HIV-protease inhibitors induce expression of suppressor of cytokine signaling-1 in insulin-sensitive tissues and promote insulin resistance and type 2 diabetes mellitus

Insulin resistance, hyperglycemia, and type 2 diabetes are among the sequelae of metabolic syndromes that occur in 60-80% of human immunodeficiency virus (HIV)-positive patients treated with HIV-protease inhibitors (PIs). Studies to elucidate the molecular mechanism(s) contributing to these changes, however, have mainly focused on acute, in vitro actions of PIs. Here, we examined the chronic (7 wk) in vivo effects of the PI indinavir (IDV) in male Zucker diabetic fatty (fa/fa) (ZDF) rats. IDV exposure accelerated the diabetic state and dramatically exacerbated hyperglycemia and oral glucose intolerance in the ZDF rats, compared with vehicle-treated ZDF rats. Oligonucleotide gene array analyses revealed upregulation of suppressor of cytokine signaling-1 (SOCS-1) expression in insulin-sensitive tissues of IDV rats. SOCS-1 is a known inducer of insulin resistance and diabetes, and immunoblotting analyses revealed increases in SOCS-1 protein expression in adipose, skeletal muscle, and liver tissues of IDV-administered ZDF rats. This was associated with increases in the upstream regulator TNF-alpha and downstream effector sterol regulatory element-binding protein-1 and a decrease in IRS-2. IDV and other PIs currently in clinical use induced the SOCS-1 signaling cascade also in L6 myotubes and 3T3-L1 adipocytes exposed acutely to PIs under normal culturing conditions and in tissues from Zucker wild-type lean control rats administered PIs for 3 wk, suggesting an effect of these drugs even in the absence of background hyperglycemia/hyperlipidemia. Our findings therefore indicate that induction of the SOCS-1 signaling cascade by PIs could be an important contributing factor in the development of metabolic dysregulation associated with long-term exposures to HIV-PIs.     

Genetic Screens Identify Host Factors for SARS-CoV-2 and Common Cold Coronaviruses

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Transport of nucleosides across the Plasmodium falciparum parasite plasma membrane has characteristics of PfENT1

Like all parasitic protozoa, the human malaria parasite Plasmodium falciparum lacks the enzymes required for de novo synthesis of purines and it is therefore reliant upon the salvage of these compounds from the external environment. P. falciparum equilibrative nucleoside transporter 1 (PfENT1) is a nucleoside transporter that has been localized to the plasma membrane of the intraerythrocytic form of the parasite. In this study we have characterized the transport of purine and pyrimidine nucleosides across the plasma membrane of 'isolated' trophozoite-stage P. falciparum parasites and compared the transport characteristics of the parasite with those of PfENT1 expressed in Xenopus oocytes. The transport of nucleosides into the parasite: (i) was, in the case of adenosine, inosine and thymidine, very fast, equilibrating within a few seconds; (ii) was of low affinity [K(m) (adenosine) = 1.45 +/- 0.25 mM; K(m) (thymidine) = 1.11 +/- 0.09 mM]; and (iii) showed 'cross-competition' for adenosine, inosine and thymidine, but not cytidine. The kinetic characteristics of nucleoside transport in intact parasites matched very closely those of PfENT1 expressed in Xenopus oocytes [K(m) (adenosine) = 1.86 +/- 0.28 mM; K(m) (thymidine) = 1.33 +/- 0.17 mM]. Furthermore, PfENT1 transported adenosine, inosine and thymidine, with a cross-competition profile the same as that seen for isolated parasites. The data are consistent with PfENT1 serving as a major route for the uptake of nucleosides across the parasite plasma membrane.     

Fatty acid composition of Simonsiella strains

Gas-liquid chromatography of methyl esters of bound fatty acids extracted from the cells of 48 Simonsiella strains showed that these aerobic, gliding, multicellular-filamentous bacteria have fatty acid profiles of the pattern considered typical of Gramnegative eubacteria. All strains contained predominantly tetradecanoic acid (29.5%), 9-hexadecenoic acid (22.2%), an unidentified acid with an equivalent chain length of approximately 20 carbon atoms (15.8%), and dodecanoic acid (11.4%).Discriminant analysis of the mean relative percentages of 12 fatty acids correctly assigned 94% of the strains to groups based on their source of origin (i.e., the oral cavities of sheep, cat, human or dog); the relative amounts of only 3 of the fatty acids (9-octadecenoic acid, hexadecanoic acid, and tetradecanoic acid) provided most of this discrimination.     

Heterologous expression of wild type and deglycosylated human sex steroid-binding protein (SBP or SHBG) in the yeast, Pichia pastoris. Characterization of the recombinant proteins.

Wild type, partially and fully-deglycosylated human sex steroid-binding protein (SBP or SHBG) cDNAs lacking the native cucaryotic signal sequence were cloned into a yeast expression vector containing the Saccharomyces cerevisiae alpha-factor for extracellular secretion. Following transformation into Pichia pastoris, the wild type and all constructed mutants were successfully expressed. The levels were lower for the deglycosylated mutants indicating that oligosaccharide side chains may play a role in SBP secretion. Under fermentation conditions, the wild type protein was expressed at a level of 4 mg/l while the fully-deglycosylated mutant T7A/N351Q/N367Q was expressed at about 1.5 mg/l. The latter was purified from several fermentation runs and was found to be completely deglycosylated, electrophoretically homogeneous and fully active. The aminoterminus was found to have the sequence NH2QSAHDPPAV- indicating that cleavage of the alpha-factor occurred at the A(+7)-Q(+8) peptide bond. The molecular mass of the subunit was determined to be 39,717.8 Da, which is in complete agreement with the amino acid sequence of the T7A/N351Q/N367/Q mutant. The equilibrium constants for the dissociation of 5alpha-dihydrotestosterone and steroid binding specificity were found to be identical to that of the human plasma protein indicating that the missing N-terminal segment NH2-LRPVLPT and the removal of oligosaccharide side chains do not affect the stability and active conformation of the protein. In conclusion, the data presented reveal that the SBP mutant T7A/N351Q/N367/Q is the protein of choice for solving the three-dimensional structure.     

