👤 Peter J O'Brien

Cerebrospinal fluid amyloid beta 42, total tau, and phosphorylated tau 181 are well accepted markers of Alzheimer's disease. These biomarkers better reflect disease pathogenesis compared to clinical diagnosis. Here, we perform a genome wide association study meta-analysis including 18,948 individuals of European ancestry and identify 12 genome-wide significant loci across all three biomarkers, eight of them novel. We replicate the association of biomarkers with APOE, CR1, GMNC/CCDC50 and C16orf95/MAP1LC3B. Novel loci include BIN1 for amyloid beta and GNA12, MS4A6A, SLCO1A2 with both total tau and phosphorylated tau 181, as well as additional loci on chr. 8, near ANGPT1 and chr. 9 near SMARCA2. We also demonstrate that these variants have significant association with Alzheimer's disease risk, disease progression and/or brain amyloidosis. The associated genes are implicated in lipid metabolism independent of APOE, coupled with autophagy and brain volume regulation driven by total tau and phosphorylated tau 181 dysregulation. Show less

Plasma biomarkers of Alzheimer's disease (AD) pathology are frequently tested in specialized research settings, which limits the generalizability of findings. Using electronic health records and banked plasma, we evaluated plasma biomarkers-phosphorylated tau 217 (p-tau Participants (n = 617; 44% Black/African American; 41% female) were selected from the University of Pennsylvania Medicine BioBank with plasma assayed using Fujirebio Lumipulse. International Classification of Diseases (ICD) Ninth and Tenth Revision codes determined AD dementia (ADD) (n = 43), mild-cognitive impairment (MCI) (n = 140), unspecified/non-AD cognitive impairment (CI) (n = 106), and cognitively normal cases (n = 328), and other medical histories. APOE ε4, body mass index (BMI), metrics of kidney function (eg, estimated glomerular filtration rate [eGFR]), and liver disease were derived from electronic health records. Multivariable models identified factors related to plasma levels. Previously established cutpoints classified AD status ("AD+," "AD-," or "Intermediate"). Plasma p-tau In this real-world dataset, we identified effects of multimorbidities on plasma biomarkers, especially kidney function. The p-tau Show less

CTNNB1-mutated hepatocellular carcinomas are characterized by a distinctive morphology and activation of the Wnt pathway. AXIN1 also plays a key role in the Wnt pathway, but the morphology of AXIN1-mutated tumors has not been examined. In addition, there are ongoing questions on the ability of AXIN1 mutations to activate the Wnt pathway in hepatocellular carcinoma. AXIN1 mutated tumors (N=18) were studied, along with control groups: CTNNB1 (N=17), APC (6), or "Other" genes in the Wnt pathway (5). Wnt pathway activation was studied by immunostains for beta-catenin and glutamine synthetase. Findings were supplemented by gene expression analysis using TCGA data. On histologic examination, the classic morphology associated with beta-catenin mutations was found in all 4 groups: 8/18 AXIN1 (44%), 10/17 CTNNB1 (59%), 4/6 APC (67%), and 1/5 Other (20%). By immunohistochemistry, Wnt pathway activation was found in 11/18 AXIN1 (61%), 15/17 CTTNB1 (88%), 6/6 APC (100%), and 5/5 (100%) of Other. In AXIN1-mutated tumors, the Wnt pathway was weakly activated. Glutamine synthetase stains also highlighted a new "progressed pattern" associated with distinct subnodules of staining. Tertiary lymphoid structures were uncommon except for cases with CTTNNB1 mutations plus additional mutations in the Wnt pathway. In summary, the classic morphology associated with CTNNB1 mutations is found in hepatocellular carcinomas with mutations in AXIN1, APC, and other Wnt genes. AXIN1 mutated tumors have Wnt activation that is detectable but at lower levels than CTNNB1 mutated tumors. As tumors progress, their level of Wnt activation can change. Show less

