Extraneuronal pathology in a canine model of CLN2 neuronal ceroid lipofuscinosis after intracerebroventricular gene therapy that delays neurological disease progression.

CLN2 neuronal ceroid lipofuscinosis is a hereditary lysosomal storage disease with primarily neurological signs that results from mutations in TPP1, which encodes the lysosomal enzyme tripeptidyl peptidase-1 (TPP1). Studies using a canine model for this disorder demonstrated that delivery of TPP1 enzyme to the cerebrospinal fluid (CSF) by intracerebroventricular administration of an AAV-TPP1 vector resulted in substantial delays in the onset and progression of neurological signs and prolongation of life span. We hypothesized that the treatment may not deliver therapeutic levels of this protein to tissues outside the central nervous system that also require TPP1 for normal lysosomal function. To test this hypothesis, dogs treated with CSF administration of AAV-TPP1 were evaluated for the development of non-neuronal pathology. Affected treated dogs exhibited progressive cardiac pathology reflected by elevated plasma cardiac troponin-1, impaired cardiac function and development of histopathological myocardial lesions. Progressive increases in the plasma activity levels of alanine aminotransferase and creatine kinase indicated development of pathology in the liver and muscles. The treatment also did not prevent disease-related accumulation of lysosomal storage bodies in the heart or liver. These studies indicate that optimal treatment outcomes for CLN2 disease may require delivery of TPP1 systemically as well as directly to the central nervous system.

Diagnostic immunohistochemistry of canine and feline intracalvarial tumors in the age of brain biopsies.

The focus of immunohistochemistry as applied to nervous system tumors is in identifying the neoplasm present and evaluating margins between normal and neoplastic tissue. Although not always utilized by specialists in neuropathology, immunohistochemistry remains useful to resolve concerns about the differentiation and rate of tumor growth. The aims of this review are to discuss the utility of immunohistochemical reagents currently used in diagnosis of canine and feline intracalvarial tumors, to indicate the applicability of some tests currently used in human nervous system tumors for domestic species, and to evaluate a few less commonly used reagents. A panel of biomarkers is usually needed to confirm a diagnosis, with groups of reagents for leptomeningeal, intraparenchymal, and ventricular neoplasms. In the future, signature genetic alterations found among feline and canine brain tumors--as correlated prospectively with diagnosis, rate of enlargement, or response to treatment--may result in new immunohistochemical reagents to simplify the task of diagnosis. Prospective studies determining the type and proportion of stem cell marker expression on patient longevity are likely to be fruitful and suggest new therapies. Due to increased frequency of biopsy or partial resection of tumors from the living patient, biomarkers are needed to serve as accurate prognostic indicators and assist in determining the efficacy of developing therapeutic options in nervous system tumors of dogs and cats.

Subclinical hypocalcemia, plasma biochemical parameters, lipid metabolism, postpartum disease, and fertility in postparturient dairy cows.

A study was conducted to evaluate the potential association between Ca status at calving and postpartum energy balance, liver lipid infiltration, disease occurrence, milk yield and quality parameters, and fertility in Holstein cows. One hundred cows were assigned to 1 of 2 groups based on whole-blood ionized Ca concentration ([iCa]) on the day of calving [d 0; hypocalcemic [iCa] <1.0 mmol/L (n=51); normocalcemic [iCa] ≥ 1.0 mmol/L (n=49)]. Cows were blocked based on calving date and parity. Blood samples were collected approximately 14 d from expected calving date (d -14), the day of calving (d 0), and on d 3, 7, 14, 21, and 35 postpartum for measurement of plasma nonesterified fatty acid, iCa, total Ca, glucose, and total and direct bilirubin concentrations, and plasma aspartate aminotransferase and gamma glutamyl transferase activities. Liver biopsies were obtained from a subset of cows on d 0, 7, and 35 for quantification of lipid content. Milk samples were collected on d 3, 7, 14, 21, and 35 postpartum for measurement of somatic cell count and percentages of protein, fat, and solids-not-fat. Data for peak test-day milk yield, services per conception, and days open were obtained from Dairy Herd Improvement Association herd records. Disease occurrence was determined based on herd treatment records. Hypocalcemic cows had significantly higher nonesterified fatty acids on d 0. Hypocalcemic cows also had significantly more lipid in hepatocytes on d 7 and 35 postpartum. However, no statistically significant differences were observed between groups for plasma aspartate aminotransferase and gamma glutamyl transferase activities or total and direct bilirubin concentrations. Milk protein percentage was lower in hypocalcemic cows on d 21 and 35. However other milk quality variables (somatic cell count, milk fat percentage, and solids-not-fat) and milk yield variables (peak test-day milk yield and 305-d mature-equivalent 4% fat-corrected milk yield) did not differ between groups. No differences were observed between groups in the occurrence of clinical mastitis, ketosis, displaced abomasum, dystocia, retained placenta, metritis, or fertility measures (percentage cycling at 50-60 d postpartum, services per conception, or days open). These data suggest that early lactation fatty acid metabolism differs between cows with subclinical hypocalcemia and their normocalcemic counterparts.

