DAVID bioinformatics resources consists of an integrated biological knowledgebase and analytic tools aimed at systematically extracting biological meaning from large gene/protein lists. This protocol explains how to use DAVID, a high-throughput and integrated data-mining environment, to analyze gene lists derived from high-throughput genomic experiments. The procedure first requires uploading a gene list containing any number of common gene identifiers followed by analysis using one or more text and pathway-mining tools such as gene functional classification, functional annotation chart or clustering and functional annotation table. By following this protocol, investigators are able to gain an in-depth understanding of the biological themes in lists of genes that are enriched in genome-scale studies.

Functional analysis of large gene lists, derived in most cases from emerging high-throughput genomic, proteomic and bioinformatics scanning approaches, is still a challenging and daunting task. The gene-annotation enrichment analysis is a promising high-throughput strategy that increases the likelihood for investigators to identify biological processes most pertinent to their study. Approximately 68 bioinformatics enrichment tools that are currently available in the community are collected in this survey. Tools are uniquely categorized into three major classes, according to their underlying enrichment algorithms. The comprehensive collections, unique tool classifications and associated questions/issues will provide a more comprehensive and up-to-date view regarding the advantages, pitfalls and recent trends in a simpler tool-class level rather than by a tool-by-tool approach. Thus, the survey will help tool designers/developers and experienced end users understand the underlying algorithms and pertinent details of particular tool categories/tools, enabling them to make the best choices for their particular research interests.

An acidic extracellular pH is a fundamental property of the malignant phenotype. In von Hippel-Lindau (VHL)-defective tumors the cell surface transmembrane carbonic anhydrase (CA) CA9 and CA12 genes are overexpressed because of the absence of pVHL. We hypothesized that these enzymes might be involved in maintaining the extracellular acidic pH in tumors, thereby providing a conducive environment for tumor growth and spread. Using Northern blot analysis and immunostaining with specific antibodies we analyzed the expression of CA9 and CA12 genes and their products in a large sample of cancer cell lines, fresh and archival tumor specimens, and normal human tissues. Expression was also analyzed in cultured cells under hypoxic conditions. Expression of CA IX and CA XII in normal adult tissues was detected only in highly specialized cells and for most tissues their expression did not overlap. Analysis of RNA samples isolated from 87 cancer cell lines and 18 tumors revealed high-to-moderate levels of expression of CA9 and CA12 in multiple cancers. Immunohistochemistry revealed high-to-moderate expression of these enzymes in various normal tissues and multiple common epithelial tumor types. The immunostaining was seen predominantly on the cell surface membrane. The expression of both genes was markedly induced under hypoxic conditions in tumors and cultured tumor cells. We conclude that the cell surface trans-membrane carbonic anhydrases CA IX and CA XII are overexpressed in many tumors suggesting that this is a common feature of cancer cells that may be required for tumor progression. These enzymes may contribute to the tumor microenvironment by maintaining extracellular acidic pH and helping cancer cells grow and metastasize. Our studies show an important causal link between hypoxia, extracellular acidification, and induction or enhanced expression of these enzymes in human tumors.

BACKGROUND

From studies of genetic polymorphisms and the rate of progression from human immunodeficiency virus type 1 (HIV-1) infection to the acquired immunodeficiency syndrome (AIDS), it appears that the strongest susceptibility is conferred by the major-histocompatibility-complex (MHC) class I type HLA-B*35,Cw*04 allele. However, cytotoxic T-lymphocyte responses have been observed against HIV-1 epitopes presented by HLA-B*3501, the most common HLA-B*35 subtype. We examined subtypes of HLA-B*35 in five cohorts and analyzed the relation of structural differences between HLA-B*35 subtypes to the risk of progression to AIDS.

METHODS

Genotyping of HLA class I loci was performed for 850 patients who seroconverted and had known dates of HIV-1 infection. Survival analyses with respect to the rate of progression to AIDS were performed to identify the effects of closely related HLA-B*35 subtypes with different peptide-binding specificities.

