Some properties of alginate gels derived from algal sodium alginate

Alginic acid in soluble sodium alginate turns to insoluble gel after contact with divalent metal ions, such as calcium ions. The sodium alginate character has an effect on the alginate gel properties. In order to prepare a suitable calcium alginate gel for use in seawater, the effects of sodium alginate viscosity and M/G ratio (the ratio of D-mannuronate to L-guluronate) on the gel strength were investigated. The wet tensile strengths of gel fibers derived from high viscosity sodium alginate were higher than those from low viscosity sodium alginate. The tensile strength increased with diminishing sodium alginate M/G ratio. Among the gel fibers tested, the gel fiber obtained from a sodium alginate I-5G (1% aqueous solution viscosity = 520 mPa·s, M/G ratio = 0.6) had the highest wet tensile strength. After 13 days treatment in seawater, the wet tensile strength of the gel fiber retained 36% of the original untreated gel strength. For sodium alginates with similar viscosities, the seawater tolerance of low M/G ratio alginate was greater than that of the high M/G ratio one. This study enables us to determine a suitable calcium alginate gel for use in seawater.     

Oxygen diffusivity in gel beads containing viable cells

This article proposes a simple steady-state method for measuring the effective diffusion coefficient of oxygen (D(e)) in gel beads entrapping viable cells. We applied this method to the measurement of D(e) in Ca- and Ba-alginate gel beads entrapping Saccharomyces cerevisiae and Pseudomonas ovalis. The diffusivity of oxygen through gel beads containing viable cells was measured within an accuracy of +/-7% and found not to be influenced by cell density (0-30 g/L gel), cell type, and cell viability in gel beads. The oxygen diffusivity in the Ca-alginate gel beads was superior to that of the Ba-alginate gel beads, and the D(e) in the Ca-alginate gel beads nearly equalled the molecular diffusion coefficient in the liquid containing the gel beads. The oxygen concentration profile in a single Ca-alginate gel bead was calculated and compared to the distribution of mycelia of Aspergillus awamori grown in that gel bead. This procedure indicated that the oxygen concentration profile is useful for the estimation of the thickness of the cell layer in a gel bead. Numerical investigation revealed that high effectiveness factors, greater than 0.8, could be obtained using microgel beads with a radius of 0.25 mm.     

Immunogenicity of synthetic peptides derived from Plasmodium falciparum proteins

To obtain antibodies suitable to be used in an antigen-capture assay, we have identified, synthesized, and evaluated a series of peptides from different Plasmodium falciparum excretory-secretory proteins: glutamate-rich protein (GLURP); histidine-rich protein 2; histidine-rich protein 3; Falciparum interspersed repeat antigen and, serine-rich antigen homologous. Conformational as well as antigenic predictions were performed using the ANTHEPROT package. Chemical synthesis was carried out by the multiple manual synthesis using the t-boc strategy. The peptides were used as antigens for the preparation of polyclonal antibodies in rabbits. Out of the 14 peptide constructs, eight by ELISA and, six by MABA elicited antibodies that showed correspondence between the predictive study and the immunogenicity obtained in rabbits. All antipeptide (GLURP, HRP2, and FIRA) antisera were found to bind to the corresponding synthetic sequence in an ELISA assay. The binding activity and specificity of antibodies were determined by Western blot with supernatant culture from P. falciparum. Anti-GLURP (IMT-94 and IMT-200) antisera bound to five molecules present in supernatant with molecular weight of 73, 82, 116, 124, and 128 kDa. Anti-HRP2 (IMT-192) antisera recognized a band of 58 kDa. In both cases, the specific molecules were inhibited by preincubation with the homologous peptide. Anti-HRP3, anti-FIRA neither anti-SERPH antisera showed reactivity. Anti-peptides HRP2 antibodies recognized the recombinant protein present in Parasight-F test. The same way, synthetic peptides from HRPII molecule were recognized by monoclonal antibody present in the Parasight-F assay. Our results confirm the potential value of synthetic peptides when inducing monospecific polyclonal antibodies for the development of diagnostic tests based on the capture of antigens.     

Oxygen diffusion through gels employed for immobilization

Methods of measuring effective O₂ diffusivity, calculation of kinetic parameters and theoretical models are reviewed. Different techniques and procedures used to overcome problems arising from limitation of O₂ diffusion through gels are also reported.     

Oxygen diffusion through gels employed for immobilization

This review reports on the O₂ diffusion in gels with microorganisms, the difficulties arising due to their presence and the solutions to overcome this problem. Among the topics discussed are the morphological behaviour of the microorganisms, the change in the profile of O₂ uptake rate during the growth of microorganisms and the importance of the effectiveness factor in determining O₂ diffusivity. Some experiments with co-immobilization, either with different microorganisms (aerobic/anaerobic) or with agents liberating O₂ are also reported and discussed.     