A search for type 1 diabetes susceptibility genes in families from the United Kingdom.

Genetic analysis of a mouse model of major histocompatability complex (MHC)-associated autoimmune type 1 (insulin-dependent) diabetes mellitus (IDDM) has shown that the disease is caused by a combination of a major effect at the MHC and at least ten other susceptibility loci elsewhere in the genome. A genome-wide scan of 93 affected sibpair families (ASP) from the UK (UK93) indicated a similar genetic basis for human type 1 diabetes, with the major genetic component at the MHC locus (IDDM1) explaining 34% of the familial clustering of the disease (lambda(s)=2.5; refs 3,4). In the present report, we have analysed a further 263 multiplex families from the same population (UK263) to provide a total UK data set of 356 ASP families (UK356). Only four regions of the genome outside IDDM1/MHC, which was still the only major locus detected, were not excluded at lambda(s)=3 and lod=-2, of which two showed evidence of linkage: chromosome 10p13-p11 (maximum lod score (MLS)=4.7, P=3x10(-6), lambda(s)=1.56) and chromosome 16q22-16q24 (MLS=3.4, P=6.5x10(-5), lambda(s)=1.6). These and other novel regions, including chromosome 14q12-q21 and chromosome 19p13-19q13, could potentially harbour disease loci but confirmation and fine mapping cannot be pursued effectively using conventional linkage analysis. Instead, more powerful linkage disequilibrium-based and haplotype mapping approaches must be used; such data is already emerging for several type 1 diabetes loci detected initially by linkage.

Haplotype tagging for the identification of common disease genes.

Genome-wide linkage disequilibrium (LD) mapping of common disease genes could be more powerful than linkage analysis if the appropriate density of polymorphic markers were known and if the genotyping effort and cost of producing such an LD map could be reduced. Although different metrics that measure the extent of LD have been evaluated, even the most recent studies have not placed significant emphasis on the most informative and cost-effective method of LD mapping-that based on haplotypes. We have scanned 135 kb of DNA from nine genes, genotyped 122 single-nucleotide polymorphisms (SNPs; approximately 184,000 genotypes) and determined the common haplotypes in a minimum of 384 European individuals for each gene. Here we show how knowledge of the common haplotypes and the SNPs that tag them can be used to (i) explain the often complex patterns of LD between adjacent markers, (ii) reduce genotyping significantly (in this case from 122 to 34 SNPs), (iii) scan the common variation of a gene sensitively and comprehensively and (iv) provide key fine-mapping data within regions of strong LD. Our results also indicate that, at least for the genes studied here, the current version of dbSNP would have been of limited utility for LD mapping because many common haplotypes could not be defined. A directed re-sequencing effort of the approximately 10% of the genome in or near genes in the major ethnic groups would aid the systematic evaluation of the common variant model of common disease.

Neuropathology and craniofacial lesions of osteopetrotic Red Angus calves.

Inherited osteopetrosis was identified in cattle herds in Wyoming, Nebraska, and Missouri in 2008 to 2010. Ten affected Red Angus calves were examined to characterize lesions in brain, teeth, and skull. Six affected aborted or stillborn calves were homozygous for the recently characterized deletion mutation in SLC4A2. Four affected calves were heterozygous for the SLC4A2 mutation and survived 1 to 7 days after birth. Gross lesions were similar in all 10 calves. Brains were rectangular and dorsoventrally compressed, with concave depressions in the parietal cortex owing to thickened parietal bone. Cerebellar hemispheres were compressed with herniation of the cerebellar vermis into the foramen magnum. Moderate bilateral chromatolysis affected multiple cranial nerve nuclei and, in some calves, the red nucleus. There was loss of retinal ganglion cells with severe atrophy of optic nerves. Periventricular corpora amylacea were in the thalamus, caudate nucleus, and midbrain. Vessels and neuropil in the dorsomedial aspect of the thalamus were mineralized. Dysplastic change in premolar and molar teeth comprised intra-alveolar intermingling of dentin, enamel, cementum, and bone, contributing to dental ankylosis. Changes in the heads of osteopetrotic calves are similar to those in children with malignant forms of homozygous recessive osteopetrosis.

