Risk factors associated with failed transfer of passive immunity in male and female dairy calves: A 2008 retrospective cross-sectional study.

This retrospective cross-sectional study was designed to identify risk factors associated with failed transfer of passive immunity (FTPI) and to determine differences in colostrum management between male and female dairy calves. Research technicians visited a total of 16 commercial dairy farms weekly. For each calf born on these farms, the farm personnel completed a birth record to document the colostrum management practices provided, level of calving assistance, calf sex, and time of birth. On the weekly visits to the farms, the technicians collected blood from calves that were 1 to 7 d of age. Serum was separated via centrifugation and the concentration of serum total protein (STP) was determined using a digital refractometer. Failed transfer of passive immunity was defined as calves having an STP of <5.2 g/dL. Data were available for 1,778 calves aged 1 to 7 d. Several differences were observed with respect to how male and female calves were managed. Male calves were more likely to receive a lower volume of colostrum, have colostrum delivered using a nipple bottle followed by an esophageal tube feeder, be fed pooled colostrum, and receive fresh colostrum rather than frozen colostrum relative to female calves. Serum total protein (STP) ranged from 3.6 to 9.7 g/dL with a mean of 5.7 g/dL (standard deviation, 0.7 g/dL) and 21.1% of the calves had FTPI. Using a mixed linear regression model, we identified that a calf being male (-0.14 g/dL), being delivered by a hard pull (-0.23 g/dL), and receiving the first feeding colostrum from a combination of a nipple bottle followed by an esophageal tube feeder (-0.12 g/dL) were associated with a lower concentration of STP. Feeding 6 L or more of colostrum in the first 24 h of life was associated with a 0.14 g/dL higher concentration of STP compared with feeding <3.9 L of colostrum. For FTPI, being delivered by a hard pull [odds ratio (OR) 2.21] and receiving the first feeding colostrum from a nipple bottle followed by an esophageal tube feeder (OR 1.83) were associated with higher odds of FTPI. Feeding >6 L of colostrum in the first 24 h of life was associated with a reduced odds (OR 0.65) of FTPI compared with feeding <3.9 L of colostrum. This study highlights the importance of certain management practices in reducing FTPI incidence and identifies discrepancies in colostrum management between male and female dairy calves.

Identification of novel genes differentially expressed in omental fat of obese subjects and obese type 2 diabetic patients.

Obesity is associated with an increased risk for developing type 2 diabetes, insulin resistance, hypertension, dyslipidemia, cardiovascular disease, respiratory dysfunction, and certain forms of cancer. Insulin resistance in many type 2 diabetic patients is the result of increased visceral adiposity. To identify novel genes implicated in type 2 diabetes and/or obesity and to elucidate the molecular mechanisms underlying both diseases, we analyzed gene expression in omental fat from lean and obese nondiabetic subjects and obese type 2 diabetic patients using mRNA differential display and subtracted library techniques. After screening over 13,800 subtracted cDNA clones and 6,912 cDNA amplification products, we identified 2,078 cDNAs that showed potential differential expression in the omental fat of lean versus obese nondiabetic subjects versus obese type 2 diabetic patients. Data analysis showed that 70.7% of these clones corresponded to unknown genes (26.7% matched express sequence tags [ESTs]) and 29.3% corresponded to known genes. Reverse Northern and classic Northern analyses further confirmed that the expression of five of these cDNA clones was elevated in obese nondiabetic subjects and obese type 2 diabetic patients. Four candidate genes were further evaluated for tissue distribution, which showed expression primarily in adipose and skeletal muscle tissue, and chromosomal localization. We concluded that both mRNA differential display and subtracted cDNA libraries are powerful tools for identifying novel genes implicated in the pathogenesis of obesity and type 2 diabetes.

Cloning and characterization of Rhesus monkey neuropeptide Y receptor subtypes.

Neuropeptide Y (NPY) is a 36 amino acid peptide that is abundant in the brain and peripheral nervous system. NPY has a variety of effects when administered into the brain including a pronounced feeding effect, anxiolysis, regulation of neuroendocrine axes and inhibition of neurotransmitter release. These effects are mediated by up to 6 G protein coupled receptors designated Y1, Y2, Y3, Y4, Y5 and y6. To better understand the phylogeny and pharmacology of NPY in non-human primates, we have cloned and expressed the NPY Y1, Y2 and Y5 receptor subtypes from the Rhesus monkey. No cDNA sequence encoding a Y4 receptor was found suggesting substantial sequence differences when compared to the human sequence. Comparison of these sequences with those from human indicated strong sequence conservation of Y1, Y2 and Y5 between the two species. The displacement of (125)I-PYY binding to the Rhesus monkey and human receptors by various peptides was compared to evaluate the pharmacology of the two species. Similar pharmacologies were noted across the species at the various receptor subtypes. These results indicate the Rhesus monkey and human NPY receptor subtypes have a close amino acid sequence conservation and that the peptide recognition domains are conserved as well.

