Energy-dependent Manner Research Articles

A Leu–Lys-rich antimicrobial peptide: activity and mechanism

To develop novel antibiotic peptides useful as therapeutic drugs, the analogues were designed to increase not only net positive charge by Lys substitution but also hydrophobic helix region by Leu substitution from cecropin A (1–8)–magainin 2 (1–12) hybrid peptide (CA–MA). In particular, CA–MA analogue P5 (P5), designed by flexible region (GIG→P) substitution, Lys (positions 4, 8, 14, 15) and Leu (positions 5, 6, 12, 13, 16, 17, 20) substitutions, showed an enhanced antimicrobial and antitumor activity without hemolysis. Confocal microscopy showed that P5 was located in the plasma membrane. The antibacterial effects of analogues were further confirmed by using 1,6-diphenyl-1,3,5-hexatriene as a plasma membrane probe. Flow cytometric analysis revealed that P5 acted in an energy-independent manner. This interaction is also independent of the ionic environment. Furthermore, P5 causes significant morphological alterations of the bacterial surfaces as shown by scanning electron microscopy and showed strong membrane disrupting activity when examined using liposomes (phosphatidyl choline/cholesterol; 10:1, w/w). Its potent antibiotic activity suggests that P5 is an excellent candidate as a lead compound for the development of novel antiinfective agents.

ABCG2 confers resistance to indolocarbazole compounds by ATP-dependent transport

The ABC half-transporter, ABCG2, is known to confer resistance to chemotherapeutic agents including indolocarbazole derivatives. MCF7 cells were introduced by either wild type ABCG2 (ABCG2-482R) or mutant ABCG2 (-482T), whose amino acid at position 482 is substituted to threonine from arginine, and their cross-resistance pattern was analyzed. Although this amino acid substitution seems to affect cross-resistance patterns, both 482T- and 482R-transfectants showed strong resistance to indolocarbazoles, confirming that ABCG2 confers resistance to them. For further characterization of ABCG2-mediated transport, we investigated indolocarbazole compound A ( Fig. 1) excretion in cell-free system. Compound A was actively transported in membrane vesicles prepared from one of the 482T- transfectants and its uptake was supported by hydrolysis of various nucleoside triphosphates. This transport was inhibited completely by the other indolocarbazole compound, but not by mitoxantrone, implying that the binding site of mitoxantrone or the transport mechanisms for mitoxantrone is different from those of indolocarbazoles. These results showed that ABCG2 confers resistance to indolocarbazoles by transporting them in an energy-dependent manner.

Influence on the plasma membrane of Candida albicans by HP (2–9)–magainin 2 (1–12) hybrid peptide

A 20-residue hybrid peptide (HP (2–9)–MA (1–12): HP–MA), incorporating 2–9 residues of Helicobacter pyroli ribosomal protein L1 (HP) and 1–12 residues of magainin 2 (MA), has more potent antibacterial activity than parent peptide HP (2–20) and magainin 2. In this study, the antifungal activity and its mechanism of HP–MA were investigated. HP–MA displayed a strong antifungal activity in an energy-dependent manner. To elucidate the antifungal mechanism(s) of HP–MA, FACScan analysis and the change in membrane dynamics using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a membrane probe of Candida albicans were examined. The results indicated that the HP–MA exerts its antifungal effect by acting on the plasma membrane. Furthermore, the peptide induced remarkable morphological change when tested for membrane disrupting activity using liposomes (PC/Cholesterol; 10:1, w/w). In C. albicans, dimorphism plays a crucial role in pathogenesis but HP–MA could disrupt the mycelial forms and exert its antifungal effect on the blastoconidia in 20% fetal bovine serum.

