Antibody Response to Serpin B13 Induces Adaptive Changes in Mouse Pancreatic Islets and Slows Down the Decline in the Residual Beta Cell Function in Children with Recent Onset of Type 1 Diabetes Mellitus

Type 1 diabetes mellitus (T1D) is characterized by a heightened antibody (Ab) response to pancreatic islet self-antigens, which is a biomarker of progressive islet pathology. We recently identified a novel antibody to clade B serpin that reduces islet-associated T cell accumulation and is linked to the delayed onset of T1D. As natural immunity to clade B arises early in life, we hypothesized that it may influence islet development during that time. To test this possibility healthy young Balb/c male mice were injected with serpin B13 mAb or IgG control and examined for the number and cellularity of pancreatic islets by immunofluorescence and FACS. Beta cell proliferation was assessed by measuring nucleotide analog 5-ethynyl-2′-deoxyuridine (5-EdU) incorporation into the DNA and islet Reg gene expression was measured by real time PCR. Human studies involved measuring anti-serpin B13 autoantibodies by Luminex. We found that injecting anti-serpin B13 monoclonal Ab enhanced beta cell proliferation and Reg gene expression, induced the generation of ∼80 pancreatic islets per animal, and ultimately led to increase in the beta cell mass. These findings are relevant to human T1D because our analysis of subjects just diagnosed with T1D revealed an association between baseline anti-serpin activity and slower residual beta cell function decline in the first year after the onset of diabetes. Our findings reveal a new role for the anti-serpin immunological response in promoting adaptive changes in the endocrine pancreas and suggests that enhancement of this response could potentially help impede the progression of T1D in humans.     

Regional Differences in Islet Distribution in the Human Pancreas - Preferential Beta-Cell Loss in the Head Region in Patients with Type 2 Diabetes

While regional heterogeneity in islet distribution has been well studied in rodents, less is known about human pancreatic histology. To fill gaps in our understanding, regional differences in the adult human pancreas were quantitatively analyzed including the pathogenesis of type 2 diabetes (T2D). Cadaveric pancreas specimens were collected from the head, body and tail regions of each donor, including subjects with no history of diabetes or pancreatic diseases (n = 23) as well as patients with T2D (n = 12). The study further included individuals from whom islets were isolated (n = 7) to study islet yield and function in a clinical setting of islet transplantation. The whole pancreatic sections were examined using an innovative large-scale image capture and unbiased detailed quantitative analyses of the characteristics of islets from each individual (architecture, size, shape and distribution). Islet distribution/density is similar between the head and body regions, but is >2-fold higher in the tail region. In contrast to rodents, islet cellular composition and architecture were similar throughout the pancreas and there was no difference in glucose-stimulated insulin secretion in islets isolated from different regions of the pancreas. Further studies revealed preferential loss of large islets in the head region in patients with T2D. The present study has demonstrated distinct characteristics of the human pancreas, which should provide a baseline for the future studies integrating existing research in the field and helping to advance bi-directional research between humans and preclinical models.     

Identification of microRNAs expressed highly in pancreatic islet‐like cell clusters differentiated from human embryonic stem cells

Type 1 diabetes is an autoimmune destruction of pancreatic islet beta cell disease, making it important to find a new alternative source of the islet beta cells to replace the damaged cells. hES (human embryonic stem) cells possess unlimited self‐renewal and pluripotency and thus have the potential to provide an unlimited supply of different cell types for tissue replacement. The hES‐T3 cells with normal female karyotype were first differentiated into EBs (embryoid bodies) and then induced to generate the T3pi (pancreatic islet‐like cell clusters derived from T3 cells), which expressed pancreatic islet cell‐specific markers of insulin, glucagon and somatostatin. The expression profiles of microRNAs and mRNAs from the T3pi were analysed and compared with those of undifferentiated hES‐T3 cells and differentiated EBs. MicroRNAs negatively regulate the expression of protein‐coding mRNAs. The T3pi showed very high expression of microRNAs, miR‐186, miR‐199a and miR‐339, which down‐regulated the expression of LIN28, PRDM1, CALB1, GCNT2, RBM47, PLEKHH1, RBPMS2 and PAK6. Therefore, these microRNAs and their target genes are very likely to play important regulatory roles in the development of pancreas and/or differentiation of islet cells, and they may be manipulated to increase the proportion of beta cells and insulin synthesis in the differentiated T3pi for cell therapy of type I diabetics.     