Hypertension is increasingly prevalent among middle-aged adults, but the impacts of physical activity and postures are not fully understood in middle-aged males and females, limiting targeted prevention. This study investigated associations between habitual physical activity and postures with hypertension, and whether they differ between sexes at the same age. 4416 participants (age: 46 years) in the 10th sweep of the 1970 British Cohort Study were used. Participants wore an activPAL to measure physical behaviors. Stage 2 hypertension was defined as >140/90 mmHg or antihypertensive medication use. Isotemporal substitution models assessed the theoretical effect of reallocating 30 min of one behavior with another. Males had higher mean body mass index (BMI: 28.5 ± 4.6 kg/m Show less

The prevalence of childhood obesity is increasing worldwide, along with the associated common comorbidities of type 2 diabetes and cardiovascular disease in later life. Motivated by evidence for a strong genetic component, our prior genome-wide association study (GWAS) efforts for childhood obesity revealed 19 independent signals for the trait; however, the mechanism of action of these loci remains to be elucidated. To molecularly characterize these childhood obesity loci, we sought to determine the underlying causal variants and the corresponding effector genes within diverse cellular contexts. Integrating childhood obesity GWAS summary statistics with our existing 3D genomic datasets for 57 human cell types, consisting of high-resolution promoter-focused Capture-C/Hi-C, ATAC-seq, and RNA-seq, we applied stratified LD score regression and calculated the proportion of genome-wide SNP heritability attributable to cell type-specific features, revealing pancreatic alpha cell enrichment as the most statistically significant. Subsequent chromatin contact-based fine-mapping was carried out for genome-wide significant childhood obesity loci and their linkage disequilibrium proxies to implicate effector genes, yielded the most abundant number of candidate variants and target genes at the Show less

Recent trials in Alzheimer's disease (AD) demonstrate encouraging outcomes. These trials target risk mechanisms identified through genetic analysis whilst directly aiming to reduce progression rates. Evidence from other neurodegenerative diseases suggests the genetics of progression is distinct from risk of disease. To expand these initial successes and improve clinical outcomes further we need to understand genetics of progression of disease. These can be deduced through rigorous analysis of meticulously phenotyped longitudinal cohorts. In this study we first looked at known genetic drivers of risk, namely polygenic risk scores for AD and A total of 387 individuals with, genetic data, amyloid positivity and in active decline (ADNI (n=222) and AIBL(n=165)) were used to perform generalised mixed effects linear model genome wide association studies of longitudinal cognitive decline as measured by mini mental state examination. The resulting summary statistics were subjected z, and colocalization analyses. Established AD risk factors, including These findings enhance our understanding of the biological underpinnings of AD progression, opening new avenues for therapeutic intervention. Show less

Platelet-like particles (PLPs), derived from megakaryocytic cell lines MEG-01 and K-562, are widely used as a surrogate to study platelet formation and function. We demonstrate by RNA-Seq that PLPs are transcriptionally distinct from platelets. Expression of key genes in signaling pathways promoting platelet activation/aggregation, such as the PI3K/AKT, protein kinase A, phospholipase C, and α-adrenergic and GP6 receptor pathways, was missing or under-expressed in PLPs. Functionally, PLPs do not aggregate following epinephrine, collagen, or ADP stimulation. While PLPs aggregated in response to thrombin, they did not display enhanced expression of surface markers P-selectin and activated α Show less

Image-Based Sexual Abuse (IBSA) represents a significant and growing public health concern, yet individuals affected by IBSA exhibit diverse experiences of risk, victimization, and resilience. This study aimed to identify distinct profiles among individuals exposed to IBSA, childhood victimization and adversity, online risk behavior, and delinquency. Using Latent Profile Analysis (LPA), we analyzed data from 2,630 young adults (ages 18-28) recruited through an online survey, oversampling sexual and gender minorities and individuals with IBSA experiences prior to the age of 18. Seven distinct profiles emerged, reflecting varying levels of risk engagement. The largest group Show less