The influence of bovine casein-derived exorphins on mast cells in rodents

The secretagogue effects of α-casein (90–95) and β-casomorphin-7 on two populations of mast cells in rodents were investigated. Mast cells of “connective tissue type” were obtained from rat peritoneal cavity (PMC), and “mucosal-like type” mast cells (BMMC) were cultured from mouse bone marrow cells. β-Hexosaminidase activity was considered to be a marker of the cells’ secretory response. No reaction of BMMC on exorphins was found at up to millimolar concentrations. β-Casomorphin-7 and α-casein (90–95) exert secretagogue effect on PMC at milli- and micromolar concentrations, respectively. For this reason, further experiments were performed with α-casein (90–95) only. The PMC were activated in a non-cytotoxic, dose-, temperature-, and energy-dependent manner. Although PMC were responsive in a calcium and magnesium free buffer, the reaction was abolished when preincubation with EDTA was performed. Preincubation with opiate antagonist, naloxone, was effective only at millimolar concentration range. The significant decrease in PMC reactivity to α-casein (90–95) after preincubation with benzalkonium chloride suggests a peptidergic pathway of activation which involves a membrane-assisted but receptor-independent stimulation of G i-like proteins.

Antifungal mechanism of SMAP-29 (1–18) isolated from sheep myeloid mRNA against Trichosporon beigelii

The antifungal activity and mechanism of SMAP-29 (1–18) (SMAP-29), a cathelicidin-derived antimicrobial peptide deduced from N-terminal sequence of sheep myeloid mRNA, were investigated. SMAP-29 displayed a strong antifungal activity against various fungi. To understand the antifungal mechanism(s) of SMAP-29, we examined the interaction of SMAP-29 with the pathogenic fungus Trichosporon beigelii. Confocal microscopy showed that SMAP-29 was localized in the plasma membrane. The antifungal effects of SMAP-29 were further confirmed by using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a plasma membrane probe. Flow cytometric analysis revealed that SMAP-29 acted in an energy-dependent manner. This interaction is also dependent on the ionic environment. Furthermore, SMAP-29 caused significant morphological changes when testing the membrane disrupting activity using liposomes (phosphatidylcholine/cholesterol; 10:1, w/w), as shown by scanning electron microscopy. The results suggest that SMAP-29 may exert its antifungal activity by disrupting the structure of cell membranes, via direct interaction with the lipid bilayers and irregularly disrupted fungal membranes in an energy- and salt-dependent manner.

Low level 809-nm diode laser-induced in vitro stimulation of the proliferation of human gingival fibroblasts.

The authors investigated the effects of low level laser irradiation on the proliferation rate of human gingival fibroblasts (HGF) in vitro. HGF were obtained from gingival connective tissue explants and cultured under standard conditions. 110 cell cultures in their logarithmic growth phase were spread on 96-well tissue culture plates and were irradiated at energy fluences of 1.96-7.84 J/cm(2). Another 110 cultures served as control. An 809-nm semiconductor laser operated at a power output of 10 mW in the cw-mode was used. The time of exposure varied between 75 and 300 seconds. Laser treatment was performed alternatively once, twice, and three times at a 24-hour interval. After lasing, incubation was continued for 24 hours. The proliferation rate was determined by means of fluorescence activity of a redox indicator added to the cell culture. Proliferation was determined 24, 48, and 72 hours after irradiation and expressed in relative fluorescence units (RFU). The irradiated cells revealed a considerably higher proliferation activity. The differences were highly significant 24 hour after irradiation (Mann-Whitney U-test, P < 0.05) but decreased in an energy-dependent manner after 48 and 72 hour after irradiation. A cellular effect of the soft laser irradiation on HGF is evident. Its duration, however, seems to be limited. These findings might be clinically relevant, indicating that repeated treatments are necessary to achieve a positive laser effect in clinical applications.

