Rodent Counterparts Research Articles

The ATRA-dependent overexpression of the glutamate transporter EAAC1 requires RARβ induction

The mechanisms underlying trafficking and membrane targeting of EAAC1, the rodent counterpart of the human EAAT3 carrier for anionic amino acids, are well characterized. In contrast, much less is known on the regulation of Slc1a1, the gene that encodes for the transporter. We have recently found that all- trans retinoic acid (ATRA) stimulates EAAC1 expression and anionic amino acid transport in C6 rat glioma cells. We report here that the ATRA effect on EAAC1 activity was inhibited by the specific RAR antagonist LE540 and mimicked by Am80, a RAR agonist, but not by the RXR agonist HX630. Moreover, the ATRA-dependent induction of Slc1a1 mRNA required the synthesis of a protein intermediate and was not associated with changes in the messenger half-life. ATRA treatment induced the expression of both Rarb mRNA and RARβ protein several hours before the induction of Slc1a1, while the mRNA for RFX1, a transcription factor recently involved in Slc1a1 transcription, was unchanged. In addition, Rarb silencing markedly inhibited the ATRA-dependent increase of both Rarb and Slc1a1 mRNAs. We conclude that in C6 glioma cells the induction of Slc1a1 by ATRA requires the synthesis of RARβ, suggesting that the receptor is involved in the regulation of the transporter gene.

Uniquely Hominid Features of Adult Human Astrocytes

Defining the microanatomic differences between the human brain and that of other mammals is key to understanding its unique computational power. Although much effort has been devoted to comparative studies of neurons, astrocytes have received far less attention. We report here that protoplasmic astrocytes in human neocortex are 2.6-fold larger in diameter and extend 10-fold more GFAP (glial fibrillary acidic protein)-positive primary processes than their rodent counterparts. In cortical slices prepared from acutely resected surgical tissue, protoplasmic astrocytes propagate Ca(2+) waves with a speed of 36 microm/s, approximately fourfold faster than rodent. Human astrocytes also transiently increase cystosolic Ca(2+) in response to glutamatergic and purinergic receptor agonists. The human neocortex also harbors several anatomically defined subclasses of astrocytes not represented in rodents. These include a population of astrocytes that reside in layers 5-6 and extend long fibers characterized by regularly spaced varicosities. Another specialized type of astrocyte, the interlaminar astrocyte, abundantly populates the superficial cortical layers and extends long processes without varicosities to cortical layers 3 and 4. Human fibrous astrocytes resemble their rodent counterpart but are larger in diameter. Thus, human cortical astrocytes are both larger, and structurally both more complex and more diverse, than those of rodents. On this basis, we posit that this astrocytic complexity has permitted the increased functional competence of the adult human brain.

Survey of the Human Pancreatic β-Cell G1/S Proteome Reveals a Potential Therapeutic Role for Cdk-6 and Cyclin D1 in Enhancing Human β-Cell Replication and Function In Vivo

OBJECTIVESTo comprehensively inventory the proteins that control the G1/S cell cycle checkpoint in the human islet and compare them with those in the murine islet, to determine whether these might therapeutically enhance human β-cell replication, to determine whether human β-cell replication can be demonstrated in an in vivo model, and to enhance human β-cell function in vivo.RESEARCH DESIGN AND METHODSThirty-four G1/S regulatory proteins were examined in human islets. Effects of adenoviruses expressing cdk-6, cdk-4, and cyclin D1 on proliferation in human β-cells were studied in both invitro and in vivo models.RESULTSMultiple differences between murine and human islets occur, most strikingly the presence of cdk-6 in human β-cells versus its low abundance in the murine islet. Cdk-6 and cyclin D1 in vitro led to marked activation of retinoblastoma protein phosphorylation and cell cycle progression with no induction of cell death. Human islets transduced with cdk-6 and cyclin D1 were transplanted into diabetic NOD-SCID mice and markedly outperformed native human islets in vivo, maintaining glucose control for the entire 6 weeks of the study.CONCLUSIONSThe human G1/S proteome is described for the first time. Human islets are unlike their rodent counterparts in that they contain easily measurable cdk-6. Cdk-6 overexpression, alone or in combination with cyclin D1, strikingly stimulates human β-cell replication, both in vitro as well as in vivo, without inducing cell death or loss of function. Using this model, human β-cell replication can be induced and studied in vivo.