Bactericidal and tumoricidal activities of synthetic peptides derived from granulysin

Granulysin, a 9-kDa protein localized to human CTL and NK cell granules, is cytolytic against tumor cells and microbes. Molecular modeling predicts that granulysin is composed of five alpha-helices separated by short loop regions. In this report, synthetic peptides corresponding to the linear granulysin sequence were characterized for lytic activity. Peptides corresponding to the central region of granulysin lyse bacteria, human cells, and synthetic liposomes, while peptides corresponding to the amino or carboxyl regions are not lytic. Peptides corresponding to either helix 2 or helix 3 lyse bacteria, while lysis of human cells and liposomes is dependent on the helix 3 sequence. Peptides in which positively charged arginine residues are substituted with neutral glutamine exhibit reduced lysis of all three targets. While reduction of recombinant 9-kDa granulysin increases lysis of Jurkat cells, reduction of cysteine-containing granulysin peptides decreases lysis of Jurkat cells. In contrast, lysis of bacteria by recombinant granulysin or by cysteine-containing granulysin peptides is unaffected by reducing conditions. Jurkat cells transfected with either CrmA or Bcl-2 are protected from lysis by recombinant granulysin or the peptides. Differential activity of granulysin peptides against tumor cells and bacteria may be exploited to develop specific antibiotics without toxicity for mammalian cells.     

The antimicrobial activity of hexapeptides derived from synthetic combinatorial libraries

S.E. BLONDELLE, E. TAKAHASHI, K.T. DINH AND R.A. HOUGHTEN. 1995. A series of peptides identified through the use of synthetic hexapeptide combinatorial libraries (represented by the formula Ac-RRWWCO-NH₂) were examined for their antimicrobial activity against five different micro-organisms. Their toxicity was also evaluated in an in vitro haemolytic assay. The peptides showed activity against the five micro-organisms, although higher activities were found against Gram-positive bacteria. Both growth inhibition and cell viability assays were carried out to demonstrate the bactericidal activities of these peptides against two of the micro-organisms tested. The dimeric cystine forms of these peptides were shown to have biological activities identical to the monomeric forms.     

Effective diffusivity of galactose in calcium alginate gels containing immobilized Zymomonas mobilis.

The effective diffusivity of galactose was measured for calcium alginate gel membranes containing immobilized live Zymomonas mobilis cells at concentrations ranging from 0 to 150 g dry wt/L of gel. Since galactose is not taken up by living Z. mobilis organisms, the diffusion of this representative six-carbon sugar could be studied independently of sugar consumption. Various immobilized biomass loadings were achieved by two different techniques: addition of biomass at known concentrations to the sodium alginate solution before membrane formation and growth of cells in the gel to various biomass concentrations. The highest immobilized cell concentration, attained by in situ growth, corresponds to the maximum of this system, as growth beyond this maximum concentration led to disintegration of the gel membrane. The galactose effective diffusivity measurements for both methods of immobilized cell loading overlap within experimental error and follow the same general monotonic decline with entrapped biomass concentration. Most of the data fall below the upper bound predicted by Hashin and Shtrikman (1962) and show good agreement with the random pore model of Wakao and Smith (1962, 1964). Available effective diffusivity data from the literature provide evidence that the random pore model is an excellent predictor of sugar effective diffusivity in gel immobilized cell systems in general.     

Entrapment of Lavandula vera cells with synthetic resin prepolymers and its application to pigment production

Summary Cultured cells of Lavandula vera were entrapped with photosensitive synthetic resin prepolymers (PVA-SbQ). PVA-SbQ-entrapped cells grew well inside gel matrices and synthesized de novo blue pigments in the presence of l-cysteine as an inducer. The entrapped cells were superior to calcium alginate-entrapped cells judging from cell growth and total pigment productivity. Release of the pigments, which were almost insoluble in water, from the gels was markedly enhanced by the increase in hydrophilicity of the cell-entrapping gels. The entrapped cells could be used repeatedly for the pigment production.Dedicated to Professor Dr. Georg Manecke on occasion of his 70th birthday     

