Kaposi Fibroblast Growth Factor Research Articles

Dual-Modified Liposome for Targeted and Enhanced Gene Delivery into Mice Brain.

The development of neuropharmaceutical gene delivery systems requires strategies to obtain efficient and effective brain targeting as well as blood-brain barrier (BBB) permeability. A brain-targeted gene delivery system based on a transferrin (Tf) and cell-penetrating peptide (CPP) dual-functionalized liposome, CPP-Tf-liposome, was designed and investigated for crossing BBB and permeating into the brain. We selected three sequences of CPPs [melittin, Kaposi fibroblast growth factor (kFGF), and penetration accelerating sequence-R8] and compared their ability to internalize into the cells and, subsequently, improve the transfection efficiency. Study of intracellular uptake indicated that liposomal penetration into bEnd.3 cells, primary astrocytes, and primary neurons occurred through multiple endocytosis pathways and surface modification with Tf and CPP enhanced the transfection efficiency of the nanoparticles. A coculture in vitro BBB model reproducing the in vivo anatomophysiological complexity of the biologic barrier was developed to characterize the penetrating properties of these designed liposomes. The dual-functionalized liposomes effectively crossed the in vitro barrier model followed by transfecting primary neurons. Liposome tissue distribution in vivo indicated superior ability of kFGF-Tf-liposomes to overcome BBB and reach brain of the mice after single intravenous administration. These findings demonstrate the feasibility of using strategically designed liposomes by combining Tf receptor targeting with enhanced cell penetration as a potential brain gene delivery vector. SIGNIFICANCE STATEMENT: Rational synthesis of efficient brain-targeted gene carrier included modification of liposomes with a target-specific ligand, transferrin, and with cell-penetrating peptide to enhance cellular internalization. Our study used an in vitro triple coculture blood-brain barrier (BBB) model as a tool to characterize the permeability across BBB and functionality of designed liposomes prior to in vivo biodistribution studies. Our study demonstrated that rational design and characterization of BBB permeability are efficient strategies for development of brain-targeted gene carriers.

Hydrophobic and electrostatic interactions between cell penetrating peptides and plasmid DNA are important for stable non-covalent complexation and intracellular delivery.

Cell penetrating peptides are useful tools for intracellular delivery of nucleic acids. Delivery of plasmid DNA, a large nucleic acid, poses a challenge for peptide mediated transport. The paper investigates and compares efficacy of five novel peptide designs for complexation of plasmid DNA and subsequent delivery into cells. The peptides were designed to contain reported DNA condensing agents and basic cell penetrating sequences, octa-arginine (R8 ) and CHK6 HC coupled to cell penetration accelerating peptides such as Bax inhibitory mutant peptide (KLPVM) and a peptide derived from the Kaposi fibroblast growth factor (kFGF) membrane translocating sequence. A tryptophan rich peptide, an analogue of Pep-3, flanked with CH3 on either ends was also a part of the study. The peptides were analysed for plasmid DNA complexation, protection of peptide-plasmid DNA complexes against DNase I, serum components and competitive ligands by simple agarose gel electrophoresis techniques. Hemolysis of rat red blood corpuscles (RBCs) in the presence of the peptides was used as a measure of peptide cytotoxicity. Plasmid DNA delivery through the designed peptides was evaluated in two cell lines, human cervical cancer cell line (HeLa) and (NIH/3 T3) mouse embryonic fibroblasts via expression of the secreted alkaline phosphatase (SEAP) reporter gene. The importance of hydrophobic sequences in addition to cationic sequences in peptides for non-covalent plasmid DNA complexation and delivery has been illustrated. An alternative to the employment of fatty acid moieties for enhanced gene transfer has been proposed. Comparison of peptides for plasmid DNA complexation and delivery of peptide-plasmid DNA complexes to cells estimated by expression of a reporter gene, SEAP. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Inhibition of Basic Secretagogue-Induced Signaling in Mast Cells by Cell Permeable Gαi-Derived Peptides

