Fatty Acid and Retinol-Binding Protein: Unusual Protein Conformational and Cavity Changes Dictated by Ligand Fluctuations.

Lipid-binding proteins (LBPs) are soluble proteins responsible for the uptake, transport, and storage of a large variety of hydrophobic lipophilic molecules including fatty acids, steroids, and other lipids in the cellular environment. Among the LBPs, fatty acid binding proteins (FABPs) present preferential binding affinities for long-chain fatty acids. While most of FABPs in vertebrates and invertebrates present similar ß-barrel structures with ligands accommodated in their central cavity, parasitic nematode worms exhibit additional unusual α-helix rich fatty acid- and retinol-binding proteins (FAR). Herein, we report the comparison of extended molecular dynamics (MD) simulations performed on the ligand-free and palmitic acid-bond states of the Necator americanus FAR-1 (Na-FAR-1) with respect to other classical ß-barrel FABPs. Principal component analysis (PCA) has been used to identify the different conformations adopted by each system during MD simulations. The α-helix fold encompasses a complex internal ligand-binding cavity with a remarkable conformational plasticity that allows reversible switching between distinct states in the holo-Na-FAR-1. The cavity can change up to one-third of its size affected by conformational changes of the protein-ligand complex. Besides, the ligand inside the cavity is not fixed but experiences large conformational changes between bent and stretched conformations. These changes in the ligand conformation follow changes in the cavity size dictated by the transient protein conformation. On the contrary, protein-ligand complex in ß-barrel FABPs fluctuates around a unique conformation. The significantly more flexible holo-Na-FAR-1 ligand-cavity explains its larger ligand multiplicity respect to ß-barrel FABPs.

Manual therapy for the management of pain and limited range of motion in subjects with signs and symptoms of temporomandibular disorder: a systematic review of randomised controlled trials.

There is a lack of knowledge about the effectiveness of manual therapy (MT) on subjects with temporomandibular disorders (TMD). The aim of this systematic review is to synthetise evidence regarding the isolated effect of MT in improving maximum mouth opening (MMO) and pain in subjects with signs and symptoms of TMD. MEDLINE(®) , Cochrane, Web of Science, SciELO and EMBASE(™) electronic databases were consulted, searching for randomised controlled trials applying MT for TMD compared to other intervention, no intervention or placebo. Two authors independently extracted data, PEDro scale was used to assess risk of bias, and GRADE (Grading of Recommendations Assessment, Development and Evaluation) was applied to synthetise overall quality of the body of evidence. Treatment effect size was calculated for pain, MMO and pressure pain threshold (PPT). Eight trials were included, seven of high methodological quality. Myofascial release and massage techniques applied on the masticatory muscles are more effective than control (low to moderate evidence) but as effective as toxin botulinum injections (moderate evidence). Upper cervical spine thrust manipulation or mobilisation techniques are more effective than control (low to high evidence), while thoracic manipulations are not. There is moderate-to-high evidence that MT techniques protocols are effective. The methodological heterogeneity across trials protocols frequently contributed to decrease quality of evidence. In conclusion, there is widely varying evidence that MT improves pain, MMO and PPT in subjects with TMD signs and symptoms, depending on the technique. Further studies should consider using standardised evaluations and better study designs to strengthen clinical relevance.

Viremia control following antiretroviral treatment and therapeutic immunization during primary SIV251 infection of macaques.

Prolonged antiretroviral therapy (ART) is not likely to eradicate human immunodeficiency virus type I (HIV-I) infection. Here we explore the effect of therapeutic immunization in the context of ART during primary infection using the simian immunodeficiency virus (SIV251) macaque model. Vaccination of rhesus macaques with the highly attenuated poxvirus-based NYVAC-SIV vaccine expressing structural genes elicited vigorous virus-specific CD4 + and CD8+ T cell responses in macaques that responded effectively to ART. Following discontinuation of a six-month ART regimen, viral rebound occurred in most animals, but was transient in six of eight vaccinated animals. Viral rebound was also transient in four of seven mock-vaccinated control animals. These data establish the importance of antiretroviral treatment during primary infection and demonstrate that virus-specific immune responses in the infected host can be expanded by therapeutic immunization.

HIV-1 recombinant poxvirus vaccine induces cross-protection against HIV-2 challenge in rhesus macaques.