RESULTS

HLA-B*35 subtypes were divided into two groups according to peptide-binding specificity: the HLA-B*35-PY group, which consists primarily of HLA-B*3501 and binds epitopes with proline in position 2 and tyrosine in position 9; and the more broadly reactive HLA-B*35-Px group, which also binds epitopes with proline in position 2 but can bind several different amino acids (not including tyrosine) in position 9. The influence of HLA-B*35 in accelerating progression to AIDS was completely attributable to HLA-B*35-Px alleles, some of which differ from HLA-B*35-PY alleles by only one amino acid residue.

CONCLUSIONS

This analysis shows that, in patients with HIV-1 infection, a single amino acid change in HLA molecules has a substantial effect on the rate of progression to AIDS. The different consequences of HLA-B*35-PY and HLA-B*35-Px in terms of disease progression highlight the importance of the epitope specificities of closely related class I molecules in the immune defense against HIV-1.

Transient antiretroviral treatment with tenofovir, (R)-9-(2-phosphonylmethoxypropyl)adenine, begun shortly after inoculation of rhesus macaques with the highly pathogenic simian immunodeficiency virus (SIV) isolate SIVsmE660, facilitated the development of SIV-specific lymphoproliferative responses and sustained effective control of the infection following drug discontinuation. Animals that controlled plasma viremia following transient postinoculation treatment showed substantial resistance to subsequent intravenous rechallenge with homologous (SIVsmE660) and highly heterologous (SIVmac239) SIV isolates, up to more than 1 year later, despite the absence of measurable neutralizing antibody. In some instances, resistance to rechallenge was observed despite the absence of detectable SIV-specific binding antibody and in the face of SIV lymphoproliferative responses that were low or undetectable at the time of challenge. In vivo monoclonal antibody depletion experiments demonstrated a critical role for CD8(+) lymphocytes in the control of viral replication; plasma viremia rose by as much as five log units after depletion of CD8(+) cells and returned to predepletion levels (as low as <100 copy Eq/ml) as circulating CD8(+) cells were restored. The extent of host control of replication of highly pathogenic SIV strains and the level of resistance to heterologous rechallenge achieved following transient postinoculation treatment compared favorably to the results seen after SIVsmE660 and SIVmac239 challenge with many vaccine strategies. This impressive control of viral replication was observed despite comparatively modest measured immune responses, less than those often achieved with vaccination regimens. The results help establish the underlying feasibility of efforts to develop vaccines for the prevention of AIDS, although the exact nature of the protective host responses involved remains to be elucidated.

Activating mutations in the Met receptor tyrosine kinase, both germline and somatic, have been identified in human papillary renal cancer. Here we report a novel germline missense Met mutation, P1009S, in a patient with primary gastric cancer. The dosage of the mutant Met DNA was elevated in the tumor when compared to its matched normal DNA. Therefore, as with hereditary renal papillary cancer, the mutant Met allele may also be selectively duplicated in the tumor. Different from previously reported Met mutations, which occur in the tyrosine kinase domain, this missense mutation is located at the juxtamembrane domain, and is not constitutively activated. However, following treatment with HGF/SF, the P1009S mutant Met protein, expressed in NIH3T3 cells, displays increased and persistent tyrosine phosphorylation compared to the wild-type Met. Importantly, these cells also form colonies in soft agar, and are highly tumorigenic in athymic nude mice. A second nucleotide change in this region of Met, T1010I, was found in a breast cancer biopsy and a large cell lung cancer cell line. Although this previously reported 'polymorphism' did not stimulate NIH3T3 cell growth in soft agar, it was more active than the wild-type Met in the athymic nude mice tumorigenesis assay, suggesting that it may have effects on tumorigenesis. Met has been shown to be highly expressed in human gastric carcinoma cell lines, and our results raise the possibility that activating missense Met mutations could contribute to tumorigenesis of gastric cancer.

