Mechanisms of arsenical and diamidine uptake and resistance in Trypanosoma brucei

Sleeping sickness, caused by Trypanosoma brucei spp., has become resurgent in sub-Saharan Africa. Moreover, there is an alarming increase in treatment failures with melarsoprol, the principal agent used against late-stage sleeping sickness. In T. brucei, the uptake of melarsoprol as well as diamidines is thought to be mediated by the P2 aminopurine transporter, and loss of P2 function has been implicated in resistance to these agents. The trypanosomal gene TbAT1 has been found to encode a P2-type transporter when expressed in yeast. Here we investigate the role of TbAT1 in drug uptake and drug resistance in T. brucei by genetic knockout of TbAT1. Tbat1-null trypanosomes were deficient in P2-type adenosine transport and lacked adenosine-sensitive transport of pentamidine and melaminophenyl arsenicals. However, the null mutants were only slightly resistant to melaminophenyl arsenicals and pentamidine, while resistance to other diamidines such as diminazene was more pronounced. Nevertheless, the reduction in drug sensitivity might be of clinical significance, since mice infected with tbat1-null trypanosomes could not be cured with 2 mg of melarsoprol/kg of body weight for four consecutive days, whereas mice infected with the parental line were all cured by using this protocol. Two additional pentamidine transporters, HAPT1 and LAPT1, were still present in the null mutant, and evidence is presented that HAPT1 may be responsible for the residual uptake of melaminophenyl arsenicals. High-level arsenical resistance therefore appears to involve the loss of more than one transporter.     

Conserved residues F316 and G476 in the concentrative nucleoside transporter 1 (hCNT1) affect guanosine sensitivity and membrane expression, respectively

The functional significance of two highly conserved amino acid residues, F316 [putative transmembrane domain (TM)7] and G476 (putative TM11), in the concentrative nucleoside transporter hCNT1 (SLC28A1) was examined by performing site-directed mutagenesis. Conservative mutations at these positions (F316A, F316Y, G476A, and G476L) were generated and expressed in Madin-Darby canine kidney (MDCK) cells as fusion polypeptides with green fluorescent protein (GFP). Unlike wild-type hCNT1, G476A-GFP and G476L-GFP were not expressed in the plasma membrane in undifferentiated or differentiated MDCK cells and had no functional activity. Like wild-type hCNT1, F316A-GFP and F316Y-GFP were expressed in the plasma membrane of undifferentiated MDCK cells and in the apical membrane of differentiated MDCK cells. Remarkably, transport of [³H]uridine by F316Y-GFP or F316A-GFP was highly sensitive to inhibition by guanosine. Furthermore, genotyping of exon 11 of hCNT1 (TM7) in a panel of 260 anonymous human DNA samples revealed a novel F316H variant (TT>CA; 1/260). When expressed in MDCK cells, [³H]uridine transport by F316H was also found to be sensitive to inhibition by guanosine (IC₅₀ = 148 µM). The effect of the F316H mutation resembles the N4 type nucleoside transporter phenotype previously reported to be present in human kidneys. We suggest that the N4 transport system is a naturally occurring variant of hCNT1, perhaps at the F316 position. Collectively, our data show that G476 is important for correct membrane targeting, folding, and/or intracellular processing of hCNT1. In addition, we have discovered that hCNT1 displays natural variation at position F316 and that the variant F316H confers on the transporter an unusual sensitivity to inhibition by guanosine. localization; inhibition; polymorphism     

Aquaporin 2 Mutations in Trypanosoma brucei gambiense Field Isolates Correlate with Decreased Susceptibility to Pentamidine and Melarsoprol