Background: Basic secretagogues of connective tissue mast cells act as receptor mimetic agents that trigger mast cells by activating G proteins. This leads to simultaneous propagation of two signaling pathways: one that culminates in exocytosis, while the other involves protein tyrosine phosphorylation and leads to release of arachidonic acid metabolites. We have previously shown that introduction of a peptide that comprises the C-terminal end of Gαi₃ into permeabilized mast cells inhibits basic secretagogue-induced exocytosis [Aridor et al., Science 1993;262:1569–1572]. We investigated whether cell-permeable peptides, composed of the C-terminus of Gαi₃ fused with importation sequences, affect mast cell function. Methods: Following preincubation with the fused peptides, rat peritoneal mast cells were activated by compound 48/80 and analyzed for histamine and prostaglandin D₂ release and protein tyrosine phosphorylations. Results: We demonstrate that out of three importation sequences tested only Gαi₃ peptide fused with the Kaposi fibroblast growth factor importation sequence (ALL1) inhibited release of histamine. ALL1 as well as a cell-permeable peptide that corresponds to Gαi₂ also blocked compound 48/80-stimulated protein tyrosine phosphorylation, though the latter did not block histamine release. ALL1 effect was G protein-specific, as it was incapable of blocking protein tyrosine phosphorylation stimulated by pervanadate. Conclusion: ALL1, a transducible Gαi₃-corresponding peptide, blocks the two signaling pathways in mast cells: histamine release and protein tyrosine phosphorylation. Cell permeable peptides that block these two signaling cascades may constitute a novel approach for preventing the onset of the allergic reaction.

Flow cytometric screening of cell-penetrating peptides for their uptake into embryonic and adult stem cells

There is an increasing appreciation of the potential of cell-penetrating peptides (CPPs) as vectors to deliver peptides, proteins, and DNA into cells. However, the absolute and relative efficacy of various CPPs for applications targeting stem cells and primary cells is unclear. In this study, we have developed a two-step loading method and a flow cytometric assay to systematically compare the cellular uptake of five CPPs into embryonic stem cells, neurospheres (NSs), primary bone marrow hematopoietic progenitor (Sca-1 +Lin −) cells, and hematopoietic cell lines (TF-1, K562, and FDCP Mix). The series of CPPs tested included three arginine-rich peptides; one was derived from HIV transactivator of transcription (TAT), one was derived from Antennapedia (Antp), and the third was a synthetic peptide known as protein transduction domain 4 (PTD4). Two hydrophobic peptides were also tested; one was derived from Kaposi fibroblast growth factor (K-FGF), and one was derived from PreS2 surface antigen of hepatitis B virus (PreS2-TLM). Our results indicate, for the first time, that arginine-rich CPPs can internalize into primary NSs and bone marrow Sca-1 +Lin − cells. In addition, in all cell types examined, the uptake of arginine-rich CPPs is significantly greater than that of hydrophobic peptides.

Protein Kinase Cζ Is Up-regulated in Osteoarthritic Cartilage and Is Required for Activation of NF-κB by Tumor Necrosis Factor and Interleukin-1 in Articular Chondrocytes

Protein kinase Czeta (PKCzeta) is an intracellular serine/threonine protein kinase that has been implicated in the signaling pathways for certain inflammatory cytokines, including interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha), in some cell types. A study of gene expression in articular chondrocytes from osteoarthritis (OA) patients revealed that PKCzeta is transcriptionally up-regulated in human OA articular cartilage clinical samples. This finding led to the hypothesis that PKCzeta may be an important signaling component of cytokine-mediated cartilage matrix destruction in articular chondrocytes, believed to be an underlying factor in the pathophysiology of OA. IL-1 treatment of chondrocytes in culture resulted in rapidly increased phosphorylation of PKCzeta, implicating PKCzeta activation in the signaling pathway. Chondrocyte cell-based assays were used to evaluate the contribution of PKCzeta activity in NF-kappaB activation and extracellular matrix degradation mediated by IL-1, TNF, or sphingomyelinase. In primary chondrocytes, IL-1 and TNF-alpha caused an increase in NF-kappaB activity resulting in induction of aggrecanase-1 and aggrecanase-2 expression, with consequent increased proteoglycan degradation. This effect was blocked by the pan-specific PKC inhibitors RO 31-8220 and bisindolylmaleimide I, partially blocked by Gö 6976, and was unaffected by the PKCzeta-sparing inhibitor calphostin C. A cell-permeable PKCzeta pseudosubstrate peptide inhibitor was capable of blocking TNFand IL-1-mediated NF-kappaB activation and proteoglycan degradation in chondrocyte pellet cultures. In addition, overexpression of a dominant negative PKCzeta protein effectively prevented cytokine-mediated NF-kappaB activation in primary chondrocytes. These data implicate PKCzeta as a necessary component of the IL-1 and TNF signaling pathways in chondrocytes that result in catabolic destruction of extracellular matrix proteins in osteoarthritic cartilage.

