Adapting controlled release technologies to the delivery of DNA has the potential to overcome extracellular barriers that limit gene therapy. Controlled release systems can enhance gene delivery and increase the extent and duration of transgene expression relative to more traditional delivery methods (e.g., injection). These systems typically deliver vectors locally, which can avoid distribution to distant tissues, decrease toxicity to nontarget cells, and reduce the immune response to the vector. Delivery vehicles for controlled release are fabricated from natural and synthetic polymers, which function either by releasing the vector into the local tissue environment or by maintaining the vector at the polymer surface. Vector release or binding is regulated by the effective affinity of the vector for the polymer, which depends upon the strength of molecular interactions. These interactions occur through nonspecific binding based on vector and polymer composition or through the incorporation of complementary binding sites (e.g., biotin-avidin). This review examines the delivery of nonviral and viral vectors from natural and synthetic polymers and presents opportunities for continuing developments to increase their applicability.

MicroRNAs (miRNAs) play important regulatory roles in animals and plants by targeting mRNAs for cleavage or translational repression. They have diverse expression patterns and might regulate various developmental and physiological processes. Profiling miRNA expression is very helpful for studying biological functions of miRNAs. We report a novel miRNA profiling microarray, in which miRNAs were directly labeled at the 3' terminus with biotin and hybridized with complementary oligo-DNA probes immobilized on glass slides, and subsequently detected by measuring fluorescence of quantum dots labeled with streptavidin bound to miRNAs through streptavidin-biotin interaction. The detection limit of this microarray for miRNA was approximately 0.4 fmol, and the detection dynamic range spanned about 2 orders of magnitude. We made a model microarray to profile 11 miRNAs from leaf and root of rice (Oryza sativa L. ssp. indica) seedlings. The analysis results of the miRNAs had a good reproducibility and were consistent with the northern blot result. To avoid using high-cost detection equipment, colorimetric detection, a method based on nanogold probe coupled with silver enhancement, was also successfully introduced into miRNA profiling microarray detection.

Antigens of human (HIV) or simian immunodeficiency viruses (SIV) were identified with polyclonal or monoclonal antibodies and avidin-biotin complex (ABC) immunohistochemistry in fixed surgical pathology and autopsy specimens of humans or monkeys with the acquired immunodeficiency syndrome. With B-5 fixative, viral antigens were readily detected in lymph nodes of 8 of 13 patients with follicular hyperplasia, but in only 1 of 12 patients with follicular atrophy. Antigen was detected in follicular dendritic reticular cells and rare blastlike cells, extracellularly, and in postcapillary venules, medullary lymphocytes, sinus histiocytes, and macrophages in some lymph nodes. In the brain at autopsy, antigen could be found in gliomesenchymal-cell nodules, astrocytes, vascular endothelial cells, multinucleated cells, and astrocytes and macrophages associated with demyelination. In contrast, 4 rhesus monkeys with experimental SIV infection had abundant antigen in sinus histiocytes, macrophages, and multinucleated giant cells of lymph nodes and spleen and in thymic epithelial cells. Brain lesions of monkeys resembled those of humans, with antigen found in macrophages and multinucleated giant cells. Antibodies to HIV also were immunoreactive in formalin-fixed tissue sections of monkeys containing SIV antigens. The ABC technique provided a fast and efficient method for localizing HIV and SIV antigens in fixed surgical and autopsy specimens. These findings are consistent with those found with in situ hybridization, ultrastructural studies, frozen sections of lymph nodes, and permanent sections of brain.

