White adipose tissue (fat) is the primary organ for energy storage and its regulation has serious implications on human health. Excess fat tissue causes significant morbidity, and adipose tissue dysfunction caused by excessive adipocyte hypertrophy has been proposed to play a significant role in the pathogenesis of metabolic disease. Studies in both humans and animal models show that metabolic dysfunction is more closely associated with visceral than subcutaneous fat accumulation. Here, we show that in mice fed a high-fat diet, visceral fat (VAT) grows mostly by hypertrophy and subcutaneous fat (SAT) by hyperplasia, providing a rationale for the different effects of specific adipose depots on metabolic health. To address whether depot expansion is controlled at the level of stem/progenitor cells, we developed a strategy to prospectively identify adipogenic progenitors (APs) from both depots. Clonogenic assays and in vivo bromodeoxyuridine (BrdU) studies show that APs are eightfold more abundant in SAT than VAT, and that AP proliferation is significantly increased in SAT but not VAT in response to high-fat diet. Our results suggest that depot-specific differences in AP abundance and proliferation underlie whether a fat depot expands by hypertrophy or hyperplasia, and thus may have important implications on the development of metabolic disease. In addition, we provide the first evidence that dietary inputs can modulate the proliferation of adipogenic progenitors in adults.

The human CDC2L5 gene encodes a protein of unknown physiological function. This protein is closely related to the cyclin-dependent kinase (Cdks) family and contains an arginine/serine-rich (RS) domain. The Cdks were first identified as crucial regulators of cell-cycle progression, more recently they were found to be involved in transcription and mRNA processing. RS domains are mainly present in proteins regulating pre-mRNA splicing, suggesting CDC2L5 having a possible role in this process. In this study, we demonstrate that CDC2L5 is located in the nucleoplasm, at a higher concentration in speckles, the storage sites for splicing factors. Furthermore, this localization is dependent on the presence of the N-terminal sequence including the RS domain. Then, we report that CDC2L5 directly interacts with the ASF/SF2-associated protein p32, a protein involved in splicing regulation. Overexpression of CDC2L5 constructs disturbs constitutive splicing and switches alternative splice site selection in vivo. These results argue in favor of a functional role of the CDC2L5 kinase in splicing regulation.

The human immunodeficiency virus type 1 (HIV-1) Tat is a 14-kDa viral protein that acts as a potent transactivator by binding to the transactivation-responsive region, a structured RNA element located at the 5 end of all HIV-1 transcripts. Tat transactivates viral gene expression by inducing the phosphorylation of the C-terminal domain of RNA polymerase II through several Tat-activated kinases and by recruiting chromatinremodeling complexes and histone-modifying enzymes to the HIV-1 long terminal repeat. Histone acetyltransferases, including p300 and hGCN5, not only acetylate histones but also acetylate Tat at lysine positions 50 and 51 in the arginine-rich motif. Acetylated Tat at positions 50 and 51 interacts with a specialized protein module, the bromodomain, and recruits novel factors having this particular domain, such as P/CAF and SWI/SNF. In addition to having its effect on transcription, Tat has been shown to be involved in splicing. In this study, we demonstrate that Tat interacts with cyclin-dependent kinase 13 (CDK13) both in vivo and in vitro. We also found that CDK13 increases HIV-1 mRNA splicing and favors the production of the doubly spliced protein Nef.In addition, we demonstrate that CDK13 acts as a possible restriction factor, in that its overexpression decreases the production of the viral proteins Gag and Env and subsequently suppresses virus production. Using small interfering RNA against CDK13, we show that silencing of CDK13 leads to a significant increase in virus production. Finally, we demonstrate that CDK13 mediates its effect on splicing through the phosphorylation of ASF/SF2.Human immunodeficiency virus type 1 (HIV-1) is a complex retrovirus that depends on alternative splicing to generate mRNAs encoding viral proteins essential for viral replication. More than 40 different alternatively spliced HIV-1 mRNAs are produced through the differential combination of four 5Ј splice donor sites (D1 to D4) and eight 3Ј splice acceptor sites (A 1 , A 2 , A 3 , A 4a , A 4b , A 4c , A 5 , and A 7 ) (49,51,58). Splicing of HIV-1 mRNA generates three classes of RNA: unspliced (US) (9 kb), including Gag and polymerase; singly spliced (4 kb), including Env, Vpu, Vif, and Vpr; and doubly spliced (2 kb), including Tat, Rev, and Nef. The expression of these different viral mRNA species is temporally regulated. While multiply spliced viral RNA species are abundant during early stages of infection, US and singly spliced viral RNA appear at the later stages of infection (32, 33).HIV-1 is highly dependent on the host splicing machinery in order to process viral transcripts into the different mRNA isoforms present in infected cells (reviewed in reference 59). Serine/ arginine-rich (SR) proteins are among the splicing factors that are essential for the operation and regulation of viral and cellular splicing (29,52). They belong to a family of a dozen different proteins characterized by one or two RNA recognition motifs at their N termini and an arginine/serine-rich (RS) motif at their C termini (12). SR prote...