Site-Specific DNA Excision in Transgenic Rice With a Cell-Permeable Cre Recombinase

The removal of selected marker genes from transgenic plants is necessary to address biosafety concerns and to carry out further experiments with transgenic organisms. In the present study, the 12-amino-acid membrane translocation sequence (MTS) from the Kaposi fibroblast growth factor (FGF)-4 was used as a carrier to deliver enzymatically active Cre proteins into living plant cells, and to produce a site-specific DNA excision in transgenic rice plants. The process, which made cells permeable to Cre recombinase-mediated DNA recombination, circumvented the need to express Cre under spatiotemporal control and was proved to be a simple and efficient system to achieve marker-free transgenic plants. The ultimate aim of the present study is to develop commercial rice cultivars free from selected marker genes to hasten public acceptance of transgenic crops.

Proapoptotic Activity of Cell-Permeable Anti-Akt Single-Chain Antibodies

We developed anti-Akt1 single-chain antibodies (scFv) by panning a mouse phage-displayed scFv recombinant antibody library. Recombinant scFv that bound glutathione S-transferase (GST)-Akt1 were screened for their ability to inhibit Akt activity in vitro in a kinase reaction containing human recombinant Akt1 and an Akt/serum glucocorticoid-inducible kinase (SGK) substrate. Michaelis-Menten analysis of kinase inhibition by a selected scFv was consistent with scFv-mediated competition with enzyme's substrate for the catalytic site of Akt. To generate a membrane-permeable version of the anti-Akt1 scFv, the scFv gene was subcloned into a GST expression vector carrying a membrane-translocating sequence (MTS) from Kaposi fibroblast growth factor. A purified GST-anti-Akt1-MTS fusion protein accumulated intracellularly in 293T, BT-474, and PyVmT cells in a dose- and time-dependent fashion. Intracellular accumulation correlated temporally with inhibition of p-Ser(473) Akt and GSK-3alpha/beta phosphorylation, suggesting that Ser(473) is an Akt autophosphorylation site. Phosphorylated (activated) phosphoinositide-dependent kinase 1, mitogen-activated protein kinase, p38, and HER2 (erbB2) were not affected, supporting Akt kinase specificity for the inhibitory scFv. Exogenously expressed constitutively active Akt2 and Akt3 were also inhibited in vitro by the anti-Akt1 fusion protein. Furthermore, GST-anti-Akt1-MTS induced apoptosis in three cancer cell lines that express constitutively active Akt. Finally, systemic treatment with the anti-Akt scFv reduced tumor volume and neovascularization and increased apoptosis in PyVmT-expressing transgenic tumors implanted in mouse dorsal window chambers. Thus, GST-anti-Akt1-MTS is a novel cell-permeable inhibitor of Akt, which selectively inhibits Akt-mediated survival in intact cells both in vitro and in vivo.

Oral immunization of Carassius auratus with modified recombinant A-layer proteins entrapped in alginate beads

This study was focused on the utilization of a recombinant expression system to produce a unique modified subunit vaccine possessing a self-contained delivery system which could potentially improve the uptake and delivery of vaccine products as well their immunogenic potential. For this purpose the A-layer protein (At-R) associated with the fish pathogen atypical Aeromonas salmonicida was cloned and modified by the genetic fusion of the protein transduction domain (MTS) derived from Kaposi fibroblast growth factor (At-MTS). The potential for these proteins to be employed as antigens for oral immunization of goldfish was examined by encapsulation of At-R, At-MTS and the control, BSA, into biodegradable alginate gel macrospheres which were fed to goldfish in place of standard pellet fish feed. The bead physical properties were modified only in the presence of At-R and the temporal release of proteins was significantly less when At-MTS was employed. Western blot analysis of serum samples collected from fish following intubation with the recombinant proteins determined that the rate of protein uptake from the digestive tract into the blood system improved considerably when MTS was fused to At-R. Experimental fish were fed one of three protein–alginate formulae on a schedule of 3 days/week or 5 days/month for a period of 2 months. After 1 month, animals fed on the 5-day protocol demonstrated increased serum antibody titers while following an additional month of feeding this level decreased and titers were found to be higher in fish maintained on the 3-day regime. Fish fed At-MTS maintained the highest titer at the end of 2-month period. To determine whether the diminished antibody titers were a result of oral tolerance fish were injected intraperitoneally with the At-R antigen. Only experimental groups which had been fed At-R or At-MTS demonstrated increased antibody titers which paralleled a typical secondary humoral response. In spite of the presence of an increased titer to A-protein, vaccinated fish did not demonstrate resistance to infection with atypical A. salmonicida.