Compressive myelopathy associated with ectasia of the vertebral and spinal arteries in a dog.

A 4-year-old dog was presented for acute, progressive tetraparesis and cervical hyperesthesia. Symmetrical tubular structures coursing along the lateroventral aspects of the spinal cord at the fourth and fifth cervical vertebrae were identified in magnetic resonance images. At necropsy, vertebral arteries and their spinal branches were severely ectatic bilaterally, and the cervical spinal cord was compressed. Histologically, the ectatic branches of the vertebral and ventral spinal arteries were surrounded by fibrosis with scant mononuclear cell infiltrates and hemorrhage. Spinal branches of the vertebral arteries had focally severe reduction in the tunica media. A thrombus was in an arterial branch. Smaller vessels in adjacent tissue had fibrinoid degeneration. Axonal degeneration was detected in the affected spinal cord and nerve roots. The segmental degenerative radiculomyelopathy in this dog was attributed to anomalous ectasia of the vertebral and ventral spinal arteries.

Deep, zonal subequatorial currents.

Large-scale, westward-extending tongues of warm (Pacific) and cold (Atlantic) water are found between 2000 and 3000 meters both north and south of the equator in the Pacific and Atlantic oceans. They are centered at 5 degrees to 8 degrees north and 10 degrees to 15 degrees south (Pacific) and 5 degrees to 8 degrees north and 15 degrees to 20 degrees south (Atlantic). They are separated in both oceans by a contrasting eastward-extending tongue, centered at about 1 degrees to 2 degrees south, in agreement with previous helium isotope observations (Pacific). Thus, the indicated deep tropical westward flows north and south of the equator and eastward flow near the equator may result from more general forcing than the hydrothermal forcing previously hypothesized.

Mediterranean outflow mixing and dynamics.

The Mediterranean Sea produces a salty, dense outflow that is strongly modified by entrainment as it first begins to descend the continental slope in the eastern Gulf of Cadiz. The current accelerates to 1.3 meters per second, which raises the internal Froude number above 1, and is intensely turbulent through its full thickness. The outflow loses about half of its density anomaly and roughly doubles its volume transport as it entrains less saline North Atlantic Central water. Within 100 kilometers downstream, the current is turned by the Coriolis force until it flows nearly parallel to topography in a damped geostrophic balance. The mixed Mediterranean outflow continues westward, slowly descending the continental slope until it becomes neutrally buoyant in the thermocline where it becomes an important water mass.

Strategies in complex disease mapping.

Dissecting the genetics of common, complex disorders remains one of the great challenges in human genetics. The acceleration of human genome sequence determination, improvements in informatics, large-scale identification of single nucleotide polymorphisms and improvements in scoring technologies have now increased the feasibility of identifying polymorphisms that predispose to common disease.

Cerebrospinal Fluid Levels of Phosphorylated Neurofilament Heavy as a Diagnostic Marker of Canine Degenerative Myelopathy.