As potential targets for human immunodeficiency virus type 1 and simian immunodeficiency virus (HIV-1 and SIV), dendritic cells (DCs) likely play a significant role in the onset and spread of infection as well as in the induction of antiviral immunity. Using the SIV-macaque system to study the very early events in DC-virus interactions, we compared chemically inactivated SIV having conformationally and functionally intact envelope glycoproteins (2,2'-dithiodipyridine [AT-2] SIV) to infectious and heat-treated SIV. Both human and macaque DCs interact similarly with SIV without detectable effects on DC viability, phenotype, or endocytic function. As assessed by measuring cell-associated viral RNA, considerable amounts of virus are captured by the DCs and this is reduced when the virus is heat treated or derived from a strain that expresses low levels of envelope glycoprotein. Immunostaining for SIV proteins and electron microscopy indicated that few intact virus particles are retained at the periphery of the endocytically active, immature DCs. This contrasts with a perinuclear localization of numerous virions in large vesicular compartments deeper within mature DCs (in which macropinocytosis is down-regulated). Both immature and mature DCs are capable of clathrin-coated pit-mediated uptake of SIV, supporting the notion that the receptor-mediated uptake of virus can occur readily in mature DCs. While large numbers of whole viruses were preferentially found in mature DCs, both immature and mature DCs contained similar amounts of viral RNA, suggesting that different uptake/virus entry mechanisms are active in immature and mature DCs. These findings have significant implications for cell-to-cell transmission of HIV-1 and SIV and support the use of AT-2 SIV, an authentic but noninfectious form of virus, as a useful tool for studies of processing and presentation of AT-2 SIV antigens by DCs.

The 97-kDa valosin-containing protein (p97-VCP) plays a role in a wide variety of cellular activities, many of which are regulated by the ubiquitin-proteasome (Ub-Pr)-mediated degradation pathway. We previously demonstrated that VCP binds to multi-ubiquitin chains and may act as a molecular chaperone that targets the ubiquitinated substrates to the proteasome for degradation. In this report, we show that although the ubiquitin chain-binding activity, carried out by the N-terminal 200 residues (N domain), is necessary for the degradation of proteasome substrates, it is not sufficient. Using in vitro degradation assays, we demonstrated that the entire VCP molecule, consisting of the N domain and two ATPase domains D1 and D2, is required for mediating the Ub-Pr degradation. The ATPase activity of VCP requires Mg(2+), and is stimulated by high temperature. Under optimal conditions, VCP hydrolyzes ATP with a K(m) of approximately 0.33 mm and a V(max) of approximately 0.52 nmol P(i) min(-1) microg(-1). At a physiological temperature, mutation in D2 significantly inhibits the ATPase activity, while that in D1 has little effect. Interestingly, mutations in D1, but not D2, abolish the heat-stimulated ATPase activity. Thus, we provide the first demonstration that the ATPase activity of VCP is required for mediating the Ub-Pr degradation, that D2 accounts for the major ATPase activity, and that D1 contributes to the heat-induced activity.

Amino acid substitutions in human immunodeficiency virus type 1 (HIV-1) Gag cleavage sites have been identified in HIV-1 isolated from patients with AIDS failing chemotherapy containing protease inhibitors (PIs). However, a number of highly PI-resistant HIV-1 variants lack cleavage site amino acid substitutions. In this study we identified multiple novel amino acid substitutions including L75R, H219Q, V390D/V390A, R409K, and E468K in the Gag protein at non-cleavage sites in common among HIV-1 variants selected against the following four PIs: amprenavir, JE-2147, KNI-272, and UIC-94003. Analyses of replication profiles of various mutant clones including competitive HIV-1 replication assays demonstrated that these mutations were indispensable for HIV-1 replication in the presence of PIs. When some of these mutations were reverted to wild type amino acids, such HIV-1 clones failed to replicate. However, virtually the same Gag cleavage pattern was seen, indicating that the mutations affected Gag protein functions but not their cleavage sensitivity to protease. These data strongly suggest that non-cleavage site amino acid substitutions in the Gag protein recover the reduced replicative fitness of HIV-1 caused by mutations in the viral protease and may open a new avenue for designing PIs that resist the emergence of PI-resistant HIV-1.

