Expression, purification, and physicochemical characterization of the N-terminal active site of human angiotensin-I converting enzyme.

We have cloned, over expressed, and purified one of the two catalytic domains (residues Ala361 to Gly468, ACE-N) of human somatic angiotensin-I converting enzyme in Escherichia coli. This construct represents the N-catalytic domain including the two binding motifs and the 23 amino acid spacers as well as some amino acid residues before and after the motifs that might help in correct conformation. The overexpressed protein was exclusively localized to insoluble inclusion bodies. Inclusion bodies were solubilized in an 8-M urea buffer. Purification was carried out by differential centrifugation and gel filtration chromatography under denaturing conditions. About 12 mg of ACE-N peptide per liter of bacterial culture was obtained. The integrity of recombinant protein domain was confirmed by ESI/MS. Structural analysis using CD spectroscopy has shown that, in the presence of TFE, the ACE-N protein fragment has taken a conformation, which is consistent with the one found in testis ACE by X-ray crystallography. This purification procedure enables the production of an isotopically labeled protein fragment for structural studying in solution by NMR spectroscopy.     

Effect of thymosin peptides on the chick chorioallantoic membrane angiogenesis model.

The effect of alpha- and beta-thymosin peptides, namely prothymosin alpha (ProT(alpha)), thymosin alpha(1) (T(alpha)1), parathymosin alpha (ParaT(alpha)), thymosin beta(4) (Tbeta4), thymosin beta(10) (Tbeta10), and thymosin beta(9) (Tbeta9), on the angiogenesis process was investigated using the chick chorioallantoic membrane as an in vivo angiogenesis model. The thymosin peptides tested were applied in 10 microl aliquots containing 0.01-4 nmoles of Tbeta4, Tbeta10 or Tbeta9, 0.016-6.66 nmoles of T(alpha)1, 4.1 pmoles-1.66 nmoles of ProT(alpha), and 4.4 pmoles-1.76 nmoles of ParaT(alpha). Phorbol 12-myristate 13-acetate and hydrocortisone were also used as positive and negative control, respectively. Tbeta4, ProT(alpha) and T(alpha)1 were found to enhance angiogenesis, while Tbeta10, Tbeta9 and ParaT(alpha) exhibited an inhibitory effect on the angiogenesis process. When mixtures of Tbeta4 and Tbeta10 containing active amounts of the two peptides at different proportions were applied, the promoting effect of Tbeta4 on angiogenesis was reversed in the presence of increasing concentrations of Tbeta10 and vice versa. The effect of Tbeta10, Tbeta9, ProT(alpha) and ParaT(alpha), in parallel with Tbeta4 and T(alpha)1, on the angiogenesis process was investigated for the first time as far as we know and the results of this study offer more insight into the biological regulatory roles of thymosin peptides, and provide helpful information about their therapeutic potential. Whether these agents could be used either as inhibitors of angiogenesis in disease states where uncontrolled angiogenesis is involved, e.g. in carcinogenesis, or as angiogenesis promoters that could be useful in wound healing, fracture repair, peptic ulcers etc., remains to be further studied.     

Folding in solution of the C‐catalytic protein fragment of angiotensin‐converting enzyme

Angiotensin‐converting enzyme (ACE) is a key molecule of the renin–angiotensin–aldosterone system which is responsible for the control of blood pressure. For over 30 years it has become the target for fighting off hypertension. Many inhibitors of the enzyme have been synthesized and used widely in medicine despite the lack of ACE structure. The last 5 years the crystal structure of ACE separate domains has been revealed, but in order to understand how the enzyme works it is necessary to study its structure in solution. We present here the cloning, overexpression in Escherichia coli, purification and structural study of the Ala₉₅₉ to Ser₁₀₆₆ region (ACE_C) that corresponds to the C‐catalytic domain of human somatic angiotensin‐I‐converting enzyme. ACE_C was purified under denatured conditions and the yield was 6 mg/l of culture. Circular dichroism (CD) spectroscopy indicated that 1,1,1‐trifluoroethanol (TFE) is necessary for the correct folding of the protein fragment. The described procedure can be used for the production of an isotopically labelled ACE_959–1066 protein fragment in order to study its structure in solution by NMR spectroscopy. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Quantitation of human immunodeficiency virus type 1 DNA forms with the second template switch in peripheral blood cells predicts disease progression independently of plasma RNA load