Controlled aggregation of primary human pancreatic islet cells leads to glucose‐responsive pseudoislets comparable to native islets

Clinical islet transplantation is a promising treatment for patients with type 1 diabetes. However, pancreatic islets vary in size and shape affecting their survival and function after transplantation because of mass transport limitations. To reduce diffusion restrictions and improve islet cell survival, the generation of islets with optimal dimensions by dispersion followed by reassembly of islet cells, can help limit the length of diffusion pathways. This study describes a microwell platform that supports the controlled and reproducible production of three‐dimensional pancreatic cell clusters of human donor islets. We observed that primary human islet cell aggregates with a diameter of 100–150 μm consisting of about 1000 cells best resembled intact pancreatic islets as they showed low apoptotic cell death (<2%), comparable glucose‐responsiveness and increasing PDX1, MAFA and INSULIN gene expression with increasing aggregate size. The re‐associated human islet cells showed an a‐typical core shell configuration with beta cells predominantly on the outside unlike human islets, which became more randomized after implantation similar to native human islets. After transplantation of these islet cell aggregates under the kidney capsule of immunodeficient mice, human C‐peptide was detected in the serum indicating that beta cells retained their endocrine function similar to human islets. The agarose microwell platform was shown to be an easy and very reproducible method to aggregate pancreatic islet cells with high accuracy providing a reliable tool to study cell–cell interactions between insuloma and/or primary islet cells.     

Updating the proteome of the uncultivable hemotrophic Mycoplasma suis in experimentally infected pigs

Mycoplasma suis belongs to the hemotrophic mycoplasmas that are associated with acute and chronic anemia in a wide range of livestock and wild animals. The inability to culture M. suis in vitro has hindered its characterization at the molecular level. Since the publication of M. suis genome sequences in 2011 only one proteome study has been published. Aim of the presented study was to significantly extend the proteome coverage of M. suis strain KI_3806 during acute infection by applying three different protein extraction methods followed by 1D SDS‐PAGE and LC‐MS/MS. A total of 404 of 795 M. suis KI_3806 proteins (50.8%) were identified. Data analysis revealed the expression of 83.7% of the predicted ORFs with assigned functions but also highlights the expression of 179 of 523 (34.2%) hypothetical proteins with unknown functions. Computational analyses identified expressed membrane‐associated hypothetical proteins that might be involved in adhesion or host–pathogen interaction. Furthermore, analyses of the expressed proteins indicated the existence of a hexose‐6‐phosphate‐transporter and an ECF transporter. In conclusion, our proteome study provides a further step toward the elucidation of the unique life cycle of M. suis and the establishment of an in vitro culture. All MS data have been deposited in the ProteomeXchange with identifier PXD002294 ( http://proteomecentral.proteomexchange.org/dataset/PXD002294 ).     

A novel technique for the in vivo imaging of autoimmune diabetes development in the pancreas by two-photon microscopy

Type 1 diabetes (T1D) is characterized by the immune-mediated destruction of beta cells in the pancreas. Little is known about the in vivo dynamic interactions between T cells and beta cells or the kinetic behavior of other immune cell subsets in the pancreatic islets. Utilizing multiphoton microscopy we have designed a technique that allows for the real-time visualization of diabetogenic T cells and dendritic cells in pancreatic islets in a live animal, including their interplay with beta cells and the vasculature. Using a custom designed stage, the pancreas was surgically exposed under live conditions so that imaging of islets under intact blood pressure and oxygen supply became possible. We demonstrate here that this approach allows for the tracking of diabetogenic leukocytes as well as vascularization phenotype of islets and accumulation of dendritic cells in islets during diabetes pathogenesis. This technique should be useful in mapping crucial kinetic events in T1D pathogenesis and in testing the impact of immune based interventions on T cell migration, extravasation and islet destruction.     