Transcriptional activation of the human CYP1A1 gene (coding for cytochrome P450 1A1) is mediated by the aryl hydrocarbon receptor (AhR). In the present study we have examined the effect of the common dietary polyphenolic compounds quercetin and kaempferol on the transcription of CYP1A1 and the function of the AhR in MCF-7 human breast cancer cells. Quercetin caused a time- and concentration-dependent increase in the amount of CYP1A1 mRNA and CYP1A1 enzyme activity in MCF-7 cells. The increase in CYP1A1 mRNA caused by quercetin was prevented by the transcription inhibitor actinomycin D. Quercetin also caused an increase in the transcription of a chloramphenicol reporter vector containing the CYP1A1 promoter. Quercetin failed to induce CYP1A1 enzyme activity in AhR-deficient MCF-7 cells. Gel retardation studies demonstrated that quercetin activated the ability of the AhR to bind to an oligonucleotide containing the xenobiotic-responsive element (XRE) of the CYP1A1 promoter. These results indicate that quercetin's effect is mediated by the AhR. Kaempferol did not affect CYP1A1 expression by itself but it inhibited the transcription of CYP1A1 induced by the prototypical AhR ligand 2,3,7, 8-tetrachlorodibenzo-p-dioxin (TCDD), as measured by a decrease in TCDD-induced CYP1A1 promoter-driven reporter vector activity, and CYP1A1 mRNA in cells. Kaempferol also abolished TCDD-induced XRE binding in a gel-shift assay. Both compounds were able to compete with TCDD for binding to a cytosolic extract of MCF-7 cells. Known ligands of the AhR are, for the most part, man-made compounds such as halogenated and polycyclic aromatic hydrocarbons. These results demonstrate that the dietary flavonols quercetin and kaempferol are natural, dietary ligands of the AhR that exert different effects on CYP1A1 transcription.

To evaluate the relationship between T cell turnover, immune activation, and CD4 recovery in HIV infection, 32 antiretroviral-naive HIV-1-infected patients were studied before and after initiation of highly active antiretroviral therapy (HAART). Elevated CD4 and CD8 T cell turnover (measured by Ki67) in HIV infection decreased with HAART in blood and lymphoid tissue. Increased peripheral CD4 T cell turnover was strongly associated with immune activation even after viral suppression to less than 50 copies/mL (R = 0.8; p <.001). Increased CD4 T cell turnover correlated strongly with CD4 cell counts both before (R = -0.6; p <.001) and after (R = -0.4; p =.05) HAART. In patients with baseline CD4 cell counts of less than 350/microL, decreases in CD4 T cell turnover with HAART significantly correlated with increases in CD4 cell counts. In addition, persistently elevated levels of CD4 T cell turnover after HAART were associated with incomplete CD4 T cell recovery despite HIV RNA levels of less than 50 copies/mL. These data suggest that immune activation is central to CD4 cell depletion in HIV infection and immune reconstitution with HAART.

Macrophages are a major source of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha), which are expressed during conditions of inflammation, infection, or injury. We identified an activity secreted by a macrophage tumor cell line that negatively regulates bacterial lipopolysaccharide (LPS)-induced expression of TNF-alpha. This activity, termed TNF-alpha-inhibiting factor (TIF), suppressed the induction of TNF-alpha expression in macrophages, whereas induction of three other proinflammatory cytokines (interleukin-1beta [IL-1beta], IL-6, and monocyte chemoattractant protein 1) was accelerated or enhanced. A similar or identical inhibitory activity was secreted by IC-21 macrophages following LPS stimulation. Inhibition of TNF-alpha expression by macrophage conditioned medium was associated with selective induction of the NF-kappaB p50 subunit. Hyperinduction of p50 occurred with delayed kinetics in LPS-stimulated macrophages but not in fibroblasts. Overexpression of p50 blocked LPS-induced transcription from a TNF-alpha promoter reporter construct, showing that this transcription factor is an inhibitor of the TNF-alpha gene. Repression of the TNF-alpha promoter by TIF required a distal region that includes three NF-kappaB binding sites with preferential affinity for p50 homodimers. Thus, the selective repression of the TNF-alpha promoter by TIF may be explained by the specific binding of inhibitory p50 homodimers. We propose that TIF serves as a negative autocrine signal to attenuate TNF-alpha expression in activated macrophages. TIF is distinct from the known TNF-alpha-inhibiting factors IL-4, IL-10, and transforming growth factor beta and may represent a novel cytokine.

