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PubMed2001

Chronic venous insufficiency and venous leg ulceration.

UNLABELLED: Venous ulcers are the most common form of leg ulcers. Venous disease has a significant impact on quality of life and work productivity. In addition, the costs associated with the long-term care of these chronic wounds are substantial. Although the exact pathogenic steps leading from venous hypertension to venous ulceration remain unclear, several hypotheses have been developed to explain the development of venous ulceration. A better understanding of the current pathophysiology of venous ulceration has led to the development of new approaches in its management. New types of wound dressings, topical and systemic therapeutic agents, surgical modalities, bioengineered tissue, matrix materials, and growth factors are all novel therapeutic options that may be used in addition to the "gold standard," compression therapy, for venous ulcers. This review discusses current aspects of the epidemiology, pathophysiology, clinical presentation, diagnostic assessment, and current therapeutic options for chronic venous insufficiency and venous ulceration. (J Am Acad Dermatol 2001;44:401-21.) LEARNING OBJECTIVE: At the conclusion of this learning activity, participants should be familiar with the 3 main types of lower extremity ulcers and should improve their understanding of the epidemiology, pathogenesis, risk factors, clinical presentation, diagnostic assessment, and current therapies for chronic venous insufficiency and venous ulcers.

Anti-Bacterial Agents↗
PubMed2003

Isolation and identification of short nucleotide sequences that affect translation initiation in Saccharomyces cerevisiae.

In previous studies, we demonstrated the sufficiency of short nucleotide sequences to facilitate internal initiation of translation in mammalian cells. By using a selection methodology, we have now identified comparable sequences in Saccharomyces cerevisiae. For these studies, a library of constructs expressing dicistronic mRNAs with the HIS3 gene as the second cistron and 18 random nucleotides in the intercistronic region was introduced into a yeast strain in which the endogenous HIS3 gene was deleted. Untransformed cells or those containing the parent construct failed to grow on medium lacking histidine. Intercistronic sequences recovered from cells that did grow were evaluated by using various criteria. Fifty-six of the 18-nt sequences (approximately 1/400,000) functioned as synthetic internal ribosome entry sites (IRESes). The 14 most active sequences allowed growth in the presence of 0.1-0.6 mM 3-amino-1,2,4-triazole, a competitive inhibitor of the HIS3 gene product. In addition, eight sequences were identified that were not IRESes, but that enhanced HIS3 expression by an alternative mechanism that depended on the 5' end of the mRNA and appeared to involve either shunting or reinitiation. Comparisons among the 56 selected IRESes identified eight significant sequence matches containing up to 10 nucleotides. Many of the selected sequences also contained extensive complementary matches to yeast 18S rRNA, some at overlapping sites. The identification of cis sequences that facilitate translation initiation in yeast enables detailed biochemical and genetic analyses of underlying mechanisms and may have practical applications for bioengineering.

Base Sequence↗
PubMed2000

A protein-based therapeutic for human cytomegalovirus infection.

Current antiviral strategies target viral gene products. Although initially successful, their severe toxicity and susceptibility to circumvention by the generation of drug-resistant variants limit their usefulness. By contrast, the central role of the host cell serine endoprotease furin in the proteolytic activation of numerous pathogens points to the endoprotease as a strategic target for therapeutics. Herein, we show that the production of infectious human cytomegalovirus is dramatically reduced by exogenous addition of a bioengineered serpin, alpha(1)-PDX. This protein is a potent and selective furin inhibitor (K(i) = 0.6 nM) and is 10-fold more effective than currently used antiherpetic agents in cell-culture models. The requirement of furin for the processing of envelope glycoproteins from many pathogenic viruses and for the activation of several bacterial toxins suggests that selective inhibitors of furin have potential as broad-based anti-pathogens.

Blotting, Western↗
PubMed2006

Time scales of membrane fusion revealed by direct imaging of vesicle fusion with high temporal resolution.