Conditional Expression of Smad7 in Pancreatic β Cells Disrupts TGF-β Signaling and Induces Reversible Diabetes Mellitus

Identification of signaling pathways that maintain and promote adult pancreatic islet functions will accelerate our understanding of organogenesis and improve strategies for treating diseases like diabetes mellitus. Previous work has implicated transforming growth factor-β (TGF-β) signaling as an important regulator of pancreatic islet development, but has not established whether this signaling pathway is required for essential islet functions in the adult pancreas. Here we describe a conditional system for expressing Smad7, a potent inhibitor of TGF-β signaling, to identify distinct roles for this pathway in adult and embryonic β cells. Smad7 expression in Pdx1 ⁺ embryonic pancreas cells resulted in striking embryonic β cell hypoplasia and neonatal lethality. Conditional expression of Smad7 in adult Pdx1 ⁺ cells reduced detectable β cell expression of MafA, menin, and other factors that regulate β cell function. Reduced pancreatic insulin content and hypoinsulinemia produced overt diabetes that was fully reversed upon resumption of islet TGF-β signaling. Thus, our studies reveal that TGF-β signaling is crucial for establishing and maintaining defining features of mature pancreatic β cells.     

In vivo characterization of transplanted human embryonic stem cell‐derived pancreatic endocrine islet cells

Abstract. Objectives: Islet‐like clusters (ILCs), differentiated from human embryonic stem cells (hESCs), were characterized both before and after transplantation under the kidney capsule of streptozotocin‐induced diabetic immuno‐incompetent mice. Materials and methods: Multiple independent ILC preparations (n = 8) were characterized by immunohistochemistry, flow cytometry and cell insulin content, with six preparations transplanted into diabetic mice (n = 42), compared to controls, which were transplanted with either a human fibroblast cell line or undifferentiated hESCs (n = 28). Results: Prior to transplantation, ILCs were immunoreactive for the islet hormones insulin, C‐peptide and glucagon, and for the ductal epithelial marker cytokeratin‐19. ILCs also had cellular insulin contents similar to or higher than human foetal islets. Expression of islet and pancreas‐specific cell markers was maintained for 70 days post‐transplantation. The mean survival of recipients was increased by transplanted ILCs as compared to transplanted human fibroblast cells (P < 0.0001), or undifferentiated hESCs (P < 0.042). Graft function was confirmed by secretion of human C‐peptide in response to an oral bolus of glucose. Conclusions: hESC‐derived ILC grafts continued to contain cells that were positive for islet endocrine hormones and were shown to be functional by their ability to secrete human C‐peptide. Further enrichment and maturation of ILCs could lead to generation of a sufficient source of insulin‐producing cells for transplantation into patients with type 1 diabetes.     

Association of DLA‐DQB1 alleles with exocrine pancreatic insufficiency in Pembroke Welsh Corgis

Exocrine pancreatic insufficiency (EPI) is a digestive disorder resulting from the insufficient secretion of enzymes from the pancreas. In dogs, this condition is often attributed to pancreatic acinar atrophy, wherein the enzyme‐producing acinar cells are believed to be destroyed through an autoimmune process. Although EPI affects many diverse breeds, to date, molecular studies have been limited to the German Shepherd dog. A recent study of major histocompatibility genes in diseased and healthy German Shepherd dogs identified both risk and protective haplotypes. Herein, we genotyped DLA‐DQB1 in Pembroke Welsh Corgis to determine whether dog leukocyte antigen alleles contribute to the pathogenesis of EPI across dog breeds. We evaluated 14 affected and 43 control Pembroke Welsh Corgis, which were selected based on an age of onset similar to German Shepherd dogs. We identified one protective allele (odds ratio = 0.13, P‐value = 0.044) and one risk allele (odds ratio = 3.8, P‐value = 0.047). As in German Shepherd dogs, the risk allele is a duplication of DLA‐DQB1 (alleles DQB1*013:03 and 017:01); however, Pembroke Welsh Corgis have acquired a single polymorphism on DQB1*017:01. Thus, the DLA‐DQB1 duplication is a risk allele for EPI in at least two breeds.     