Here we present a novel technique for the alignment of flexible proteins. The method does not require an a priori knowledge of the flexible hinge regions. The FlexProt algorithm simultaneously detects the hinge regions and aligns the rigid subparts of the molecules. Our technique is not sensitive to insertions and deletions. Numerous methods have been developed to solve rigid structural comparisons. Unlike FlexProt, all previously developed methods designed to solve the protein flexible alignment require an a priori knowledge of the hinge regions. The FlexProt method is based on 3-D pattern-matching algorithms combined with graph theoretic techniques. The algorithm is highly efficient. For example, it performs a structural comparison of a pair of proteins with 300 amino acids in about 7 s on a 400-MHz desktop PC. We provide experimental results obtained with this algorithm. First, we flexibly align pairs of proteins taken from the database of motions. These are extended by taking additional proteins from the same SCOP family. Next, we present some of the results obtained from exhaustive all-against-all flexible structural comparisons of 1329 SCOP family representatives. Our results include relatively high-scoring flexible structural alignments between the C-terminal merozoite surface protein vs. tissue factor; class II aminoacyl-tRNA synthase, histocompatibility antigen vs. neonatal FC receptor; tyrosine-protein kinase C-SRC vs. haematopoetic cell kinase (HCK); tyrosine-protein kinase C-SRC vs. titine protein (autoinhibited serine kinase domain); and tissue factor vs. hormone-binding protein. These are illustrated and discussed, showing the capabilities of this structural alignment algorithm, which allows un-predefined hinge-based motions.

Trophic factor withdrawal induces cell death by mechanisms that are incompletely understood. Previously we reported that withdrawal of interleukin-7 (IL-7) or IL-3 produced a rapid intracellular alkalinization, disrupting mitochondrial metabolism and activating the death protein Bax. We now observe that this novel alkalinization pathway is mediated by the pH regulator NHE1, as shown by the requirement for sodium, blocking by pharmacological inhibitors or use of an NHE1-deficient cell line, and the altered phosphorylation of NHE1. Alkalinization also required the stress-activated p38 mitogen-activated protein kinase (MAPK). Inhibition of p38 MAPK activity with pharmacological inhibitors or expression of a dominant negative kinase prevented alkalinization. Activated p38 MAPK directly phosphorylated the C terminus of NHE1 within a 40-amino-acid region. Analysis by mass spectroscopy identified four phosphorylation sites on NHE1, Thr 717, Ser 722, Ser 725, and Ser 728. Thus, loss of trophic cytokine signaling induced the p38 MAPK pathway, which phosphorylated NHE1 at specific sites, inducing intracellular alkalinization.

OBJECTIVE

To determine the maximum-tolerated dose, toxicities, and pharmacokinetic profile of the farnesyl protein transferase inhibitor R115777 when administered orally bid for 5 days every 2 weeks.

METHODS

Twenty-seven patients with a median age of 58 years received 85 cycles of R115777 using an intrapatient and interpatient dose escalation schema. Drug was administered orally at escalating doses as a solution (25 to 850 mg bid) or as pellet capsules (500 to 1300 mg bid). Pharmacokinetics were assessed after the first dose and the last dose administered during cycle 1.

RESULTS

Dose-limiting toxicity of grade 3 neuropathy was observed in one patient and grade 2 fatigue (decrease in two performance status levels) was seen in four of six patients treated with 1,300 mg bid. The most frequent clinical grade 2 or 3 adverse events in any cycle included nausea, vomiting, headache, fatigue, anemia, and hypotension. Myelosuppression was mild and infrequent. Peak plasma concentrations of R115777 were achieved within 0.5 to 4 hours after oral drug administration. The elimination of R115777 from plasma was biphasic, with sequential half-lives of about 5 hours and 16 hours. There was little drug accumulation after bid dosing, and steady-state concentrations were achieved within 2 to 3 days. The pharmacokinetics were dose proportional in the 25 to 325 mg/dose range for the oral solution. Urinary excretion of unchanged R115777 was less than 0.1% of the oral dose. One patient with metastatic colon cancer treated at the 500-mg bid dose had a 46% decrease in carcinoembryonic antigen levels, improvement in cough, and radiographically stable disease for 5 months.

CONCLUSIONS

R115777 is bioavailable after oral administration and has an acceptable toxicity profile. Based upon pharmacokinetic data, the recommended dose for phase II trials is 500 mg orally bid (total daily dose, 1, 000 mg) for 5 consecutive days followed by 9 days of rest. Studies of continuous dosing and studies of R115777 in combination with chemotherapy are ongoing.

