed using Steven's Power Law to allow comparison across modalities. Obtained results and their implications for use as in-vehicle alerts and warnings are discussed.

Mitochondrial hyperpolarization inhibits the electron transport chain and increases incomplete reduction of oxygen, enabling production of reactive oxygen species (ROS). The consequence is mitochondrial damage that eventually causes cell death. Uncoupling proteins (UCPs) are inner mitochondrial membrane proteins that dissipate the mitochondrial proton gradient by transporting H(+) across the inner membrane, thereby stabilizing the inner mitochondrial membrane potential and reducing the formation of ROS. The role of UCP2 in neuroprotection is still in debate. This study seeks to clarify the role of UCP2 in transient focal ischemia (tFI) and to further understand the mechanisms of ischemic brain damage. Both wild-type and UCP2-knockout mice were subjected to tFI. Knocking out UCP2 significantly increased the infarct volume to 61% per hemisphere as compared with 18% in wild-type animals. Knocking out UCP2 suppressed antioxidant, cell-cycle, and DNA repair genes, including Sod1 and Sod2, Gstm1, and cyclins. Furthermore, knocking out UCP2 significantly upregulated the protein levels of the inflammatory cytokines, including CTACK, CXCL16, Eotaxin-2, fractalkine, and BLC. It is concluded that knocking out the UCP2 gene exacerbates neuronal death after cerebral ischemia with reperfusion and this detrimental effect is mediated by alteration of antioxidant genes and upregulation of inflammatory mediators.

The structural features responsible for the activities of hepatic lipase (HL) can be clarified by in vivo comparisons of naturally occurring variants. The coding sequence of HL from C57BL/6J (B6) and SPRET/EiJ (SPRET) mice differs by four amino acids (S106N, A156V, L416V, S480T); however, these changes are not predicted to influence HL function. To test for allelic effects, we generated SPRET-HL transgenics with physiological levels of HL mRNA and HL activity that was parallel in female transgenics and about 70% higher in male transgenics, toward tri-[3H]oleate, compared with B6 controls. We found no correlation between activity levels and plasma lipids. However, significant allelic effects on plasma lipids were observed. Compared with B6-HL, SPRET-HL mediated reductions in total cholesterol (TC) and VLDL-, LDL- and HDL-cholesterol and HDL-triglyceride (TG) in fed males, and SPRET-HL decreased total TG and VLDL- and HDL-TG levels in fasted males. Fasted female transgenics had reduced TC compared with controls. We also found allele and sex effects on lipoprotein particle size. Male transgenic mice had increased VLDL and decreased LDL size, and female transgenic mice had decreased HDL size compared with control animals. These findings demonstrate highly divergent effects of naturally occurring HL coding sequence variants on lipid and lipoprotein metabolism.

A 15-year-old female Simmental cross-breed cow was presented to the Purdue University Veterinary Teaching Hospital for evaluation of a perifemoral soft tissue mass. Impression smears made from an excisional biopsy contained a population of pleomorphic mesenchymal cells with abundant, periodic acid-Schiff-positive (PAS), intracytoplasmic granular material, and rare elongated multinucleated cells consistent with strap-like cells. A second population of small round cells suggestive of lymphocytes or progenitor cells was also noted. A cytologic diagnosis of sarcoma was made, with rhabdomyosarcoma considered most likely based on the large amount of PAS-positive material (presumed to be glycogen) and the rare strap-like cells. Histopathologic sections contained an unencapsulated, densely cellular neoplasm composed of haphazardly arranged highly pleomorphic mesenchymal cells and a few small round cells. The mesenchymal cells were positive for vimentin, non-specific muscle actin, and myoglobin, and negative for phosphotungstic acid-hematoxylin, smooth muscle actin, and desmin. Glycogen granules were confirmed by transmission electron microscopy. A diagnosis of pleomorphic rhabdomyosarcoma was made. While cytologic findings may suggest rhabdomyosarcoma, cytologic features can be highly variable, and a definitive diagnosis usually requires cytochemical and immunohistochemical staining.