Membrane fusion is a vital process of life involved, for example, in cellular secretion via exocytosis, signaling between nerve cells, and virus infection. In both the life sciences and bioengineering, controlled membrane fusion has many possible applications, such as drug delivery, gene transfer, chemical microreactors, or synthesis of nanomaterials. Until now, the fusion dynamics has been elusive because direct observations have been limited to time scales that exceed several milliseconds. Here, the fusion of giant lipid vesicles is induced in a controlled manner and monitored with a temporal resolution of 50 micros. Two different fusion protocols are used that are based on synthetic fusogenic molecules and electroporation. For both protocols, the opening of the fusion necks is very fast, with an average expansion velocity of centimeters per second. This velocity indicates that the initial formation of a single fusion neck can be completed in a few hundred nanoseconds.

Animals↗
PubMed2006

Engineering broad root-knot resistance in transgenic plants by RNAi silencing of a conserved and essential root-knot nematode parasitism gene.

Secreted parasitism proteins encoded by parasitism genes expressed in esophageal gland cells mediate infection and parasitism of plants by root-knot nematodes (RKN). Parasitism gene 16D10 encodes a conserved RKN secretory peptide that stimulates root growth and functions as a ligand for a putative plant transcription factor. We used in vitro and in vivo RNA interference approaches to silence this parasitism gene in RKN and validate that the parasitism gene has an essential function in RKN parasitism of plants. Ingestion of 16D10 dsRNA in vitro silenced the target parasitism gene in RKN and resulted in reduced nematode infectivity. In vivo expression of 16D10 dsRNA in Arabidopsis resulted in resistance effective against the four major RKN species. Because no known natural resistance gene has this wide effective range of RKN resistance, bioengineering crops expressing dsRNA that silence target RKN parasitism genes to disrupt the parasitic process represents a viable and flexible means of developing novel durable RKN-resistant crops and could provide crops with unprecedented broad resistance to RKN.

Animals↗
PubMed2001

Molecular immunolabeling with recombinant single-chain variable fragment (scFv) antibodies designed with metal-binding domains.

To study the molecular structure and function of gene products in situ, we developed a molecular immunolabeling technology. Starting with cDNA from hybridomas producing monoclonal antibodies against biotin, catalase, and superoxide dismutase, we bioengineered recombinant single-chain variable fragment antibodies (scFv) and their derivatives containing metal-binding domains (scFv:MBD). As tested with surface plasmon resonance and enzyme-linked immunosorbent assay, affinity binding constants of the scFv (5.21 x 10(6) M(-1)) and scFv:MBD (4.17 x 10(6) M(-1)) were close to those of Fab proteolytic fragments (9.78 x 10(6) M(-1)) derived from the parental IgG antibodies. After saturation of MBD with nickel or cobalt, scFv:MBD was imaged with electron spectroscopic imaging at each element's specific energy loss, thus generating the element's map. Immunolabeling with scFv:MBD resulted in a significant improvement of the labeling fidelity over that obtained with Fab or IgG derivatives, as it produced a much heavier specific labeling and label-free background. As determined with radioimmunoassay, labeling effectiveness with scFv:MBD was nearly the same as with scFv, but much higher than with scFv conjugated to colloidal gold, Nanogold, or horseradish peroxidase. This technology opens possibilities for simultaneous imaging of multiple molecules labeled with scFv:MBD at the molecular resolution within the same sample with electron spectroscopic imaging. Moreover, the same scFv:MBD can also be imaged with fluorescence resonance energy transfer and lifetime imaging as well as positron emission tomography and magnetic resonance imaging. Therefore, this technology may serve as an integrative factor in life science endeavors.

Animals↗
PubMed1995

Modification of the substrate specificity of an acyl-acyl carrier protein thioesterase by protein engineering.