There are several forms of human immunodeficiency virus type 1 (HIV-1) DNA in peripheral blood T cells and lymph nodes in untreated HIV-1-infected individuals and in patients whose plasma HIV-1 RNA levels are suppressed by long-term combination antiretroviral therapy. However, it remains to be established whether the concentration of HIV-1 DNA in cells predicts the clinical outcome of HIV-1 infection. In this report, we measured the concentration of HIV-1 DNA forms which has undergone the second template switch (STS DNA) and 2-long-terminal-repeat DNA circles in peripheral blood mononuclear cell (PBMC) samples. To do this, we used molecular-beacon-based real-time PCR assays and studied 130 patients with hemophilia in the Multicenter Hemophilia Cohort Study. We assessed the influence of baseline HIV-1 STS DNA levels on the progression of HIV-1 disease in the absence of combination antiretroviral therapy by Kaplan-Meier and Cox regression analysis. Among the patients who progressed to AIDS, the median levels (interquartile ranges) of STS HIV-1 DNA in PBMC were significantly higher than those of patients who remained AIDS free during the 16 years of follow-up (1,017 [235 to 6,059] and 286 [31 to 732] copies per 10(6) PBMC, respectively; P < 0.0001). Rates of progression to death and development of AIDS varied significantly (log rank P < 0.001) by quartile distribution of HIV-1 STS DNA levels. After adjustment for age at seroconversion, baseline CD4(+) T-cell counts, plasma viral load, and T-cell-receptor excision circles, the relative hazards (RH) of death and AIDS were significantly increased with higher HIV-1 STS DNA levels (adjusted RH, 1.84 [95% confidence interval (CI), 1.30 to 2.59] and 2.62 [95% CI, 1.75 to 3.93] per 10-fold increase per 10(6) PBMC, respectively). HIV-1 STS DNA levels in each individual remained steady in longitudinal PBMC samples during 16 years of follow-up. Our findings show that the concentration of HIV-1 STS DNA in PBMC complements the HIV-1 RNA load in plasma in predicting the clinical outcome of HIV-1 disease. This parameter may have important implications for understanding the virological response to combination antiretroviral therapy.     

Use of Coreceptors Other Than CCR5 by Non-Syncytium-Inducing Adult and Pediatric Isolates of Human Immunodeficiency Virus Type 1 Is Rare In Vitro

We have tested a panel of pediatric and adult human immunodeficiency virus type 1 (HIV-1) primary isolates for the ability to employ the following proteins as coreceptors during viral entry: CCR1, CCR2b, CCR3, CCR4, CCR5, CCR8, CXCR4, Bonzo, BOB, GPR1, V28, US28, and APJ. Most non-syncytium-inducing isolates could utilize only CCR5. All syncytium-inducing viruses used CXCR4, some also employed V28, and one (DH123) used CCR8 and APJ as well. A longitudinal series of HIV-1 subtype B isolates from an infected infant and its mother utilized Bonzo efficiently, as well as CCR5. The maternal isolates, which were syncytium inducing, also used CXCR4, CCR8, V28, and APJ.     

Analysis of binding parameters of HIV-1 integrase inhibitors: Correlates of drug inhibition and resistance

This study undertook an exploratory data analysis of the binding parameters of HIV-1 integrase inhibitors. The study group involved inhibitors in preclinical development from the diketo acid, pyrroloquinoline and naphthyridine carboxamide families and the most advanced inhibitors Raltegravir and Elvitegravir. Distinct differences were observed in the energetics of binding between the studied classes of inhibitors that also correlated with drug resistant patterns. Quantitative-property–activity-relationships correlated experimental IC₅₀ values to the binding energy and the logarithm of the partition coefficient between n-octanol and water (clog P). The approach followed here serves as an improved basis for the development of ‘second generation’ integrase inhibitors.     