Chronic airway inflammation, one of the pathophysiologic features of bronchial asthma, is suspected to be responsible for irreversible pathological changes of airways, called airway remodeling. To examine the mechanisms of airway remodeling in asthma, we investigated the expression of epidermal growth factor (EGF) and its receptor immunohistochemically in asthmatic human airways. Airway specimens from seven patients with asthma were obtained from autopsied and surgically resected lungs. Control specimens were obtained from lungs of eight subjects without asthma and other pulmonary complications at autopsy. We stained those specimens by the avidin-biotin-peroxidase complex (ABC) method with anti-human polyclonal EGF antibody and monoclonal EGF receptor antibodies. Three different portions of airways-large bronchi (about 1 cm in diameter), small bronchi (about 3 mm in diameter), and peripheral airways (less than 2 mm in diameter)-were examined. The thickness of the bronchial smooth muscle and basement membrane was significantly greater in the asthmatic airways than in controls. Clear immunoreactivities of EGF were widely observed on bronchial epithelium, glands, and smooth muscle in asthmatic airways. In the controls, the bronchial epithelium and the bronchial glands partially expressed faint EGF immunoreactivity. For the EGF receptor, clear immunoreactivities were also observed on bronchial epithelium, glands, smooth muscle, and basement membrane in asthmatic airways. In control airways, only part of the bronchial epithelium and smooth muscle weakly expressed EGF receptor immunoreactivity. These results suggest a possible contribution of EGF to the pathophysiology of bronchial asthma, including airway remodeling.

P2X receptors contain 10 conserved cysteines in the extracellular loop. To investigate whether these residues form disulfide bonds, we created a series of single and double cysteine-alanine mutants in the human P2X(1) receptor. Mutants were expressed in Xenopus laevis oocytes and effects on ATP potency, cell-surface expression, and N-biotinoylaminoethyl methanethiosulfonate (MTSEA-Biotin) labeling of free cysteines were determined. For the majority of single mutants, only a modest decrease (2- to 5-fold) in ATP potency was recorded. For mutants C261A and C270A, the peak current amplitudes were reduced by 93.6 +/- 2.0 and 95.0 +/- 1.0%, respectively; this was a result of low cell-surface expression of these mutant receptors. Wild-type receptors showed no labeling with MTSEA-biotin suggesting that all 10 cysteine residues in the extracellular loop are disulfide-bonded. Mutation of cysteines at positions 126, 132, 149, 159, 217, and 227 resulted in MTSEA-biotinylation of a free cysteine residue created by the disruption of a disulfide bond and provides direct biochemical evidence for at least three disulfide bonds. Based on phenotypic comparisons of single and double cysteine mutants, we propose the following disulfide bond pairs in the human P2X(1) receptor: C117-C165, C126-C149, C132-C159, C217-C227, and C261-C270. None of these bonds are individually essential for channel function. However, trafficking of the receptor to the cell membrane is severely reduced by disruption of the C261-C270 disulfide bond or disruption of C117-C165 together with another bond.

Previous studies have indicated that white blood cells possess DNA on their outer membranes. In this study we set out to determine whether exogenous DNA bound to cells in a fashion compatible with a ligand receptor union. Purified populations of white blood cells; neutrophils (polymorphonuclear leukocytes, PMN), adherent mononuclear cells (ADMC), rosetting lymphocytes (E+ cells), and nonrosetting lymphocytes (E- cells) were incubated with radiolabeled lambda phage DNA in increasing concentrations. Binding of [3H]DNA was a saturable process and was inhibited by excess cold DNA and prior trypsinization of the cells. Rate zonal density centrifugation of purified cell membrane preparations confirmed that DNA was binding to the outer cell surface. The dissociation constant for all four cell types was approximately 10(-9) M, and from 0.81 X 10(3) to 2.6 X 10(3) molecules of lambda phage DNA bound to each cell depending upon cell type. Binding was not competitively inhibited by RNA, polydeoxyadenylic acid-polydeoxythymidylic acid (poly [d(A).d(T)]), or mononucleotides. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE)-separated proteins from PMN, ADMC, E+, and E- cells were electrophoretically blotted onto nitrocellulose sheets; a probe of biotin-labeled DNA indicated a single species of DNA-binding molecule migrating in a position consistent with a molecular weight of 30,000. Isotopic and immunofluorescent studies indicate that DNA is internalized and degraded to oligonucleotides; this process is inhibited by cycloheximide. These results support the notion that there is a common binding site for DNA on white blood cells, that the stoichiometry of the association is compatible with a ligand receptor relationship, and that this apparent receptor is responsible for the endocytosis and degradation of exogenous DNA.