Inhibition of human renin by synthetic peptides derived from its prosegment

The primary structure of human preprorenin has recently been determined from its cDNA sequence. It includes a 46-amino acid NH2-terminal prosegment. Six peptides corresponding to the entire prosegment (9-40), except for the NH2-terminal (1-8) and COOH-terminal (41-46) ends have been synthesized. These peptides were tested for their inhibitory effect on human plasma renin activity. Boc-Tyr-Thr-Thr-Phe-Lys-Arg-Ile-Phe-Leu-Lys-Arg-Met-Pro-OMe (where Boc represents t-butoxycarbonyl and OMe represents methoxy) (h Y(9-20) and its fragment Boc-Leu-Lys-Arg-Met-Pro-OMe h (16-20) were the most potent inhibitors with IC50 values of 2 X 10(-4) and 3 X 10(-4)M, respectively. Peptides located near the COOH-terminus were less inhibitory. The inhibitory capacity of h (16-20) was studied further on highly purified human renin acting on either pure human angiotensinogen or a synthetic human tetradecapeptide substrate. In both of these assays its inhibitory potency was about 10-fold greater than that found on plasma renin activity. Peptide h (16-20) was 3-6 times less potent in inhibiting human renin than its mouse counterpart m (15-19) was in inhibiting mouse renin. Kinetic studies carried out with h (16-20) showed a mixed type of inhibition. When human angiotensinogen was used as substrate, Ki and K'i values were 17.7 +/- 3.9 and 2.9 +/- 0.9 microM, respectively. These studies showed that human renin, like mouse renin and pepsin, can be inhibited by peptides derived from its prosegment. In addition, as in the case of pepsin, they suggest that the NH2-terminal part of the prosegment interacts more strongly with the active enzyme.     

Protein determination of cells immobilized in cross-linked synthetic gels

Summary An assay for the determination of the protein content of whole cells immobilized in cross-linked synthetic gels was developed. The assay is based on a three step procedure: a) methanol dehydration, b) protein extraction by 1.0 M alkali at 125°C c) colorimetric assay of the extracted protein according to Bradford's procedure (Bradford M. M. (1976), Anal. Biochem. 72:248–254). The procedure worked out was found adequate for the determination of the protein content of microbial cells immobilized in synthetic and native polymer-gel-systems.     

Markers derived from amplified fragment length polymorphism gels for plant ecology and evolution studies

We describe the types of polymerase chain reaction (PCR) markers that we have isolated using amplified fragment length polymorphisms (AFLP) in closely related taxa from diverse plant genera. With these markers, both inter- and intraspecific differences have been identified. The characterization of the nucleotide sequences and fragment length polymorphisms of such AFLP-derived PCR markers is promising for investigating the ecology and evolution of closely related plant taxa.     

Formation and degradation of a synthetic humic acid derived from 3-fluorocatechol

A synthetic fluorinated humic acid (FHA) was prepared by the spontaneous oxidative polymerization of 3-fluorocatechol. The ¹³C-solid-state NMR spectrum showed signals in the region for aromatic carbons with different substituents (aryl-H, aryl-C, aryl-O carbons) and for carboxyl-carbon. The latter indicated the formation of carboxylic groups, probably caused by ring cleavages during the polymerization process. An indication of the formation of carboxylic groups was also found in the infrared spectrum (band at 1715 cm⁻¹). The dissolved FHA was degraded with active mycelium of the agaric white-rot fungus Nematoloma frowardii as well as with its isolated manganese peroxidase. In both cases, decolorization of the brownish FHA solution and partial defluorination (45–60%) took place. Degradation proceeded via formation of lower-molecular-mass fulvic acid-like substances. The results demonstrate that halogenated humic substances, e.g., resulting from the humification of xenobiotic compounds (bound residues), can in principle be eliminated by ligninolytic fungi (e.g., soil colonizing litter decomposers) and their manganese peroxidase system. Received: 28 June 1999 / Received revision: 14 October 1999 / Accepted: 16 October 1999     

Immunosuppressive properties of synthetic peptides derived from CD4 and HLA-DR antigens

Synthetic peptides derived from the beta 1 domain of HLA-DR antigens containing RFDS and a peptide derived from the immunoglobulin-like amino-terminal domain of CD4 and containing RADS were shown to exhibit specific dose-dependent inhibitory effects on antigen-induced HLA class II-restricted T-cell proliferation and in vitro antibody synthesis. These inhibitory activities are similar to those exhibited by anti-CD4 and HLA-DR antibodies, respectively. The peptides derived from HLA-DR or CD4 and anti-CD4 or anti-HLA-DR antibodies acted together in synergy to inhibit these responses when the relevant cell populations were incubated with infrainhibitory concentrations of the reagents. In contrast, these peptides were shown to exert no inhibitory activity on nonspecific T-cell activation mediated by ionomycin, phorbol myristate acetate, and interleukin-2.     