The purpose of this study was to investigate the longitudinal association of replacing stationary time (ST) with either light (LPA) or moderate-to-vigorous (MVPA) physical activity and replacing LPA with MVPA on 2-year clinical outcomes in individuals with varying severities of knee osteoarthritis. This retrospective cohort study used isotemporal substitution models to investigate the association of replacing 10-60 min of ST with LPA or MVPA and of LPA with MVPA on Western Ontario and McMaster Universities Osteoarthritis Index pain, stiffness, function, and gait speed at 2 years. Device-based stationary and physical activities were monitored using accelerometry within the Osteoarthritis Initiative cohort (n = 848). All analyses were completed separately for mild-to-moderate (Kellgren-Lawrence grade = 1-2) and severe (Kellgren-Lawrence grade = 3-4) knee osteoarthritis groups. In individuals with mild-to-moderate osteoarthritis, substituting 30-60 min of ST or LPA with MVPA improved pain and gait speed at 2 years but worsened stiffness. Replacing 60 min of ST with LPA improved Western Ontario and McMaster Universities Osteoarthritis Index function. For severe osteoarthritis, substituting 30-60 min of ST or LPA with MVPA led to worsened Western Ontario and McMaster Universities Osteoarthritis Index pain and function at 2 years, whereas substituting ST or LPA with MVPA minimally impacted stiffness. Improvements in gait speed were observed with 10-to-60-min ST or LPA substitutions with MVPA. Stationary and physical activity substitutions had varying longitudinal effects in individuals with mild-to-moderate versus severe knee osteoarthritis. Individuals with mild-to-moderate and severe knee osteoarthritis may require differing physical activity prescription to improve functional and overall health outcomes. Significance/Implications: These findings underscore the importance of severity-specific physical activity recommendations to support clinical outcomes in knee osteoarthritis management. Show less

The prevalence of childhood obesity is increasing worldwide, along with the associated common comorbidities of type 2 diabetes and cardiovascular disease in later life. Motivated by evidence for a strong genetic component, our prior genome-wide association study (GWAS) efforts for childhood obesity revealed 19 independent signals for the trait; however, the mechanism of action of these loci remains to be elucidated. To molecularly characterize these childhood obesity loci we sought to determine the underlying causal variants and the corresponding effector genes within diverse cellular contexts. Integrating childhood obesity GWAS summary statistics with our existing 3D genomic datasets for 57 human cell types, consisting of high-resolution promoter-focused Capture-C/Hi-C, ATAC-seq, and RNA-seq, we applied stratified LD score regression and calculated the proportion of genome-wide SNP heritability attributable to cell type-specific features, revealing pancreatic alpha cell enrichment as the most statistically significant. Subsequent chromatin contact-based fine-mapping was carried out for genome-wide significant childhood obesity loci and their linkage disequilibrium proxies to implicate effector genes, yielded the most abundant number of candidate variants and target genes at the Show less

This study aimed to establish variants in CBX1, encoding heterochromatin protein 1β (HP1β), as a cause of a novel syndromic neurodevelopmental disorder. Patients with CBX1 variants were identified, and clinician researchers were connected using GeneMatcher and physician referrals. Clinical histories were collected from each patient. To investigate the pathogenicity of identified variants, we performed in vitro cellular assays and neurobehavioral and cytological analyses of neuronal cells obtained from newly generated Cbx1 mutant mouse lines. In 3 unrelated individuals with developmental delay, hypotonia, and autistic features, we identified heterozygous de novo variants in CBX1. The identified variants were in the chromodomain, the functional domain of HP1β, which mediates interactions with chromatin. Cbx1 chromodomain mutant mice displayed increased latency-to-peak response, suggesting the possibility of synaptic delay or myelination deficits. Cytological and chromatin immunoprecipitation experiments confirmed the reduction of mutant HP1β binding to heterochromatin, whereas HP1β interactome analysis demonstrated that the majority of HP1β-interacting proteins remained unchanged between the wild-type and mutant HP1β. These collective findings confirm the role of CBX1 in developmental disabilities through the disruption of HP1β chromatin binding during neurocognitive development. Because HP1β forms homodimers and heterodimers, mutant HP1β likely sequesters wild-type HP1β and other HP1 proteins, exerting dominant-negative effects. Show less