Rhesus macaques were immunized with attenuated vaccinia or canarypox human immunodeficiency virus type 1 (HIV-1) recombinants and boosted with HIV-1 protein subunits formulated in alum. Following challenge with HIV-2SBL6669, three out of eight immunized macaques resisted infection for six months and another exhibited significantly delayed infection, whereas all three naive controls became infected. Immunizations elicited both humoral and cellular immune responses; however, no clear correlates of protection were discerned. Although more extensive studies are now called for, this first demonstration of cross-protection between HIV-1 and -2 suggests that viral variability may not be an insurmountable problem in the design of a global AIDS vaccine.

Mucosal AIDS vaccine reduces disease and viral load in gut reservoir and blood after mucosal infection of macaques.

Given the mucosal transmission of HIV-1, we compared whether a mucosal vaccine could induce mucosal cytotoxic T lymphocytes (CTLs) and protect rhesus macaques against mucosal infection with simian/human immunodeficiency virus (SHIV) more effectively than the same vaccine given subcutaneously. Here we show that mucosal CTLs specific for simian immunodeficiency virus can be induced by intrarectal immunization of macaques with a synthetic-peptide vaccine incorporating the LT(R192G) adjuvant. This response correlated with the level of T-helper response. After intrarectal challenge with pathogenic SHIV-Ku2, viral titers were eliminated more completely (to undetectable levels) both in blood and intestine, a major reservoir for virus replication, in intrarectally immunized animals than in subcutaneously immunized or control macaques. Moreover, CD4+ T cells were better preserved. Thus, induction of CTLs in the intestinal mucosa, a key site of virus replication, with a mucosal AIDS vaccine ameliorates infection by SHIV in non-human primates.

Monoclonal antibody against human T cell leukemia virus p19 defines a human thymic epithelial antigen acquired during ontogeny.

Using monoclonal antibody 12/1-2 against a 19,000-dalton human T cell leukemia virus (HTLV) protein (anti-p19), previously demonstrated to be reactive with HTLV-infected human cells, but not in numerous other uninfected cells, we found a reactive antigen to be expressed on the neuroendocrine component of human thymic epithelial cells but not on any other normal epithelial or neuroendocrine human tissues. Moreover, this reactive antigen is acquired on neuroendocrine thymic epithelium during thymic ontogeny--first appearing on fetal thymic epithelial cells between 8 and 15 wk gestation. While only a portion of thymic epithelial cells in the subcapsular cortical region of 15- and 24-wk fetal thymuses contained anti-p19+ epithelial cells, the entire subcapsular cortical region of newborn thymus epithelium was anti-p19+. By age 3 yr, normal subjects' entire subcapsular cortical and medullary thymic epithelium was anti-p19+. Using antibody against HTLV core protein, p24, and c-DNA probes for HTLV DNA, neither HTLV-specific p24 protein nor proviral DNA could be demonstrated in anti-p19+ thymic epithelial tissue. However, thymic epithelial extracts, disrupted HTLV extracts, as well as purified HTLV p19 antigen all inhibited the binding of anti-p19 antibody to thymic epithelium. Thus, anti-p19 may recognize a determinant on an HTLV-encoded 19,000-dalton structural protein that is shared by human thymic epithelium. Alternatively, anti-p19 defines a host encoded protein that is selectively expressed by normal thymic epithelium, and is induced to be expressed in HTLV-infected malignant T cells.

A FABP4-PPARγ signaling axis regulates human monocyte responses to electrophilic fatty acid nitroalkenes.

Nitro-fatty acids (NO2-FA) are electrophilic lipid mediators derived from unsaturated fatty acid nitration. These species are produced endogenously by metabolic and inflammatory reactions and mediate anti-oxidative and anti-inflammatory responses. NO2-FA have been postulated as partial agonists of the Peroxisome Proliferator-Activated Receptor gamma (PPARγ), which is predominantly expressed in adipocytes and myeloid cells. Herein, we explored molecular and cellular events associated with PPARγ activation by NO2-FA in monocytes and macrophages. NO2-FA induced the expression of two PPARγ reporter genes, Fatty Acid Binding Protein 4 (FABP4) and the scavenger receptor CD36, at early stages of monocyte differentiation into macrophages. These responses were inhibited by the specific PPARγ inhibitor GW9662. Attenuated NO2-FA effects on PPARγ signaling were observed once cells were differentiated into macrophages, with a significant but lower FABP4 upregulation, and no induction of CD36. Using in vitro and in silico approaches, we demonstrated that NO2-FA bind to FABP4. Furthermore, the inhibition of monocyte FA binding by FABP4 diminished NO2-FA-induced upregulation of reporter genes that are transcriptionally regulated by PPARγ, Keap1/Nrf2 and HSF1, indicating that FABP4 inhibition mitigates NO2-FA signaling actions. Overall, our results affirm that NO2-FA activate PPARγ in monocytes and upregulate FABP4 expression, thus promoting a positive amplification loop for the downstream signaling actions of this mediator.