Detection of interactions of the β‐amyloid peptide with small molecules employing transferred NOEs

The interaction of pineal hormone melatonin, the histological dye thioflavin T, and the olive tree polyphenol oleuropein, with the 28 amino acid residue N‐terminal fragment of the β‐amyloid peptide (β‐AP) of Alzheimer's disease, [β‐AP(1‐28)], was detected in solution through the observation of transferred NOEs (trNOEs) in 1D and 2D NOE spectroscopy (NOESY) experiments. The trNOE method is applied for the first time in the detection of interactions of soluble β‐AP(1‐28) with small molecules and may provide a means of screening for the identification of possible inhibitors of the formation of neurotoxic β‐AP assemblies. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Cellular HIV-1 DNA Levels in Drug Sensitive Strains Are Equivalent to Those in Drug Resistant Strains in Newly-Diagnosed Patients in Europe

Background

HIV-1 genotypic drug resistance is an important threat to the success of antiretroviral therapy and transmitted resistance has reached 9% prevalence in Europe. Studies have demonstrated that HIV-1 DNA load in peripheral blood mononuclear cells (PBMC) have a predictive value for disease progression, independently of CD4 counts and plasma viral load.

Methodology/Principal Findings

Molecular-beacon-based real-time PCR was used to measure HIV-1 second template switch (STS) DNA in PBMC in newly-diagnosed HIV-1 patients across Europe. These patients were representative for the HIV-1 epidemic in the participating countries and were carrying either drug-resistant or sensitive viral strains. The assay design was improved from a previous version to specifically detect M-group HIV-1 and human CCR5 alleles. The findings resulted in a median of 3.32 log₁₀ HIV-1 copies/10⁶ PBMC and demonstrated for the first time no correlation between cellular HIV-1 DNA load and transmitted drug-resistance. A weak association between cellular HIV-1 DNA levels with plasma viral RNA load and CD4⁺ T-cell counts was also reconfirmed. Co-receptor tropism for 91% of samples, whether or not they conferred resistance, was CCR5. A comparison of pol sequences derived from RNA and DNA, resulted in a high similarity between the two.

Conclusions/Significance

An improved molecular-beacon-based real-time PCR assay is reported for the measurement of HIV-1 DNA in PBMC and has investigated the association between cellular HIV-1 DNA levels and transmitted resistance to antiretroviral therapy in newly-diagnosed patients from across Europe. The findings show no correlation between these two parameters, suggesting that transmitted resistance does not impact disease progression in HIV-1 infected individuals. The CCR5 co-receptor tropism predominance implies that both resistant and non-resistant strains behave similarly in early infection. Furthermore, a correlation found between RNA- and DNA-derived sequences in the pol region suggests that genotypic drug-resistance testing could be carried out on either template.     

Treatment-associated polymorphisms in protease are significantly associated with higher viral load and lower CD4 count in newly diagnosed drug-naive HIV-1 infected patients

Background

Bone marrow stromal cell antigen 2 (BST-2) is a cellular factor that restricts the egress of viruses such as human immunodeficiency virus (HIV-1) from the surface of infected cells, preventing infection of new cells. BST-2 is variably expressed in most cell types, and its expression is enhanced by cytokines such as type I interferon alpha (IFN-??). In this present study, we used the beta-retrovirus, mouse mammary tumor virus (MMTV) as a model to examine the role of mouse BST-2 in host infection in vivo.

Results

By using RNA interference, we show that loss of BST-2 enhances MMTV replication in cultured mammary tumor cells and in vivo. In cultured cells, BST-2 inhibits virus accumulation in the culture medium, and co-localizes at the cell surface with virus structural proteins. Furthermore, both scanning electron micrograph (SEM) and transmission electron micrograph (TEM) show that MMTV accumulates on the surface of IFN??-stimulated cells.

Conclusions

Our data provide evidence that BST-2 restricts MMTV release from naturally infected cells and that BST-2 is an antiviral factor in vivo.