A structure-activity study of the inhibition of HIV-1 Tat-dependent trans-activation by mixmer 2'-O-methyl oligoribonucleotides containing locked nucleic acid (LNA), alpha-L-LNA, or 2'-thio-LNA residues.

The HIV-1 trans-activation responsive element (TAR) RNA stem-loop interacts with the HIV trans-activator protein Tat and other cellular factors to stimulate transcriptional elongation from the viral long terminal repeat (LTR). Inhibitors of these interactions block full-length transcription and, hence, would potentially inhibit HIV replication. We have studied structure-activity relationships in inhibition of trans-activation by steric block 2'-O-methyl (OMe) oligonucleotides chimeras (mixmers) containing locked nucleic acid (LNA) units. Inhibition was measured both in Tat-dependent in vitro transcription from an HIV-1 DNA template directed by HeLa cell nuclear extract and in a robust HeLa cell reporter assay that involves use of stably integrated plasmids to express firefly luciferase Tat dependently and Renilla luciferase Tat-independently. OMe oligonucleotides with optimally 40%-50% LNA units and a minimum of 12 residues in length were active in the cellular assay when delivered with cationic gemini surfactant GS11 at 50% inhibitory concentrations of 230 +/- 40 nM, whereas activity in the in vitro transcription assay was observed down to 9 residues. No cellular activity was observed for OMe oligonucleotides of 12 or 16 residues, which was shown to be due to poor cellular uptake. Both 12-mer mixmers containing alpha -L-LNA or 2'-thio-LNA (S-LNA) were also active in in vitro transcription and the former in cellular reporter inhibition assays, demonstrating that the property of promotion of cellular uptake by LNA is not due to specific sugar conformational effects. Covalent conjugates of OMe/LNA chimeras with Kaposi-fibroblast growth factor (K-FGF) or Transportan peptides failed to enter HeLa cells without a delivery agent but were fully active when delivered by cationic gemini surfactant, showing that in principle, peptide conjugation does not interfere with cellular activity. Thus, OMe/LNA mixmers are powerful reagents for use as steric block inhibitors of gene expression regulated by protein-RNA interactions within HeLa cell nuclei.

A quantitative validation of fluorophore-labelled cell-permeable peptide conjugates: fluorophore and cargo dependence of import

Cell-permeable peptides were evaluated for a quantitatively controlled import of small molecules. The dependence of the import efficiency on the fluorophore, on the position of the fluorophore as well as on the nature of the cargo were addressed. Cellular uptake was quantitated by flow cytometry and fluorescence correlation microscopy (FCM). Fluorophores with different spectral characteristics, covering the whole visible spectral range, were selected in order to enable the simultaneous detection of several cell-permeable peptide constructs. The transcytosis sequences were based either on the sequence of the Antennapedia homeodomain protein (AntpHD)-derived penetratin peptide or the Kaposi fibroblast growth factor (FGF)-derived membrane translocating sequence (MTS)-peptide. In general, the AntpHD-derived peptides had a three- to fourfold higher import efficiency than the MTS-derived peptides. In spite of the very different physicochemical characteristics of the fluorophores, the import efficiencies for analogues labelled at different positions within the sequence of the import peptides showed a strong positive correlation. However, even for peptide cargos of very similar size, pronounced differences in import efficiency were observed. The use of cell-permeable peptide/cargo constructs for intracellular analyses of structure–function relationships therefore requires the determination of the intracellular concentrations for each construct individually.