Proteomic Analysis of Disease Stratified Human Pancreas Tissue Indicates Unique Signature of Type 1 Diabetes

Type 1 diabetes (T1D) and type 2 diabetes (T2D) are associated with functional beta cell loss due to ongoing inflammation. Despite shared similarities, T1D is an autoimmune disease with evidence of autoantibody production, as well as a role for exocrine pancreas involvement. Our hypothesis is that differential protein expression occurs in disease stratified pancreas tissues and regulated proteins from endocrine and exocrine tissues are potential markers of disease and potential therapeutic targets. The study objective was to identify novel proteins that distinguish the pancreas from donors with T1D from the pancreas from patients with T2D, or autoantibody positive non-diabetic donors. Detailed quantitative comprehensive proteomic analysis was applied to snap frozen human pancreatic tissue lysates from organ donors without diabetes, with T1D-associated autoantibodies in the absence of diabetes, with T1D, or with T2D. These disease-stratified human pancreas tissues contain exocrine and endocrine tissues (with dysfunctional islets) in the same microenvironment. The expression profiles of several of the proteins were further verified by western blot. We identified protein panels that are significantly and uniquely upregulated in the three disease-stratified pancreas tissues compared to non-disease control tissues. These proteins are involved in inflammation, metabolic regulation, and autoimmunity, all of which are pathways linked to, and likely involved in, T1 and T2 diabetes pathogenesis. Several new proteins were differentially upregulated in prediabetic, T1D, and T2D pancreas. The results identify proteins that could serve as novel prognostic, diagnostic, and therapeutic tools to preserve functional islet mass in Type 1 Diabetes.     

SEL1L deficiency impairs growth and differentiation of pancreatic epithelial cells

Background  

The vertebrate pancreas contains islet, acinar and ductal cells. These cells derive from a transient pool of multipotent pancreatic progenitors during embryonic development. Insight into the genetic determinants regulating pancreatic organogenesis will help the development of cell-based therapies for the treatment of diabetes mellitus. Suppressor enhancer lin12/Notch 1 like (Sel1l) encodes a cytoplasmic protein that is highly expressed in the developing mouse pancreas. However, the morphological and molecular events regulated by Sel1l remain elusive.     

Human Islets Have Fewer Blood Vessels than Mouse Islets and the Density of Islet Vascular Structures Is Increased in Type 2 Diabetes

Human and rodent islets differ substantially in several features, including architecture, cell composition, gene expression and some aspects of insulin secretion. Mouse pancreatic islets are highly vascularized with interactions between islet endothelial and endocrine cells being important for islet cell differentiation and function. To determine whether human islets have a similar high degree of vascularization and whether this is altered with diabetes, we examined the vascularization of islets from normal human subjects, subjects with type 2 diabetes (T2D), and normal mice. Using an integrated morphometry approach to quantify intra-islet capillary density in human and mouse pancreatic sections, we found that human islets have five-fold fewer vessels per islet area than mouse islets. Islets in pancreatic sections from T2D subjects showed capillary thickening, some capillary fragmentation and had increased vessel density as compared with non-diabetic controls. These changes in islet vasculature in T2D islets appeared to be associated with amyloid deposition, which was noted in islets from 8/9 T2D subjects (and occupied 14% ± 4% of islet area), especially around the intra-islet capillaries. The physiological implications of the differences in the angioarchitecture of mouse and human islets are not known. Islet vascular changes in T2D may exacerbate β cell/islet dysfunction and β cell loss.     