Thymic-deficient hosts rely primarily on antigen-driven expansion to restore the peripheral T-cell compartment following T-cell depletion (TCD). The degree to which this thymic-independent pathway can restore immune competence remains poorly understood but has important implications for a number of clinical conditions including stem cell transplantation and human immunodeficiency virus (HIV) infection. A model of HY-mediated skin graft rejection by athymic, TCD mice was used to show that restoration of naive and recall responses via peripheral expansion requires transfer of only 25 x 10(6) lymph node (LN) cells representing approximately 10% of the T-cell repertoire. Constitutive expression of bcl-2 in the expanding inocula restored recall responses to HY at a substantially lower LN cell dose (1 x 10(6)), which is normally insufficient to induce HY-mediated graft rejection in athymic hosts. Interestingly, bcl-2 had no effect on primary responses. Interleukin-7 (IL-7) potently enhanced thymic-independent peripheral expansion and led to HY graft rejection using an LN cell dose of 1 x 10(6) in both primary and recall models. The restoration of immune competence by IL-7 appeared to be mediated through a combination of programmed cell death inhibition, improved costimulation, and modulation of antigen-presenting cell (APC) function. These results show that immune competence for even stringent antigens such as HY can be restored in the absence of thymic function and identify IL-7 as a potent modulator of thymic-independent T-cell regeneration.

Several norindenoisoquinolines substituted with methoxy or methylenedioxy groups have been prepared and their anticancer properties evaluated in cancer cell cultures and in topoisomerase I inhibition assays. 2,3-Dimethoxy-8,9-methylenedioxy-11H-indeno[1,2-c]isoquinoline hydrochloride (14) is a strong topoisomerase I inhibitor and also displays very high cytotoxicity in the NCI cancer cell culture screen (mean graph midpoint of 50 nM). The X-ray crystal structure of norindenoisoquinoline 14 in complex with topoisomerase I and DNA has been solved, providing insight into the structure-activity relationships within this class of new anticancer agents. The number and position of the norindenoisoquinoline substituents have a significant influence on biological activity and demonstrate that substitution on the nitrogen atom is not an absolute requirement for the antitumor effect of the indenoisoquinolines. Removal of the 11-keto group from the lead compound 1 and replacement of the N-alkyllactam with an unsubstituted pyridine ring causes the indenoisoquinoline ring system to flip over in the DNA-enzyme-inhibitor ternary complex. This allows the nitrogen atom to assume the hydrogen bond acceptor role of the 11-keto group, resulting in hydrogen bonding to Arg364.

Kidney cancer is a devastating disease; however, biological therapies have achieved some limited success. The murine renal cancer Renca has been used as a model for developing new preclinical approaches to the treatment of renal cell carcinoma. Successful cytokine-based approaches require CD8(+) T cells, but the exact mechanisms by which T cells mediate therapeutic benefit have not been completely identified. After successful biological therapy of Renca in BALB/c mice, we generated CTLs in vitro using mixed lymphocyte tumor cultures. These CTL mediated tumor-specific H-2K(d)-restricted lysis and production of IFN-gamma, TNF-alpha, and Fas ligand (FasL) in response to Renca. CTL used both granule- and FasL-mediated mechanisms to lyse Renca, although granule-mediated killing was the predominant lytic mechanism in vitro. The cytokines IFN-gamma and TNF-alpha increased the sensitivity of Renca cells to CTL lysis by both granule- and FasL-mediated death pathways. Adoptive transfer of these anti-Renca CTL into tumor-bearing mice cured most mice of established experimental pulmonary metastases, and successfully treated mice were immune to tumor rechallenge. Interestingly, we were able to establish Renca-specific CTL from mice gene targeted for perforin (pfp(-/-)) mice. Although these pfp(-/-) CTL showed reduced cytotoxic activity against Renca, their IFN-gamma production in the presence of Renca targets was equivalent to that of wild-type CTL, and adoptive transfer of pfp(-/-) CTL was as efficient as wild-type CTL in causing regression of established Renca pulmonary metastases. Therefore, although granule-mediated killing is of paramount importance for CTL-mediated lysis in vitro, some major in vivo effector mechanisms clearly are independent of perforin.