The predominant mechanism of drug resistance in African trypanosomes is decreased drug uptake due to loss-of-function mutations in the genes for the transporters that mediate drug import. The role of transporters as determinants of drug susceptibility is well documented from laboratory-selected Trypanosoma brucei mutants. But clinical isolates, especially of T. b. gambiense, are less amenable to experimental investigation since they do not readily grow in culture without prior adaptation. Here we analyze a selected panel of 16 T. brucei ssp. field isolates that (i) have been adapted to axenic in vitro cultivation and (ii) mostly stem from treatment-refractory cases. For each isolate, we quantify the sensitivity to melarsoprol, pentamidine, and diminazene, and sequence the genomic loci of the transporter genes TbAT1 and TbAQP2. The former encodes the well-characterized aminopurine permease P2 which transports several trypanocides including melarsoprol, pentamidine, and diminazene. We find that diminazene-resistant field isolates of T. b. brucei and T. b. rhodesiense carry the same set of point mutations in TbAT1 that was previously described from lab mutants. Aquaglyceroporin 2 has only recently been identified as a second transporter involved in melarsoprol/pentamidine cross-resistance. Here we describe two different kinds of TbAQP2 mutations found in T. b. gambiense field isolates: simple loss of TbAQP2, or loss of wild-type TbAQP2 allele combined with the formation of a novel type of TbAQP2/3 chimera. The identified mutant T. b. gambiense are 40- to 50-fold less sensitive to pentamidine and 3- to 5-times less sensitive to melarsoprol than the reference isolates. We thus demonstrate for the first time that rearrangements of the TbAQP2/TbAQP3 locus accompanied by TbAQP2 gene loss also occur in the field, and that the T. b. gambiense carrying such mutations correlate with a significantly reduced susceptibility to pentamidine and melarsoprol.     

TbLpn,a key enzyme in lipid droplet formation and phospholipid metabolism,is essential for mitochondrial integrity and growth of Trypanosoma brucei

Mammalian phosphatidic acid phosphatases, also called lipins, show high amino acid sequence identity to Saccharomyces cerevisiae Pah1p and catalyze the dephosphorylation of phosphatidic acid (PA) to diacylglycerol. Both the substrate and product of the reaction are key precursors for the synthesis of phospholipids and triacylglycerol (TAG). We now show that expression of the Trypanosoma brucei lipin homolog TbLpn is essential for parasite survival in culture. Inducible down‐regulation of TbLpn in T. brucei procyclic forms increased cellular PA content, decreased the numbers of lipid droplets, reduced TAG steady‐state levels and inhibited in vivo [³H]TAG formation after labeling trypanosomes with [³H]glycerol. In addition, fluorescence and transmission electron microscopy revealed that depletion of TbLpn caused major alterations in mitochondrial morphology and function, i.e., the appearance of distorted mitochondrial matrix, and reduced ATP production via oxidative phosphorylation. Effects of lipin depletion on mitochondrial integrity have previously not been reported. N‐ and C‐terminally tagged forms of TbLpn were localized in the cytosol.     

Genotypic Status of the TbAT1/P2 Adenosine Transporter of Trypanosoma brucei gambiense Isolates from Northwestern Uganda following Melarsoprol Withdrawal

Background

The development of arsenical and diamidine resistance in Trypanosoma brucei is associated with loss of drug uptake by the P2 purine transporter as a result of alterations in the corresponding T. brucei adenosine transporter 1 gene (TbAT1). Previously, specific TbAT1 mutant type alleles linked to melarsoprol treatment failure were significantly more prevalent in T. b. gambiense from relapse patients at Omugo health centre in Arua district. Relapse rates of up to 30% prompted a shift from melarsoprol to eflornithine (α-difluoromethylornithine, DFMO) as first-line treatment at this centre. The aim of this study was to determine the status of TbAT1 in recent isolates collected from T. b. gambiense sleeping sickness patients from Arua and Moyo districts in Northwestern Uganda after this shift in first-line drug choice.

Methodology and results

Blood and cerebrospinal fluids of consenting patients were collected for DNA preparation and subsequent amplification. All of the 105 isolates from Omugo that we successfully analysed by PCR-RFLP possessed the TbAT1 wild type allele. In addition, PCR/RFLP analysis was performed for 74 samples from Moyo, where melarsoprol is still the first line drug; 61 samples displayed the wild genotype while six were mutant and seven had a mixed pattern of both mutant and wild-type TbAT1. The melarsoprol treatment failure rate at Moyo over the same period was nine out of 101 stage II cases that were followed up at least once. Five of the relapse cases harboured mutant TbAT1, one had the wild type, while no amplification was achieved from the remaining three samples.