DNA polymerase zeta (Pol zeta), a heterodimer of Rev3 and Rev7, is essential for DNA damage provoked mutagenesis in eukaryotes. DNA polymerases that function in a processive complex with the replication clamp proliferating cell nuclear antigen (PCNA) have been shown to possess a close match to the consensus PCNA-binding motif QxxLxxFF. This consensus motif is lacking in either subunit of Pol zeta, yet its activity is stimulated by PCNA. In particular, translesion synthesis of UV damage-containing DNA is dramatically stimulated by PCNA such that translesion synthesis rates are comparable with replication rates by Pol zeta on undamaged DNA. PCNA also stimulated translesion synthesis of a model abasic site by Pol zeta. Efficient PCNA stimulation required that PCNA was prevented from sliding off the damage-containing model oligonucleotide template-primer through the use of biotin-streptavidin bumpers or other blocks. Under those experimental conditions, facile bypass of the abasic site was also detected by DNA polymerase delta or eta (Rad30). The yeast DNA damage checkpoint clamp, consisting of Rad17, Mec3, and Ddc1, and an ortholog of human 9-1-1, has been implicated in damage-induced mutagenesis. However, this checkpoint clamp did not stimulate translesion synthesis by Pol zeta or by DNA polymerase delta.

Streptavidin binds biotin conjugates with exceptional stability but dissociation does occur, limiting its use in imaging, DNA amplification and nanotechnology. We identified a mutant streptavidin, traptavidin, with more than tenfold slower biotin dissociation, increased mechanical strength and improved thermostability; this resilience should enable diverse applications. FtsK, a motor protein important in chromosome segregation, rapidly displaced streptavidin from biotinylated DNA, whereas traptavidin resisted displacement, indicating the force generated by Ftsk translocation.

Streptavidin forms two-dimensional crystals when specifically bound to layers of biotinylated lipids at the air/water interface. The three-dimensional structure of streptavidin determined from the crystals by electron crystallography corresponds well with the structure determined by x-ray crystallography. Comparison of the electron and x-ray crystallographic structures reveals the occurrence of free biotin-binding sites on the surface of the two-dimensional crystals facing the aqueous solution. The free biotin-binding sites could be specifically labeled with biotinylated ferritin. The streptavidin/biotinylated lipid system may provide a general approach for the formation of two-dimensional crystals of biotinylated macromolecules.

In this report, we present a fluorescence-based approach to the assessment of cellular gene expression and transcription rates. Nuclear run-on was performed by supplying biotin-16-UTP to nuclei, and labeled transcripts were bound to streptavidin-coated magnetic beads. Total cDNA was then synthesized by means of random hexamer primed reverse transcription of captured molecules. To monitor transcript abundance in cDNA, both from nuclear run-on and total RNA, we propose a semiquantitative PCR approach based on the use of fluorescent primers.

Xenopus egg extracts, which support nuclear assembly and DNA replication, were functionally depleted of lamin LIII by inoculating them with monoclonal anti-lamin antibodies. Phase-contrast microscopy and electron-microscopy studies indicated that lamin-depleted extracts supported efficient chromatin decondensation, and assembly of double membrane structures and nuclear pores on demembranated sperm heads. Immunofluorescence microscopy suggests that lamin-antibody complexes are transported across the nuclear membrane but do not assemble into a lamina. These findings were confirmed by immunoblotting analysis of isolated nuclei. Metabolic labelling studies with either biotin-11-dUTP or [32P]dCTP, revealed that nuclei lacking a lamina were unable to initiate DNA replication and that, although such nuclei could import proteins required for DNA replication (e.g. PCNA), these proteins were apparently not organized into replicon clusters.