We assessed the spatiotemporal GLP-1 and GIP receptor signaling, trafficking, and recycling dynamics of GIPR mono-agonists, GLP-1R mono-agonists including semaglutide, and GLP-1/GIP dual-agonists MAR709 and tirzepatide. Receptor G protein recruitment and internalization/trafficking dynamics were assessed using bioluminescence resonance energy transfer (BRET)-based technology and live-cell HILO microscopy. Relative to native and acylated GLP-1 agonists, MAR709 and tirzepatide showed preserved maximal cAMP production despite partial Gα Our data indicated that MAR709 and tirzepatide induce unique spatiotemporal GLP-1 and GIP receptor signaling, trafficking, and recycling dynamics relative to native peptides, semaglutide, and matched mono-agonist controls. These findings support the hypothesis that the structure of GLP-1/GIP dual-agonists confer a biased agonism that, in addition to its influence on intracellular signaling, uniquely modulates receptor trafficking. Show less

OBJECTIVE: Niacin therapy fails to reduce cardiovascular events in statin-treated subjects even though it increases plasma HDL-C (HDL [high-density lipoprotein] cholesterol) and decreases LDL-C (LDL [low-density lipoprotein] cholesterol) and triglyceride levels. To investigate potential mechanisms for this lack of cardioprotection, we quantified the HDL proteome of subjects in 2 niacin clinical trials: the CPC study (Carotid Plaque Composition) and the HDL Proteomics substudy of the AIM-HIGH trial (Atherothrombosis Intervention in Metabolic Syndrome with Low HDL/High Triglycerides). APPROACH AND RESULTS: Using targeted proteomics, we quantified levels of 31 HDL proteins from 124 CPC subjects and 120 AIM-HIGH subjects. The samples were obtained at baseline and after 1 year of statin monotherapy or niacin-statin combination therapy. Compared with statin monotherapy, niacin-statin combination therapy did not reduce HDL-associated apolipoproteins APOC1, APOC2, APOC3, and APOC4, despite significantly lowering triglycerides. In contrast, niacin markedly elevated HDL-associated PLTP (phospholipid transfer protein), CLU (clusterin), and HP/HPR (haptoglobin/haptoglobinrelated proteins; P≤0.0001 for each) in both the CPC and AIM-HIGH cohorts. CONCLUSIONS: The addition of niacin to statin therapy resulted in elevated levels of multiple HDL proteins linked to increased atherosclerotic risk, which might have compromised the cardioprotective effects associated with higher HDL-C levels and lower levels of LDL-C and triglycerides. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT00715273; NCT00880178; NCT00120289. Show less

Regulator of G protein signaling (RGS) proteins have attracted attention as a result of their primary role in directing the specificity as well as the temporal and spatial aspects of G protein-coupled receptor signaling. In addition, alterations in RGS protein expression have been observed in a number of disease states, including certain cancers. In this area, RGS17 is of particular interest. It has been demonstrated that, while RGS17 is expressed primarily in the central nervous system, it has been found to be inappropriately expressed in lung, prostate, breast, cervical, and hepatocellular carcinomas. Overexpression of RGS17 leads to dysfunction in inhibitory G protein signaling and an overproduction of the intracellular second messenger cAMP, which in turn alters the transcription patterns of proteins known to promote various cancer types. Suppressing RGS17 expression with RNA interference (RNAi) has been found to decrease tumorigenesis and sufficiently prevents cancer cell migration, leading to the hypothesis that pharmacological blocking of RGS17 function could be useful in anticancer therapies. We have identified small-molecule fragments capable of binding the RGS homology (RH) domain of RGS17 by using a nuclear magnetic resonance fragment-based screening approach. By chemical shift mapping of the two-dimensional Show less

Islet vascularization is essential for intact islet function and glucose homeostasis. We have previously shown that primary cilia directly regulate insulin secretion. However, it remains unclear whether they are also implicated in islet vascularization. At eight weeks, murine Show less

Non-alcoholic fatty liver disease (NAFLD) is a very common indication for liver transplantation. How fat-rich diets promote progression from fatty liver to more damaging inflammatory and fibrotic stages is poorly understood. Here, we show that disrupting phosphorylation at Ser196 (S196A) in the liver X receptor alpha (LXRα, NR1H3) retards NAFLD progression in mice on a high-fat-high-cholesterol diet. Mechanistically, this is explained by key histone acetylation (H3K27) and transcriptional changes in pro-fibrotic and pro-inflammatory genes. Furthermore, S196A-LXRα expression reveals the regulation of novel diet-specific LXRα-responsive genes, including the induction of Ces1f, implicated in the breakdown of hepatic lipids. This involves induced H3K27 acetylation and altered LXR and TBLR1 cofactor occupancy at the Ces1f gene in S196A fatty livers. Overall, impaired Ser196-LXRα phosphorylation acts as a novel nutritional molecular sensor that profoundly alters the hepatic H3K27 acetylome and transcriptome during NAFLD progression placing LXRα phosphorylation as an alternative anti-inflammatory or anti-fibrotic therapeutic target. Show less