The integrity of the alpha-helical domain of intestinal fatty acid binding protein is essential for the collision-mediated transfer of fatty acids to phospholipid membranes.

Intestinal FABP (IFABP) and liver FABP (LFABP), homologous proteins expressed at high levels in intestinal absorptive cells, employ markedly different mechanisms of fatty acid transfer to acceptor model membranes. Transfer from IFABP occurs during protein-membrane collisional interactions, while for LFABP transfer occurs by diffusion through the aqueous phase. In addition, transfer from IFABP is markedly faster than from LFABP. The overall goal of this study was to further explore the structural differences between IFABP and LFABP which underlie their large functional differences in ligand transport. In particular, we addressed the role of the alphaI-helix domain in the unique transport properties of intestinal FABP. A chimeric protein was engineered with the 'body' (ligand binding domain) of IFABP and the alphaI-helix of LFABP (alpha(I)LbetaIFABP), and the fatty acid transfer properties of the chimeric FABP were examined using a fluorescence resonance energy transfer assay. The results showed a significant decrease in the absolute rate of FA transfer from alpha(I)LbetaIFABP compared to IFABP. The results indicate that the alphaI-helix is crucial for IFABP collisional FA transfer, and further indicate the participation of the alphaII-helix in the formation of a protein-membrane "collisional complex". Photo-crosslinking experiments with a photoactivable reagent demonstrated the direct interaction of IFABP with membranes and further support the importance of the alphaI helix of IFABP in its physical interaction with membranes.

Three distinct genes in human DNA related to the transforming genes of mammalian sarcoma retroviruses.

Southern blot hybridization was used to identify human and other vertebrate DNA sequences that were homologous to cloned DNA fragments containing the oncogenic nucleic acid sequences of three different type C mammalian retroviruses (simian sarcoma virus, the Snyder-Theilen strain of feline sarcoma virus, and the Harvey strain of murine sarcoma virus). Each onc gene counterpart has a single genetic locus, which probably contains non-onc intervening sequences. The human DNA sequences may represent genes important to cell growth or cell differentiation, or both. Their identification and isolation may allow elucidation of their role in these processes and in neoplasias.

Identification of HTLV-III/LAV sor gene product and detection of antibodies in human sera.

The nucleotide sequence of the genome of HTLV-III, the infectious agent etiologically associated with the acquired immune deficiency syndrome, predicts a small open reading frame, termed sor, located between the pol and env genes. A DNA segment containing 82 percent of the sor region was inserted into a prokaryotic expression vector, pJL6, to determine whether sor encodes a viral protein and to gain some insight into its possible function. The bacterially synthesized sor protein reacted with sera from individuals infected with HTLV-III, indicating that sor is expressed as a protein product or products that are immunogenic in vivo. Antibodies to the purified, bacterially synthesized sor protein were found to react specifically with the same protein and also with a protein of molecular weight 23,000 (23K) in HTLV-III-infected H9 cell extracts. The 23K protein comigrated with a protein immunoprecipitated by the serum of a hemophiliac patient with antibodies to HTLV-III, suggesting that this protein is probably the sor gene product.

Constitutively activated Jak-STAT pathway in T cells transformed with HTLV-I.

Human T cell lymphotropic virus I (HTLV-I) is the etiological agent for adult T cell leukemia and tropical spastic paraparesis (also termed HTLV-I-associated myelopathy). HTLV-I-infected peripheral blood T cells exhibit an initial phase of interleukin-2 (IL-2)-dependent growth; over time, by an unknown mechanism, the cells become IL-2-independent. Whereas the Jak kinases Jak1 and Jak3 and the signal transducer and activator of transcription proteins Stat3 and Stat5 are activated in normal T cells in response to IL-2, this signaling pathway was constitutively activated in HTLV-I-transformed cells. In HTLV-I-infected cord blood lymphocytes, the transition from IL-2-dependent to IL-2-independent growth correlated with the acquisition of a constitutively activated Jak-STAT pathway, which suggests that this pathway participates in HTLV-I-mediated T cell transformation.

Identification of the fusion peptide of primate immunodeficiency viruses.