Cyclical and Dose-Dependent Responses of Adult Human Mature Oligodendrocytes to Fingolimod

Fingolimod is a sphingosine-1-phosphate (S1P) analogue that has been used in clinical trials as a systemic immunomodulatory therapy for multiple sclerosis. Fingolimod readily accesses the central nervous system, raising the issue of its direct effects on neural cells. We assessed the effects of active fingolimod on dissociated cultures of mature, myelin-producing oligodendrocytes (OLGs) derived from adult human brain. Human OLGs express S1P receptor transcripts in relative abundance of S1P5>S1P3>S1P1, with undetectable levels of S1P4. Low doses of fingolimod (100 pmol/L to 1 nmol/L) induced initial membrane elaboration (2 days), subsequent retraction (4 days), and recurrence of extension with prolonged treatment (8 days). Higher doses (10 nmol/L to 1 mumol/L) caused the opposite modulation of membrane dynamics. Retraction was rescued by co-treatment with the S1P3/S1P5 pathway antagonist, suramin, and was associated with RhoA-mediated cytoskeletal signaling. Membrane elaboration was mimicked using the S1P1 agonist SEW2871. Fingolimod rescued human OLGs from serum and glucose deprivation-induced apoptosis, which was reversed with suramin co-treatment and mimicked using an S1P5 agonist. High doses of fingolimod induced an initial down-regulation of S1P5 mRNA levels relative to control (4 hours), subsequent up-regulation (2 days), and recurrent down-regulation (8 days). S1P1 mRNA levels were inversely regulated compared with S1P5. These results indicate that fingolimod modulates maturity- and species-specific OLG membrane dynamics and survival responses that are directly relevant for myelin integrity.

GABA in the female brain — Oestrous cycle-related changes in GABAergic function in the periaqueductal grey matter

In many women, aversive psychological and somatic symptoms develop during the late luteal phase of the menstrual cycle, when progesterone levels fall sharply. Following intravenous administration in anaesthetised rats, the progesterone metabolite allopregnanolone readily gained access to the periaqueductal grey (PAG), a region involved in generating panic-like anxiety, and inhibited neural activity via actions at GABA A receptors. Withdrawal of female rats from prolonged systemic dosing with progesterone leads to increased numbers of α4, β1 and δ GABA A receptor subunit-immunoreactive neurones in the PAG. In naturally cycling rats a similar upregulation occurred during late dioestrus, when progesterone levels fall. Functional experiments revealed that upregulation of α4β1δ receptor subunit expression was associated with a decrease in GABAergic tone in the PAG and increased responsiveness to a panicogenic CCK 2 receptor agonist. The oestrous cycle-linked plasticity of GABA receptors was absent in rats housed in quiet conditions in an isolated room suggesting that environmental factors may be able to influence the central response to hormonal changes. In susceptible animals, i.e. those housed in a communal animal holding room, oestrous cycle-related changes in GABAergic circuits may underlie the development of increased anxiety levels that represent a rodent counterpart to premenstrual syndrome in women.