The objective was to quantify the effect of furosemide and carbazochrome on exercise-induced pulmonary hemorrhage (EIPH) in Standardbred horses using red blood cell count and hemoglobin concentration in bronchoalveolar lavage (BAL) fluid. Six healthy Standardbred horses with prior evidence of EIPH performed a standardized treadmill test 4 h after administration of placebo, furosemide, or furosemide-carbazochrome combination. Red blood cell (RBC) counts and hemoglobin concentrations were determined on the BAL fluid. The RBC count in BAL ranges were (2903-26,025 cells/microL), (45-24,060 cells/microL), and (905-3045 cells/microL) for placebo, furosemide, and furosemide-carbazochrome, respectively. Hemoglobin concentration ranges were (0.03-0.59 mg/mL), (0.01-0.55 mg/mL), and (0.007-0.16 mg/mL) for placebo, furosemide, and furosemide-carbazochrome groups, respectively. No significant differences were detected among treatments. However, there was great variability among horses, suggesting that a larger sample size or better selection of horses was needed.

Objective: To determine whether platelet growth factors are preserved in supernatants obtained from rehydrated trehalose-stabilized, freeze-dried (lyophilized) equine platelets and whether those growth factors stimulate fibroblast proliferation and migration and enhance fibroblast-associated contraction in a collagen gel assay., Animals: 6 clinically normal adult horses., Procedures: Blood samples were obtained from 6 horses, and washed platelets were prepared via differential centrifugation. Washed platelets were freeze-dried in a physiologic buffer with a mixture of trehalose and polyethylene glycol 4000. Rehydrated platelet supernatants and releasates prepared from fresh washed platelets stimulated with thrombin or platelet-activating factor were evaluated for transforming growth factor beta1 and platelet-derived growth factor-BB by use of ELISAs. Effects of rehydrated freeze-dried platelet supernatants on fibroblast proliferation, migration, and collagen gel contraction were compared with effects of 1%, 2.5%, or 10% fetal bovine serum (FBS)., Results: Supernatants from freeze-dried platelets contained similar amounts of growth factors as thrombin- and platelet-activating factor-stimulated platelet releasates. The supernatants significantly enhanced fibroblast proliferation and migration in a scratch assay, compared with FBS-free control or low (1%) FBS conditions. Additionally, supernatants from freeze-dried platelets enhanced contraction of fibroblast-seeded collagen gels, compared with the effect of 1% FBS., Conclusions and Clinical Relevance: The preparation technique preserved platelet growth factors, enhanced fibroblast proliferation and migration, and improved fibroblastseeded collagen gel contraction under conditions of low FBS concentration; these platelet supernatant preparations may prove useful as an aid to conventional wound management.

A 16-year-old castrated male Arabian horse was presented to the Purdue University Veterinary Teaching Hospital with a 4-hour history of colic. Initial examinations provided strong evidence for small intestinal obstruction. Abdominal surgery revealed a strangulating lipoma, and 25 feet of small intestine were resected. Postoperatively, the horse developed obstructive ileus due to adhesion formation, which required a second laparotomy. During and after surgery, the abdomen was lavaged with sodium carboxymethylcellulose (CMC). One week after the second surgery, evaluation of peritoneal fluid revealed an inflammatory exudate, with many macrophages containing amorphous to granular, pink to magenta phagocytosed material. Extracellular aggregates of the material were also observed. The material was consistent with CMC. To our knowledge, this report is the first to demonstrate the phagocytosis of CMC by peritoneal fluid macrophages.