The plant acyl-acyl carrier protein (ACP) thioesterases (TEs) are of biochemical interest because of their roles in fatty acid synthesis and their utilities in the bioengineering of plant seed oils. When the FatB1 cDNA encoding a 12:0-ACP TE (Uc FatB1) from California bay, Umbellularia californica (Uc) was expressed in Escherichia coli and in developing oilseeds of the plants Arabidopsis thaliana and Brassica napus, large amounts of laurate (12:0) and small amounts of myristate (14:0) were accumulated. We have isolated a TE cDNA from camphor (Cinnamomum camphorum) (Cc) seeds that shares 92% amino acid identity with Uc FatB1. This TE, Cc FatB1, mainly hydrolyzes 14:0-ACP as shown by E. coli expression. We have investigated the roles of the N- and C-terminal regions in determining substrate specificity by constructing two chimeric enzymes, in which the N-terminal portion of one protein is fused to the C-terminal portion of the other. Our results show that the C-terminal two-thirds of the protein is critical for the specificity. By site-directed mutagenesis, we have replaced several amino acids in Uc FatB1 by using the Cc FatB1 sequence as a guide. A double mutant, which changes Met-197 to an Arg and Arg-199 to a His (M197R/R199H), turns Uc FatB1 into a 12:0/14:0 TE with equal preference for both substrates. Another mutation, T231K, by itself does not effect the specificity. However, when it is combined with the double mutant to generate a triple mutant (M197R/R199H/T231K), Uc FatB1 is converted to a 14:0-ACP TE. Expression of the double-mutant cDNA in E. coli K27, a strain deficient in fatty acid degradation, results in accumulation of similar amounts of 12:0 and 14:0. Meanwhile the E. coli expressing the triple-mutant cDNA produces predominantly 14:0 with very small amounts of 12:0. Kinetic studies indicate that both wild-type Uc FatB1 and the triple mutant have similar values of Km,app with respect to 14:0-ACP. Inhibitory studies also show that 12:0-ACP is a good competitive inhibitor with respect to 14:0-ACP in both the wild type and the triple mutant. These results imply that both 12:0- and 14:0-ACP can bind to the two proteins equally well, but in the case of the triple mutant, the hydrolysis of 12:0-ACP is severely impaired. The ability to modify TE specificity should allow the production of additional "designer oils" in genetically engineered plants.

Amino Acid Sequence↗
PubMed1999

Acid-catalyzed lactonization of alpha2,8-linked oligo/polysialic acids studied by high performance anion-exchange chromatography.

Recent studies from many laboratories revealed remarkable structural, distributional, and functional diversities of oligo/polysialic acids (OSA/PSA) that exist in organisms ranging from bacteria to man. These diversities are further complicated by the fact that OSA/PSA spontaneously form lactones under even mildly acidic conditions. By using high performance anion-exchange chromatography (HPAEC) with nitrate eluents, we found that lactonization of alpha2,8-linked OSA/PSA (oligo/poly-Neu5Ac, oligo/poly-Neu5Gc and oligo/poly-KDN) proceeds readily, and the lactonization process displays three discrete stages. The initial stage is characterized by limited lactonization occurring between two internal sialic acid residues, reflected by a regular pattern of lactone peaks interdigitated with non-lactonized peaks on HPAEC. In the middle stage, multiple lactonized species are formed from a molecule with a given degree of polymerization (DP), in which the maximum number of lactone rings formed equals DP minus 2. At the final stage, completely lactonized species become the major components, resulting in drastic changes in the physicochemical properties of the sample. Interestingly, the smallest lactonizable OSA are tetramer, trimer, and dimer at the initial, middle, and final stages, respectively. At any of the stages, OSA/PSA of higher DP lactonize more rapidly, but all the lactone rings rapidly open up when exposed to mild alkali. Lactonized OSA/PSA are resistant to both enzyme- and acid-catalyzed glycosidic bond cleavage. The latter fact was utilized to obtain more high DP oligo/poly(alpha2,8-Neu5Gc) chains from a polysialoglycoprotein. Our results should be useful in preparation, storage, and analysis of OSA/PSA. Possible biological significance and bioengineering potentials of lactonization are discussed.

Chromatography, High Pressure Liquid↗
PubMed2000

Identification of the mechanism responsible for the increased fibrin specificity of TNK-tissue plasminogen activator relative to tissue plasminogen activator.