Tissue Proteome Signatures Associated with Five Grades of Prostate Cancer and Benign Prostatic Hyperplasia

The histology‐based Gleason score (GS) of prostate cancer (PCa) tissue biopsy is the most accurate predictor of disease aggressiveness and an important measure to guide treatment strategies and patient management. The variability associated with PCa tumor sampling and the subjective determination of the GS are challenges that limit accurate diagnostication and prognostication. Thus, novel molecular signatures are needed to distinguish between indolent and aggressive forms of PCa for better patient management and outcomes. Herein, label‐free LC‐MS/MS proteomics is used to profile the proteome of 50 PCa tissues spanning five grade groups (n = 10 per group) relative to tissues from individuals with benign prostatic hyperplasia (BPH). Over 2000 proteins are identified albeit at different levels between and within the patient groups, revealing biological processes associated with specific grades. A panel of 11 prostate‐derived proteins including IGKV3D‐20, RNASET2, TACC2, ANXA7, LMOD1, PRCP, GYG1, NDUFV1, H1FX, APOBEC3C, and CTSZ display the potential to stratify patients from low and high PCa grade groups. Parallel reaction monitoring of the same sample cohort validate the differential expression of LMOD1, GYG1, IGKV3D‐20, and RNASET2. The four proteins associated with low and high PCa grades reported here warrant further exploration as candidate biomarkers for PCa aggressiveness.     

Roles of cytokines in the pathogenesis and therapy of type 1 diabetes

Type 1 diabetes (T1D) results from autoimmune destruction of the insulin-producing β-cells in the pancreatic islets of Langerhans by autoreactive T helper 1 (Th1) cells characterized by their cytokine secretory products, interleukin-2 (IL-2) and interferon γ (IFNγ). Th1-type cytokines (IL-2 and IFNγ) correlate with T1D, whereas Th2 (IL-4 and IL-10), Th3 (transforming growth factor beta [TGFβ]), and T regulatory cell-type cytokines (IL-10 and TGFβ) correlate with protection from T1D. Paradoxically, however, administrations of Th1-type cytokines (IL-2 and IFNγ) and immunotherapies that induce Th1-type cytokine responses actually prevent T1D, at least in animal models. Therefore, immunotherapies that inhibit IL-2 production/action will block Th1 cell/cytokine-driven effector mechanisms of pancreatic islet β-cell destruction; however, anti-IL-2 therapy will not allow immune tolerance to be established. In contrast, immunotherapies that increase IL-2 production/action may correct an immunodeficiency in IL-2 production that appears to underlie the autoimmunity of T1D, thereby restoring immune tolerance to islet β-cells and prevention of T1D.     

Altered islet composition and disproportionate loss of large islets in patients with type 2 diabetes

Human islets exhibit distinct islet architecture with intermingled alpha- and beta-cells particularly in large islets. In this study, we quantitatively examined pathological changes of the pancreas in patients with type 2 diabetes (T2D). Specifically, we tested a hypothesis that changes in endocrine cell mass and composition are islet-size dependent. A large-scale analysis of cadaveric pancreatic sections from T2D patients (n = 12) and non-diabetic subjects (n = 14) was carried out combined with semi-automated analysis to quantify changes in islet architecture. The method provided the representative islet distribution in the whole pancreas section that allowed us to examine details of endocrine cell composition in individual islets. We observed a preferential loss of large islets (>60 µm in diameter) in T2D patients compared to non-diabetic subjects. Analysis of islet cell composition revealed that the beta-cell fraction in large islets was decreased in T2D patients. This change was accompanied by a reciprocal increase in alpha-cell fraction, however total alpha-cell area was decreased along with beta-cells in T2D. Delta-cell fraction and area remained unchanged. The computer-assisted quantification of morphological changes in islet structure minimizes sampling bias. Significant beta-cell loss was observed in large islets in T2D, in which alpha-cell ratio reciprocally increased. However, there was no alpha-cell expansion and the total alpha-cell area was also decreased. Changes in islet architecture were marked in large islets. Our method is widely applicable to various specimens using standard immunohistochemical analysis that may be particularly useful to study large animals including humans where large organ size precludes manual quantitation of organ morphology.     