Objective: To measure the frequency and magnitude of reduced fibrinogen binding in a population of horses from a Thoroughbred breeding farm., Animals: 444 Thoroughbred horses, 1 to 27 years old, including 316 females, 72 geldings, and 56 sexually intact males., Procedures: Blood was collected from horses into tubes containingacid citrate dextrose adenine, and washed platelets were examined by use of flow cytometry for their ability to bind fibrinogen., Results: Data regarding fibrinogen binding to activated platelets were normally distributed, with nearly identical amounts of variation regardless of sex. In 3 horses, fibrinogen binding to platelets was reduced from 67.6% to 83.4%, compared with normal platelets, which indicated an inability of platelets to aggregate in response to thrombin (0.1 U/mL)., Conclusions and Clinical Relevance: Platelet fibrinogen binding of the affected horses identified in this study was characteristic of a reported heritable bleeding disorder in which the reduction in fibrinogen binding correlated with prolonged bleeding times in template bleeding assays. The bleeding disorder is distinct from Glanzmann thrombasthenia, in which platelets fail to bind fibrinogen because of lack of alphallb-beta3 integrin on their surface. The prevalence of affected horses within the small sample population studied here (0.7% [n = 3]) is considerably higher than the prevalence of bleeding disorders within more genetically diverse groups.

Background: Bleeding in racing horses associated with exercise appears to be multifactorial, and clinical investigation into severe cases rarely occurs. Previously, we reported a severe bleeding diathesis in a Thoroughbred mare. Herein, we describe the cellular physiology of this defect, provide a diagnostic tool for identifying it, and demonstrate that the dysfunction is heritable., Hypothesis: The subject has a heritable defect in platelet secretion that reduces thrombin generation in the absence of additional plasma factors and delays the onset of thrombin production even in the presence of these factors., Animals: The study included 3 clinically normal Thoroughbred horses: the subject and her offspring., Methods: Washed platelets were examined for their ability to (1) translocate phosphatidylserine to the outer leaflet of the platelet membrane as determined by annexin-V binding, (2) generate thrombin as assessed by the activity of the prothrombinase enzyme complex, and (3) bind fibrinogen and form aggregates as determined by flow cytometry., Results: Subject and offspring platelets created procoagulant surfaces by translocating phosphatidylserine. The subject's platelets demonstrated reduced prothrombinase activity, resulting in decreased production of thrombin relative to control platelets. Subject and offspring platelets bound less fibrinogen than control platelets when stimulated with thrombin., Conclusions and Clinical Importance: The subject mare has a transmissible defect that involves reduced generation of thrombin by activated platelets, resulting in decreased aggregation and ineffective clotting. A flow cytometric assay of fibrinogen binding to washed platelets discriminates individuals with this platelet dysfunction and may be useful for discerning subclinical congenital or acquired platelet dysfunctions.

This paper describes the clinical, laboratory and histological findings in three horses with immune-mediated polysynovitis; they had lost weight, suffered intermittent fever, were lethargic and stiff, and had effusions in several joints. Laboratory abnormalities included anaemia, leucocytosis, hyperfibrinogenaemia and hyperglobulinaemia. The diagnosis was based on the presence of a suppurative, non-septic inflammation in at least two different joints in each of the horses and the presence of immunoglobulins in the synovial membrane of one of them. The horses were treated with a combination of dexamethasone and azathioprine, and responded well to the initial treatment. Twenty months after its last re-evaluation, the first horse was being maintained on azathioprine because similar clinical signs had recurred after the cytotoxic drug was discontinued; the second horse was finishing a tapering course of prednisolone 15 months after its first examination, and the third horse was euthanased five months after it was first examined as a result of an unrelated injury.

Objective: To determine clinical signs, results of diagnostic testing, and outcome in horses with internal Corynebacterium pseudotuberculosis infection., Design: Retrospective study., Animals: 30 horses., Procedure: Information pertaining to clinical data, results of diagnostic tests, and costs of hospitalization and treatment was extracted from medical records of affected horses., Results: Internal C. pseudotuberculosis infection was diagnosed on the basis of clinical signs, diagnostic imaging, and clinicopathologic data, including results of serologic tests and bacterial culture. The most common clinical signs were concurrent external abscesses, anorexia, fever, lethargy, weight loss, and signs of respiratory tract disease or abdominal pain. Clinicopathologic abnormalities included a geometric mean reciprocal serum synergistic hemolysin inhibition titer > or = 512, leukocytosis with neutrophilia, hyperglobulinemia, hyperfibrinogenemia, and anemia. Specific organ involvement was diagnosed in 27 of 30 horses. Affected organs included the liver (18 horses), lungs (12), kidneys (7), and spleen (3); multiple organs were affected in 10 horses. Treatment with antimicrobials for a median of 36 days (range, 7 to 97 days) was usually successful, yielding an overall survival rate of 71%., Conclusions and Clinical Relevance: Early diagnosis and long-term antimicrobial treatment were important for a successful outcome in horses with internal C. pseudotuberculosis infection. Ultrasonographic imaging was an important technique for identifying specific organs affected, aiding in obtaining samples for a definitive diagnosis, and monitoring response to treatment. Pregnant mares with internal infections are at risk for fetal loss. Preexisting chronic organ disease may be associated with a poor prognosis.