Interest in developing diverse nanoparticle (NP)-biological composite materials continues to grow almost unabated. This is motivated primarily by the desire to simultaneously exploit the properties of both NP and biological components in new hybrid devices or materials that can be applied in areas ranging from energy harvesting and nanoscale electronics to biomedical diagnostics. The utility and effectiveness of these composites will be predicated on the ability to assemble these structures with control over NP/biomolecule ratio, biomolecular orientation, biomolecular activity, and the separation distance within the NP-bioconjugate architecture. This degree of control will be especially critical in creating theranostic NP-bioconjugates that, as a single vector, are capable of multiple functions in vivo, including targeting, image contrast, biosensing, and drug delivery. In this review, a perspective is given on current and developing chemistries that can provide improved control in the preparation of NP-bioconjugates. The nanoscale properties intrinsic to several prominent NP materials are briefly described to highlight the motivation behind their use. NP materials of interest include quantum dots, carbon nanotubes, viral capsids, liposomes, and NPs composed of gold, lanthanides, silica, polymers, or magnetic materials. This review includes a critical discussion on the design considerations for NP-bioconjugates and the unique challenges associated with chemistry at the biological-nanoscale interface-the liabilities of traditional bioconjugation chemistries being particularly prominent therein. Select bioorthogonal chemistries that can address these challenges are reviewed in detail, and include chemoselective ligations (e.g., hydrazone and Staudinger ligation), cycloaddition reactions in click chemistry (e.g., azide-alkyne cyclyoaddition, tetrazine ligation), metal-affinity coordination (e.g., polyhistidine), enzyme driven modifications (e.g., HaloTag, biotin ligase), and other site-specific chemistries. The benefits and liabilities of particular chemistries are discussed by highlighting relevant NP-bioconjugation examples from the literature. Potential chemistries that have not yet been applied to NPs are also discussed, and an outlook on future developments in this field is given.

Conjugation of drugs with antibodies to surface endothelial antigens is a potential strategy for drug delivery to endothelium. We studied antibodies to platelet-endothelial adhesion molecule 1 (PECAM-1, a stably expressed endothelial antigen) as carriers for vascular immunotargeting. Although 125I-labeled anti-PECAM bound to endothelial cells in culture, the antibody was poorly internalized by the cells and accumulated poorly after intravenous administration in mice and rats. However, conjugation of biotinylated anti-PECAM (b-anti-PECAM) with streptavidin (SA) markedly stimulated uptake and internalization of anti-PECAM by endothelial cells and by cells expressing PECAM. In addition, conjugation with streptavidin markedly stimulated uptake of 125I-labeled b-anti-PECAM in perfused rat lungs and in the lungs of intact animals after either intravenous or intraarterial injection. The antioxidant enzyme catalase conjugated with b-anti-PECAM/SA bound to endothelial cells in culture, entered the cells, escaped intracellular degradation, and protected the cells against H2O2-induced injury. Anti-PECAM/SA/125I-catalase accumulated in the lungs after intravenous injection or in the perfused rat lungs and protected these lungs against H2O2-induced injury. Thus, modification of a poor carrier antibody with biotin and SA provides an approach for facilitation of antibody-mediated drug targeting. Anti-PECAM/SA is a promising candidate for vascular immunotargeting of bioactive drugs.

The existence of auxotrophic mutants of Saccharomyces cerevisiae having an absolute requirement for the long-chain base (lcb) component of sphingolipids suggests that sphingolipids are crucial for viability and growth. One mutant, termed the lcb1-1 mutant, lacks the activity of serine palmitoyltransferase, the first enzyme in the pathway for long-chain base synthesis. Here, we present evidence that LCB1 has been molecularly cloned. The size of the LCB1 transcript, the direction of transcription, and transcription initiation sites were determined. In addition, the coding region and its 5' and 3' flanking regions were sequenced. Analysis of the DNA sequence revealed a single open reading frame of 1,674 nucleotides, encoding a predicted peptide of 558 amino acids. The hydropathy profile of the predicted peptide suggests a hydrophobic, globular, membrane-associated protein with two potential transmembrane helices. Comparison of the predicted amino acid sequence to known protein sequences revealed homology to 5-aminolevulinic acid synthase and to 2-amino-3-ketobutyrate coenzyme A ligase. These homologies, the similarity of the chemical reactions catalyzed by the three enzymes, and the finding that LCB1 restores serine palmitoyltransferase activity to an lcb1-defective strain indicate that serine palmitoyltransferase or a subunit of the enzyme is the most likely product of LCB1. Homology of the LCB1 predicted protein to the Escherichia coli biotin synthetase was also observed, but the biological significance of this observation is not clear. A role for sphingolipids in sporulation is implicated by our finding that diploids homozygous for lcb1 failed to sporulate.