ISOLATION OF QUIESCENT (NORMAL) HUMAN PANCREATIC STELLATE CELLS (HPSCS)

Activated pancreatic stellate cells (PSCs) play a central role in pancreatic fibrosis. Most of our knowledge of PSC biology has been obtained from rat PSC cultures, with studies of human PSCs (hPSCs) being limited to pre-activated cancer- or inflammation-associated cells isolated by the outgrowth method from resected pancreatic specimens. Thus, an in vitro model ofhPSCs in their normal, quiescent state has been lacking. The aims of this study were: i) to isolate quiescent hPSCs using density gradient centrifugation; ii) to assess their morphology and their expression of stellate cell selective markers; and iii) to evaluate their response to the mitogen platelet derived growth factor (PDGF). Methods: hPSCs were isolated from normal pancreatic tissue (n = 4, obtained during resections for non-malignant and non-inflammatory pancreatic pathologies), using a Nycodenz density gradient. Freshly isolated and cultured cells were examined by phase contrast microscopy. Cells were immunostained for the stellate cell selective markers [glial fibrillary acidic protein (GFAP) and desmin] and α smooth muscle actin (an activation marker). hPSCs were incubated for 24h with and without PDGF (20ng/ml) and cell proliferation assessed by measuring 3H-thymidine incorporation into cellular DNA. PDGF receptor β expression by hPSCs was assessed by western blotting. Results: Freshly isolated hPSCs displayed a polygonal appearance with multiple refringent lipid droplets in the cytoplasm. These droplets were progressively lost in culture although ~10% of cells retained their droplets until the first passage at 10 days. Human PSCs expressed GFAP, desmin, αSMA and PDGF receptor β. Notably, hPSCs responded to PDGF by significantly increased proliferation [data expressed as % of control, mean ± SE: 138.3 ± 9.9; p < 0.05; n = 3 separate hPSC preparations]. Conclusions: Isolation of (normal) quiescent human PSCs is feasible. Freshly isolated and early culture hPSCs exhibit the presence of lipid droplets similar to quiescent rat PSCs and display the same markers as their rodent and human cancer-associated counterparts. In addition, they respond toPDGF in a manner similar to rat PSCs. Implication: The ability to isolate normal human PSCs from normal pancreatic tissue provides a useful in vitro tool to study their biology in their physiological, non-activated state.

Differential expression of HNK‐1 and p75NTR in adult canine Schwann cells and olfactory ensheathing cells in situ but not in vitro

Olfactory ensheathing cells (OECs) are promising candidates for autologous cell transplantation therapies of nervous system injury and disease. Large animal models are relevant for transferring experimental data into clinical practice. In vivo studies have suggested that adult canine OECs may display similar regenerating capacities as their rodent counterpart. However, data on their molecular phenotype required for generating pure cell preparations are still scarce. In the present study, we comparatively analyzed expression of the carbohydrate HNK-1 epitope and the neurotrophin receptor p75(NTR) in adult canine Schwann cells and olfactory ensheathing cells in situ and in vitro. Myelinating and nonmyelinating Schwann cells in situ exclusively expressed HNK-1 and p75(NTR), respectively, whereas OECs were negative for both markers. In vitro, OECs and Schwann cells shared cell surface expression of p75(NTR) but not of HNK-1, which could be detected transiently in intracellular vesicles. This suggests that Schwann cells and OECs in vitro phagozytose HNK-1+ cellular debris. The cultivation-induced downregulation of HNK-1 expression in Schwann cells and upregulation of p75(NTR) in OECs argues for the possibility that axonal signals control the expression of both markers in situ. Whereas HNK-1 expression in Schwann cells is most likely controlled by signals inducing myelination, e.g., neuregulin, the mechanisms that may suppress p75(NTR) expression in OECs in situ remain to be elucidated. Interestingly, HNK-1 expression in the adult dog was found in both sensory and motor nerve myelinating Schwann cells. This is reminiscent of humans and differs from rodents; it also underscores the importance of large animal models for translational research.

Advanced glycation end products decrease mesangial cell MMP-7: A role in matrix accumulation in diabetic nephropathy?