TNK-tissue plasminogen activator (TNK-t-PA), a bioengineered variant of tissue-type plasminogen activator (t-PA), has a longer half-life than t-PA because the glycosylation site at amino acid 117 (N117Q, abbreviated N) has been shifted to amino acid 103 (T103N, abbreviated T) and is resistant to inactivation by plasminogen activator inhibitor 1 because of a tetra-alanine substitution in the protease domain (K296A/H297A/R298A/R299A, abbreviated K). TNK-t-PA is more fibrin-specific than t-PA for reasons that are poorly understood. Previously, we demonstrated that the fibrin specificity of t-PA is compromised because t-PA binds to (DD)E, the major degradation product of cross-linked fibrin, with an affinity similar to that for fibrin. To investigate the enhanced fibrin specificity of TNK-t-PA, we compared the kinetics of plasminogen activation for t-PA, TNK-, T-, K-, TK-, and NK-t-PA in the presence of fibrin, (DD)E or fibrinogen. Although the activators have similar catalytic efficiencies in the presence of fibrin, the catalytic efficiency of TNK-t-PA is 15-fold lower than that for t-PA in the presence of (DD)E or fibrinogen. The T and K mutations combine to produce this reduction via distinct mechanisms because T-containing variants have a higher K(M), whereas K-containing variants have a lower k(cat) than t-PA. These results are supported by data indicating that T-containing variants bind (DD)E and fibrinogen with lower affinities than t-PA, whereas the K and N mutations have no effect on binding. Reduced efficiency of plasminogen activation in the presence of (DD)E and fibrinogen but equivalent efficiency in the presence of fibrin explain why TNK-t-PA is more fibrin-specific than t-PA.

Alanine↗
PubMed2000

Polyarginines are potent furin inhibitors.

The ubiquitous serine endoprotease furin has been implicated in the activation of bacterial toxins and viral glycoproteins as well as in the metastatic progression of certain tumors. Although high molecular mass bioengineered serpin inhibitors have been well characterized, no small nontoxic nanomolar inhibitors have been reported to date. Here we describe the identification of such inhibitors using positional scanning amidated and acetylated synthetic l- and d-hexapeptide combinatorial libraries. The results indicated that l-Arg or l-Lys in all positions generated the most potent inhibitors. However, further investigation revealed that the peptide terminating groups hindered inhibition. Consequently, a series of non-amidated and acetylated polyarginines was synthesized. The most potent inhibitor identified, nona-l-arginine, had a K(i) for furin of 40 nm. The K(i) values for the related convertases PACE4 and prohormone convertase-1 (PC1) were 110 nm and 2.5 microm, respectively. Although nona-l-arginine was cleaved by furin, the major products after a 6-h incubation at 37 degrees C were hexa- and hepta-l-arginines, both of which retained the great majority of their potency and specificity against furin. Hexa-d-arginine was as potent and specific a furin inhibitor as hexa-l-arginine (K(i) values of hexa-d-arginine: 106 nm, 580 nm, and 13.2 microm for furin, PACE4, and PC1, respectively). PC2 was not inhibited by any polyarginine tested; indeed, PC2 showed an increase in activity of up to 140% of the control in the presence of l-polyarginines. Data are also presented that show extended subsite recognition by furin and PC2. Whereas N-terminal acetylation was found to reduce the inhibitory potency of the l-hexapeptide LLRVKR against furin 8-fold, C-terminal amidation reduced the potency < 2-fold. Conversely, N-terminal acetylation increased the potency against PC2 nearly 3-fold, whereas C-terminal amidation of the same peptide increased the potency by a factor of 1.6. Our data indicate that non-acetylated, poly-d-arginine-derived molecules may represent excellent lead compounds for the development of therapeutically useful furin inhibitors.

Animals↗
PubMed2003

Role of the connecting peptide in insulin biosynthesis.