The differential expression of the ras oncogene product p21 in the primary tumor, regional nodes, and distant metastatic sites in patients with disseminated breast cancer was examined to define the biologic and clinical significance of the ras oncogene in the progression of breast cancer. The avidin-biotin peroxidase complex method was used on formalin-fixed, paraffin-embedded tissues from 16 patients with metastatic disease. The primary antibody used in this protocol was RAP-5, an anti-p21 murine monoclonal IgG2a. p21 antigen staining was similar in the primary tumor and regional nodes from the same patient (P less than 0.05), but the staining of distant metastases was more variable. Expression of ras p21 was consistently increased in invasive components of the primary tumor as compared with intraductal tumor. In addition, a high level of p21 expression was seen in tumor emboli in lymphatics and blood vessels as compared with contiguous tumor in parenchymal tissue. Although p21 staining is present in aggressive primary breast cancers and most metastatic sites, our findings indicate that markedly enhanced p21 expression is associated with the earlier stages (invasion and dissemination) of aggressive breast cancers.

We have used fiberoptic bronchoscopy to obtain endobronchial biopsies in which mast cells and eosinophils were enumerated using monoclonal antibodies directed against mast cell tryptase (AA1) and the eosinophil cationic protein (EG2). Eleven symptomatic atopic asthmatics treated with beta 2-agonists alone and six normal subjects were studied. Over a period of 2 wk prior to bronchoscopy, patients recorded asthma symptom scores, bronchodilator usage, and twice-daily peak expiratory flow. Five days before bronchoscopy, methacholine responsiveness was assessed. Two biopsies were taken from the subcarinae, one of which was processed into araldite for immunostaining by the streptavidin biotin immunoperoxidase method and the other into Spurr resin for electron microscopy. The number of AA1 staining mast cells present in the bronchial mucosa was not significantly different in the epithelium or submucosa between the asthmatic and the normal subjects. However, in the biopsies from asthmatics, there were significantly greater numbers of EG2-staining eosinophils in the epithelium (median, 1.2/mm versus zero; p less than 0.005) and in the submucosa (median, 50/mm2 versus 1/mm2; p less than 0.001). Electron microscopy showed morphologic features of mast cell and eosinophil degranulation in the asthmatics. No correlation could be established between mast cell or eosinophil numbers and indices of disease activity of PC20 methacholine, which points to the complexity of mechanisms responsible for the symptoms and the airway hyperresponsiveness of asthma.

Intermittent episodes of febrile illness are the most benign and recognized symptom of infection with malaria parasites, although the effects on parasite survival and virulence remain unclear. In this study, we identified the molecular factors altered in response to febrile temperature by measuring differential expression levels of individual genes using high-density oligonucleotide microarray technology and by performing biological assays in asexual-stage Plasmodium falciparum parasite cultures incubated at 37 degrees C and 41 degrees C (an elevated temperature that is equivalent to malaria-induced febrile illness in the host). Elevated temperature had a profound influence on expression of individual genes; 336 of approximately 5,300 genes (6.3% of the genome) had altered expression profiles. Of these, 163 genes (49%) were upregulated by twofold or greater, and 173 genes (51%) were downregulated by twofold or greater. In-depth sensitive sequence profile analysis revealed that febrile temperature-induced responses caused significant alterations in the major parasite biologic networks and pathways and that these changes are well coordinated and intricately linked. One of the most notable transcriptional changes occurs in genes encoding proteins containing the predicted Pexel motifs that are exported into the host cytoplasm or inserted into the host cell membrane and are likely to be associated with erythrocyte remodeling and parasite sequestration functions. Using our sensitive computational analysis, we were also able to assign biochemical or biologic functional predictions for at least 100 distinct genes previously annotated as "hypothetical." We find that cultivation of P. falciparum parasites at 41 degrees C leads to parasite death in a time-dependent manner. The presence of the "crisis forms" and the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling-positive parasites following heat treatment strongly support the notion that an apoptosis-like cell death mechanism might be induced in response to febrile temperatures. These studies enhance the possibility of designing vaccines and drugs on the basis of disruption in molecules and pathways of parasite survival and virulence activated in response to febrile temperatures.