The mouse lectin-related Ly49 family and the human killer cell Ig-like receptor (KIR) family represent structurally distinct, yet functionally analogous, class I MHC receptors that are expressed on natural killer cells and some T cells. The functional similarity of these two families has been borne out by the demonstration of identical signal transduction pathways associated with each receptor family. The Ly49 family therefore provides a useful model system to study the role of this dass of receptors in the regulation of the immune system. Recent data relating to the Ly49 repertoire in several mouse strains has revealed an additional evolutionary parallel between KIR and Ly49 receptor families. There is now an appreciation of the variation in the number and type of Ly49s expressed in different mouse strains, similar to the previously demonstrated differences in the number of KIR genes found in humans. This review summarizes the current members of the Ly49 gene family, their MHC class I recognition and associated signal transduction pathways.

All retroviruses (except the spumaretroviruses) contain a nucleocapsid (NC) protein that encodes one or two copies of the Zn2+-finger sequence -Cys-X2-Cys-X4-His-X4-Cys-. This region has been shown to be essential for recognition and packaging of the genomic RNA during virion particle assembly. Additionally, this region has been shown to be involved in early infection events in a wide spectrum of retroviruses, including mammalian type C [e.g., murine leukemia virus (MuLV)], human immunodeficiency virus type 1 (HIV-1), Rous sarcoma virus, and other retroviruses. Mutations in the two Zn2+-fingers of the NC protein of simian immunodeficiency virus strain Mne [SIV(Mne)] have been generated. The resulting virions contained the normal complement of processed viral proteins with densities indistinguishable from wild-type SIV(Mne). All of the mutants had electron micrograph morphologies similar to those of immature particles observed in wild-type preparations. RNA packaging was less affected by mutations in the NC protein of SIV(Mne) than has been observed for similar mutants in the MuLV and HIV-1 systems. Nevertheless, in vitro replication of SIV(Mne) NC mutants was impaired to levels comparable to those observed for MuLV and HIV-1 NC mutants; replication defective NC mutants are typically 10(5)- to 10(6)-fold less infectious than similar levels of wild-type virus. One mutant, DeltaCys33-Cys36, was also found to be noninfectious in vivo when mutant virus was administered intravenously to a pig-tailed macaque. NC mutations can therefore be used to generate replication defective virions for candidate vaccines in the SIV macaque model for primate lentiviral diseases.

LAMP3 (DC-LAMP, TSC403, CD208) was originally isolated as a gene specifically expressed in lung tissues. LAMP3 is located on a chromosome 3q segment that is frequently amplified in some human cancers, including uterine cervical cancer. Because two other members of the LAMP family of lysosomal membrane glycoproteins, LAMP1 and LAMP2, were previously implicated in potentially modulating the interaction of vascular endothelial and cancer cells, we hypothesized that LAMP3 might also play an important part in metastasis. To clarify the metastatic potential of LAMP3 in cervical cancers, we transfected a LAMP3 expression vector into a human uterine cervical cancer cell line, TCS. In an in vitro invasion assay, the migration of LAMP3-overexpressing TCS cells was significantly higher than in control TCS cells. In an in vivo metastasis assay, distant metastasis was detected in 9 of 11 LAMP3-overexpressing TCS cell-injected mice and in only 1 of 11 control mice. Histologic study showed that LAMP3-overexpressing cells readily invaded into the lymph-vascular space. In clinical samples, quantitative real-time reverse transcription-PCR (RT-PCR) analyses showed that LAMP3 mRNA was significantly up-regulated in 47 of 47 (100%) cervical cancers and in 2 of 15 (13%) cervical intraepithelial neoplasias, compared with a low level of LAMP3 mRNA expressed in normal uterine cervixes. Interestingly, high LAMP3 expression was significantly correlated with the overall survival of patients with stage I/II cervical cancers. These findings indicate that LAMP3 overexpression is associated with an enhanced metastatic potential and may be a prognostic factor for cervical cancer.