Decreased expression of apM1 in omental and subcutaneous adipose tissue of humans with type 2 diabetes.

We have screened a subtracted cDNA library in order to identify differentially expressed genes in omental adipose tissue of human patients with Type 2 diabetes. One clone (#1738) showed a marked reduction in omental adipose tissue from patients with Type 2 diabetes. Sequencing and BLAST analysis revealed clone #1738 was the adipocyte-specific secreted protein gene apM1 (synonyms ACRP30, AdipoQ, GBP28). Consistent with the murine orthologue, apM1 mRNA was expressed in cultured human adipocytes and not in preadipocytes. Using RT-PCR we confirmed that apM1 mRNA levels were significantly reduced in omental adipose tissue of obese patients with Type 2 diabetes compared with lean and obese normoglycemic subjects. Although less pronounced, apM1 mRNA levels were reduced in subcutaneous adipose tissue of Type 2 diabetic patients. Whereas the biological function of apM1 is presently unknown, the tissue specific expression, structural similarities to TNFalpha, and the dysregulated expression observed in obese Type 2 diabetic patients suggest that this factor may play a role in the pathogenesis of insulin resistance and Type 2 diabetes.

Synthesis and in vitro biological activity of 4alpha-(2-propenyl)-5alpha-cholest-24-en-3alpha,12 alpha-diol, a 12alpha-hydroxyl analog of 4alpha-(2-propenyl)-5alpha-cholest-24-en-3alpha-ol: the latter is a potent activator of the low-density lipoprotein receptor promoter.

4alpha-(2-Propenyl)-5alpha-cholest-24-en-3alpha-ol (3) was shown recently in a Chinese hamster ovary (CHO) cell-based low-density lipoprotein receptor/luciferase (LDLR/Luc) assay to be a potent transcriptional activator of the LDL receptor promoter in the presence of 25-hydroxycholesterol. Because of the involvement of 12alpha-hydroxylation in the metabolism of cholesterol, we are interested in investigating the effect of introducing a 12alpha-hydroxyl group to 3 on the transcriptional activity of the LDL receptor promoter. Thus 4alpha-(2-propenyl)-5alpha-cholest-24-en-3alpha,12a lpha-diol (14), a 12alpha-hydroxyl analog of 3, was synthesized from deoxycholic acid via the formation of 12alpha-[[(tertbutyl)dimethylsilyl]oxy]-4alpha-( 2-propenyl)-5alpha-cholest-24-en-3-one (11). Test results show that 14 is inactive at concentrations of up to 20 microg/ml, compared to 3 with an EC30 value of 2.6 microM, in the CHO cell-based LDLR/Luc assay. Apparently introduction of a 12alpha-hydroxyl group abolishes the capability of 3alpha-sterol 14 to activate the transcription of the LDL receptor promoter. However, in the [1-14C-acetate]cholesterol biosynthesis inhibition assay in CHO cells, 14 at 10 microg/ml (23 microM) is shown to inhibit the cholesterol biosynthesis by 51% relative to the control cells. Our previous studies indicated that 3 showed a 38% inhibition, but 4alpha-(2-propenyl)-5alpha-cholestan-3alpha-ol (1) exhibited no inhibition in the same assay at 10 microg/ml. In summary the results indicate that, in addition to the 24,25-unsaturation, the 12alpha-hydroxyl group in 14 has also conferred an inhibitory effect on cholesterol biosynthesis in CHO cells; however, the inhibition of cholesterol biosynthesis by 14 does not lead to the transcriptional activation of the LDL receptor promoter.

Synthesis and in vitro biological activity of 4alpha-(2-propenyl)-5alpha-cholest-24-en-3alpha-ol: a 24,25-dehydro analog of the hypocholesterolemic agent 4alpha-(2-propenyl)-5alpha-cholestan-3alpha-ol.