We describe a method, termed reverse chromosome painting, which allows the rapid analysis of the content and breakpoints of aberrant chromosomes. The method involves the sorting of small numbers of the aberrant chromosome from short term blood culture preparations or cell lines by using bivariate flow karyotype analysis. The sorted chromosomes are amplified and biotin labelled enzymatically using a degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR), the product annealed to metaphase spreads from normal subjects, and hybridisation detected using fluorescence in situ hybridisation (FISH). We show the usefulness of this method for routine clinical cytogenetics by the analysis of cases involving an insertion, a deletion, a translocation, and two cases of a chromosome with additional material of unknown origin. The method has particular application for the rapid resolution of the origin of de novo unbalanced chromosome duplications.

Hyaluronate is actively synthesized by cultured epidermis and dermis, but no direct histological data have been available about its localization in normal human skin. A hyaluronate-specific biotinylated probe, prepared from the hyaluronate binding region of cartilage proteoglycan, was applied to human skin sections and visualized using the biotin-avidin-peroxidase system. The specificity of this staining was confirmed by hyaluronidase predigestion and by hyaluronate-derived oligosaccharides added to the staining solution. All dermis showed diffuse binding of the probe, but the highest staining intensity was observed in the epidermal intercellular spaces. The stainability extended from basal cells to the middle layers of the epidermis, whereas the granular layer and stratum corneum were completely negative. Also, the basal side of basal cells (basement membrane) did not bind the hyaluronate probe. The abundance of hyaluronate on surfaces and intercellular spaces of the spinous cells is suggested to have an important role in the physiology of human epidermis.

CRM1 plays an important role in the nuclear export of cargo proteins bearing nuclear exporting signal sequences. Leptomycin B (LMB), a well-known CRM1 inhibitor, possesses strong antitumor properties. However, its toxicity prevents it from being clinically useful. In this study, we demonstrate that a novel compound, CBS9106, inhibits CRM1-dependent nuclear export, causing arrest of the cell cycle and inducing apoptosis in a time- and dose-dependent manner for a broad spectrum of cancer cells, including multiple myeloma cells. CBS9106 reduces CRM1 protein levels significantly without affecting CRM1 mRNA expression. This effect could be reversed by adding bortezomib or LMB. Moreover, CBS9106-biotin allows capture of CRM1 protein by streptavidin beads in a competitive manner with LMB and vice versa. Mass spectrometric analysis shows that CBS9106 reacts with a synthetic CRM1 peptide that contains Cys528 but not with a Cys528 mutant peptide. Oral administration of CBS9106 significantly suppresses tumor growth and prolongs survival in mice bearing tumor xenograft without a significant loss in body weight. A reduced level of CRM1 protein is also observed in tumor xenografts isolated from mice treated with CBS9106. Taken together, these results indicate that CBS9106 is a novel reversible CRM1 inhibitor and a promising clinical candidate.

Monoclonal antibody HHF35 has previously been characterized biochemically as recognizing isotypes of actin (alpha and gamma) which are specific to muscle cells. In this study, the authors have investigated the normal and pathologic tissue distribution of HHF35-positive cells using the avidin-biotin immunoperoxidase method on methacarn-fixed, paraffin-embedded sections of human tissue. In addition to muscle tissues (smooth, skeletal, and cardiac) the antibody localizes to myoepithelium, as well as most of the capsular cells of several parenchymal organs, including liver, kidney, and spleen, with extension of the latter cells into the splenic trabeculaes. In pathologic tissues, the antibody localizes to cells, identified by some investigators as "myofibroblasts," in the stroma of certain tumors, within hyperplastic fibrous tissue responses ("fibromatoses") such as Dupuytren's contracture, and within fibrotic lung tissue. HHF35 also localizes to cells that proliferate within the intima in lesions of atherosclerosis and to a unique population of reactive mesothelial and submesothelial cells. Among tumors, it is positive only on leiomyomas, leiomyosarcomas, and rhabdomyosarcomas, and negative on all nonmuscle sarcomas. This antibody thus shows great potential utility as a diagnostic reagent in various pathologic conditions, most especially in the diagnosis of tumors of muscle origin.