Secondary-Site Mutation Restores the Transport Defect Caused by the Transmembrane Domain Mutation of the Xenobiotic Transporter MexB in Pseudomonas aeruginosa

It has been suggested that the MexB subunit of the MexAB-OprM efflux transporter of Pseudomonas aeruginosa exports xenobiotics in an energy-dependent manner. To investigate the role of the transmembrane segments (TMS) of MexB in the transporter activity, we isolated 24 spontaneous mutants showing hypersusceptibility to antibiotics. Among them, three mutations were located at TMS-3, TMS-4, and TMS-10 having amino acid substitution Leu376vPro, Gly397vVal, and Val928vGly, respectively. A secondary mutation, which suppressed the defect caused by the Val928vGly mutation in TMS-10, was found at the 403rd amino acid residue in TMS-4 with a change of glycine to serine, suggesting that TMS-4 and TMS-10 may be in close proximity. This result provided strong support for the recent notion that negatively charged residues in TMS-4 might form a salt-bridge with a positive charge in TMS-10 (Guan, L., and Nakae, T. (2001) J. Bacteriol. 183, 1734–1739). The transporter function impaired by the Gly397vVal mutation in TMS-4 was recovered by the secondary mutation, Gln998vHis, in the loop between TMS-11 and TMS-12, thereby suggesting that TMS-4 and TMS-11 or TMS-12 might also be in close proximity. Thus, it is most likely that TMS-4, TMS-10, and TMS-11 or TMS-12 are packed close three dimensionally.

Effect of simulated CO2 and GaAlAs laser surface decontamination on temperature changes in Ti-plasma sprayed dental implants.

To investigate and compare temperature elevations at the implant-bone interface during simulated implant surface decontamination with a CO2 and a GaAlAs laser. Stepped cylinder implants (Frialit 2) Friadent GmbH, Mannheim, Germany) with a Titanium plasma sprayed surface were inserted into bone blocks cut from pig femurs. An artificial periimplant bone defect provided access for laser irradiation in the coronal third. Both lasers were operated at 1.0-2.5 W in the cw-mode. The bone block was placed into a 37 degrees C water bath in order to simulate in vivo thermal conductivity and diffusitivity of heat. K-type thermocouples connected to a digital meter were used to register temperature changes at the periimplant bone. In mean, the critical threshold of 47 degrees C was exceeded after 8 seconds at a power output of 2.5 W, 13 seconds at 2.0 W, 18 seconds at 1.5 W, and 42 seconds at 1.0 W with the GaAlAs laser and 15 seconds (2.5 W), 23 seconds (2.0 W), 35 seconds (1.5 W), and 56 seconds (1.0 W) with the CO2 laser. At equal energy fluence, GaAlAs laser irradiation induced significantly higher temperature elevations than CO2 laser irradiation. In an energy dependent manner implant surface decontamination with both laser types must be limited in time to allow the implant and bone to cool down. Clinical guidelines are presented to avoid tissue damage.

Biochemical characterization of horA-independent hop resistance mechanism in Lactobacillus brevis

We isolated a strain from hop-resistant Lactobacillus brevis ABBC45, which had lost a plasmid (pRH45) harboring a putative hop resistance gene, horA. The hop resistance level of this horA-deficient strain, named ABBC45 C, was initially low but gradually induced by repeated growth in media containing progressively increasing levels of hop compounds. Although the hop resistance level was substantially lower than that of the hop-adapted wild type strain, hop-adapted ABBC45 C (ABBC45 CR) was still capable of growing in beer, suggesting ABBC45 possesses at least two hop resistance mechanisms. Hop resistance acquired by ABBC45 CR gradually diminished to the pre-adapted level, when the strain was grown repeatedly in the absence of hop compounds. ABBC45 CR was found to be cross-resistant to several structurally unrelated drugs, including ethidium bromide, daunomycin and nisin. In addition, ABBC45 CR was shown to extrude ethidium in an energy-dependent manner, while ABBC45 C did not show such activity. This indicates that the efflux pump was induced by adaptation to hop compounds. The efflux activity of ethidium was reduced by the addition of hop compounds, suggesting hop compounds are also the substrate of the efflux pump. It was also shown that the efflux activity was completely dissipated with the abolition of proton motive force (PMF). These results, taken together, suggest the hop resistance mechanism of ABBC45 C is mediated by PMF-dependent multidrug efflux pump.

Nucleocytoplasmic shuttling of polypyrimidine tract-binding protein is uncoupled from RNA export.