Increased extracellular matrix material is a pathological hallmark of diabetic nephropathy. In addition to collagens, a variety of non-collagenous glycoproteins such as fibronectin also accumulate in the kidney of diabetics. The effect of diabetes on degradative pathways, in particular those involving non-collagenous proteins, are relatively unexplored. In this study, we determined the expression of the major matrix metalloproteinase (MMP) responsible for degrading the non-collagenous matrix glycoprotein fibronectin. Furthermore, the modulation of these MMPs by advanced glycation end products (AGE), a key factor in the diabetic milieu, was explored. Exposure of mesangial cells to AGEs led to a significant reduction in MMP-7, but not MMP-3 or -10. MMP-7 expression was normalized by both aminoguanidine, an inhibitor of glycation product formation, or by a neutralizing anti-transforming growth factor-beta (TGF-beta) antibody. In streptozotocin-induced diabetic rats, the diminution in MMP-7 expression and excessive fibronectin accumulation were attenuated by aminoguanidine. Humans with type 2 diabetes and nephropathy displayed similar alterations in MMP-7 to their rodent counterparts. Our findings suggest that diminished expression of the glycoprotein-degrading enzyme, MMP-7, may play a role in fibronectin accumulation in the diabetic kidney in response to AGEs and/or TGF-beta.

The structural and functional analysis of the human HSPA2 gene promoter region.

HSPA2 is a human counterpart of the testis-specific rodent Hst70/Hsp70.2 gene. In contrast to the latter, the expression of the human HSPA2 gene is not limited to the testis, and recent data show that human tumor cells can express this gene at significant levels. The characteristics of HSPA2 expression suggests that it can influence the phenotype and survival of cancer cells similarly as overexpression of major members of the HSP70 gene family. Until now, neither the structure of the transcription unit of the human HSPA2 gene has been established nor a functional analysis of its promoter performed. In this study we established that the human HSPA2 gene, in contrast to its rodent counterparts, is intronless and has a single transcription start site. We also show that the same type of HSPA2 transcripts are synthesized in the testes and in cancer cell lines. In order to perform a functional study of the HSPA2 promoter, we used a transient transfection assay and found that the 392 bp fragment upstream of the ATG codon was a minimal region required for efficient transcription, while a 150 bp deletion from the 5' end of this region dramatically reduced the promoter activity. Delineation of the minimal promoter is a basic step toward identifying the cis and trans elements involved in the regulation of the HSPA2 gene expression in cancer cells.

Identification of 5-HT3A and 5-HT3B receptor subunits in human hippocampus

The pentameric 5-HT3 receptor complex is a ligand-gated ion channel that mediates fast synaptic transmission in the brain. Expression of two subunits (5-HT3A and 5-HT3B subunits) gives rise to at least two receptor isoforms (homomeric 5-HT3A and heteromeric 5-HT3A/3B receptors), which differ in their biophysical characteristics, although expression of these proteins has not been investigated in human brain. The expression of h5-HT3A and 5-HT3B subunits in the human hippocampus was investigated using selective polyclonal antibodies (SDS-PAGE/Western blotting, immunohistochemistry), with expression of each subunit verified by PCR detection of subunit transcripts. 5-HT3A and 5-HT3B subunit immunoreactivity was identified within the human hippocampus. The cellular pattern of expression for each subunit was similar, with predominant immunoreactivity associated with pyramidal neurones in CA fields 2 and 3, and also the relatively large neurones within the hilus (CA4 field). Transcripts for each subunit were also identified in human hippocampal tissue. These findings indicate that human hippocampal neurones are capable of forming at least two, functionally different, isoforms of the 5-HT3 receptor. Furthermore the expression pattern of 5-HT3A and 5-HT3B subunits in human hippocampus appears to differ with the rodent counterpart, which may underlie the differences in some of the behavioural effects of 5-HT3 receptor antagonists between these species.