Regulator of G protein signaling (RGS) proteins are negative regulators of G protein-coupled receptor (GPCR) signaling through their ability to act as GTPase-activating proteins (GAPs) for activated Gα subunits. Members of the RZ subfamily of RGS proteins bind to activated Gα Show less

It is unknown if adult human skeletal muscle has an epigenetic memory of earlier encounters with growth. We report, for the first time in humans, genome-wide DNA methylation (850,000 CpGs) and gene expression analysis after muscle hypertrophy (loading), return of muscle mass to baseline (unloading), followed by later hypertrophy (reloading). We discovered increased frequency of hypomethylation across the genome after reloading (18,816 CpGs) versus earlier loading (9,153 CpG sites). We also identified AXIN1, GRIK2, CAMK4, TRAF1 as hypomethylated genes with enhanced expression after loading that maintained their hypomethylated status even during unloading where muscle mass returned to control levels, indicating a memory of these genes methylation signatures following earlier hypertrophy. Further, UBR5, RPL35a, HEG1, PLA2G16, SETD3 displayed hypomethylation and enhanced gene expression following loading, and demonstrated the largest increases in hypomethylation, gene expression and muscle mass after later reloading, indicating an epigenetic memory in these genes. Finally, genes; GRIK2, TRAF1, BICC1, STAG1 were epigenetically sensitive to acute exercise demonstrating hypomethylation after a single bout of resistance exercise that was maintained 22 weeks later with the largest increase in gene expression and muscle mass after reloading. Overall, we identify an important epigenetic role for a number of largely unstudied genes in muscle hypertrophy/memory. Show less

Adipose tissue dysfunction is associated with inflammation, metabolic syndrome and other diseases in aging. Recent work has demonstrated that compromised autophagy activity in aging adipose tissue promotes ER stress responses, contributing to adipose tissue and systemic inflammation in aging. Phosphatidylinositol 3-kinase catalytic subunit type 3 (Pik3c3) is an 887 amino acid lipid kinase that regulates intracellular membrane trafficking and autophagy activity. To address the mechanistic link between autophagy and ER stress response in aging adipose tissue, we generated a line of adipose tissue-specific Show less

Regulator of G protein signaling (RGS) proteins temporally regulate heterotrimeric G protein signaling cascades elicited by G protein-coupled receptor activation and thus are essential for cell homeostasis. The dysregulation of RGS protein expression has been linked to several pathologies, spurring discovery efforts to identify small-molecule inhibitors of these proteins. Presented here are the results of a high-throughput screening (HTS) campaign targeting RGS17, an RGS protein reported to be inappropriately upregulated in several cancers. A screen of over 60,000 small molecules led to the identification of five hit compounds that inhibit the RGS17-Gα Show less

Synaptic processes and plasticity of synapses are mediated by large suites of proteins. In most cases, many of these proteins are tethered together by synaptic scaffold proteins. Scaffold proteins have a large number and typically a variety of protein interaction domains that allow many different proteins to be assembled into functional complexes. Because each scaffold protein has a different set of protein interaction domains and a unique set of interacting partners, the presence of synaptic scaffolds can provide insight into the molecular mechanisms that regulate synaptic processes. In studies of rabbit retina, we found SAP102 and Chapsyn110 selectively localized in the tips of B-type horizontal cell processes, where they contact cone and rod photoreceptors. We further identified some known SAP102 binding partners, kainate receptor GluR6/7 and inward rectifier potassium channel Kir2.1, closely associated with SAP102 in photoreceptor invaginations. The kainate receptor occupies a position distinct from that of the majority of AMPA receptors that dominate the horizontal cell postsynaptic response. GluR6/7 and Kir2.1 presumably are involved in synaptic processes that govern cell-to-cell communication and could both contribute in different ways to synaptic currents that mediate feedback signaling. Notably, we failed to find evidence for the presence of Cx57 or Cx59 that might be involved in ephaptic feedback signaling in this complex. The presence of SAP102 and its binding partners in both cone and rod invaginating synapses suggests that whatever mechanism is supported by this protein complex is present in both types of photoreceptors. J. Comp. Neurol. 525:850-867, 2017. © 2016 Wiley Periodicals, Inc. Show less