Polypyrimidine tract binding protein, PTB/hnRNP I, is involved in pre-mRNA processing in the nucleus and RNA localization and translation in the cytoplasm. In this report, we demonstrate that PTB shuttles between the nucleus and cytoplasm in an energy-dependent manner. Deletion mutagenesis demonstrated that a minimum of the N terminus and RNA recognition motifs (RRMs) 1 and 2 are necessary for nucleocytoplasmic shuttling. Deletion of RRM3 and 4, domains that are primarily responsible for RNA binding, accelerated the nucleocytoplasmic shuttling of PTB. Inhibition of transcription directed by either RNA polymerase II alone or all RNA polymerases yielded similar results. In contrast, selective inhibition of RNA polymerase I did not influence the shuttling kinetics of PTB. Furthermore, the intranuclear mobility of GFP-PTB, as measured by fluorescence recovery after photobleaching analyses, increased significantly in transcriptionally inactive cells compared with transcriptionally active cells. These observations demonstrate that nuclear RNA transcription and export are not necessary for the shuttling of PTB. In addition, binding to nascent RNAs transcribed by RNA polymerase II and/or III retards both the nuclear export and nucleoplasmic movement of PTB. The uncoupling of PTB shuttling and RNA export suggests that the nucleocytoplasmic shuttling of PTB may also play a regulatory role for its functions in the nucleus and cytoplasm.

Effect of Creatine Supplementation on Metabolite Levels in ALS Motor Cortices

Mitochondrial pathology is an early observation in motor neurons and skeletal muscle of patients with amyotrophic lateral sclerosis (ALS). To clarify the relevance of this finding, we determined the effects of a 1-month oral administration of creatine on 1H NMR-visible metabolites in the motor cortices of 15 controls and 15 patients with sporadic ALS, most of whom had mitochondrial pathology in skeletal muscle. In the motor cortex of the ALS group the N-acetylaspartate (NAA)/creatine (Crt) metabolite ratio was lower than in our control group, indicating NAA loss. Upon creatine supplementation we observed in the controls a decline in the NAA/Crt, NAA/choline (Cho), glutamate + glutamine (Glx)/Crt, and Glx/Cho metabolite ratios. In contrast, in the ALS patient group the NAA/Crt and the NAA/Cho metabolite ratios remained unchanged, while the Glx/Crt and Glx/Cho metabolite ratios decreased. These data are compatible with the interpretation that creatine supplementation causes an increase in the diminished NAA levels in ALS motor cortex as well as an increase of choline levels in both ALS and control motor cortices. Because NAA is synthesized by mitochondria in an energy-dependent manner and the NAA/Cho metabolite ratios in the ALS motor cortices were found to be correlated to the degree of mitochondrial pathology in ALS skeletal muscle, our results can be explained by a deficiency of enzymes of mitochondrial respiratory chain in the ALS motor cortex which might affect motor neuron survival.

Cargo delivery kinetics of cell-penetrating peptides

A diversity of cell-penetrating peptides (CPPs), is known, but so far the only common denominator for these peptides is the ability to gain cell entry in an energy-independent manner. The mechanism used by CPPs for cell entry is largely unknown, and data comparing the different peptides are lacking. In order to gain more information about the cell-penetrating process, as well as to quantitatively compare the uptake efficiency of different CPPs, we have studied the cellular uptake and cargo delivery kinetics of penetratin, transportan, Tat (48–60) and MAP (KLAL). The respective CPPs (labelled with the fluorescence quencher, 3-nitrotyrosine) are coupled to small a pentapeptide cargo (labelled with the 2-amino benzoic acid fluorophore) via a disulfide bond. The cellular uptake of the cargo is registered as an increase in fluorescence intensity when the disulfide bond of the CPP–S–S–cargo construct is reduced in the intracellular milieu. Our data show that MAP has the fastest uptake, followed by transportan, Tat(48–60) and, last, penetratin. Similarly, MAP has the highest cargo delivery efficiency, followed by transportan, Tat (48–60) and, last, penetratin. Since some CPPs have been found to be toxic at high concentration, we characterized the influence of CPPs on cellular 2-[ 3H]deoxyglucose-6-phosphate leakage. Measurements on this system show that the membrane-disturbing potential appears to be correlated with the hydrophobic moment of the peptides. In summary, the yield and kinetics of cellular cargo delivery for four different CPPs has been quantitatively characterized.