Sperm-binding fibronectin type II-module proteins are genetically linked and functionally related

Fibronectin type II (Fn2) module-containing proteins in the male genital tract are characterized by different numbers of Fn2 modules. Predominantly two classes exist which are distinct by having either two or four Fn2 modules. Minor variants with three Fn2 modules were also found in the human and the porcine epididymis. To reveal their relationship, mRNAs and proteins of representatives of these classes were studied in human, in Sus scrofa, and in rodents. Adult boars expressed members of both classes, i.e. ELSPBP1 and pB1, in subsequent regions of the epididymis, and both were under androgenic control. Human and rodent epididymides, on the other hand, alternatively contained only representatives of one of these two classes, i.e. ELSPBP1 in the human and two different pB1-related counterparts in rodents. ELSPBP1 and pB1-related genomic sequences were closely linked in chromosomal regions HSA 19q and SSC 6 q11–q21; conserved synteny between these regions is well established. On the other hand, in a syntenic region on mouse chromosome 7, ELSPBP1-related sequences were lacking. Tight binding to the sperm membrane via a choline-mediated mechanism was a common feature of the two classes of Fn2-module proteins, suggesting related function(s). However, differences in their regionalized expression patterns along the male genital tract as well as in association sites on the sperm surface suggested a species-specific sequential order in sperm binding.

Functional Assessment of Human Alcohol Dehydrogenase Family in Ethanol Metabolism: Significance of First‐Pass Metabolism

Alcohol dehydrogenase (ADH) is the principal enzyme responsible for ethanol metabolism in mammals. Human ADH constitutes a unique complex enzyme family with no equivalent counterpart in experimental rodents. This study was undertaken to quantitatively assess relative contributions of human ADH isozymes and allozymes to hepatic versus gastric metabolism of ethanol in the context of the entire family. Kinetic parameters for ethanol oxidation for recombinant human class I ADH1A, ADH1B1, ADH1B2, ADH1B3, ADH1C1, and ADH1C2; class II ADH2; class III ADH3; and class IV ADH4 were determined in 0.1 M sodium phosphate at pH 7.5 over a wide range of substrate concentrations in the presence of 0.5 mM NAD+. The composite numerical formulations for organ steady-state ethanol clearance were established by summing up the kinetic equations of constituent isozymes/allozymes with the assessed contents in livers and gastric mucosae with different genotypes. In ADH1B*1 individuals, ADH1B1 and ADH1C allozymes were found to be the major contributors to hepatic-alcohol clearance; ADH2 made a significant contribution only at high ethanol levels (> 20 mM). ADH1B2 was the major hepatic contributor in ADH1B*2 individuals. ADH1C allozymes were the major contributor at low ethanol (< 2 mM), whereas ADH1B3 the major form at higher levels (> 10 mM) in ADH1B*3 individuals. For gastric mucosal-alcohol clearance, the relative contributions of ADH1C allozymes and ADH4 were converse as ethanol concentration increased. It was assessed that livers with ADH1B*1 may eliminate approximately 95% or more of single-passed ethanol as inflow sinusoidal alcohol reaches approximately 1 mM and that stomachs with different ADH1C genotypes may remove 20% to 30% of single-passed alcohol at the similar level in mucosal cells. This work provides just a model, but a strong one, for quantitative assessments of ethanol metabolism in the human liver and stomach. The results indicate that the hepatic-alcohol clearance of ADH1B*2 individuals is higher than that of the ADH1B*1 and those of the ADH1B*3 versus the ADH1B*1 vary depending on sinusoidal ethanol levels. The maximal capacity for potential alcohol first-pass metabolism in the liver is greater than in the stomach.