Conclusions/significance

The apparent disappearance of mutant alleles at Omugo may correlate with melarsoprol withdrawal as first-line treatment. Our results suggest that melarsoprol could successfully be reintroduced following a time lag subsequent to its replacement. A field-applicable test to predict melarsoprol treatment outcome and identify patients for whom the drug can still be beneficial is clearly required. This will facilitate cost-effective management of HAT in rural resource-poor settings, given that eflornithine has a much higher logistical requirement for its application.     

Expression of A1 adenosine receptors in the developing avian retina: in vivo modulation by A2A receptors and endogenous adenosine

Little is known about the mechanisms that regulate the expression of adenosine receptors during CNS development. We demonstrate here that retinas from chick embryos injected in ovo with selective adenosine receptor ligands show changes in A1 receptor expression after 48 h. Exposure to A1 agonist N⁶‐cyclohexyladenosine (CHA) or antagonist 8‐Cyclopentyl‐1, 3‐dipropylxanthine (DPCPX) reduced or increased, respectively, A1 receptor protein and [³H]DPCPX binding, but together, CHA+DPCPX had no effect. Interestingly, treatment with A_2A agonist 3‐[4‐[2‐[[6‐amino‐9‐[(2R,3R,4S,5S)‐5‐(ethylcarbamoyl)‐3,4‐dihydroxy‐oxolan‐2‐yl]purin‐2‐yl]amino] ethyl]phenyl] propanoic acid (CGS21680) increased A1 receptor protein and [³H]DPCPX binding, and reduced A_2A receptors. The A_2A antagonists 7‐(2‐phenylethyl)‐5‐amino‐2‐(2‐furyl)‐pyrazolo‐[4,3‐e]‐1,2,4‐trizolo[1,5‐c] pyrimidine (SCH58261) and 4‐(2‐[7‐amino‐2‐[2‐furyl][1,2,4]triazolo[2,3‐a][1,3,5]triazo‐5‐yl‐amino]ethyl)phenol (ZM241385) had opposite effects on A1 receptor expression. Exposure to CGS21680 + CHA did not change A1 receptor levels, whereas CHA + ZM241385 or CGS21680 + DPCPX had no synergic effect. The blockade of adenosine transporter with S‐(4‐nitrobenzyl)‐6‐thioinosine (NBMPR) also reduced [³H]DPCPX binding, an effect blocked by DPCPX, but not enhanced by ZM241385. [³H]DPCPX binding kinetics showed that treatment with CHA reduced and CGS21680 increased the Bmax, but did not affect Kd values. CHA, DPCPX, CGS21680, and ZM241385 had no effect on A1 receptor mRNA. These data demonstrated an in vivo regulation of A1 receptor expression by endogenous adenosine or long‐term treatment with A1 and A_2A receptors modulators.     

Effects of Methylglyoxal bis(Guanylhydrazone) on Trypanosomatid Flagellates: Inhibition of Growth and Nucleoside Incorporation in Trypanosoma brucei*

SYNOPSIS. Methylglyoxal bis(guanylhydrazone) (MGBG) at 0.5 mm had little effect in vitro on Blastocrithidia culicis, Crithidia oncopelti, and Leishmania spp., but completely inhibited growth of Trypanosoma brucei. Inhibition became irreversible after a 3-h exposure of T. brucei culture procyclics. Treated organisms remained motile, but failed to divide. Polyamines, spermidine, and spermine, did not reverse the anti-trypanosome action of MGBG (preloading of cells or concurrent administration). Two intraperitoneal injections of the drug at a concentration of 50 mg kg body weight at a 1-day interval greatly reduced the parasitemia of T. brucei and T. congolense in rats. Trypanosome infections, however, relapsed and killed the animals in 6 days after treatment. It was evident from the results of tracer experiments with T. brucei that MGBG significantly lowered incorporation of [³H]thymidine by culture procyclics and of [³H]uridine by bloodstream forms; in both stages [³H]leucine incorporation was only slightly inhibited. It is suggested that MGBG interferes with nucleoside incorporation by Trypanosoma and that its mode of action is different in bloodstream and culture procyclics.     