4Alpha-(2-propenyl)-5alpha-cholestan-3alpha-ol (LY295427) was previously identified from a Chinese hamster ovary (CHO) cell-based low density lipoprotein receptor/luciferase (LDLR/Luc) assay to be a potent transcriptional activator of the LDL receptor promoter in the presence of 25-hydroxycholesterol. To investigate the effect of the 24,25-unsaturation in the D-ring side chain (desmosterol D-ring side chain) on antagonizing the repressing effect of 25-hydroxycholesterol, 4alpha-(2-propenyl)-5alpha-cholest-24-en-3alpha-ol (17), a 24,25-dehydro analog of LY295427, was thus synthesized from lithocholic acid via the formation of 3alpha-[[(1,1-dimethylethyl)dimethylsilyl]oxy]-4alpha- (2-propenyl)-5alpha-cholan-24-al (15). Test results showed that 17 had an EC30 value of 2.6 microM, comparable to 2.9 microM of LY295427, in the CHO cell-based LDLR/Luc assay in the presence of 25-hydroxycholesterol. Apparently, the built-in 24,25-unsaturation in the D-ring side chain of 17 had added little effect to antagonizing the repressing effect of 25-hydroxycholesterol. In the [1-14C-acetate]cholesterol biosynthesis inhibition assay, 17 at 10 microg/ml (23 microM) has been shown to inhibit the cholesterol biosynthesis in CHO cells by 38% relative to the vehicle control; whereas LY295427 showed no inhibition in the same assay in our previous studies. In contrast to LY295427, the built-in 24,25-unsaturation in the D-ring side chain of 17 has conferred an inhibitory effect on cholesterol biosynthesis in CHO cells. In summary, the observed LDL receptor promoter activity of 17 is related to its ability to prevent 25-hydroxycholesterol from exerting the repressing effect via an undetermined mechanism and, in part, to inhibit the cholesterol biosynthesis.

Effects of LY295427, a low-density lipoprotein (LDL) receptor up-regulator, on LDL receptor gene transcription and cholesterol metabolism in normal and hypercholesterolemic hamsters.

The action of LY295427 [(3alpha,4alpha, 5alpha)-4-(2-propenylcholestan-3-ol)], a compound that derepresses low-density lipoprotein receptor (LDL-R) expression in a cell-based model, was examined in hamsters. It was found that the compound does not have an effect in normal chow-fed hamsters, in which LDL-R levels are not repressed, but exerts a marked hypocholesterolemic effect (>70% decrease) in cholesterol-coconut oil-fed hamsters, in which LDL-R is repressed. In this model, there is a dose-response for cholesterol lowering with an approximate ED50 value of 40 mg/kg/day and an inverse relationship between serum cholesterol and serum LY295427 levels. LDL-R mRNA is increased (2-fold) and liver cholesterol ester content is decreased (>90%). Unlike the 3-hydroxy-3-methylglutarylcoenzyme A reductase inhibitor lovastatin, the decreased serum cholesterol is confined to the non-high-density lipoprotein fraction. Furthermore, LY295427 does not affect cholesterol biosynthesis, and it does not have a significant effect on cholesterol absorption. These data suggest that LY295427 acts in the hypercholesterolemic hamster by derepressing LDL-R transcription, thereby enhancing cholesterol clearance from the blood. The results with LY295427 suggest that compounds that act to increase LDL-R may represent a novel approach in the pharmacotherapy for hypercholesterolemia.

Characterization of the neuropeptide Y5 receptor in the human hypothalamus: a lack of correlation between Y5 mRNA levels and binding sites.

Neuropeptide Y (NPY) is a 36-amino-acid peptide that appears to play a central role in the control of feeding behavior. Recently, a cDNA encoding a novel NPY receptor subtype (Y5) was cloned from the rat and human hypothalamus, and shown to have a pharmacology consistent with NPY-induced feeding. We have subsequently cloned this cDNA from human hypothalamus and stably expressed it in CHO cells. Consistent with earlier reports, hY5 has a high affinity for NPY, [Leu31, Pro34]NPY, and NPY(3-36), but low affinity for larger C-terminal deletions of NPY and BIBP3226. High levels of hY5 mRNA were found in the human testis, brain, spleen and pancreas, with lower levels in several other tissues. In the human brain, hY5 mRNA levels were typically higher than hY2, but lower in comparison to hY1 receptor mRNA. To quantify the relative amounts of hY1, hY2 and hY5 mRNA in the human hypothalamus, we employed competitive RT-PCR. Interestingly, the relative amount of hY5 mRNA was substantially higher than either hY1 or hY2. However, pharmacological characterization of NPY binding sites in human hypothalamus membranes revealed predominantly the hY2 subtype. These data establish that while hY5 mRNA levels are very high in the human hypothalamus, conventional radioligand binding techniques do not detect hY5-like binding site. Whether hY5-like binding sites exist in the other human tissues that express hY5 mRNA (and what function hY5 has in those tissues) awaits future investigation.