Internalization of tenecin 3 by a fungal cellular process is essential for its fungicidal effect on Candida albicans.

Tenecin 3 is a glycine-rich, antifungal protein of 78 residues isolated from the insect Tenebrio molitor larva. As an initial step towards understanding the antifungal mechanism of tenecin 3, we examined how this protein interacts with the pathogenic fungus Candida albicans to exert its antifungal action. Tenecin 3 did not induce the release of a fluorescent dye trapped in the artificial membrane vesicles and it did not perturb the membrane potential of C. albicans by the initial interaction. Fluorescence confocal microscopy and flow cytometric analysis revealed that tenecin 3 is rapidly internalized into the cytoplasmic space in energy-dependent and temperature-dependent manners. This internalization is also dependent on the ionic environment and cellular metabolic states. These results suggest that the internalization of tenecin 3 into the cytoplasm of C. albicans is mediated by a fungal cellular process. The internalized tenecin 3 is dispersed in the cytoplasm, and the loss of cell viability occurs after this internalization.

Nucleotide binding domain 1 of the human retinal ABC transporter functions as a general ribonucleotidase.

Members of the ATP binding cassette (ABC) superfamily are transmembrane proteins that are found in a variety of tissues which transport substances across cell membranes in an energy-dependent manner. The retina-specific ABC protein (ABCR) has been linked through genetic studies to a number of inherited visual disorders, including Stargardt macular degeneration and age-related macular degeneration (ARMD). Like other ABC transporters, ABCR is characterized by two nucleotide binding domains and two transmembrane domains. We have cloned and expressed the 522-amino acid (aa) N-terminal cytoplasmic region (aa 854-1375) of ABCR containing nucleotide binding domain 1 (NBD1) with a purification tag at its amino terminus. The expressed recombinant protein was found to be soluble and was purified using single-step affinity chromatography. The purified protein migrated as a 66 kDa protein on SDS-PAGE. Analysis of the ATP binding and hydrolysis properties of the NBD1 polypeptide demonstrated significant differences between NBD1 and NBD2 [Biswas, E. E., and Biswas, S. B. (2000) Biochemistry 39, 15879-15886]. NBD1 was active as an ATPase, and nucleotide inhibition studies suggested that nucleotide binding was not specific for ATP and all four ribonucleotides can compete for binding. Further analysis demonstrated that NBD1 is a general nucleotidase capable of hydrolysis of ATP, CTP, GTP, and UTP. In contrast, NBD2 is specific for adenosine nucleotides (ATP and dATP). NBD1 bound ATP with a higher affinity than NBD2 (K(mNBD1) = 200 microm vs K(mNBD2) = 631 microm) but was less efficient as an ATPase (V(maxNBD1) = 28.9 nmol min(-)(1) mg(-)(1) vs V(maxNBD2) = 144 nmol min(-)(1) mg(-)(1)). The binding efficiencies for CTP and GTP were comparable to that observed for ATP (K(mCTP) = 155 microm vs K(mGTP) = 183 microm), while that observed for UTP was decreased 2-fold (K(mUTP) = 436 microm). Thus, the nucleotide binding preference of NBD1 is as follows: CTP > GTP > ATP >> UTP. These studies demonstrate that NBD1 of ABCR is a general nucleotidase, whereas NBD2 is a specific ATPase.

Differential properties of two putative nuclear localization sequences found in the carboxyl-terminus of human ifn-gamma.