Some Operational Characteristics of Glycine Release in Rat Retina: The Role of Reverse Mode Operation of Glycine Transporter Type-1 (GlyT-1) in Ischemic Conditions

Rat posterior eyecups containing the retina were prepared, loaded with [³H]glycine and superfused in order to determine its release originated from glycinergic amacrine cells and/or glial cells. Deprivation of oxygen and glucose from the Krebs-bicarbonate buffer used for superfusion evoked a marked increase of [³H]glycine release, an effect that was found to be external Ca²⁺-independent. Whereas oxygen and glucose deprivation increased [³H]glycine release, its uptake was reduced suggesting that energy deficiency shifts glycine transporter type-1 operation from normal to reverse mode. The increased release of [³H]glycine evoked by oxygen and glucose deprivation was suspended by addition of the non-competitive glycine transporter type-1 inhibitor NFPS and the competitive inhibitor ACPPB further suggesting the involvement of this transporter in the mediation of [³H]glycine release. Oxygen and glucose deprivation also evoked [³H]glutamate release from rat retina and the concomitantly occurring release of the NMDA receptor agonist glutamate and the coagonist glycine makes NMDA receptor pathological overstimulation possible in hypoxic conditions. [³H]Glutamate release was suspended by addition of the excitatory amino acid transporter inhibitor TBOA. Sarcosine, a substrate inhibitor of glycine transporter type-1, also increased [³H]glycine release probably by heteroexchange shifting transporter operation into reverse mode. This effect of sarcosine was also external Ca²⁺-independent and could be suspended by NFPS. Energy deficiency in retina induced by ouabain, an inhibitor of the Na⁺–K⁺-dependent ATPase, and by rotenone, a mitochondrial complex I inhibitor added with the glycolytic inhibitor 2-deoxy-d-glucose, led to increase of retinal [³H]glycine efflux. These effects of ouabain and rotenone/2-deoxy-d-glucose could also be blocked by NFPS pointed to the preferential reverse mode operation of glycine transporter type-1 as a consequence of impaired cellular energy homeostasis. Immunohistochemical studies revealed that glycine transporter type-1, of which reverse mode operation assures [³H]glycine release, is expressed in amacrine cells in the inner nuclear and plexiform layers of the retina and also in Müller macroglia cells. We conclude that disruption of the balanced normal/reverse mode operation of glycine transporter type-1 is likely a significant factor contributing to neurotoxic processes of the retina. The possibility to inhibit glycine transporter type-1 mediated glycine efflux by drugs more potently than glycine uptake might offer some therapeutic potential for the treatment of various neurodegenerative disorders of the retina.     

CD38 deficiency suppresses adipogenesis and lipogenesis in adipose tissues through activating Sirt1/PPARγ signaling pathway

It has been recently reported that CD38 was highly expressed in adipose tissues from obese people and CD38‐deficient mice were resistant to high‐fat diet (HFD)‐induced obesity. However, the role of CD38 in the regulation of adipogenesis and lipogenesis is unknown. In this study, to explore the roles of CD38 in adipogenesis and lipogenesis in vivo and in vitro, obesity models were generated with male CD38^?/? and WT mice fed with HFD. The adipocyte differentiations were induced with MEFs from WT and CD38^?/? mice, 3T3‐L1 and C3H10T1/2 cells in vitro. The lipid accumulations and the alternations of CD38 and the genes involved in adipogenesis and lipogenesis were determined with the adipose tissues from the HFD‐fed mice or the MEFs, 3T3‐L1 and C3H10T1/2 cells during induction of adipocyte differentiation. The results showed that CD38^?/? male mice were significantly resistant to HFD‐induced obesity. CD38 expressions in adipocytes were significantly increased in WT mice fed with HFD, and the similar results were obtained from WT MEFs, 3T3‐L1 and C3H10T1/2 during induction of adipocyte differentiation. The expressions of PPARγ, AP2 and C/EBPα were markedly attenuated in adipocytes from HFD‐fed CD38^?/? mice and CD38^?/? MEFs at late stage of adipocyte differentiation. Moreover, the expressions of SREBP1 and FASN were also significantly decreased in CD38^?/? MEFs. Finally, the CD38 deficiency‐mediated activations of Sirt1 signalling were up‐regulated or down‐regulated by resveratrol and nicotinamide, respectively. These results suggest that CD38 deficiency impairs adipogenesis and lipogenesis through activating Sirt1/PPARγ‐FASN signalling pathway during the development of obesity.     