Plastid Ontogeny during Petal Development in Arabidopsis

Imaging of chlorophyll autofluorescence by confocal microscopy in intact whole petals of Arabidopsis thaliana has been used to analyze chloroplast development and redifferentiation during petal development. Young petals dissected from unopened buds contained green chloroplasts throughout their structure, but as the upper part of the petal lamina developed and expanded, plastids lost their chlorophyll and redifferentiated into leukoplasts, resulting in a white petal blade. Normal green chloroplasts remained in the stalk of the mature petal. In epidermal cells the chloroplasts were normal and green, in stark contrast with leaf epidermal cell plastids. In addition, the majority of these chloroplasts had dumbbell shapes, typical of dividing chloroplasts, and we suggest that the rapid expansion of petal epidermal cells may be a trigger for the initiation of chloroplast division. In petals of the Arabidopsis plastid division mutant arc6, the conversion of chloroplasts into leukoplasts was unaffected in spite of the greatly enlarged size and reduced number of arc6 chloroplasts in cells in the petal base, resulting in few enlarged leukoplasts in cells from the white lamina of arc6 petals.     

A RNA interference screen identifies the protein phosphatase 2A subunit PR55gamma as a stress-sensitive inhibitor of c-SRC

Protein Phosphatase type 2A (PP2A) represents a family of holoenzyme complexes with diverse biological activities. Specific holoenzyme complexes are thought to be deregulated during oncogenic transformation and oncogene-induced signaling. Since most studies on the role of this phosphatase family have relied on the use of generic PP2A inhibitors, the contribution of individual PP2A holoenzyme complexes in PP2A-controlled signaling pathways is largely unclear. To gain insight into this, we have constructed a set of shRNA vectors targeting the individual PP2A regulatory subunits for suppression by RNA interference. Here, we identify PR55gamma and PR55delta as inhibitors of c-Jun NH(2)-terminal kinase (JNK) activation by UV irradiation. We show that PR55gamma binds c-SRC and modulates the phosphorylation of serine 12 of c-SRC, a residue we demonstrate to be required for JNK activation by c-SRC. We also find that the physical interaction between PR55gamma and c-SRC is sensitive to UV irradiation. Our data reveal a novel mechanism of c-SRC regulation whereby in response to stress c-SRC activity is regulated, at least in part, through loss of the interaction with its inhibitor, PR55gamma.     

Identification, cloning, and functional expression of three glutathione transferase genes from Aspergillus fumigatus

Analysis of the genome of the human pathogen, Aspergillus fumigatus, revealed the presence of several putative glutathione transferase (GST) open reading frames. Three A. fumigatus GST genes, termed gstA, B, and C, were cloned and recombinant proteins expressed in Escherichia coli. Functional analysis of recombinant gstA-C confirms that the enzymes exhibit GST activity and glutathione peroxidase activity. RT-PCR confirmed low basal expression of gstA and gstC which was markedly up-regulated (at least 4x-10x) in the presence of either H2O2 or 1-chloro-2,4-dinitrobenzene (CDNB). GstB expression was only observed in the presence of CDNB. These results demonstrate for the first time the existence of three functional GSTs in A. fumigatus and strongly suggest a role for these enzymes in the response of the organism to both oxidative stress and xenobiotic presence.     

Characterization of Herpes Simplex Virus Type 1 RNA Present in the Absence of De Novo Protein Synthesis

We used herpes simplex virus type 1 (HSV-1) DNA and restriction fragments of HSV-1 DNA covalently coupled to cellulose as a reagent to isolate for further characterization the major and minor HSV-1 immediate-early mRNA species in HeLa cells infected and maintained in the absence of de novo protein synthesis. Five major and several minor immediate-early mRNA species were characterized. One major species was a 4.2-kilobase mRNA mapping in the TR(S)/IR(S) region with its 3' end distal to the U(S) region; this mRNA encoded a 170,000-dalton polypeptide in vitro. A 2.8-kilobase mRNA, encoding a 120,000-dalton polypeptide, was mapped in the TR(L)/IR(L) region with its 3' end directed toward the U(L) region. Three 1.8-kilobase mRNA species were mapped. One, mapping in the IR(S) region with its 3' end in the U(S), encoded a 68,000-dalton polypeptide. One mapped in the TR(S) region and had its 3' end in the U(S) region; the third one encoded a 64,000-dalton polypeptide and mapped in the U(L) region near the IR(L) region. One minor species 5.2 kilobases in size was clearly detectable mapping in the U(L) region. Furthermore, there were indications that one or more immediate-early mRNA species approximately 3 kilobases in size hybridized to regions near the TR(L) and in or near the TR(S)/IR(S) regions. Nuclear immediate-early RNA mapped only in those regions where polyribosomal immediate-early mRNA mapped, although minor differences were seen. Finally, we demonstrated that at least three major immediate-early mRNA's-4.2 kilobases, 2.8 kilobases, and the 1.8-kilobase one mapping in the IR(S)/U(S) region-continued to appear on polyribosomes as functional mRNA late after infection.