Membrane fusion induced by the envelope glycoproteins of human and simian immunodeficiency viruses (HIV and SIVmac) is a necessary step for the infection of CD4 cells and for the formation of syncytia after infection. Identification of the region in these molecules that mediates the fusion events is important for understanding and possibly interfering with HIV/SIVmac infection and pathogenesis. Amino acid substitutions were made in the 15 NH2-terminal residues of the SIVmac gp32 transmembrane glycoprotein, and the mutants were expressed in recombinant vaccinia viruses, which were then used to infect CD4-expressing T cell lines. Mutations that increased the overall hydrophobicity of the gp32 NH2-terminus increased the ability of the viral envelope to induce syncytia formation, whereas introduction of polar or charged amino acids in the same region abolished the fusogenic function of the viral envelope. Hydrophobicity in the NH2-terminal region of gp32 may therefore be an important correlate of viral virulence in vivo and could perhaps be exploited to generate a more effective animal model for the study of acquired immunodeficiency syndrome.

Expanded HIV-1 cellular tropism by phenotypic mixing with murine endogenous retroviruses.

In view of the current interest in in vivo murine models for acquired immunodeficiency syndrome (AIDS), the interaction between human immunodeficiency virus type 1 (HIV-1) and endogenous murine leukemia virus (MuLV)-related retroviruses was investigated with a human leukemic T cell line (PF-382x) that acquired xenotropic MuLV (X-MuLV) after in vivo passage in immunosuppressed mice. Despite similar levels of membrane CD4 expression and HIV-1 125I-labeled gp 120 binding, a dramatic acceleration in the time course of HIV-1 infection was observed in PF-382x compared to its X-MuLV-negative counterpart (PF-382). Moreover, PF-382 cells coinfected by X-MuLV and HIV-1 generated a progeny of phenotypically mixed viral particles, enabling HIV-1 to productively infect a panel of CD4- human cells, including B lymphoid cells and purified normal peripheral blood CD4-/CD8+ T lymphocytes. Mixed viral phenotypes were also produced by human CD4+ T cells coinfected with an amphotropic MuLV-related retrovirus (A-MuLV) and HIV-1. These data show that endogenous MuLV acquired by human cells transplanted into mice can significantly interact with HIV-1, thereby inducing important alterations of HIV-1 biological properties.

Dioctophyme renale in a domestic cat (Felis catus): Renal location and nephrectomy.

Dioctophymosis is caused by Dioctophyme renale, nematode with indirect life cycle. Its intermediate host is a freshwater oligochaete and its definitive host is a wild or household carnivore. The adult nematode develops in the definite host, generally locating itself in the kidney. This article was meant to describe the first nephrectomy performed in a domestic cat due to renal dioctophymosis in Argentina. The subject showed a non-specific appearance of generally feeling ill, hematuria and mild diarrhea. It was diagnosed through abdominal ultrasound, followed by exploratory celiotomy and nephrectomy. After verifying absence of free specimens, the right kidney was removed. This organ was found to be enlarged in a spheroidal manner in contrast to the left kidney, with significant thickening of the renal capsule, excessive congestion of vessels and adhesions involving the caudal vena cava. An adult nematode was removed from the right kidney and identified as Dioctophyme renale. Reports of feline dioctophymosis are scarce being most of them necropsy findings. In this we are presenting a confirmed case of D. renale removed by surgery from a live cat. The results presented here reinforces the fact that cats are also appropriate definitive hosts for this parasite.

The natural history and evolution of human and simian T cell leukemia/lymphotropic viruses.

The past five years have seen significant advances in understanding the origin and evolution of human T-cell leukemia/lymphotropic virus types I and II. The highlights include the identification of human T-cell leukemia/lymphotropic virus types I and II genotypic variants in remote human populations and the discovery of widely divergent simian T-cell leukemia virus in African and Asian non-human primates.

Identification of the human T cell lymphoma virus in B cell lines established from patients with adult T cell leukemia.

Cell lines were established from the peripheral blood of two patients with adult T cell leukemia. In contrast to our previous experience, where all such lines expressed T cell markers, these two cell lines expressed B cell antigens and Ig light chains (kappa on CF-2, lambda on HS). Human T cell lymphoma proviral (HTLV) sequences were demonstrated in both cell lines. Since only a portion of the cells in culture expressed Ig light chains, experiments were carried out to exclude the possibility that the cultures were not a mixture of B and T or non-B cells. Cells that expressed kappa- or lambda-light chains were separated by cell sorting from kappa- or lambda-negative cells and replaced in culture. Light chain negative cells reexpressed light chains after time in culture. After 5-azacytidine treatment of the cell lines, all cells expressed Ig light chains. These studies show that the human retrovirus HTLV, which has been demonstrated to be associated with certain T cell malignancies, can infect B cells or B cell precursors.