Rag1 immunodeficiency‐induced early aging and senescence in zebrafish are dependent on chronic inflammation and oxidative stress

In mammals, recombination activating gene 1 (RAG1) plays a crucial role in adaptive immunity, generating a vast range of immunoglobulins. Rag1^?/? zebrafish (Danio rerio) are viable and reach adulthood without obvious signs of infectious disease in standard nonsterile conditions, suggesting that innate immunity could be enhanced to compensate for the lack of adaptive immunity. By using microarray analysis, we confirmed that the expression of immunity‐ and apoptosis‐related genes was increased in the rag1^?/? fish. This tool also allows us to notice alterations of the DNA repair and cell cycle mechanisms in rag1^?/? zebrafish. Several senescence and aging markers were analyzed. In addition to the lower lifespan of rag1^?/? zebrafish compared to their wild‐type (wt) siblings, rag1^?/? showed a higher incidence of cell cycle arrest and apoptosis, a greater amount of phosphorylated histone H2AX, oxidative stress and decline of the antioxidant mechanisms, an upregulated expression and activity of senescence‐related genes and senescence‐associated β‐galactosidase, respectively, diminished telomere length, and abnormal self‐renewal and repair capacities in the retina and liver. Metabolomic analysis also demonstrated clear differences between wt and rag1^?/? fish, as was the deficiency of the antioxidant metabolite l ‐acetylcarnitine (ALCAR) in rag1^?/? fish. Therefore, Rag1 activity does not seem to be limited to V(D)J recombination but is also involved in senescence and aging. Furthermore, we confirmed the senolytic effect of ABT‐263, a known senolytic compound and, for the first time, the potential in vivo senolytic activity of the antioxidant agent ALCAR, suggesting that this metabolite is essential to avoid premature aging.     

Complement selectively elicits glutamate release from nerve endings in different regions of mammal central nervous system

Our study was aimed at investigating whether complement, a complex of soluble and membrane‐associated serum proteins, could, in addition to its well‐documented post‐synaptic activity, also pre‐synaptically affect the release of classic neurotransmitters in central nervous system (CNS). Complement (dilution 1 : 10 to 1 : 10000) elicited the release of preloaded [³H]‐d ‐aspartate ([³H]d ‐ASP) and endogenous glutamate from mouse cortical synaptosomes in a dilution‐dependent manner. It also evoked [³H]d ‐ASP release from mouse hippocampal, cerebellar, and spinal cord synaptosomes, as well as from rat and human cortical nerve endings, but left unaltered the release of GABA, [³H]noradrenaline or [³H]acetylcholine. Lowering external Na⁺ (from 140 to 40 mM) or Ca²⁺ (from 1.2 to 0.1 mM) ions prevented the 1 : 300 complement‐evoked [³H]d ‐ASP release from mouse cortical synaptosomes. Complement‐induced releasing effect was unaltered in synaptosomes entrapped with the Ca²⁺ ions chelator 1,2‐bis‐(2‐aminophenoxy) ethane‐N,N,N’,N’, tetra‐acetic acid or with pertussis toxin. Nifedipine,/ω‐conotoxin GVIA/ω‐conotoxin MVIIC mixture as well as the vesicular ATPase blocker bafilomycin A1 were also inefficacious. The excitatory amino acid transporter blocker DL‐threo‐ß‐benzyloxyaspartic acid, on the contrary, reduced the complement‐evoked releasing effect in a concentration‐dependent manner. We concluded that complement‐induced releasing activity is restricted to glutamatergic nerve endings, where it was accounted for by carrier‐mediated release. Our observations afford new insights into the molecular events accounting for immune and CNS crosstalk.