Coupling growth of Lolium perenne L. in sterile solution culture with steady-state (13)CO(2) labelling allowed quantification of the contribution of C, assimilated either before or after a specific time point, both to plant compartments and root exudates. Plants were grown for 27 days in atmospheres containing CO(2) with delta(13)C signatures of either -13.5 or -36.1 per thousand. Air supplies to plants were then reciprocally switched to the opposing signature (day 0), plants were destructively harvested and root exudates collected over the next 8 days. Following the switch, C assimilated after day 0 and transported to the roots initially only appeared in root tips, later appearing in both tip and non-tip material. The delta(13)C signature of the root exudate changed exponentially with time. Assimilation pre- and post-day 0 contributed equally to exudate C at 4.5 days. Beyond day 8, assimilation pre-day 0 still contributed 41.7% of exudate C. Over all 8 days, a linear relationship existed between the delta(13)C signatures of root tips and exudate, suggesting that all newly assimilated C in the exudate was from root tips. Results imply pulse-labelling approaches to study root exudates are discriminative in the sources of exudates labelled and in the sites from which exudation occurs.

•  This study examined morphological and topological responses of nodal root axes to defoliation in a fast- and a slow-growing grass species. •  Vegetative tillers of both Lolium perenne and Festuca ovina were grown on slant boards and either left intact or subjected to repeated defoliation, under both a high nitrogen (N) and a low N supply. Root length, diameter and branching characteristics were measured on individual nodal root axes. •  The total axis root length of F. ovina was less when plants had been defoliated. Root axis weight, primary root axis length and primary root diameter were also less with defoliation than an undefoliated control, under high N. Under low N conditions the root axes of F. ovina had a more randomly branched topology without defoliation. For L. perenne under low N conditions, the length of the primary root axis was longer with defoliation than in an undefoliated control, while the primary root axis diameter decreased. By contrast to F. ovina, the root axes of L. perenne had a more randomly branched topology without defoliation only when supplied with high N. •  The greatest plasticity in branching caused by defoliation was observed under high N for L. perenne and under low N for F. ovina. Although grass root axis topology has, in general, a herringbone in structure, the nodal root system can alter root axis structure in response to defoliation.

A 13-year-old Morgan gelding was evaluated because of a mass in the caudal region of the abdomen. The horse had been presumed to be a gelding, but necropsy findings revealed a retained testis in the right retroperitoneal space. Histologically, the retained testis contained neoplastic cells; metastases were identified in the liver, spleen, lungs, and sublumbar lymph nodes. Immunohistochemical examination of the testis and metastatic tissues confirmed the diagnosis of malignant Sertoli cell tumor. Testicular neoplasms are infrequently reported in stallions. Seminomas are most commonly reported, whereas Sertoli cell tumors are considered to be rare. Typical biological behavior of Sertoli cell tumors in horses is unknown. To the authors' knowledge, there have been 2 reports of Sertoli cell tumors in horses; the tumors developed in descended testes, and 1 tumor was malignant.