Oregano essential oil has long been known for its health-promoting benefits. Here, we report its activity against viral replication. Oregano oil was found to specifically inhibit lentiviruses, such as human and simian immunodeficiency viruses (HIV and SIV), irrespective of virus tropism, but not hepatitis C virus, adenovirus 5 (ADV5), Zika virus, and influenza (H1N1) virus. Oregano oil’s most abundant components, carvacrol and its isomer, thymol, were shown to block virus-target cell fusion while not perturbing other stages of the virus life cycle. We detected changes in virus particle density, suggesting that cholesterol depletion from the HIV-1 envelope membrane reduces virus entry. Furthermore, infection was rescued by adding exogenous cholesterol. The evolution of viral resistance to carvacrol supported this mechanism of action with the identification of mutations in the viral gp41 fusion protein that counteracted cholesterol depletion. In addition, resistance to carvacrol emerged later than typically observed for other clinically used drugs, strengthening its antiviral potential. Structure-activity relationship studies revealed key motifs of carvacrol and thymol required for HIV neutralization and identified previously unknown active analogs. Carvacrol was also shown to additively cooperate with antiretroviral therapy. In sum, oregano oil and improved carvacrol and thymol analogs could be considered to supplement current HIV therapeutics. IMPORTANCE Oregano essential oil has multiple benefits in traditional medicine, cosmetics, and food industries. Carvacrol and its analog, thymol, are well-described components of oregano oil. Here, we show that these compounds inhibit HIV-target cell fusion independently of viral tropism. Our results suggest that carvacrol and thymol alter the cholesterol content of the viral membrane, blocking HIV-1 entry into the target cell. Resistance to carvacrol has selected for viruses with mutations in the viral envelope glycoprotein, gp41. This protein is known for its interaction with cholesterol present in membrane lipid rafts. Together, these results demonstrate the potential of therapies targeting the viral envelope membrane, and oregano oil is a safe supplement to antiretrovirals, potentially delaying disease progression and resistance development.

Recent findings suggested that the role of cysteine proteases would not be limited to protein degradation in lysosomes but would also play regulatory functions in more specific cell mechanisms. We analyzed here the role of these enzymes in the control of cell cycle during embryogenesis. The addition of the potent cysteine protease inhibitor E64d to newly fertilized sea urchin eggs disrupted cell cycle progression, affecting nuclear as well as cytoplasmic characteristic events. Monitoring BrdU incorporation in E64d treated eggs demonstrated that DNA replication is severely disturbed. Moreover, this drug treatment inhibited male histones degradation, a step that is necessary for sperm chromatin remodeling and precedes the initiation of DNA replication in control eggs. This inhibition likely explains the DNA replication disturbance and suggests that S phase initiation requires cysteine protease activity. In turn, activation of the DNA replication checkpoint could be responsible for the consecutive block of nuclear envelope breakdown (NEB). However, in sea urchin early embryos this checkpoint doesn't control the mitotic cytoplasmic events that are not tightly coupled with NEB. Thus the fact that microtubule spindle is not assembled and cyclin B-cdk1 not activated under E64d treatment more likely rely on a distinct mechanism. Immunofluorescence experiments indicated that centrosome organization was deficient in absence of cysteine protease activity. This potentially accounts for mitotic spindle disruption and for cyclin B mis-localization in E64d treated eggs. We conclude that cysteine proteases are essential to trigger S phase and to promote M phase entry in newly fertilized sea urchin eggs.

The experiments reported here were motivated by our interest to express in stably-transfected cells large amounts of recombinant rat GABAA receptors. For this, we developed an original two step selection strategy, in which the first step consisted of transfecting HEK 293 cells with rat GABAA receptor alpha and beta subunits. G 418 resistant colonies isolated at this step were screened for [3H] muscimol binding to select for those that coexpressed alpha- and beta-subunits. The best alpha and beta subunit expressing colony was then supertransfected with a plasmid coding for the gamma rat GABAA receptor subunit and a mutant DHFR gene. After a second round of selection, this time in presence of methotrexate, those colonies that coexpressed ternary alpha beta gamma GABAA receptor combinations were distinguished using [3H] flumazenil as a probe. This strategy was applied to the isolation of 3 GABAA receptor clones, alpha 1 beta 2 gamma 2s, alpha 3 beta 2 gamma 2s and alpha 5 beta 3 gamma 2s, that expressed relatively high levels of these proteins. These 3 cell lines exhibited pharmacological and functional properties similar to cells transiently-transfected with equivalent subunit combinations. These cell lines therefore provide attractive models with which to evaluate the intrinsic activity and potency of compounds at recombinant GABAA receptor subtypes.