     

Diminazene aceturate associated with sodium selenite and vitamin E in the treatment of Trypanosoma evansi infection in rats

The aim of this study was to evaluate the utilization of a standard treatment with diminazene aceturate against the infection caused by Trypanosoma evansi, associated to sodium selenite and vitamin E. In vitro tests showed trypanocidal effect related to the treatment with diminazene aceturate and sodium selenite, but vitamin E had no harmful effect on the trypanosomes. In vivo experiments utilized a total of 72 adult outbreed females rats, separated into 9 groups (A, B, C, D, E, F, G, H and I), 8 animals each. Group A was the uninfected group; groups B to I were infected with 0.2 mL of blood containing 10⁶ trypanosomes. Parasitemia was estimated daily by microscopic examination of blood smears. Group B served as positive control; group C was treated with diminazene aceturate; group D with sodium selenite; group E with vitamin E; group F received an association of diminazene aceturate and sodium selenite; group G received an association of diminazene aceturate and vitamin E; group H received an association of diminazene aceturate, sodium selenite and vitamin E, and group I received an association of sodium selenite and vitamin E. Diminazene aceturate was administrated in a single dose on the 3rd day post infection (PI). Sodium selenite and vitamin E were administered at the 3rd and 23rd day PI. In vivo tests showed increase of longevity in groups treated with diminazene aceturate associated with sodium selenite (groups F and H). No difference was found between groups C and E, thus the vitamin E did not increase the efficacy of treatment against T. evansi when associated to diminazene aceturate. The curative efficacy of treatments was 37.5, 87.7, 37.7 and 75% to the groups C, F, G and H, respectively. Other treatments showed no efficacy. The sodium selenite when combined with chemotherapy may represent an alternative in the treatment of trypanosomosis.     

Blockade of cannabinoid 1 receptor improves glucose responsiveness in pancreatic beta cells

Cannabinoid 1 receptors (CB1Rs) are expressed in peripheral tissues, including islets of Langerhans, where their function(s) is under scrutiny. Using mouse β‐cell lines, human islets and CB1R‐null (CB1R^?/?) mice, we have now investigated the role of CB1Rs in modulating β‐cell function and glucose responsiveness. Synthetic CB1R agonists diminished GLP‐1‐mediated cAMP accumulation and insulin secretion as well as glucose‐stimulated insulin secretion in mouse β‐cell lines and human islets. In addition, silencing CB1R in mouse β cells resulted in an increased expression of pro‐insulin, glucokinase (GCK) and glucose transporter 2 (GLUT2), but this increase was lost in β cells lacking insulin receptor. Furthermore, CB1R^?/? mice had increased pro‐insulin, GCK and GLUT2 expression in β cells. Our results suggest that CB1R signalling in pancreatic islets may be harnessed to improve β‐cell glucose responsiveness and preserve their function. Thus, our findings further support that blocking peripheral CB1Rs would be beneficial to β‐cell function in type 2 diabetes.     

Deletion of glutamate dehydrogenase 1 (Glud1) in the central nervous system affects glutamate handling without altering synaptic transmission

Glutamate dehydrogenase (GDH), encoded by GLUD1, participates in the breakdown and synthesis of glutamate, the main excitatory neurotransmitter. In the CNS, besides its primary signaling function, glutamate is also at the crossroad of metabolic and neurotransmitter pathways. Importance of brain GDH was questioned here by generation of CNS‐specific GDH‐null mice (CnsGlud1^?/?); which were viable, fertile and without apparent behavioral problems. GDH immunoreactivity as well as enzymatic activity were absent in Cns‐Glud1^?/? brains. Immunohistochemical analyses on brain sections revealed that the pyramidal cells of control animals were positive for GDH, whereas the labeling was absent in hippocampal sections of Cns‐Glud1^?/? mice. Electrophysiological recordings showed that deletion of GDH within the CNS did not alter synaptic transmission in standard conditions. Cns‐Glud1^?/? mice exhibited deficient oxidative catabolism of glutamate in astrocytes, showing that GDH is required for Krebs cycle pathway. As revealed by NMR studies, brain glutamate levels remained unchanged, whereas glutamine levels were increased. This pattern was favored by up‐regulation of astrocyte‐type glutamate and glutamine transporters and of glutamine synthetase. Present data show that the lack of GDH in the CNS modifies the metabolic handling of glutamate without altering synaptic transmission.     