FUNCTIONAL EXPRESSION OF SINUSOIDAL DRUG TRANSPORTERS IN PRIMARY HUMAN AND RAT HEPATOCYTES

Primary hepatocyte cultures are considered as a useful in vitro system for pharmacological/toxicological studies. Although expression of drug-metabolizing enzymes and canalicular drug transporters has been well documented in this cellular model, less information is available about sinusoidal drug transporter activities. This has led us to investigate functional expression of the major sinusoidal transporters in primary human and rat hepatocytes. Using radiolabeled substrates and chemical transporter inhibitors, activities of organic cation transporter 1, organic anion-transporting polypeptides, organic anion transporter 2, and Na(+)-taurocholate cotransporter were detected in cultured human and rat hepatocytes. In parallel, mRNA expression of these transporters was demonstrated using reverse transcriptase-quantitative polymerase chain reaction assays. Functional expression of sinusoidal transport proteins markedly decreased with time in primary rat hepatocyte cultures; by contrast, it remained relatively constant in primary human hepatocytes all along the culture, illustrating the fact that liver-specific functions, including drug-detoxifying pathways, are usually better preserved in cultured human hepatocytes than in their rodent counterparts. Primary hepatocytes, especially human hepatocytes, thus exhibit a pattern of sinusoidal transporter expression close to that found in vivo, highlighting the interest of hepatocyte cultures for drug detoxification studies.

Parallel Regulation of Membrane Trafficking and Dominant-negative Effects by Misrouted Gonadotropin-releasing Hormone Receptor Mutants

Gonadotropin-releasing hormone (GnRH) receptor mutants from patients with hypogonadotropic hypogonadism are frequently misrouted proteins that exert a dominant-negative (DN) effect on human (h) wild-type (WT) receptor, due to oligomerization and retention in the endoplasmic reticulum. Pharmacologic chaperones restore correct folding, rescuing mutants and WT receptor from this oligomer. Rat WT retains the ability to oligomerize (since human and mouse mutants exert a DN effect on rat (r) WT sequence) but, unlike human or mouse, escapes the DN effect of GnRH receptor (Gn-RHR) mutants because rGnRHR mutants route to the plasma membrane with higher efficiency than mouse or human mutants. These distinct behaviors of mouse and rat GnRHRs (distinguished by only four semi- or non-conservative amino acid differences) led us to assess the role of each amino acid. The difference in both routing and the DN effect appears mediated primarily by Ser(216) in the rGnRHR. The homologous amino acid in the hGn-RHR is also Ser and is compensated for by the primate-unique insertion of Lys(191) that, alone, dramatically decreases routing of the receptor. These studies establish the relation between the DN effect and altered receptor trafficking and explain why hGnRHR is more susceptible to defective trafficking by disease-related point mutations than rodent counterparts.

Isolation and Characterization of Multipotent Skin‐Derived Precursors from Human Skin

We have previously isolated, expanded, and characterized a multipotent precursor cell from mammalian dermis (termed skin-derived precursors [SKPs]) that can differentiate into both neural and mesodermal progeny. In this study, we report the isolation, expansion, and characterization of a similar precursor cell from neonatal human foreskin tissue. Like their rodent counterparts, human SKPs grew in suspension as spheres in the presence of the mitogens fibroblast growth factor 2 and epidermal growth factor and expressed nestin, fibronectin, vimentin, and characteristic embryonic transcription factors. Human SKPs could be maintained in culture for long periods of time and would still differentiate into neurons, glia, and smooth muscle cells, including cells with the phenotype of peripheral neurons and Schwann cells. Clonal analysis indicated that single SKP cells were multipotent and could give rise to all of these progeny. Moreover, human SKPs apparently derive from an endogenous precursor within human foreskin; a subpopulation of dissociated primary foreskin cells could differentiate into neurons, a cell type never seen in skin, and the initial spheres to develop from skin expressed the same markers and had the same potential as do passaged SKPs. Together, these data indicate that SKPs are an endogenous multipotent precursor cell present in human skin that can be isolated and expanded and differentiate into both neural and mesodermal cell types.