Synthesis of 4 alpha-(2-propenyl)-5,6-secocholestan-3 alpha-ol, a novel B-ring seco analog of the hypocholesterolemic agent 4 alpha-(2-propenyl)-5 alpha-cholestan-3 alpha-ol.

4 alpha-(2-Propenyl)-5 alpha-cholestan-3 alpha-ol (LY295427) was previously identified from a CHO cell-based assay to be a potent LDL receptor up-regulator and had demonstrated to be an effective agent in lowering plasma cholesterol levels in hypercholesterolemic hamsters. In order to investigate the effect of flexibility of the 3 alpha-hydroxy-bearing A-ring on the activity, 4 alpha-(2-propenyl)-5,6-secocholestan-3 alpha-ol (11), a B-ring seco analog of LY295427, is thus synthesized from cholest-4-en-3-one. Test results indicate that 11 is not active in the CHO cell-based LDL receptor/luciferase assay at concentrations up to 20 micrograms/mL. The result underlines the importance of maintaining the A-B-C-D ring rigidity of the 3 alpha-sterols in terms of binding to the putative oxysterol receptor.

[125I]Leu31, Pro34-PYY is a high affinity radioligand for rat PP1/Y4 and Y1 receptors: evidence for heterogeneity in pancreatic polypeptide receptors.

Cloned receptors for the PP-fold peptides are subdivided into Y1, Y2, PP1/Y4, Y5 and Y6. NPY and PYY have similar affinity for Y1, Y2, Y5 and Y6 receptors while PP has highest affinity for PP1. Pro34-substituted analogs of NPY and PYY have selectivity for Y1 and Y1-like receptors over Y2 receptors. In the present study, we found the putative Y1-selective radioligand, [125I]Leu31, Pro34-PYY, also binds with high affinity to the rat PP1 receptor in cell lines expressing the receptor. However, in rat brain sections, [125I]Leu31, Pro34-PYY does not appear to bind to the interpeduncular nucleus, a brain region containing a high density of [125I]-bPP binding sites. Therefore, it appears there is additional heterogeneity in receptors recognizing PP.

The neuropeptide Y Y1 receptor selective radioligand, [125I][Leu31,Pro34]peptide YY, is also a high affinity radioligand for human pancreatic polypeptide 1 receptors.

A number of receptors for the pancreatic polypeptide-fold peptides are proposed based on findings from pharmacology and molecular biology studies. Neuropeptide Y and peptide YY have similar affinity for neuropeptide Y Y1 and neuropeptide Y Y2 while pancreatic polypeptide has highest affinity for pancreatic polypeptide 1. Pro34-substituted analogs of neuropeptide Y and peptide YY have selectivity for neuropeptide Y Y1 over neuropeptide Y Y2 receptors. In the present study, we found that one such 'neuropeptide Y Y1-selective' radioligand, [125I][Leu31,Pro34]peptide YY, also binds with high affinity to the pancreatic polypeptide 1 receptor. Therefore, caution needs to be exercised when using Pro34-analogs to define the neuropeptide Y Y1 receptor in vivo and using tissue preparations.

Characterization of the peptide binding requirements for the cloned human pancreatic polypeptide-preferring receptor.

Traditionally, neuropeptide Y (NPY) receptors have been divided into Y1 and Y2 subtypes based on peptide pharmacology and synaptic localization. Other receptor subtypes have been proposed based on preferences for NPY, peptide YY (PYY), or pancreatic polypeptide (PP). Recently, we discovered a novel human member of this receptor family exhibiting high affinity for PP and PYY. In the current study, we expressed a DNA clone encoding this human PP-preferring receptor [hPP1 (or Y4)] in Chinese hamster ovary cells and performed a peptide structure-activity study. [125I]pPYY bound to homogenates of hPP1-Chinese hamster ovary cells with a Kd of 0.064 +/- 0.006 nM and a Bmax of 244 +/- 12 fmol/mg protein. Human PP inhibited binding with a Ki of 0.023 nM, whereas human PYY (Ki = 0.31 nM) and human NPY Ki = 12 nM) were significantly less potent. Rat, porcine, and bovine PP inhibited binding with similar affinities to human PP, whereas avian PP was substantially less potent (Ki = 1 nM). Deletion of the first four amino acids reduced the affinity of bovine PP to 1 nM. Carboxyl-terminal fragments of NPY and PYY also had reduced potency compared with the native peptides. In addition, deletion of Tyr36-amide produced a substantial reduction in affinity. Pro34-substituted NPY and PYY had modestly increased affinity compared with the native peptides, although Gln34-bPP had similar affinity compared with bovine PP. The carboxyl-terminally derived Y1 antagonist 1229U91 was a very potent (Ki = 0.042 nM) inhibitor of binding to hPP1. Thus, the carboxyl-terminal region of PP seems to be the most important part of the peptide for high affinity binding to hPP1. A few key residues (amino acids 2 and 3) in the amino-terminal region of PP contribute to the high affinity of the native peptide. Thus, features required for peptide recognition by the hPP1 receptor seem to be distinct from the Y1 and Y2 receptor.