The F box protein S phase kinase-associated protein 2 regulates adipose mass and adipocyte number in vivo

Objective: The etiology of some obesity may involve adipocyte hyperplasia. However, the role of adipocyte number in establishing adipose mass is unclear. Cyclin‐dependent kinase inhibitor p27 regulates activity of cyclin/cyclin‐dependent kinase complexes responsible for cell cycle progression. This protein is critical for establishing adult adipocyte number, and p27 knockout increases adult adipocyte number. The SCF (for Skp1‐Cullin‐F‐box protein) complex targets proteins such as p27 for ubiquitin‐proteosome degradation; the F box protein S phase kinase‐associated protein 2 (Skp2), a component of the SCF complex, specifically recognizes p27 for degradation. We used Skp2 knockout (Skp2^?/?) mice to test whether Skp2 loss decreased adipose mass and adipocyte number. Research Methods and Procedures: We measured body weight, adipose mass, adipocyte diameter and number, and glucose tolerance in wild‐type (WT), Skp2^?/?, and p27^?/?Skp2^?/? mice. Mouse embryo fibroblasts (MEFs) from WT and Skp2^?/? fetuses were differentiated to determine whether Skp2 directly affected adipogenesis. Results: Skp2^?/? mice had a 50% decrease in both subcutaneous and visceral fat pad mass and adipocyte number; these decreases exceeded those in body weight, kidney, or muscle. To test the hypothesis that Skp2 effects on adipocyte number involved p27 accumulation, we used p27^?/?Skp2^?/? double knockout mice. The Skp2^?/? decrements in adipocyte number and fat pad mass were totally reversed in p27^?/?Skp2^?/? mice. Adipogenesis was inhibited in MEFs from Skp2^?/? vs. WT mice, and this inhibition was absent in MEFs from p27^?/?Skp2^?/? mice. Discussion: Our results indicate that Skp2 regulates adipogenesis and ultimate adipocyte number in vivo; thus, Skp2 may contribute to obesity involving adipocyte hyperplasia.     

Leukocyte Migration in Adipose Tissue of Mice Null for ICAM‐1 and Mac‐1 Adhesion Receptors

Objective: To determine whether the leukocyte adhesion receptors ICAM‐1 and Mac‐1, regulators of immune cell migration, have an intrinsic role within adipose tissue by 1) analyzing the expression of ICAM‐1 in adipose tissue, 2) identifying leukocyte populations within adipose tissue, and 3) determining whether ICAM‐1 and Mac‐1 mutant mice exhibit abnormal numbers of adipose tissue leukocytes. Research Methods and Procedures: Wild‐type, ICAM‐1^?/?, and Mac‐1^?/? mice were fed a long‐term high‐fat diet. ICAM‐1 expression was analyzed by Northern blot and immunohistochemistry. Leukocytes within adipose tissue were identified by immunohistochemistry and flow cytometry. Results: ICAM‐1 was expressed in adipose tissue and localized to the vascular endothelium. Macrophages and lymphocytes were prevalent within the stromal‐vascular cell fraction of adipose tissue, and gender‐specific differences were observed, with adipose tissue from female mice containing significantly more macrophages than tissue from male mice. Numbers of leukocytes in ICAM‐1^?/? and Mac‐1^?/? mice were not different from wild‐types, however, indicating that these adhesion receptors are not required for leukocyte migration into adipose tissue. Discussion: Our results documented leukocyte populations within adipose tissue, which may be involved in the development of heightened inflammation that is characteristic of obesity.     

Deletion of mitochondrial associated ubiquitin fold modifier protein Ufm1 in Leishmania donovani results in loss of β‐oxidation of fatty acids and blocks cell division in the amastigote stage

Recently, we described the existence of the ubiquitin fold modifier 1 (Ufm1) and its conjugation pathway in Leishmania donovani. We demonstrated the conjugation of Ufm1 to proteins such as mitochondrial trifunctional protein (MTP) that catalyses β‐oxidation of fatty acids in L. donovani. To elucidate the biological roles of the Ufm1‐mediated modifications, we made an L. donovani Ufm1 null mutant (Ufm1^?/?). Loss of Ufm1 and consequently absence of Ufm1 conjugation with MTP resulted in diminished acetyl‐CoA, the end‐product of the β‐oxidation in the Ufm1^?/? amastigote stage. The Ufm1^?/? mutants showed reduced survival in the amastigote stage in vitro and ex vivo in human macrophages. This survival was restored by re‐expression of wild‐type Ufm1 with concomitant induction of acetyl‐CoA but not by re‐expressing the non‐conjugatable Ufm1, indicating the essential nature of Ufm1 conjugation and β‐oxidation. Both cell cycle analysis and ultrastructural studies of Ufm1^?/? parasites confirmed the role of Ufm1 in amastigote growth. The defect in vitro growth of amastigotes in human macrophages was further substantiated by reduced survival. Therefore, these studies suggest the importance of Ufm1 in Leishmania pathogenesis with larger impact on other organisms and further provide an opportunity to test Ufm1^?/? parasites as drug and vaccine targets.     