The polyunsaturated aldehydes are highly reactive products of fatty acid peroxidation and combustion of organic materials, and they have been documented to have diverse cyctotoxic and genotoxic effects. The alpha,beta,gamma,delta-unsaturated aldehyde 2-trans-4-trans-decadienal is produced by marine microalgae, and it is known to inhibit cell proliferation and induce apoptosis in several different cell types. However, the molecular basis for the cell cycle arrest is not fully understood. We used sea urchin embryos to examine how some of the key events of the mitotic cell division were influenced by this polyunsaturated aldehyde. We found that cell divisions in embryos of Sphaerechinus granularis were inhibited by 2-trans-4-trans-decadienal in a dose dependent manner with an EC50 of 1.3 microM. Mitotic events in the nondividing eggs were characterized using immunofluorescent staining. DNA labelling revealed that pronuclear migration was inhibited, and a total absence of incorporation of the DNA-base analogue 5-bromo-2-deoxyuridine indicated that no DNA replication had occurred. Staining of alpha-tubulin subunits showed that tubulin-polymerization was disrupted and aberrations were induced in mitotic spindles. Furthermore, we monitored the activity of the G2-M promoting complex cyclin B-Cdk1 in newly fertilized sea urchin eggs, and found that this complex was not activated in embryos treated with 2-trans-4-trans-decadienal despite the accumulation of cyclin B.

Strategies for expanding hematopoietic stem cells (HSCs) could have significant utility for transplantation-based therapies. However, deleterious consequences of such manipulations remain unknown. Here we examined the impact of HSC self-renewal divisions in vitro and in vivo on their subsequent regenerative and continuing ability to sustain blood cell production in the absence of telomerase. HSC expansion in vitro was obtained using a NUP98-HOXA10hd transduction strategy and, in vivo, using a serial transplant protocol. We observed ϳ 10kb telomere loss in leukocytes produced in secondary mice transplanted with HSCs regenerated in primary recipients of NUP98-HOXA10hd-transduced and in vitro-expanded Tert ؊/؊ HSCs 6 months before. The second generation leukocytes also showed elevated expression of ␥H2AX (relative to control) indicative of greater accumulating DNA damage. In contrast, significant telomere shortening was not detected in leukocytes produced from freshly isolated, serially transplanted wild-type (WT) or Tert ؊/؊ HSCs, suggesting that HSC replication posttransplant is not limited by telomere shortening in the mouse. These findings document a role of telomerase in telomere homeostasis, and in preserving HSC functional integrity on prolonged self-renewal stimulation. (Blood. 2011;118(7):1766-1773) IntroductionThe existence of HSCs with a capacity for sustained self-renewal is essential for lifelong blood cell production. Expansion of the stem cell pool requires the stimulation of symmetric self-renewal divisions and is critical both during early development 1 and in later life, 2,3 as well as after transplantation or afterhematopoietic injury. When small numbers of HSCs are transplanted into myeloablated or pre-immune hosts, the increases in HSC numbers that follow may be even larger than those seen during development. [4][5][6] These findings document the high replicative potential of HSCs. 7 In mice, retroviral marking studies and, more recently, reconstitution studies starting from a single transplanted cell have shown that a single HSC can reestablish the hematopoiesis of a mouse, by continual creation of new HSCs capable of regenerating the system. 8,9 The self-renewal function of HSCs and their ability to reestablish hematopoiesis permanently in a myelosuppressed host is the basis of an increasing range of therapies for (BM) failure, malignant and genetic disorders. 10,11 Broader use (eg, from cord blood sources) and improved safety (eg, by accelerating recovery) of such transplant therapies would be greatly facilitated by the development of methods for achieving significant prior expansion of HSC numbers ex vivo. [12][13][14] However, it remains unclear, whether HSC self-renewal activity, provoked by either extrinsically or intrinsically induced mechanisms would at some point have deleterious consequences (eg, by inducing HSC senescence or an impairment of some critical aspect of HSC function). Studies reported to date indicate that despite detectable levels of telomerase expression, 15 the telome...