Long chain polyunsaturated fatty acids (LCPUFA) are bioactive components of membrane phospholipids and serve as substrates for signaling molecules. LCPUFA can be obtained directly from animal foods or synthesized endogenously from 18 carbon precursors via the FADS2 coded enzyme. Vegans rely almost exclusively on endogenous synthesis to generate LCPUFA and we hypothesized that an adaptive genetic polymorphism would confer advantage. The rs66698963 polymorphism, a 22-bp insertion-deletion within FADS2, is associated with basal FADS1 expression, and coordinated induction of FADS1 and FADS2 in vitro. Here, we determined rs66698963 genotype frequencies from 234 individuals of a primarily vegetarian Indian population and 311 individuals from the US. A much higher I/I genotype frequency was found in Indians (68%) than in the US (18%). Analysis using 1000 Genomes Project data confirmed our observation, revealing a global I/I genotype of 70% in South Asians, 53% in Africans, 29% in East Asians, and 17% in Europeans. Tests based on population divergence, site frequency spectrum, and long-range haplotype consistently point to positive selection encompassing rs66698963 in South Asian, African, and some East Asian populations. Basal plasma phospholipid arachidonic acid (ARA) status was 8% greater in I/I compared with D/D individuals. The biochemical pathway product-precursor difference, ARA minus linoleic acid, was 31% and 13% greater for I/I and I/D compared with D/D, respectively. This study is consistent with previous in vitro data suggesting that the insertion allele enhances n-6 LCPUFA synthesis and may confer an adaptive advantage in South Asians because of the traditional plant-based diet practice. Show less

Amyloid A (AA) amyloidosis is a debilitating, often fatal, systemic amyloid disease associated with chronic inflammation and persistently elevated serum amyloid A (SAA). Elevated SAA is necessary but not sufficient to cause disease and the risk factors for AA amyloidosis remain poorly understood. Here we identify an extraordinarily high prevalence of AA amyloidosis (34%) in a genetically isolated population of island foxes (Urocyon littoralis) with concurrent chronic inflammatory diseases. Amyloid deposits were most common in kidney (76%), spleen (58%), oral cavity (45%), and vasculature (44%) and were composed of unbranching, 10 nm in diameter fibrils. Peptide sequencing by mass spectrometry revealed that SAA peptides were dominant in amyloid-laden kidney, together with high levels of apolipoprotein E, apolipoprotein A-IV, fibrinogen-α chain, and complement C3 and C4 (false discovery rate ≤ 0.05). Reassembled peptide sequences showed island fox SAA as an 111 amino acid protein, most similar to dog and artic fox, with 5 unique amino acid variants among carnivores. SAA peptides extended to the last two C-terminal amino acids in 5 of 9 samples, indicating that near full length SAA was often present in amyloid aggregates. These studies define a remarkably prevalent AA amyloidosis in island foxes with widespread systemic amyloid deposition, a unique SAA sequence, and the co-occurrence of AA with apolipoproteins. Show less

Glucose-dependent insulinotropic polypeptide (GIP) is an endogenous hormonal factor (incretin) that, upon binding to its receptor (GIPr; a class B G-protein-coupled receptor), stimulates insulin secretion by beta cells in the pancreas. There has been a lack of potent inhibitors of the GIPr with prolonged in vivo exposure to support studies on GIP biology. Here we describe the generation of an antagonizing antibody to the GIPr, using phage and ribosome display libraries. Gipg013 is a specific competitive antagonist with equally high potencies to mouse, rat, dog, and human GIP receptors with a Ki of 7 nm for the human GIPr. Gipg013 antagonizes the GIP receptor and inhibits GIP-induced insulin secretion in vitro and in vivo. A crystal structure of Gipg013 Fab in complex with the human GIPr extracellular domain (ECD) shows that the antibody binds through a series of hydrogen bonds from the complementarity-determining regions of Gipg013 Fab to the N-terminal α-helix of GIPr ECD as well as to residues around its highly conserved glucagon receptor subfamily recognition fold. The antibody epitope overlaps with the GIP binding site on the GIPr ECD, ensuring competitive antagonism of the receptor. This well characterized antagonizing antibody to the GIPr will be useful as a tool to further understand the biological roles of GIP. Show less