Activation of the Reg family genes by pancreatic-specific IGF-I gene deficiency and after streptozotocin-induced diabetes in mouse pancreas

We have recently reported that Pdx1-Cre-mediated whole pancreas inactivation of IGF-I gene [in pancreatic-specific IGF-I gene-deficient (PID) mice] results in increased beta-cell mass and significant protection against both type 1 and type 2 diabetes. Because the phenotype is unlikely a direct consequence of IGF-I deficiency, the present study was designed to explore possible activation of proislet factors in PID mice by using a whole genome DNA microarray. As a result, multiple members of the Reg family genes (Reg2, -3alpha, and -3beta, previously not known to promote islet cell growth) were significantly upregulated in the pancreas. This finding was subsequently confirmed by Northern blot and/or real-time PCR, which exhibited 2- to 8-fold increases in the levels of these mRNAs. Interestingly, these Reg family genes were also activated after streptozotocin-induced beta-cell damage and diabetes (wild-type T1D mice) when islet cells were undergoing regeneration. Immunohistochemistry revealed increased Reg proteins in exocrine as well as endocrine pancreas and suggested their potential role in beta-cell neogenesis in PID or T1D mice. Previously, other Reg proteins (Reg1 and islet neogenesis-associated protein) have been shown to promote islet cell replication and neogenesis. These uncharacterized Reg proteins may play a similar but more potent role, not only in normal islet cell growth in PID mice, but also in islet cell regeneration after T1D.     

Genetic variants entail type 2 diabetes as an innate immune disorder

Type 2 Diabetes (T2D) is characterized by alteration in the circulatory levels of key inflammatory proteins, where our body strives to eliminate the perturbing factor through inflammation as a final resort to restore homeostasis. Plasma proteins play a crucial role to orchestrate this immune response. Over the past two decades, rigorous genetic efforts taken to comprehend T2D physiology have been partially successful and have left behind a dearth of knowledge of its causality. Here, we have investigated how the reported genetic variants of T2D are associated with circulatory levels of key plasma proteins. We identified 99 T2D genetic variants that serve as strong pQTL (protein Quantitative Trait Loci) for 72 plasma proteins, of which 4 proteins namely Small nuclear ribonucleoprotein F [SNRPF] (p = 2.99 × 10⁻¹⁴), Platelet endothelial cell adhesion molecule [PECAM1] (p = 1.9 × 10⁻⁴⁵), Trypsin-2 [PRSS2] (p = 7.6 × 10⁻⁴³) and Trypsin-3 [PRSS3] (p = 5.7 × 10⁻⁸) were previously not reported for association to T2D. The genes that encode these 72 proteins were observed to be highly expressed in at least one of the four T2D relevant tissues - liver, pancreas, adipose and whole blood. Comparative analysis of interactions of the studied proteins amongst these four tissues revealed distinct molecular connectivity. Assessment of biological function by gene-set enrichment highlighted innate immune system as the lead process enacted by the identified proteins (FDR q = 3.7 × 10⁻¹⁶). To validate the findings, we analyzed Coronary Artery Disease (CAD) and Rheumatoid Arthritis (RA) individually and as expected, we observed innate immune system as a top enriched pathway for RA but not for CAD. Our study illuminates strong regulation of plasma proteome by the established genetic variants of T2D.     