In single-chain insulins (SCIs), the C terminus of the insulin B-chain is contiguous with the N terminus of the A-chain, connected by a short bioengineered linker sequence. SCIs have been proposed to offer potential benefit for gene therapy of diabetes (Lee, H. C., Kim, S. J., Kim, K. S., Shin, H. C., and Yoon, J. W. (2000) Nature 408, 483-488) yet relatively little is known about their folding, intracellular transport, or secretion from mammalian cells. Because SCIs can be considered as mutant proinsulin (with selective shortening of the 35-amino acid connecting peptide that normally includes two sets of flanking dibasic residues), they offer insights into understanding the role of the connecting peptide in insulin biosynthesis. Herein we have explored the relationship of the linker sequence to SCI biosynthesis, folding, and intracellular transport in transiently transfected HEK293 or Chinese hamster ovary cells or in stably transfected AtT20 cells. Despite previous reports that direct linkage of B- and A-chains produces a structure isomorphous with authentic two-chain insulin, we find that constructs with short linkers tend to be synthesized at lower levels, with a significant fraction of molecules exhibiting improper disulfide bonding. Nevertheless, disulfide-mispaired isoforms from a number of different SCI constructs are secreted. While this suggests that a novel folded state goes unrecognized by secretory pathway quality control, we find that misfolded SCIs are detected at higher levels in Chinese hamster ovary cells with artificially activated unfolded protein response mediated by inducible overexpression of active ATF-6. Such a maneuver allows analysis of more seriously misfolded mutants with further foreshortening of the linker sequence or loss (by mutation) of the insulin interchain disulfide bonds.

Animals↗
PubMed2003

Understanding substrate specificity of polyketide synthase modules by generating hybrid multimodular synthases.

Modular polyketide biosynthesis can be harnessed to generate rationally designed complex natural products through bioengineering. A detailed understanding of the features that govern transfer and processing of polyketide biosynthetic intermediates is crucial to successfully engineer new polyketide pathways. Previous studies have shown that substrate stereochemistry and protein-protein interactions between polyketide synthase modules are both important factors in this process. Here we investigated the substrate tolerance of different polyketide modules and assessed the relative importance of inter-module chain transfer versus chain elongation activity of some of these modules. By constructing a variety of hybrid modular polyketide synthase systems and assaying their ability to generate polyketide products, it was determined that the substrate tolerance of each individual ketosynthase domain is an important parameter for the successful recombination of polyketide synthase modules. Surprisingly, however, failure by a module to process a candidate substrate was not due to its inability to bind to it. Rather, it appeared to result from a blockage in carbon-carbon bond formation, suggesting that proper orientation of the initially formed acyl thioester in the ketosynthase active site was important for the enzyme-catalyzed decarboxylative condensation reaction.

Macrolides↗
PubMed2005

A dataset of human cornea proteins identified by Peptide mass fingerprinting and tandem mass spectrometry.

Diseases of the cornea are extremely common and cause severe visual impairment worldwide. To explore the basic molecular mechanisms involved in corneal health and disease, the present study characterizes the proteome of the normal human cornea. All proteins were extracted from the central 7-mm region of 12 normal human donor corneas containing all layers: epithelium, Bowman's layer, stroma, Descemet's membrane, and endothelium. Proteins were fractionated and identified using two different procedures: (i) two-dimensional gel electrophoresis and protein identification by MALDI-MS and (ii) strong cation exchange or one-dimensional SDS gel electrophoresis followed by LC-MS/MS. All together, 141 distinct proteins were identified of which 99 had not previously been identified in any mammalian corneas by direct protein identification methods. The characterized proteins are involved in many processes including antiangiogenesis, antimicrobial defense, protection from and transport of heme and iron, tissue protection against UV radiation and oxidative stress, cell metabolism, and maintenance of intracellular and extracellular structures and stability. This proteome study of the healthy human cornea provides a basis for further analysis of corneal diseases and the design of bioengineered corneas.

Chemical Fractionation↗
PubMed1998

Podophyllotoxin-induced acantholysis and cytolysis in a skin equivalent model.