The recently discovered apolipoprotein A5 (ApoA5) is fast gaining attention as a key regulator of serum triglyceride concentrations. An ApoA5 mouse knock-out model produced an approximately fourfold increase in serum triglycerides, whereas a knock-in model with human ApoA5 produced 50-70% lower concentrations of mouse serum triglycerides. In addition, peroxisome proliferator-activated receptor-alpha agonists, which are used clinically to lower serum triglyceride concentrations, cause increased ApoA5 mRNA expression. Despite these compelling molecular biology data, relatively little is known about ApoA5 protein in human serum. To better understand circulating concentrations and lipoprotein particle distribution of ApoA5, we expressed the recombinant human ApoA5 protein and raised antibodies against both the NH(2) and COOH termini. Using the above reagents, we demonstrate for the first time that ApoA5 is present in human serum, although at much lower concentrations than other apolipoproteins such as ApoA1. Using a dual-antibody sandwich ELISA that we developed, we observed ApoA5 concentrations in human serum ranging from 24 to 406 microg/L compared with approximately 1 g/L for ApoA1. We also examined the lipoprotein particle distribution of ApoA5 and found that ApoA5 was detectable in VLDL, HDL, and chylomicrons, but not LDL. These data demonstrate for the first time that ApoA5 is a secreted protein present in human serum and is associated with specific lipoprotein particles. In addition, our data indicate that the circulating concentration of human ApoA5 is very low compared with other apolipoproteins. Show less

A panel of 42 rodent X cat somatic cell hybrids segregating individual cat chromosomes in different combinations was used to assign five isozyme structural loci to cat chromosomes. The feline homolog for glutathione reductase (GSR) was mapped to chromosome C2. Adenosine deaminase (ADA) and inosine triphosphatase (ITPA) were located on chromosome A3. Lactate dehydrogenase-A (LDHA) and acid phosphatase-2 (ACP2) were reassigned to chromosome D1. Localization of these genes increases the known feline genetic map and extends the known syntenic homologies between the cat and other mammalian species. Show less

A procedure is described which permits the isolation from the prepuberal mouse testis of highly purified populations of primitive type A spermatogonia, type A spermatogonia, type B spermatogonia, preleptotene primary spermatocytes, leptotene and zygotene primary spermatocytes, pachytene primary spermatocytes and Sertoli cells. The successful isolation of these prepuberal cell types was accomplished by: (a) defining distinctive morphological characteristics of the cells, (b) determining the temporal appearance of spermatogenic cells during prepuberal development, (c) isolating purified seminiferous cords, after dissociation of the testis with collagenase, (d) separating the trypsin-dispersed seminiferous cells by sedimentation velocity at unit gravity, and (e) assessing the identity and purity of the isolated cell types by microscopy. The seminiferous epithelium from day 6 animals contains only primitive type A spermatogonia and Sertoli cells. Type A and type B spermatogonia are present by day 8. At day 10, meiotic prophase is initiated, with the germ cells reaching the early and late pachytene stages by 14 and 18, respectively. Secondary spermatocytes and haploid spermatids appear throughout this developmental period. The purity and optimum day for the recovery of specific cell types are as follows: day 6, Sertoli cells (purity>99 percent) and primitive type A spermatogonia (90 percent); day 8, type A spermatogonia (91 percent) and type B spermatogonia (76 percent); day 18, preleptotene spermatocytes (93 percent), leptotene/zygotene spermatocytes (52 percent), and pachytene spermatocytes (89 percent), leptotene/zygotene spermatocytes (52 percent), and pachytene spermatocytes (89 percent). Show less