Systemic administration of IL-12 and intermittent doses of IL-2 induce complete regression of metastatic murine renal carcinoma. Here, we show that overt tumor regression induced by IL-12/pulse IL-2 is preceded by recruitment of CD8(+) T cells, vascular injury, disrupted tumor neovascularization, and apoptosis of both endothelial and tumor cells. The IL-12/IL-2 combination synergistically enhances cell surface FasL expression on CD8(+) T lymphocytes in vitro and induces Fas and FasL expression within tumors via an IFN-gamma-dependent mechanism in vivo. This therapy also inhibits tumor neovascularization and induces tumor regression by mechanisms that depend critically on endogenous IFN-gamma production and an intact Fas/FasL pathway. The ability of IL-12/pulse IL-2 to induce rapid destruction of tumor-associated endothelial cells and regression of established metastatic tumors is ablated in mice with a dysregulated Fas/FasL pathway. The common, critical role for endogenous IFN-gamma and the Fas/FasL pathway in early antiangiogenic effects and in antitumor responses suggests that early, cytokine-driven innate immune mechanisms and CD8(+) T cell-mediated responses are interdependent. Definition of critical early molecular events engaged by IL-12/IL-2 may provide new perspective into optimal therapeutic engagement of a productive host-antitumor immune response.

Neurodegeneration observed in lentiviral-associated encephalitis has been linked to viral-infected and -activated central nervous system macrophages. We hypothesized that lentivirus, macrophages, or both lentivirus and macrophages within distinct microenvironments mediate synaptic damage. Using the simian immunodeficiency virus (SIV)-infected macaque model, we assessed the relationship between virus, macrophages, and neurological damage in multiple brain regions using laser confocal microscopy. In SIV-infected macaques with SIV encephalitis (SIVE), brain tissue concentrations of SIV RNA were 5 orders of magnitude greater than that observed in nonencephalitic animals. In SIVE, staining for postsynaptic protein microtubule-associated protein-2 was significantly decreased in the caudate, hippocampus, and frontal cortical gray matter compared to nonencephalitic controls, whereas staining for presynaptic protein synaptophysin was decreased in SIV-infected macaques with and without encephalitis. These data suggest that presynaptic damage occurs independent of pathological changes associated with SIVE, whereas postsynaptic damage is more tightly linked to regional presence of both activated and infected macrophages.

How is the native structure encoded in the amino acid sequence? For the traditional backbone centric view, the dominant forces are hydrogen bonds (backbone) and phi-psi propensity. The role of hydrophobicity is non-specific. For the side-chain centric view, the dominant force of protein folding is hydrophobicity. In order to understand the balance between backbone and side-chain forces, we have studied the contributions of three components of a beta-hairpin peptide: turn, backbone hydrogen bonding and side-chain interactions, of a 16-residue fragment of protein G. The peptide folds rapidly and cooperatively to a conformation with a defined secondary structure and a packed hydrophobic cluster of aromatic side-chains. Our strategy is to observe the structural stability of the beta-hairpin under systematic perturbations of the turn region, backbone hydrogen bonds and the hydrophobic core formed by the side-chains, respectively. In our molecular dynamics simulations, the peptides are solvated. with explicit water molecules, and an all-atom force field (CFF91) is used. Starting from the original peptide (G41EWTYDDATKTFTVTE56), we carried out the following MD simulations. (1) unfolding at 350 K; (2) forcing the distance between the C(alpha) atoms of ASP47 and LYS50 to be 8 A; (3) deleting two turn residues (Ala48 and Thr49) to form a beta-sheet complex of two short peptides, GEWTYDD and KTFTVTE; (4) four hydrophobic residues (W43, Y45, F52 and T53) are replaced by a glycine residue step-by-step; and (5) most importantly, four amide hydrogen atoms (T44, D46, T53, and T55, which are crucial for backbone hydrogen bonding), are substituted by fluorine atoms. The fluorination not only makes it impossible to form attractive hydrogen bonding between the two beta-hairpin strands, but also introduces a repulsive force between the two strands due to the negative charges on the fluorine and oxygen atoms. Throughout all simulations, we observe that backbone hydrogen bonds are very sensitive to the perturbations and are easily broken. In contrast, the hydrophobic core survives most perturbations. In the decisive test of fluorination, the fluorinated peptide remains folded under our simulation conditions (5 ns, 278 K). Hydrophobic interactions keep the peptide folded, even with a repulsive force between the beta-strands. Thus, our results strongly support a side-chain centric view for protein folding.