Interferon-gamma (IFN-gamma), a protein that uses the Jak-Stat pathway for signal transduction, translocates rapidly to the nucleus in cells treated extracellularly with the cytokine. A nuclear localization sequence (NLS) has been identified and characterized in the C-terminus of IFN-gamma. Both human and murine IFN-gamma contain this NLS. We show in this report that human IFN-gamma (HuIFN-gamma) contains a second NLS at an upstream site, as determined in standard import assays using digitonin-permeabilized HeLa cells. The primary sequence, analogous with the NLS sequence identified in murine IFN-gamma, representing amino acids 122-132 of HuIFN-gamma was capable of mediating the nuclear import of the autofluorescent protein allophycocyanin (APC) in an energy-dependent manner. The second sequence, representing amino acids 78-92 of HuIFN-gamma, was also capable of mediating the nuclear import of APC in an energy-dependent manner but to a greatly reduced extent. The nuclear import of both sequences conjugated to APC was strongly blocked by competition with unconjugated HuIFN-gamma(122-132). Competition by the sequence HuIFN-gamma(78-92) effectively blocked the import of APC-conjugated HuIFN-gamma(78-92) but, at the same concentration, was not capable of inhibiting the nuclear import of APC-conjugated HuIFN-gamma(122-132), suggesting that HuIFN-gamma(78-92) was a less efficient NLS than HuIFN-gamma(122-132). This is consistent with >90% loss of antiviral activity of HuIFN-gamma lacking the downstream NLS in 122-132. The nuclear import of APC-conjugated HuIFN-gamma(122-132) was inhibited by a peptide containing the prototypical polybasic NLS of the SV40 T NLS, which suggests that the same Ran/importin cellular machinery is used in both cases.

Sphingomyelin trafficking in Chlamydia pneumoniae-infected cells.

Chlamydia pneumoniae is a bacterial obligate intracellular parasite with a developmental cycle common to all members of the genus Chlamydia. Like other chlamydiae, the developmental cycle of C. pneumoniae occurs entirely within a membrane-bound intracellular vacuole, termed an inclusion, that is non-fusogenic with endosomal or lysosomal compartments. To characterize the vesicular interactions of the C. pneumoniae inclusion, we used a fluorescent analogue of ceramide, (N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]-6-aminocaproyl-Derythro-sphingosine (C6-NBD-Cer), that has previously been used to characterize the endogenous synthesis and transport of sphingolipids from the Golgi apparatus to Chlamydia trachomatis and Chlamydia psittaci inclusions. Sphingolipids are trafficked to C. pneumoniae inclusions in a time-, temperature- and energy-dependent manner with properties very similar to the delivery of sphingomyelin to C. trachomatis inclusions. These results indicate that interactions of the inclusion with a subset of sphingomyelin-containing exocytic vesicles is a property common to all species of chlamydiae.

Characterization of the amino acid transport of new immortalized choroid plexus epithelial cell lines: a novel in vitro system for investigating transport functions at the blood-cerebrospinal fluid barrier.

To establish and characterize a choroid plexus epithelial cell line (TR-CSFB) from a new type of transgenic rat harboring the temperature-sensitive simian virus 40 (ts SV 40) large T-antigen gene (Tg rat). Choroid plexus epithelial cells were isolated from the Tg rat and cultured on a collagen-coated dish at 37 degrees C during the first period of 3 days. Cells were subsequently cultured at 33 degrees C to activate large T-antigen. At the third passage, cells were cloned by colony formation and isolated from other cells using a penicillin cup. Five immortalized cell lines of choroid plexus epithelial cells (TR-CSFB 1 approximately 5) were obtained from two Tg rats. These cell lines had a polygonal cell morphology, expressed the typical choroid plexus epithelial cell marker, transthyretin, and possessed Na+, K+-ATPase on their apical side. TR-CSFBs cells expressed a large T-antigen and grew well at 33 degrees C with a doubling-time of 35 approximately 40 hr. [3H]-L-Proline uptake by TR-CSFB cells took place in an Na+-dependent, ouabain-sensitive, energy-dependent, and concentration-dependent manner. It was also inhibited by alpha-methylaminoisobutylic acid, suggesting that system A for amino acids operates in TR-CSFB cells. When [3H]-L-proline uptake was measured using the Transwell device, the L-proline uptake rate following application to the apical side was five-fold greater than that following application to the basal side. In addition, both Na+-dependent and Na+-independent L-glutamic acid (L-Glu) uptake processes were present in TR-CSFB cells. Immortalized choroid plexus epithelial cell lines were successfully established from Tg rats and have the properties of choroid plexus epithelial cells, and amino acid transport activity was observed in vivo.