1036. Engraftment of side population Stem Cells in Canine Recipients

Side population (SP) cells, isolated on the basis of efflux of the fluorescent dye Hoechst 33342, contain most of the hematopoietic stem cells of adult bone marrow. In addition to their ability to reconstitute the hematopoietic system, marrow-derived SP cells may contribute to the regeneration of nonhematopoietic tissues including cardiac and skeletal muscle, vascular endothelium and depleted osteoprogenitor pools. SP cells have also been identified in heart, mammary gland and liver where they demonstrated stem cell activity. Given the potential of SP cells to generate the entire hematopoietic repertoire and their possible capacity to differentiate into multiple tissues, SP cells may provide an ideal source of stem cells for use in regenerative medicine. To date, however, demonstration of the in vivo functional potential of SP cells has been limited to murine studies. Before SP cells can be used in the clinical setting, their utility and safety must be demonstrated in large animal models. Inbred dogs with genetic disorders homologous with human diseases have historically been used as models in preclinical studies to develop novel strategies for bone marrow transplantation and gene and stem cell-based therapies. Dogs share many biochemical and physiologic characteristics with humans and thus often exhibit a greater resemblance to human diseases than do their rodent counterparts. We previously demonstrated that the frequency and phenotype of canine and murine bone marrow-derived SP cells were similar and that canine SP cells efficiently engraft NOD/SCID mice. We now report the engraftment potential of male canine marrow-derived SP cells after injection into day 38 fetal pups. Twenty-seven days after injection, 9 female pups were delivered by elective C-section with 7 born alive and healthy. One female was stillborn and another was euthanized 1 month after delivery due to congenital defects. Monthly peripheral blood and bone marrow aspirates were collected and used to monitor donor cell engraftment by Y chromosome-specific polymerase chain reaction. Relatively constant engraftment levels of 0.01% in peripheral blood and bone marrow mononuclear cells have been observed out to 7 months after birth. Analysis of multiple tissues from the deceased puppies also showed the presence of Y chromosome marked cells in the blood, bone marrow, spleen, liver, kidney, muscle, and heart. These data support the hypothesis that canine SP cells offer a highly enriched source of stem cells with the potential to reconstitute the hematopoietic system and be found in multiple nonhematopoietic tissues. Current studies are underway to address questions of transdifferentiation verses fusion and to boost levels of engraftment by re-infusing recipients with donor marrow.

Valine 738 and lysine 735 in the fifth transmembrane domain of rTas1r3 mediate insensitivity towards lactisole of the rat sweet taste receptor

BackgroundThe sweet taste inhibitor lactisole acts on the human sweet taste receptor heteromer TAS1R2-TAS1R3 but not on its rodent counterpart. Recently, it was shown that the lactisole sensitivity of the human sweet taste receptor involves the part of TAS1R3 encompassing the seven transmembrane regions but not the huge N-terminal domain. Using mutational analysis we investigated which amino acid residues distinguish lactisole insensitive rat from sensitive human T1R3 receptors.ResultsThe functional analysis of specific receptor mutants in HEK293T cells revealed that the exchange of valine 738 in the fifth transmembrane domain of rTas1r3 by an alanine is sufficient to confer lactisole sensitivity to the rat sweet taste receptor. The sensitivity of this receptor mutant is ~2 fold lower than the sensitivity of the human sweet taste receptor. Additional substitution of lysine 735 by phenylalanine in rTas1r3 results in a rat sweet taste receptor that is as sensitive to lactisole as its human counterpart. The exchange of valine 738 to alanine was accompanied by a ~50% reduction in receptor efficacy. This effect was seen with all six different sweet compounds examined.ConclusionThe lactisole insensitivity of rat sweet taste receptor is caused by only two amino acids in transmembrane region five, which is critical for the interaction of lactisole with the sweet taste receptor. The observation that the mutant receptor simultaneously displays a generally reduced sensitivity towards all agonists suggests that the lactisole insensitivity of the rodent receptor might be more likely caused by the inaccessibility of the lactisole binding site rather then by its direct disruption.