Nuclear factor (NF)-κB blockade attenuates but does not abrogate LPS-mediated interleukin (IL)-1β biosynthesis in alveolar epithelial cells

The role that the nuclear factor (NF)-κB plays in regulating the biosynthesis of interleukin (IL)-1β, an inflammatory cytokine, has been investigated in vitro. Irreversible inhibition of the proteasome complex by carbobenzoxy- l-leucyl- l-leucyl- l-leucinal (MG-132; 1– 50 μM ) had no inhibitory effect on lipopolysaccharide (LPS)-mediated IL-1β biosynthesis. Furthermore, selective inhibition of NF-κB by the action of caffeic acid phenylethyl ester (CAPE; 1– 100 μM ) and sulfasalazine (SSA; 0.1–10 mM), a potent and irreversible inhibitor of NF-κB, partially attenuated but did not abolish LPS-dependent IL-1β secretion. Incorporation of a selectively permeant inhibitor of NF-κB, SN-50 (1– 20 μM ), a peptide which contains the nuclear localization sequence (NLS) for the p50 NF-κB subunit and the amino-terminal sequence of Kaposi fibroblast growth factor to promote cell permeability, attenuated in a dose-dependent manner LPS-mediated release of IL-1β. It is concluded that the NF-κB pathway is partially implicated and its blockade attenuates but does not abrogate LPS-dependent IL-1β biosynthesis in alveolar epithelial cells.

Ability of the hydrophobic FGF and basic TAT peptides to promote cellular uptake of recombinant Cre recombinase: a tool for efficient genetic engineering of mammalian genomes.

Conditional mutagenesis is a powerful tool to analyze gene functions in mammalian cells. The site-specific recombinase Cre can be used to recombine loxP-modified alleles under temporal and spatial control. However, the efficient delivery of biologically active Cre recombinase to living cells represents a limiting factor. In this study we compared the potential of a hydrophobic peptide modified from Kaposi fibroblast growth factor with a basic peptide derived from HIV-TAT to promote cellular uptake of recombinant Cre. We present the production and characterization of a Cre protein that enters mammalian cells and subsequently performs recombination with high efficiency in a time- and concentration-dependent manner. Histidine-tagged Cre recombinase transduced inefficiently unless fused to a nuclear localization signal (NLS). Fusion of NLS-Cre to the fibroblast growth factor transduction peptide did not improve the transducibility, whereas fusion with the TAT peptide significantly enhanced cellular uptake and subsequent recombination. More than 95% recombination efficiency in fibroblast cells, as well as murine embryonic stem cells, was achieved after His-TAT-NLS-Cre transduction. Efficient recombination could also be obtained in primary splenocytes ex vivo. We expect that application of His-TAT-NLS-Cre, which can be produced readily in large quantities from a bacterial source, will expand our abilities to manipulate mammalian genomes.

Nuclear Factor-κB-Independent Regulation of Lipopolysaccharide-Mediated Interleukin-6 Biosynthesis

The possible involvement of nuclear factor (NF)-κB in mediating the regulation of interleukin (IL)-6 biosynthesis in response to E. coli-derived lipopolysaccharide-endotoxin (LPS) was investigated in vitro. In alveolar epithelial cells, irreversible inhibition of the proteasome complex by carbobenzoxy- l-leucyl- l-leucyl- l-leucinal (MG-132; 1–50 μM) did not affect LPS-mediated IL-6 secretion. Whereas the selective inhibition of the NF-κB pathway by the action of caffeic acid phenyl ethyl ester (CAPE; 1–100 μM) attenuated LPS-dependent IL-6 production at 100 μM, sulfasalazine (SSA; 0.1–10 mM), a potent and irreversible inhibitor of NF-κB, did not inhibit LPS-dependent IL-6 secretion. Incorporation of a selectively permeant inhibitor of NF-κB, SN-50 (1–20 μM), a peptide which contains the nuclear localization sequence (NLS) for the p50 NF-κB subunit and the amino-terminal sequence of Kaposi fibroblast growth factor to promote cell permeability, did not reduce LPS-mediated release of IL-6. These data indicate a NF-κB-independent pathway mediating LPS-dependent regulation of IL-6 biosynthesis in the airway epithelium.

Epigenetic regulation of gene structure and function with a cell-permeable Cre recombinase

Studies of mammalian gene function are hampered by temporal limitations in which phenotypes occurring at one stage of development interfere with analysis at later stages. Moreover, phenotypes resulting from altered gene activity include both direct and indirect effects that may be difficult to distinguish. In the present study, recombinant fusion proteins bearing the 12 amino acid membrane translocation sequence (MTS) from the Kaposi fibroblast growth factor (FGF-4) were used to transduce enzymatically active Cre proteins directly into mammalian cells. High levels of recombination were observed in a variety of cultured cell types and in all tissues examined in mice following intraperitoneal administration. This represents the first use of protein transduction to induce the enzymatic conversion of a substrate in living cells and animals and provides a rapid and efficient means to manipulate mammalian gene structure and function.