Skin equivalent models are used for a wide variety of pharmacotoxicological trials. The present study was performed to assess morphologically the effect of podophyllotoxin on human bioengineered skin. The untreated model exhibited many resemblances with the parent tissues, although the epidermal differentiation was slightly impaired at the ultrastructural level. The penetration of podophyllotoxin and its biological effects inside the model appeared largely increased compared to the clinical experience with the drug. Acantholysis and cytolytic changes were prominent mimicking the effect of cantharidin. The exaggerated response of many skin equivalents to various compounds shed some doubts on the validity of the model when it is used to show efficacy rather than toxicity. This might apply to claims of efficacy for cosmetic compounds. The effect of cosmetic additives cannot be validated by such approach alone.

Acantholysis↗
PubMed2001

The impact of globalisation, free trade and technology on food and nutrition in the new millennium.

The millennium promises a dramatic politicisation of the food question. In addition to the prominent issues of food security, hunger and nutrition, bioengineering, food safety and quality, there are related issues of environmental sustainability, power, sovereignty and rights. All these issues are deeply implicated in the current corporate form of globalisation, which is transforming historic global arrangements by subordinating public institutions and the question of food security to private solutions. The present paper questions the self-evident association between globalisation and nutritional improvement.

Biotechnology↗
PubMedDate not supplied

Efficacy of a new class of perfluoropolyethers in the prevention of irritant contact dermatitis.

Perfluoropolyethers (Fomblin HC products) are chemical non-reactive polymers with special physico-chemical properties that recently showed promise as protective preparations in the prevention of irritant contact dermatitis. We evaluated the efficacy of a new class of perfluoropolyethers (perfluoropolyether phosphate, Fomblin HC/P2) in the prevention of experimentally induced cumulative irritant contact dermatitis if applied prior to irritation. A panel of 20 healthy volunteers was tested with a repetitive irritation test using 4 standard irritants (sodium lauryl sulphate of highest purity, sodium hydroxide, lactic acid and toluene) in a randomized double-blind study. Application sites were assessed clinically and by the use of bioengineering techniques (transepidermal water loss and chromametry). Three gel preparations each containing 5% perfluoropolyether phosphate showed significant efficacy against irritation due to sodium lauryl sulphate and sodium hydroxide, while one test preparation containing 2% showed inferior benefit, indicating a dose-related effect. Preparations containing perfluoropolyether phosphates can be recommended for workplaces with water-soluble irritants. Further studies under real workplace conditions are indicated.

Adolescent↗
PubMedDate not supplied

Tandem application of sodium lauryl sulfate and n-propanol does not lead to enhancement of cumulative skin irritation.

Irritant contact dermatitis has a broad spectrum of clinical features and is a leading cause of occupational disease worldwide. It has been shown previously that a combination of chemically different irritants may cause an additive effect compared to single application of these substances. In this study, tandem application of sodium lauryl sulfate and n-propanol was investigated in 20 human volunteers using non-invasive bioengineering methods, such as measurement of transepidermal water loss and chromametry. N-propanol did not enhance cumulative skin irritation when used with sodium lauryl sulfate, as has been reported for toluene. As n-propanol is the active ingredient in many disinfectants, this is of particular interest regarding occupational skin irritation in health care workers.

1-Propanol↗
PubMed2001

Effect of moisturizers on skin susceptibility to irritants.

Moisturizers are used for the treatment of dry and irritated skin. The benefit of moisturizers when used on normal skin has recently been challenged, since an earlier study indicated that the increased hydration that follows long-term use of moisturizers on normal skin may facilitate penetration of irritants. The aim of the present study was to evaluate short-term use of 2 different moisturizers used on normal skin: cream A (high lipid content) and B (moderate/low lipid content). Nineteen healthy volunteers applied the moisturizers on the upper arm/forearm 3 times daily for 5 days, while the other upper arm/forearm served as symmetrical control. The day after moisturizer treatment was stopped the skin was challenged with a patch test of sodium lauryl sulphate. Skin reactions were evaluated by bioengineering measuring methods and clinical scoring. Skin response to sodium lauryl sulphate was increased on moisturizer-treated arms compared to controls for one of the moisturizer (cream A), while this was not statistically significant for the other moisturizer (cream B). Data confirm previous indications that some moisturizers when used on normal skin may increase skin susceptibility to irritants.

Administration, Topical↗