Apoptosis after experimental stroke: fact or fashion?

This review examines the appearance of hallmarks of apoptosis following experimental stroke. The reviewed literature leaves no doubt that ischemic cell death in the brain is active, that is, requires energy; is gene directed, that is, requires new gene expression; and is capase-mediated, that is, uses apoptotic proteolytic machinery. However, sufficient differences to both classical necrosis and apoptosis exist which prevent easy mechanistic classification. It is concluded that ischemic cell death in the brain is neither necrosis nor apoptosis but is a chimera which appears on a continuum that has apoptosis and necrosis at the poles. The position on this continuum could be modulated by the intensity of the ischemic injury, the consequent availability of ATP and new protein synthesis, and both the age and context of the neuron in question. Thus the ischemic neuron may look necrotic but have actively died in an energy dependent manner with new gene expression and destruction via the apoptotic proteolytic machinery.

Proteomics of rat liver Golgi complex: minor proteins are identified through sequential fractionation.

The discovery of novel proteins resident to the Golgi complex will fuel our future studies of Golgi structure/function and provide justification for proteomic analysis of this organelle. Our approach to Golgi proteomics was to first isolate and characterize the intact organelle free of proteins in transit by use of tissue pretreated with cycloheximide. Then the stacked Golgi fraction was fractionated into biochemically defined subfractions: Triton X-114 insoluble, aqueous, and detergent phases. The aqueous and detergent phases were further fractionated by anion-exchange column chromatography. In addition, radiolabeled cytosol was incubated with stacked Golgi fractions containing proteins in transit, and the proteins bound to the Golgi stacks in an energy-dependent manner were characterized. All fractions were analyzed by two-dimensional (2-D) gel electrophoresis and identification numbers were given to 588 unique 2-D spots. Tandem mass spectrometry was used to analyze 93 of the most abundant 2-D spots taken from preparative Triton X-114 insoluble, aqueous and detergent phase 2-D gels. Fifty-one known and 22 unknown proteins were identified. This study represents the first installment in the mammalian Golgi proteome database. Our data suggest that cell fractionation followed by biochemical dissection of specific classes of molecules provides a significant advantage for the identification of low abundance proteins in organelles.

Novel cell permeable motif derived from the PreS2-domain of hepatitis-B virus surface antigens.

Efficient transfer of proteins or nucleic acids across cellular membranes is a major problem in cell biology. Recently the existence of a fusogenic sequence was predicted in the junction area of the PreS2- and S-domain of the hepatitis-B virus surface antigens. We have identified cell permeability as a novel property of the PreS2-domain. Cell permeability of PreS2 is not restricted to hepatocytes. PreS2 translocates in an energy-independent manner into cells and is evenly distributed over the cytosol. Detailed analysis revealed that cell-permeability is mediated by an amphipatic alpha-helix between amino acids 41 and 52 of PreS2. Destruction of this translocation motif (PreS2-TLM) abolishes cell permeability. PreS2-TLM per se can act as a shuttle for peptides and functional proteins (such as EGFP). This permits the highly specific modulation of intracellular signal transduction by transfer of peptides competing protein-protein interactions as demonstrated by specific inhibition of TNFalpha-dependent activation of c-Raf-1 kinase. Moreover in vivo functionality was demonstrated by PreS2-TLM-dependent protein transfer into primary bone marrow cells and into the liver. The amphipatic motif is conserved between the different hepatitis-B virus subtypes, and the surface proteins of avian and rodent hepadnaviruses exhibit similar amphipatic peptide sequences. In respect to hepatitis-B virus-infection, the PreS2-TLM could represent the postulated fusion peptide and play a crucial role in the internalization of the viral particle.