Apical and basolateral 4F2hc and the amino acid exchange of L-DOPA in renal LLC-PK1 cells

Summary. The present study aimed to examine the presence and define the role of 4F2hc, a glycoprotein associated with the LAT2 amino acid transporter, in L-DOPA handling by LLC-PK₁ cells. For this purpose we have measured the activity of the apical and basolateral inward and outward transport of [¹⁴C] L-DOPA in cell monolayers and examined the influence of 4F2hc antisense oligonucleotides on [¹⁴C] L-DOPA handling. The basal-to-apical transepithelial flux of [¹⁴C] L-DOPA progressively increased with incubation time and was similar to the apical-to-basal transepithelial flux. The spontaneous and the L-DOPA-stimulated apical fractional outflow of [¹⁴C] L-DOPA were identical to that through the basal cell side. The L-DOPA-induced fractional outflow of [¹⁴C] L-DOPA through the apical or basal cell side was accompanied by marked decreases in intracellular levels of [¹⁴C] L-DOPA. In cells treated with an antisense oligonucleotide complementary to 4F2hc mRNA for 72 h, [¹⁴C] L-DOPA inward transport and 4F2hc expression were markedly reduced. Treatment with the 4F2hc antisense oligonucleotide markedly decreased the spontaneous fractional outflow of [¹⁴C] L-DOPA through the apical or the basal cell side. It is likely that the Na⁺-independent and pH-sensitive uptake of L-DOPA include the hetero amino acid exchanger LAT2/4F2hc, which facilitates the trans-stimulation of L-DOPA and its outward transfer at both the apical and basal cell sides.     

Heat shock factor 1 deficiency via its downstream target gene αB‐crystallin (Hspb5) impairs p53 degradation

Heat shock factor Hsf1 regulates the stress‐inducibility of heat shock proteins (Hsps) or molecular chaperones. One of the functions attributed to Hsps is their participation in folding and degradation of proteins. We recently showed that hsf1^?/? cells accumulate ubiquitinated proteins. However, a direct role for Hsf1 in stability of specific proteins such as p53 has not been elucidated. We present evidence that cells deficient in hsf1 accumulate wild‐type p53 protein. We further show that hsf1^?/? cells express lower levels of αB‐crystallin and cells deficient in αB‐crystallin also accumulate p53 protein. Reports indicate that αB‐crystallin binds to Fbx4 ubiquitin ligase, and they target cyclin D1 for degradation through a pathway involving the SCF (Skp1‐Cul1‐F‐box) complex. Towards determining a mechanism for p53 degradation involving αB‐crystallin and Hsf1, we have found that ectopic expression of Fbx4 in wild‐type mouse embryo fibroblasts (MEFs) expressing mutant p53 (p53R175H) leads to increase in its degradation, while MEFs deficient in hsf1 or αBcry are defective in degradation of this p53 protein. In addition, immunoprecipitated p53R175H from wild‐type MEFs is able to pull‐down both αB‐crystallin and Fbx4. Finally, immunoprecipitated wild‐type p53 from doxorubicin treated U2OS cells can pull‐down endogenous αB‐crystallin and Fbx4. These results indicate that hsf1‐ and αBcry‐deficient cells accumulate p53 due to reduced levels of αB‐crystallin in these cells. Elevated levels of p53 in hsf1‐ and αBcry‐deficient cells lead to their increased sensitivity to DNA damaging agents. These data reveal a novel mechanism for protein degradation through Hsf1 and αB‐crystallin. J. Cell. Biochem. 107: 504–515, 2009. © 2009 Wiley‐Liss, Inc.