Involvement of CC chemokine ligand 18 (CCL18) in normal and pathological processes

CC chemokine ligand 18 (CCL18) was originally discovered as pulmonary and activation-regulated chemokine (PARC), dendritic cell (DC)-chemokine 1 (DC-CK1), alternative macrophage activation-associated CC chemokine-1 (AMAC-1), and macrophage inflammatory protein-4 (MIP-4). CCL18 primarily targets lymphocytes and immature DC, although its agonistic receptor remains unknown so far. CCL18 is mainly expressed by a broad range of monocytes/macrophages and DC. A more profound understanding of the various activation programs and functional phenotypes of these producer cells might give a better insight in the proinflammatory versus anti-inflammatory role of this CC chemokine. It is interesting that CCL18 is constitutively present at high levels in human plasma and likely contributes to the physiological homing of lymphocytes and DC and to the generation of primary immune responses. Furthermore, enhanced CCL18 production has been demonstrated in several diseases, including various malignancies and inflammatory joint, lung, and skin diseases. The lack of a rodent counterpart for human CCL18 sets all hope on primate animal models to further elucidate the importance of CCL18 in vivo. This review will address these different aspects in more detail.

P57Kip2 (cdkn1c): sequence, splice variants and unique temporal and spatial expression pattern in the rat pancreas

The cyclin-dependent kinase (CDK) inhibitor p57Kip2 (CDKN1C) is a negative regulator of cell proliferation, binding to a variety of cyclin-CDK complexes and inhibiting their kinase activities in vitro. The p57Kip2 gene is imprinted and the maternal allele is expressed in terminally differentiated cells, including human β-cells. Somatic loss of p57Kip2 expression is associated with increased β-cell proliferation in the focal form of Hyperinsulinism of Infancy. We cloned and sequenced the rat ortholog of p57Kip2, and demonstrate that it is highly homologous to the mouse gene. However, the human and rodent genes are quite divergent. Despite having highly homologous C- and N-terminal domains, the mid-portion of the human gene is entirely different from that of its rodent counterparts. Expression of p57Kip2 was evaluated during fetal and postnatal development, and a highly cell-specific, temporal and spatial expression profile was found. In contrast to other tissues, the expression pattern in rat pancreas was entirely opposite from that previously reported in man, with high levels of expression in rodent exocrine cells, but no expression in β-cells during any stage of development. These findings demonstrate that p57Kip2 expression is highly regulated. In the pancreas, the functional significance of this gene appears to be quite different in humans when compared with rodents, suggesting that a better understanding of the function of this protein may provide new insights into the mechanisms involved in the control of human β-cell mass.

Partition-Variant Desferrithiocin Analogues: Organ Targeting and Increased Iron Clearance

Altering the lipophilicity (log P(app)) of desferrithiocin analogues can change the organ distribution of the chelators and lead to enhanced iron clearance. For example, alkylation of (S)-2-(2,4-dihydroxyphenyl)-4,5-dihydro-4-methyl-4-thiazolecarboxylic acid [(S)-4'-(HO)-DADFT] and its analogues to more lipophilic compounds, such as (S)-4,5-dihydro-2-(2-hydroxy-4-methoxyphenyl)-4-methyl-4-thiazolecarboxylic acid [(S)-4'-(CH3O)-DADFT], provides ligands that achieved between a 3- and 8-fold increase in chelator concentrations in the heart, liver, and pancreas (the organs most at risk in iron-overload disease) of treated rodents. The 4'-O-methylated compounds are demethylated to their hydroxylated counterparts in rodents; furthermore, this O-demethylation takes place in both rodent and human liver microsomes. The relationship between chelator lipophilicity and iron-clearing efficacy in the iron-overloaded Cebus apella primate is further underscored by a comparison of the iron-clearing efficiency of (S)-2-(2,3-dihydroxyphenyl)-4,5-dihydro-4-methyl-4-thiazolecarboxylic acid [(S)-3'-(HO)-DADFT] and its 3'-(CH3O) counterpart. Finally, these DFT analogues are shown to be both inhibitors of the iron-mediated oxidation of ascorbate as well as effective radical scavengers.