Synthesis and biological evaluation of C‐terminal hydroxamide analogues of bombesin

Bombesin pseudo‐peptide analogues containing a hydroxamide function on the C‐terminal part of the molecule, e.g. H‐D‐Phe‐Gln‐Trp‐Ala‐Val‐Gly‐His‐Leu‐NHOBzl 1 and H‐D‐Phe‐Gln‐Trp‐Ala‐Val‐Gly‐His‐Leu‐NHOH 2 were synthesized. These compounds were tested for their ability to recognize the bombesin receptor on rat pancreatic acini and on 3T3 cells, to stimulate (i) amylase secretion from rat pancreatic acini and (ii) accumulation of tritiated thymidine in 3T3 cells. Compounds 1 and 2 were able to recognize bombesin receptors on both models with high affinity (K_i=7±2 and 5.8±0.9 nm on rat pancreatic acini, and K_i=4.1±1.2 and 7.7±1.9 nm on 3T3 cells, respectively). Interestingly, compound 1 behaved as a potent agonist in stimulating amylase secretion from rat pancreatic acini and is able to stimulate thymidine accumulation in 3T3 cells, while compound 2 was able to potently antagonize bombesin‐stimulated amylase secretion (K_i=22±5 nm ) in rat pancreatic acini and had no proper effect on 3T3 cells; however, it was able to inhibit bombesin‐stimulated thymidine accumulation in 3T3 cells with high potency (K_i=1.6±0.6 nm ). Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Differential proteomic analysis of lymphatic,venous, and arterial endothelial cells extracted from bovine mesenteric vessels

Differential protein profiling by 2‐D PAGE is generally useful in biomarker discovery, proteome analysis and routine sample preparation prior to analysis by MS. The goal of this study was to compare 2‐D PAGE‐resolved protein profile of lymphatic endothelial cells to those of venous, and arterial endothelial cells isolated from lymphatic and blood vessels of bovine mesentery (bm). Three 2‐D PAGE electrophoretograms were produced for each of the three cell types and quantitatively analyzed. Protein identification by LC‐MS/MS was performed to identify 39 proteins found to be present at statistically significantly different levels in the three cell types (p<0.05). Most of the 39 proteins have not been previously reported in EC proteomic studies of 2‐D PAGE electrophoretograms. Three proteins, HSPA1B (HSP70 family member), HSPB1 (HSP27 family member), and UBE2D3 (a member of E2 ubiquitin‐conjugating enzymes) found to be at highest levels in bm arterial endothelial cells, bm venous endothelial cells, and bm lymphatic endothelial cells, respectively, were validated by immunoblotting with appropriate antibodies. The lack of substantial overlap between our results and those of other groups' comparative studies are discussed. Functional implications of differences in levels of various proteins identified in the three cell types are also discussed.     

Proteome analysis of functionally differentiated bovine (Bos indicus) mammary epithelial cells isolated from milk

Mammary gland is made up of a branching network of ducts that end in alveoli. Terminally differentiated mammary epithelial cells (MECs) constitute the innermost layer of aveoli. They are milk‐secreting cuboidal cells that secrete milk proteins during lactation. Little is known about the expression profile of proteins in the metabolically active MECs during lactation or their functional role in the lactation process. In the present investigation, we have reported the proteome map of MECs in lactating cows using 2DE MALDI‐TOF/TOF MS and 1D‐Gel‐LC‐MS/MS. MECs were isolated from milk using immunomagnetic beads and confirmed by RT‐PCR and Western blotting. The 1D‐Gel‐LC‐MS/MS and 2DE‐MS/MS based approaches led to identification of 431 and 134 proteins, respectively, with a total of 497 unique proteins. Proteins identified in this study were clustered into functional groups using bioinformatics tools. Pathway analysis of the identified proteins revealed 28 pathways (p < 0.05) providing evidence for involvement of various proteins in lactation function. This study further provides experimental evidence for the presence of many proteins that have been predicted in annotated bovine genome. The data generated further provide a set of bovine MEC‐specific proteins that will help the researchers to understand the molecular events taking place during lactation.