BACKGROUND: No definitive, antemortem diagnostic test for canine degenerative myelopathy (DM) is available. Phosphorylated neurofilament heavy (pNF-H) is a promising biomarker for nervous system diseases. HYPOTHESIS/OBJECTIVE: Cerebrospinal fluid (CSF) and serum pNF-H is a detectable biological marker for diagnosis of canine DM. ANIMALS: Fifty-three DM-affected, 27 neurologically normal, 7 asymptomatic at-risk, and 12 DM mimic dogs. METHODS: Archived CSF and serum pNF-H concentrations were determined by a commercially available ELISA. A receiver-operating characteristic (ROC) curve was generated with CSF values. RESULTS: Compared with old control dogs, median CSF pNF-H concentration was increased in all stages of DM; old dogs 5.1 ng/mL (interquartile range [IQR] 1.4-9.3) versus DM stage 1 23.9 ng/mL (IQR 20.8-29.6; P < .05) versus DM stage 2 36.8 ng/mL (IQR 22.9-51.2; P < .0001) versus DM stage 3 25.2 ng/mL (IQR 20.2-61.8; P < .001) versus DM stage 4 38.0 ng/mL (IQR 11.6-59.9; P < .01). Degenerative myelopathy stage 1 dogs had increased median CSF pNF-H concentrations compared with asymptomatic, at-risk dogs (3.4 ng/mL [IQR 1.5-10.9; P < .01]) and DM mimics (6.6 ng/mL [IQR 3.0-12.3; P < .01]). CSF pNF-H concentration >20.25 ng/mL was 80.4% sensitive (confidence interval [CI] 66.09-90.64%) and 93.6% specific (CI 78.58-99.21%) for DM. Area under the ROC curve was 0.9467 (CI 0.92-0.9974). No differences in serum pNF-H concentration were found between control and DM-affected dogs. CONCLUSIONS AND CLINICAL IMPORTANCE: pNF-H concentration in CSF is a sensitive biomarker for diagnosis of DM. Although there was high specificity for DM in this cohort, further study should focus on a larger cohort of DM mimics, particularly other central and peripheral axonopathies.

A Homozygous RAB3GAP1:c.743delC Mutation in Rottweilers with Neuronal Vacuolation and Spinocerebellar Degeneration.

BACKGROUND: A variety of presumed hereditary, neurologic diseases have been reported in young Rottweilers. Overlapping ages of onset and clinical signs have made antemortem diagnosis difficult. One of these diseases, neuronal vacuolation and spinocerebellar degeneration (NVSD) shares clinical and histological features with polyneuropathy with ocular abnormalities and neuronal vacuolation (POANV), a recently described hereditary disease in Black Russian Terriers (BRTs). Dogs with POANV harbor mutations in RAB3GAP1 which codes for a protein involved in membrane trafficking. HYPOTHESIS: Rottweilers with NVSD will be homozygous for the RAB3GAP1:c.743delC allele associated with POANV in BRTs. ANIMALS: Eight Rottweilers with NVSD confirmed at necropsy, 128 Rottweilers without early onset neurologic signs, and 468 randomly selected dogs from 169 other breeds. METHODS: Retrospective case-control study. Dogs were genotyped for the RAB3GAP1:c.743delC allele with an allelic discrimination assay. RESULTS: All 8 NVSD-affected dogs were homozygous for the RAB3GAP1:c.743delC allele while the 128 NVSD-free Rottweilers were either homozygous for the reference allele (n = 105) or heterozygous (n = 23) and the 468 genotyped dogs from other breeds were all homozygous for the reference allele. CONCLUSIONS AND CLINICAL IMPORTANCE: The RAB3GAP1:c.743delC mutation is associated with a similar phenotype in Rottweilers and BRTs. Identification of the mutation permits a DNA test that can aid in the diagnosis of NVSD and identify carriers of the trait so that breeders can avoid producing affected dogs. Disruption of membrane trafficking could explain the neuronal vacuolation seen in NVSD and other spongiform encephalopathies.

Degenerative Encephalopathy in Nova Scotia Duck Tolling Retrievers Presenting with a Rapid Eye Movement Sleep Behavior Disorder.

BACKGROUND: Neurodegenerative diseases are a heterogeneous group of disorders characterized by loss of neurons and are commonly associated with a genetic mutation. HYPOTHESIS/OBJECTIVES: To characterize the clinical and histopathological features of a novel degenerative neurological disease affecting the brain of young adult Nova Scotia Duck Tolling Retrievers (NSDTRs). ANIMALS: Nine, young adult, related NSDTRs were evaluated for neurological dysfunction and rapid eye movement sleep behavior disorder. METHODS: Case series review. RESULTS: Clinical signs of neurological dysfunction began between 2 months and 5 years of age and were progressive in nature. They were characterized by episodes of marked movements during sleep, increased anxiety, noise phobia, and gait abnormalities. Magnetic resonance imaging documented symmetrical, progressively increasing, T2-weighted image intensity, predominantly within the caudate nuclei, consistent with necrosis secondary to gray matter degeneration. Abnormalities were not detected on clinicopathological analysis of blood and cerebrospinal fluid, infectious disease screening or urine metabolite screening in most cases. Postmortem examination of brain tissue identified symmetrical malacia of the caudate nuclei and axonal dystrophy within the brainstem and spinal cord. Genealogical analysis supports an autosomal recessive mode of inheritance. CONCLUSIONS AND CLINICAL IMPORTANCE: A degenerative encephalopathy was identified in young adult NSDTRs consistent with a hereditary disease. The prognosis is guarded due to the progressive nature of the disease, which is minimally responsive to empirical treatment.