In defoliated grasses, where photosynthesis is reduced due to removal of leaf material, it is well established that remobilization of nitrogen occurs from both older remaining leaves and roots towards the younger growing leaves. In contrast, little is known about the movement of nitrogen within intact grass plants experiencing prolonged inhibition of photosynthesis. We tested the following hypotheses in Festuca rubra L. ssp. rubra cv. Boreal: that both reduction of the atmospheric CO2 concentration and defoliation (1) induce mobilization of nitrogen from roots and older leaves towards growing leaves and (2) elicit similar directional change in the abundance of proteins in roots and older leaves relevant to the process of nitrogen mobilization including, glutamine synthetase (GS), EC 6.3.1.2; papain, EC 3.4.22.2; chymopapain, EC 3.4.22.6; ribulose bisphosphate carboxylase/oxygenase (Rubisco), EC 4.1.1.39; and the light harvesting complex of photosystem II (LHCPII). After growth at ambient atmospheric CO2 concentration, plants of F. rubra were subject to atmospheres containing either ambient (350 micro l l-1) or deplete (< 20 micro l l-1) CO2. Concurrently, plants were either left intact or defoliated on one occasion. Steady state 15N labelling coupled with a series of destructive harvests over a 7-day period enabled changes in the nitrogen dynamics of the plants to be established. Proteins pertinent to the process of nitrogen mobilization were quantified by immunoblotting. Irrespective of defoliation, plants in ambient CO2 mobilized nitrogen from older to growing leaves. This mobilization was inhibited by deplete CO2. Greater concentration of Rubisco and reduced chymopapain abundance in older remaining leaves of intact plants, in deplete compared with ambient CO2, suggested the inhibition of mobilization was due to inhibition of protein degradation, rather than to the export of degradation products. Both deplete CO2 and defoliation induced nitrogen mobilization from roots to growing leaves. In plants at ambient CO2, defoliation did not affect nitrogen uptake or its allocation. Therefore in F. rubra nitrogen mobilization can occur independently of any downregulation of nitrogen uptake. This suggests either different signal compounds may act to downregulate uptake and upregulate mobilization, or if one particular signalling compound is used its concentration threshold differs for induction of mobilization and downregulation of uptake. The abundance of the cysteine proteases papain and chymopapain was low in roots suggesting that they were not involved in protein degradation in this tissue.

In retrospective series, 17.0 to 25.5 per cent of those patients with carcinoma of the colon or rectum have been found to have concurrent inguinal herniation. This observation prompted the recommendation that patients with hernias be thoroughly screened for disease of the colon and rectum. That recommendation remains controversial in part because it has not previously been prospectively evaluated. In a one year period, 202 consecutive patients with inguinal hernias completed a preoperative protocol which included: 1, gastrointestinal history; 2, rectal examination; 3, testing of at least two stool specimens with the Hemoccult (Smith Kline Diagnostics) device; 4, sigmoidoscopy, and 5, barium enema. Malignant and premalignant diseases were discovered in five patients, only one of whom had gastrointestinal symptoms. Each malignant lesion was localized (Dukes' A or B) and, therefore, highly curable. Benign disease was found in 49 patients, including polyps in eight and one instance of tight sigmoid stricture. All of the malignant and 91 per cent of the benign lesions were in patients 50 years of age and older. The association between inguinal herniation and colorectal disease was previously attributed to luminal obstruction. In the present series, however, the sole obstructing lesion was a benign stricture. Another explanation for this association must, therefore, be sought. We conclude, on the basis of the results of this experience, however, that screening for pathologic factors of the colon and rectum is warranted in otherwise asymptomatic patients who are more than 50 years old and who present with inguinal hernias.

A 71-year-old patient had recurrent urinary tract infections for 7 years after sigmoid colectomy via a Hartmann procedure. Extensive radiological and endoscopic tests were inconclusive as to the cause of bacteriuria. Chronic back pain led to performance of a radionuclide bone scan with the incidental demonstration of a vesicoenteric fistula, confirmed at exploration. Appendectomy with resection of the involved bladder resulted in cessation of bacteriuria.

A radioreceptor assay using [3H]2-beta-endorphin (B-EP) was used to examine binding in the brains of rats that had been exercised on a treadmill for 5 months. The binding of [3H]2-beta-EP to brain homogenates gave an average Kd of 0.830 +/- 0.055 nM for the controls and 0.826 + 0.022 nM for the runners, and respective Bmax's of 166 +/- 20 pM and 159 +/- 27 pM.