A previous study from this laboratory on adoptively transferred experimental allergic encephalomyelitis (EAE) induced by myelin basic protein-responsive (MBP+) 11C-labeled lymphocytes showed that MBP+ cells entered the central nervous system (CNS) before signs, migrated through the endothelium, and remained within the perivascular space. The majority of cells effecting CNS damage were nonradiolabeled and appeared to be host-derived and non-CNS antigen specific. The present study defined the immunocytochemical and initial structural events occurring between lymphocytes and endothelial cells (EC) on CNS blood vessels during EAE induced with MBP+ lymph node cells or T-cell lines. Monoclonal antibodies against lymphocyte function-associated molecule LFA-1 and its ligand, intercellular adhesion molecule-1 (ICAM-1), and the addressin MECA-325, a marker of mouse lymph node high endothelial venules, were tested on frozen sections in combination with the avidin-biotin-complex technique. The attachment and infiltration of lymphocytes correlated with the onset of signs and the appearance in the CNS of MECA-325 and ICAM-1 on vessels with cellular infiltrates and sometimes with plump EC. The cellular infiltrates were composed largely of LFA-1+ lymphocytes. Ultrastructurally, pseudopodia from lymphocytes were seen to attach to and penetrate EC in the CNS and form small gap junction-like contacts. On the EC surface, some processes from lymphocytes made larger synapse-like contacts while others were associated with coated pits suggestive of receptor mediation. The results are in accord with specific homing and attachment of lymphocytes to the CNS vasculature being early features of the disease process in EAE and with some CNS vessels acquiring properties of lymph node elements. Understanding of the mechanisms underlying these lymphocyte/EC interactions has therapeutic import for multiple sclerosis, for which EAE is the prime model.

Nuclear factor (NF)-kappaB transcription factors are involved in the control of a large number of normal cellular and organismal processes, such as immune and inflammatory responses, developmental processes, cellular growth, and apoptosis. Transcription of the human immunodeficiency virus type 1 (HIV-1) genome depends on the intracellular environment where the integrate viral DNA is regulated by a complex interplay among viral regulatory proteins, such as Tat, and host cellular transcription factors, such as NF-kappaB, interacting with the viral long terminal repeat region. CBP (CREB-binding protein) and p300, containing an intrinsic histone acetyltransferase (HAT) activity, have emerged as coactivators for various DNA-binding transcription factors. Here, we show that the p50 subunit as well as the p50/p65 of NF-kappaB, and not other factors such as SP1, TFIIB, polymerase II, TFIIA, or p65, can be acetylated by CBP/p300 HAT domain. Acetylation of p50 was completely dependent on the presence of both HAT domain and Tat proteins, implying that Tat influences the transcription machinery by aiding CBP/p300 to acquire new partners and increase its functional repertoire. Three lysines, Lys-431, Lys-440, and Lys-441 in p50 were all acetylated in vitro, and a sequence similarity among p50, p53, Tat, and activin receptor type I on these particular lysines was observed. All proteins have been shown to be acetylated by the CBP/p300 HAT domain. Acetylated p50 increases its DNA binding properties, as evident by streptavidin/biotin pull-down assays when using labeled NF-kappaB oligonucleotides. Increased DNA binding on HIV-1 long terminal repeat coincided with increases in the rate of transcription. Therefore, we propose that acetylation of the DNA binding domain of NF-kappaB aids in nuclear translocation and enhanced transcription and also suggest that the substrate specificity of CBP/p300 can be altered by small peptide molecules, such as HIV-encoded Tat.

To define the enzymes involved in the etiology of Alzheimer's disease, we compared in mouse and human brain the mRNA levels and cellular localization of the ubiquitous beta-amyloid precursor protein (beta-APP) with those of the putative alpha-secretases ADAM10 and ADAM17 and the beta-secretases BACE and BACE2. In situ hybridization performed in mice during prenatal and postnatal development and in adulthood revealed the coexpression of beta-APP, BACE, and ADAM10. The patterns of BACE2 and ADAM17 only partially overlapped with that of beta-APP. beta-APP, BACE, and ADAM10 mRNAs have also been detected by northern blot in human brain cortex of normal subjects and in Alzheimer's disease subjects. In situ hybridization performed using combined biotin- and radiolabeled riboprobes provided evidence for the coexpression of beta-APP with BACE and ADAM10 in human cortical neurons. Our data provide cytochemical evidence supporting the role of ADAM10 and BACE as authentic alpha- and beta-secretases.