Changes in Ocean Heat, Carbon Content, and Ventilation: A Review of the First Decade of GO-SHIP Global Repeat Hydrography.

Global ship-based programs, with highly accurate, full water column physical and biogeochemical observations repeated decadally since the 1970s, provide a crucial resource for documenting ocean change. The ocean, a central component of Earth's climate system, is taking up most of Earth's excess anthropogenic heat, with about 19% of this excess in the abyssal ocean beneath 2,000 m, dominated by Southern Ocean warming. The ocean also has taken up about 27% of anthropogenic carbon, resulting in acidification of the upper ocean. Increased stratification has resulted in a decline in oxygen and increase in nutrients in the Northern Hemisphere thermocline and an expansion of tropical oxygen minimum zones. Southern Hemisphere thermocline oxygen increased in the 2000s owing to stronger wind forcing and ventilation. The most recent decade of global hydrography has mapped dissolved organic carbon, a large, bioactive reservoir, for the first time and quantified its contribution to export production (∼20%) and deep-ocean oxygen utilization. Ship-based measurements also show that vertical diffusivity increases from a minimum in the thermocline to a maximum within the bottom 1,500 m, shifting our physical paradigm of the ocean's overturning circulation.

Increased insulin resistance in obese, glucose-intolerant Southwestern American Indians: evidence for a defect not explained by obesity.

Increased in vivo resistance to insulin-mediated glucose disposal has been observed in obese subjects with normal glucose tolerance and in nonobese subjects with glucose intolerance. To determine whether the insulin resistance of glucose-intolerant obese subjects can be accounted for by obesity alone, insulin-mediated glucose disposal was measured in 14 glucose-intolerant and 21 nondiabetic. Southwestern American Indians with similar degrees of obesity. A mixture of insulin, glucose, and somatostatin was infused which delivered the same quantity of glucose and achieved similar plasma insulin concentrations in all subjects. Despite similar steady state plasma insulin levels, the mean steady state plasma glucose concentration was higher in the glucose-intolerant subjects than in weight-matched subjects with normal glucose tolerance (226 +/- 10 vs. 136 +/- 13 mg/dl; P < 0.0001). This increased resistance to insulin action was found in the presence of similar insulin binding to mononuclear cells (measured in 8 glucose-intolerant subjects and 9 subjects with normal glucose tolerance). In obese Southwestern American Indians with glucose intolerance, abnormalities beyond the site of insulin binding to its receptor may explain the observed increase in in vivo insulin resistance.

Hypergastrinemia in obese noninsulin-dependent diabetes: a possible reflection of high prevalence of vagal dysfunction.

To elucidate the relation of noninsulin-dependent (type II) diabetes mellitus to plasma levels of gastrin, pepsinogen I, and pepsinogen II, gastric acid secretion, and gastric emptying, we studied diabetic and nondiabetic obese Pima Indian subjects. Fasting and postprandial plasma gastrin concentrations were significantly higher (P less than 0.02) in diabetic than in nondiabetic subjects, but gastric acid outputs basally, after an acaloric liquid meal, and in response to betazole were similar in the two groups. Plasma pepsinogen I and pepsinogen II levels were also similar in both groups. A significant negative relation (r = -0.84; P less than 0.01) was found between basal gastrin levels and gastric acid production in nondiabetic Indians, but not in diabetic Pimas. The fractional gastric emptying rate of an acaloric liquid meal was significantly decreased in diabetic Pimas (P less than 0.01); and at least one test showing abnormal vagal function, as estimated by the Valsalva maneuver, heart rate changes between deep expiration and inspiration, and postural hypotension, was found in every diabetic subject. These findings suggest that hypergastrinemia in type II diabetes is not related to hypochlorhydria, but, instead, results from autonomic dysfunction with slow gastric emptying.

Albumin leakage into cerebrospinal fluid of dogs lethally infected with R252 canine distemper virus.