Expression cloning of a human brain neuropeptide Y Y2 receptor.

The 36-amino acid peptide, neuropeptide Y (NPY), is a member of a peptide family that includes the endocrine peptides, peptide YY (PYY), and pancreatic polypeptide (PP). NPY receptors have been broadly subdivided into postsynaptic Y1 receptors and presynaptic Y2 receptors based on the preference of Pro34-substituted analogues for the Y1 receptors and carboxyl-terminal fragments for the Y2. A Y1 receptor has been cloned, and this receptor appears to mediate several effects of NPY, including vasoconstriction and anxiolysis in animal models. We report the cloning of a human brain Y2 receptor from a human brain library. Pools of clones were transiently expressed in COS-1 cells, and 125I-PYY binding pools were identified by autoradiography. After a single positive pool was detected in the original screening, a single clone was isolated by four rounds of sequential enrichment. The clone encoded a 381-amino acid protein of the heptahelix (seven TM) type. Amino acid identity of this receptor with the Y1 receptor was 31% overall with 40% identity in the TM regions. Comparison with the human PP1 receptor indicated 33% overall amino acid identity with 42% identity in the TM regions. Pharmacologically, the receptor exhibited high affinity for NPY, PYY, and carboxyl-terminal fragments of NPY and PYY. In addition, Pro34-substituted analogues had very low affinity. With the use of Northern blot analysis, high levels of Y2 mRNA were detected in a variety of brain regions with little expression in peripheral tissues. Thus, the receptor protein has the pharmacological properties and distribution of the human Y2 receptor.

Synthesis and biological evaluation of a new series of sterols as potential hypocholesterolemic agents.

A new series of sterols was synthesized and tested in a CHO cell-based LDL receptor/luciferase (LDLR/Luc) assay to investigate the capability of derepressing the transcription of LDL receptor promoter in the presence of 25-hydroxycholesterol. The effect of various substitutions on antagonizing the repressing effect mediated by 25-hydroxycholesterol was also studied in terms of regio- and stereochemistry, lipophilicity, steric bulk, and pi-electron density. Except 12, compounds active in the primary LDLR/Luc assay were not active in the secondary simian virus 40/luciferase (SV40/Luc) assay, demonstrating the specificity of their in vitro activity. Eight active compounds of various structural types were selected and screened in a [1-14C-acetate]cholesterol biosynthesis inhibition assay; none has shown any interference with the cholesterol biosynthesis in CHO cells. In hypercholesterolemic hamsters, generally, compounds that were active in vitro were active in vivo and vice versa, with the exception of three in vitro inactive compounds: 3 beta-ols 3a' and 3c' as well as 3-ketone 2a. Experimental results from the livers of hamsters revealed that the in vivo conversion of 3a' or 2a to 3a has in part contributed to the observed in vivo activity, and it is also anticipated that 3c' may similarly be converted to 3c in hamsters.

Molecular cloning and characterization of a human adenocarcinoma/epithelial cell surface antigen complementary DNA.

A human adenocarcinoma-associated antigen (KSA) defined by the monoclonal antibody KS1/4 has become the focus of several site-directed strategies for tumor therapy. KSA, a 40,000 Da cell surface glycoprotein antigen, is found at a high density in all adenocarcinomas examined to date and in corresponding normal epithelial tissues. Here we describe the cloning and sequencing of overlapping complementary DNA clones which encode the entire KSA as expressed in UCLA-P3, a human lung adenocarcinoma cell line. We have deduced the 314-amino acid sequence and have compared it to the N-terminal amino acid sequence data of the affinity-purified antigen. The KSA is synthesized as a 314-residue-long preproprotein that is then processed to a 232-residue-long antigen. KSA appears to have a single transmembrane domain of 23 residues that separates the highly charged 26-residue cytoplasmic domain from the extracellular domain. The N-terminal region of the propeptide is rich in cysteines and contains three potential N-glycosylation sites. Computer-assisted analyses at both the DNA and protein levels have found no significant similarities of this protein to known sequences, but a GC-rich 5' terminus is evident. Northern blot analysis shows that transcription of KSA can be detected in RNA isolated from normal colon but not in RNA isolated from normal lung, prostate, or liver.