Inhibition of beta-amyloid-induced neurotoxicity by imidazopyridoindoles derived from a synthetic combinatorial library

Alzheimer's disease is a progressive neurodegenerative disorder characterized by the deposit of amyloid fibrils in the brain that result from the self-aggregative polymerization of the beta-amyloid peptide (Abeta). Evidence of a direct correlation between the ability of Abeta to form stable aggregates in aqueous solution and its neurotoxicity has been reported. The cytotoxic effects of Abeta have been attributed to the aggregation properties of a domain corresponding to the peptide fragment Abeta25-35. In an effort to generate novel inhibitors of Abeta neurotoxicity and/or aggregation, a mixture-based synthetic combinatorial library composed of 23 375 imidazopyridoindoles was generated and screened for inhibition of Abeta25-35 neurotoxicity toward the rat pheochromocytoma PC-12 cell line. The effect of the identified lead compounds on Abeta25-35 aggregation was then evaluated by means of circular dichroism (CD) and thioflavin-T fluorescence spectroscopy. Their activity against Abeta1-42 neurotoxicity toward the PC-12 cell line was also determined. The most active imidazopyridoindoles inhibited both Abeta25-35 and Abeta1-42 neurotoxicity in the low- to mid-micromolar range. Furthermore, inhibition of the random coil to beta-sheet transition and self-aggregation of Abeta25-35 was observed by CD and fluorescence spectroscopy, supporting the relationship between inhibition of the Abeta aggregation process and neurotoxicity.     

Enhanced antimicrobial activity of novel synthetic peptides derived from vejovine and hadrurin

Background

Microbial antibiotic resistance is a challenging medical problem nowadays. Two scorpion peptides displaying antibiotic activity: hadrurin and vejovine were taken as models for the design of novel shorter peptides with similar activity.

Methods

Using the standard Fmoc-based solid phase synthesis technique of Merrifield twelve peptides (18 to 29 amino acids long) were synthesized, purified and assayed against a variety of multi-drug resistant Gram-negative bacteria from clinical isolates. Hemolytic and antiparasitic activities of the peptides and their possible interactions with eukaryotic cells were verified. Release of the fluorophore calcein from liposomes treated with these peptides was measured.

Results

A peptide with sequence GILKTIKSIASKVANTVQKLKRKAKNAVA), and three analogs: Δ(Α29), Δ(K12-Q18; Ν26−Α29), and K4N Δ(K12-Q18; Ν26−Α29) were shown to inhibit the growth of Gram-negative (E. coli ATCC25922) and Gram-positive bacteria (S. aureus), as well as multi-drug resistant (MDR) clinical isolated. The antibacterial and antiparasitic activities were found with peptides at 0.78 to 25 μM and 5 to 25 μM concentration, respectively. These peptides have low cytotoxic and hemolytic activities at concentrations significantly exceeding their minimum inhibitory concentrations (MICs), showing values between 40 and 900 μM for their EC₅₀, compared to the parent peptides vejovine and hadrurin that at the same concentration of their MICs lysed more than 50% of human erythrocytes cells.

Conclusions

These peptides promise to be good candidates to combat infections caused by Gram-negative bacteria from nosocomial infections.

General significance

Our results confirm that well designed synthetic peptides can be an alternative for solving the lack of effective antibiotics to control bacterial infections.     

New broad-spectrum resistance to septoria tritici blotch derived from synthetic hexaploid wheat

Septoria tritici blotch (STB), caused by the ascomycete Mycosphaerella graminicola, is one of the most devastating foliar diseases of wheat. We screened five synthetic hexaploid wheats (SHs), 13 wheat varieties that represent the differential set of cultivars and two susceptible checks with a global set of 20 isolates and discovered exceptionally broad STB resistance in SHs. Subsequent development and analyses of recombinant inbred lines (RILs) from a cross between the SH M3 and the highly susceptible bread wheat cv. Kulm revealed two novel resistance loci on chromosomes 3D and 5A. The 3D resistance was expressed in the seedling and adult plant stages, and it controlled necrosis (N) and pycnidia (P) development as well as the latency periods of these parameters. This locus, which is closely linked to the microsatellite marker Xgwm494, was tentatively designated Stb16q and explained from 41 to 71% of the phenotypic variation at seedling stage and 28–31% in mature plants. The resistance locus on chromosome 5A was specifically expressed in the adult plant stage, associated with SSR marker Xhbg247, explained 12–32% of the variation in disease, was designated Stb17, and is the first unambiguously identified and named QTL for adult plant resistance to M. graminicola. Our results confirm that common wheat progenitors might be a rich source of new Stb resistance genes/QTLs that can be deployed in commercial breeding programs.