Cerebrospinal fluid (CSF) form nine lethally infected and three convalescent gnotobiotic dogs infected with the R252 strain of canine distemper virus (CDV) was evaluated prior to and following infection. Lethally infected dogs had a mean seven-fold increase in CSF albumin concentration compared to the preinoculation value, not present in dogs destined to survive. Immunochemical examination of tissue from these dogs revealed prominent perivascular localization of albumin. Examination of CSF cells demonstrated mild leukocytosis in both groups at the time when encephalopathic deaths occurred, with decreased lymphocyte percentages, particularly Thy-1-bearing lymphocytes, in lethally infected dogs. These dogs also had more extensive expression of viral antigens in CSF and peripheral blood leukocytes at the time of death than did surviving dogs, and failed to make antibody to viral antigens. The findings link terminal breakdown of the blood-brain barrier and extensive viral antigen expression in CSF leukocytes with experimental CDV infection resulting in death.

Production of immunoglobulin G and increased antiviral antibody in cerebrospinal fluid of dogs with delayed-onset canine distemper viral encephalitis.

Sera and cerebrospinal fluid (CSF) from four dogs with delayed-onset canine distemper viral (CDV) encephalitis (old dog encephalitis) were compared with samples from dogs with acute CDV and from recently vaccinated controls. Dogs with old dog encephalitis (ODE) had elevated CSF IgG concentrations (122 micrograms/ml) compared to controls (13 micrograms/ml) without elevated CSF albumin; their CSF IgG index was significantly greater. CSF proteins banding in the alkaline region of isoelectric focusing gels were immunochemically identified as IgG. Detectable viral neutralizing antibody was present in ODE CSF, and formed a larger proportion of IgG in CSF than in serum. Serum samples containing 2 mg IgG bound to all viral polypeptides of both R252 and Onderstepoort CDV isolates by immunoblot analysis. CSF samples of ODE patients bound viral antigens when diluted to contain as little as 5-40 micrograms IgG, while patient serum could be diluted to 40-200 micrograms IgG content compared to serum IgG of 100 micrograms/ml in vaccinated controls. Serial CSF dilutions consistently bound to H and NP polypeptides at the highest dilutions, similar to the binding of serums from recently vaccinated dogs. Thus, dogs with delayed-onset CDV encephalitis have elevated concentrations of CSF IgG, much of which is virus-specific, with an antigen binding pattern similar to that of sera of recently immunized dogs.

Inflammatory central nervous system disease in lupus-prone MRL/lpr mice: comparative histologic and immunohistochemical findings.

The brains of pathogen-free autoimmune MRL/lpr, NZBWF1 and NZB mice were examined for central nervous system (CNS) inflammation in premoribund 8-week-old animals and at ages when active systemic lupus erythematosus (SLE) was present. CNS inflammation was observed only in MRL/lpr mice. Immunohistochemical studies of brains from young MRL/lpr mice found that infiltrates were composed primarily of CD4+ cells. Older MRL/lpr mice (22 and 26 weeks of age) had CD4+ cells predominantly, but CD8+ and B220+ cells were also present. Perivascular leakage of IgG was a prominent and unexpected finding in the MRL/lpr model. Congenic MRL/+ mice with late-onset autoimmunity had no inflammatory cells in brain tissue, and there was no perivascular staining with IgG or albumin. Our findings suggest that MRL/lpr mice are a useful model for studies of lupus-associated CNS inflammatory disease, and perivascular leakage may be a primary mechanism for entry of IgG into the brain.

Phosphorylated variant of bovine prolactin.

Bovine pituitary explants and cell cultures were incubated with [32P]orthophosphate. Extracts were prepared from the explants and analyzed by sodium dodecyl sulfate-containing acrylamide gel electrophoresis and autoradiography revealing a phosphoprotein that co-migrated with authentic bovine prolactin. Clonal antibodies to bovine prolactin were produced, purified and used to prepare affinity columns. Extracts of [32P]orthophosphate-labeled explants and cells or media were applied to prolactin affinity columns and a radiolabeled protein was eluted with a pH 2.8 wash. The eluted protein was identified as prolactin by co-migration with standard on gel electrophoresis and by amino acid analysis. Treatment of immunoaffinity-purified pituitary prolactin with alkaline phosphatase reduced the phosphate associated with prolactin in a time-dependent manner, indicating a covalent phosphate linkage. Autoradiography of gels revealed prolactin from explants, cells and their associated media to be a phosphoprotein. A phosphorylated variant of bovine prolactin is synthesized and secreted in both explant and cell cultures.