Nuclear Factor-κB Blockade Attenuates but Does Not Abrogate Lipopolysaccharide-Dependent Tumor Necrosis Factor-α Biosynthesis in Alveolar Epithelial Cells

We have investigated the role that the nuclear factor (NF)-κB plays in regulating the biosynthesis of tumor necrosis factor (TNF)-α, an inflammatory cytokine. Irreversible inhibition of the proteasome complex by carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG-132; 1–50 μM) had no inhibitory effect on LPS-mediated TNF-α biosynthesis. Furthermore, selective inhibition of NF-κB by the action of caffeic acid phenylethyl ester (CAPE; 1–100 μM) and sulfasalazine (SSA; 0.1–10 mM), a potent and irreversible inhibitor of NF-κB, partially attenuated, but did not abolish, LPS-dependent TNF-α secretion. Incorporation of a selectively permeant inhibitor of NF-κB, SN-50 (1–20 μM), a peptide which contains the nuclear localization sequence (NLS) for the p50 NF-κB subunit, and the amino-terminal sequence of Kaposi fibroblast growth factor to promote cell permeability, attenuated in a dose-dependent manner LPS-mediated release of TNF-α. It is concluded that the NF-κB pathway is partially implicated and that its blockade attenuates, but does not abrogate, LPS-dependent TNF-α biosynthesis in alveolar epithelial cells.

Dissecting intracellular signaling pathways with membrane-permeable peptides.

Peptides can be designed that mimic protein interaction motifs and thus, can be used to specifically and selectively block particular steps in signal transduction cascades where protein interactions have been previously identified. This protocol describes methods to synthesize peptides coupled to a membrane-permeable sequence (MPS), designed from the signal sequence of Kaposi fibroblast growth factor, which has been previously shown to translocate covalently attached cargo peptides across the cell membrane. To increase efficiency, yield, and versatility in the preparation of these membrane-permeable peptides, a modular synthesis strategy based on two unprotected peptide segments was designed. The modular synthesis strategy allows the MPS and functional peptides to be synthesized separately. In this manner, the functional domain of a peptide or protein, synthesized by traditional fluoroenylmethyloxy-carbonyl (Fmoc) chemistry or derived from recombinant expression, may be purchased commercially to expedite synthesis. Subsequently, the MPS domain may be attached to any functional domain using a one-step conjugation reaction. This protocol provides detailed methods for peptide synthesis, activation of the MPS, and the subsequent conjugation protocol.

Altered basic fibroblast growth factor expression in common epidermal neoplasms: Examination with in situ hybridization and immunohistochemistry

Background: The fibroblast growth factor family consists of acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), and Kaposi fibroblast growth factor (kFGF). The distribution of these growth factors in skin disease has not been determined. Objective: The purpose of this study was to determine the distribution of these growth factors in keratinocytic lesions and normal skin. Methods: Skin sections from common disorders of keratinocytes were examined by in situ hybridization with specific probes for aFGF, bFGF, and kFGF, and immunohistochemistry. Results: Of the growth factors studied, only bFGF was present in skin. bFGF messenger RNA was highly expressed in both normal epidermis and benign and malignant epithelial neoplasms. In normal skin, bFGF was expressed predominantly in a suprabasal fashion, whereas in epithelial neoplasms, homogeneous high-level expression of bFGF was observed. Conclusion: bFGF is the primary member of the fibroblast growth factor expressed in the skin. The source of synthesis of bFGF is keratinocytes. Immunoreactivity for bFGF appears to be primarily confined to upper layers of the epidermis in normal skin, but is expressed at all layers of the epidermis in both benign and malignant neoplastic conditions. Genetic changes that promote epithelial tumors may also promote translation of bFGF messenger RNA into protein. Specific inhibition of bFGF activity may have application in the treatment of common skin diseases. (J Am Acad Dermatol 2000;42:973–7.)