Chromosomal mapping of murine c-fes and c-src genes.

The murine homologs of two viral oncogenes associated with tyrosine-specific kinase activity have been assigned to different loci in the mouse genome. The segregation of restriction site polymorphisms, as detected by probes that are specific for endogenous c-fes and c-src sequences, was followed in the DNA of recombinant inbred strains. The c-fes gene was mapped to the proximal portion of chromosome 7, very close to the Gpi-1 locus, whereas c-src was linked to the Psp locus on the distal half of chromosome 2.

Human gene (c-fes) related to the onc sequences of Snyder-Theilen feline sarcoma virus.

The onc gene (v-fes) of the acutely transforming feline sarcoma virus (Snyder-Theilen strain) has homologous cellular sequences (c-fes) in all vertebrate species, including humans. We isolated from a human DNA library recombinant phages containing overlapping c-fes sequences. The human c-fes locus spans a region of 3.4 kilobases and contains 1.4 kilobases of DNA homologous to the viral onc sequence interspersed with three intervening sequences.

Human T-cell lymphotropic virus type 1 Tax represses c-Myb-dependent transcription through activation of the NF-kappaB pathway and modulation of coactivator usage.

The proto-oncogene c-myb is essential for a controlled balance between cell growth and differentiation. Aberrant c-Myb activity has been reported for numerous human cancers, and enforced c-Myb transcription can transform cells of lymphoid origin by stimulating cellular proliferation and inhibiting apoptotic pathways. Here we demonstrate that activation of the NF-kappaB pathway by the HTLV-1 Tax protein leads to transcriptional inactivation of c-Myb. This conclusion was supported by the fact that Tax mutants unable to stimulate the NF-kappaB pathway could not inhibit c-Myb transactivating functions. In addition, inhibition of Tax-mediated NF-kappaB activation by coexpression of IkappaBalpha restored c-Myb transcription, and Tax was unable to block c-Myb transcription in a NEMO knockout cell line. Importantly, physiological stimuli, such as signaling with the cellular cytokines tumor necrosis factor alpha, interleukin 1 beta (IL-1beta), and lipopolysaccharide, also inhibited c-Myb transcription. These results uncover a new link between extracellular signaling and c-Myb-dependent transcription. The mechanism underlying NF-kappaB-mediated repression was identified as sequestration of the coactivators CBP/p300 by RelA. Interestingly, an amino-terminal deletion form of p300 lacking the C/H1 and KIX domains and unable to bind RelA retained the ability to stimulate c-Myb transcription and prevented NF-kappaB-mediated repression.

Molecular biology of human T-lymphotropic retroviruses.

The generic name for a family of human T-lymphotropic retroviruses is HTLV. Two of the three members in this family have been linked etiologically to human diseases: HTLV-I with adult T-cell leukemia and HTLV-III with the acquired immunodeficiency syndrome. In addition to their T-cell tropism and a number of other common biological and biochemical properties, the most unique common features of these viruses from a molecular biological point of view are the presence of the x-lor gene towards the 3' end of the genome and the phenomenon of a virus-induced trans-acting factor in activation of transcription initiated in the viral long terminal repeat. These features may not only be key in understanding the mechanism of transformation or cell killing by these viruses, but they also provide a basis for new classification of retroviruses. In spite of these similarities among HTLV-I, -II, -III, and bovine leukemia virus, the genome of HTLV-III is only distantly related to these other viruses. Instead, it shows greater homology to members of the Lentivirus family. Therefore, all these viruses may have a common progenitor. Two other salient features arose from the analyses of HTLV-III and acquired immunodeficiency syndrome. (a) HTLV-III frequently infects the brain of acquired immunodeficiency syndrome patients who suffer from central nervous system disorders. This not only identifies HTLV-III as the direct candidate in these central nervous system disorders but also poses the problem of crossing the blood-brain barrier in therapy strategies to eradicate the virus. (b) Different HTLV-III isolates comprise a spectrum of related viruses, with the degree of divergence varying from virtual identity to 10-15% difference. The most divergent region resides in the envelope gene. Whether this finding has implications in the development of an effective vaccine for acquired immunodeficiency syndrome remains to be determined.