Altered basic fibroblast growth factor expression in common epidermal neoplasms: Examination with in situ hybridization and immunohistochemistry

Background: The fibroblast growth factor family consists of acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), and Kaposi fibroblast growth factor (kFGF). The distribution of these growth factors in skin disease has not been determined. Objective: The purpose of this study was to determine the distribution of these growth factors in keratinocytic lesions and normal skin. Methods: Skin sections from common disorders of keratinocytes were examined by in situ hybridization with specific probes for aFGF, bFGF, and kFGF, and immunohistochemistry. Results: Of the growth factors studied, only bFGF was present in skin. bFGF messenger RNA was highly expressed in both normal epidermis and benign and malignant epithelial neoplasms. In normal skin, bFGF was expressed predominantly in a suprabasal fashion, whereas in epithelial neoplasms, homogeneous high-level expression of bFGF was observed. Conclusion: bFGF is the primary member of the fibroblast growth factor expressed in the skin. The source of synthesis of bFGF is keratinocytes. Immunoreactivity for bFGF appears to be primarily confined to upper layers of the epidermis in normal skin, but is expressed at all layers of the epidermis in both benign and malignant neoplastic conditions. Genetic changes that promote epithelial tumors may also promote translation of bFGF messenger RNA into protein. Specific inhibition of bFGF activity may have application in the treatment of common skin diseases. (J Am Acad Dermatol 2000;42:973–7.)

Inhibition of Calpain Blocks Platelet Secretion, Aggregation, and Spreading

Previous studies have indicated that the Ca(2+)-dependent protease, calpain, is activated in platelets within 30-60 s of thrombin stimulation, but specific roles of calpain in platelets remain to be identified. To directly test the functions of calpain during platelet activation, a novel strategy was developed for introducing calpain's specific biological inhibitor, calpastatin, into platelets prior to activation. This method involves treatment of platelets with a fusion peptide, calpastat, consisting of the cell-penetrating signal sequence from Kaposi's fibroblast growth factor connected to a calpain-inhibiting consensus sequence derived from calpastatin. Calpastat specifically inhibits thrombin peptide (SFLLR)-induced alpha-granule secretion (IC(50) = 20 microM) during the first 30 s of activation, thrombin-induced platelet aggregation (IC(50) = 50 microM), and platelet spreading on glass surfaces (IC(50) = 34 microM). Calpastat-Ala, a mutant peptide in which alanine is substituted at conserved calpastatin residues, lacks calpain inhibitory activity and fails to inhibit secretion, aggregation, or spreading. The peptidyl calpain inhibitors calpeptin, MDL 28,170 (MDL) and E64d also inhibit secretion, aggregation and spreading, but require 3-10-fold higher concentrations than calpastat for biological activity. Together, these findings demonstrate that calpain regulates platelet secretion, aggregation, and spreading and indicate that calpain plays an earlier role in platelet activation following thrombin receptor stimulation than had been previously detected.

5-Lipoxygenase Compartmentalization in Granulocytic Cells Is Modulated by an Internal Bipartite Nuclear Localizing Sequence and Nuclear Factor κB Complex Formation

A region of basic amino acids spanning residues 639–656 in the human 5-lipoxygenase sequence resembles a consensus bipartite nuclear localizing sequence. A synthetic peptide consisting of the Kaposi fibroblast growth factor signal sequence fused to the 5-lipoxygenase639–656bipartite nuclear localizing sequence has a prominent inhibitory effect on 5-lipoxygenase catalysis in granulocytic HL-60 cells activated by calcium ionophor A23187. Recombinant 5-lipoxygenase was not affected by the peptide. The peptide also inhibited redistribution of 5-lipoxygenase from the cytosol to the nuclear membrane of HL-60 cells stimulated by A23187. 5-Lipoxygenase protein was detected in nuclear factor κB (NF-κB) p65 subunit immunoprecipitate fractions prepared from HL-60 cell lysates. The amount of 5-lipoxygenase protein coimmunoprecipitated by NF-κB antiserum was increased following A23187 stimulation. In cells treated with agents that block 5-lipoxygenase translocation to the nucleus, 5-lipoxygenase protein appearing in the NF-κB immunoprecipitate was diminished. Our results implicate an internal bipartite nuclear localizing sequence as a regulatory domain that modulates 5-lipoxygenase redistribution and catalysis in granulocytic cells. Additionally, our results suggest that molecular determinants which govern 5-lipoxygenase and NF-κB redistribution to the nucleus may be coordinately controlled in granulocytic cells.