Range of activity and metabolic stability of synthetic antibacterial glycopeptides from insects

Antibacterial glycopeptides isolated from insects are exciting bio-oligomers because they represent a family of compounds in which the structural and functional effects of incorporating short O-linked sugars to protein fragments can be studied. Additionally, their high activity in vitro warrants detailed further drug development efforts. Due to the limited availability of the isolated material, we used synthetic glycopeptides and some analogs to investigate the range of activity of drosocin and pyrrhocoricin. While addition of the Gal-GalNAc disaccharide to the natural mid-chain position generally increased the antibacterial activity of drosocin, pyrrhocoricin lacking sugar appeared to be more potent, with an IC50 against Escherichia coli D22 of 150 nM. Although glycosylated drosocin was active against E. coli in the low microM range in vitro, this peptide was completely inactive when injected into mice. The lack of in vivo activity of drosocin could be explained by the unusually high degradation rate of the peptides in mammalian sera. The early degradation products were inactive in vitro. In contrast, the peptides were considerably more stable in insect hemolymph, where their natural activity is manifested.     

Glycoproteines sulfates des membranes de l'oeuf de poule et de l'oviducte Isolement et caracterisation de glycopeptides sulfatesSulfated glycoproteins form egg shell membranes and hen oviduct. Isolation and characterization of sulfated glycopeptides

Sulfated glycopeptides were isolated from pronaisc and tryptic digests of egg shell membranes and hen oviduct. They were precipitated by cationic detergents and separated by preparative electrophoresis, after removal of small quantities of glucuronoglycosaminoglycans detected only in the oviduct (isthmus and magnum). The principal isolated sulfated glycopeptides were divided according to increasing electrophoretic mobilities into two groups A and B. The homogeneity of the purified glycopeptides was confirmed by gel filtration and polyacrylamide gel electrophoresis.Glycopeptides from pool preparation of tissue are analysed and carbohydrate and amino acids average values are estimated. Hexosamines (mainly N-acetylglucosamine), hexoses (galactose, glucose, mannose) and fucose were found in Glycopeptides A. The molar ratio of hexose/hexosamine was 0.4. N-Acetylneuraminic acid and sulfate were also present in Glycopeptides A. The molar ratio of sulfate/hexosamine ranged from 0.1 to 0.25. The Glycopeptides A composition indicated the presence of chains with many glycosyl groups and a few of amino acids residues. The carbohydrate components of Glycopeptides B from egg shell membranes and magnum were found to be hexosamines (N-acetylgalactosamine and N-acetylglucosamine in equimolar proportions), hexoses (galactose mainly and glucose), N-acetylneuraminic acid, and fucose. The molar ratio of hexose/hexosamine was 1. Sulfate was also present and the molar ratio of N-acetylneuraminic acid and sulfate to hexosamine was ranged from 0.8 to 1. The main amino acid residues in these glycopeptides were serine and threonine with destruction of these hydroxyamino acids during alkali treatment. Glycopeptides B probably consist of short carbohydrate chains, linked to the polypeptide through O-glycosidic bonds involving N-acetylgalactosamine and serine and threonine. Approximately 40% of the amino acid residues were linked to carbohydrate chains.Glycopeptides B from egg shell membranes magnum and egg white were very similar in their carbohydrate and amino acid composition and in their properties.Gylcopeptides A from egg shell membranes, isthmus and magnum showed similarities and divergences especially in the amino acid composition. These results suggest that magnum and isthmus in oviduct are both concerned with the synthesis of egg shell membrane glycoproteins.     

Partial characterization of the oligosaccharides of mouse thymocyte Thy-1 glycoprotein.

Four glycopeptides (I, IIA, IIB, III) with different oligosaccharide structures were isolated from purified mouse thymocyte Thy-1 glycoprotein. The glycoprotein was digested with Pronase, and the glycopeptide fraction was isolated by gel filtration and acetylated with [3H]acetic anhydride. The different glycan structures were separated by affinity chromatography on concanavalin A-Sepharose 4B and lentil lectin-Sepharose 4B. Size determinations of intact and exoglycosidase- and endoglycosidase-digested glycopeptides were performed by gel filtration on Bio-Gel P-6, calibrated with glycopeptides of known structure. On the basis of these experiments and on the behaviour of the glycopeptides on the lectin columns, the following structures of the oligosaccharide chains were proposed: I, triantennary 'complex-type' with terminal fucose; IIA, biantennary 'complex-type' without fucose; IIB, biantennary 'complex-type' with fucose; III, a mixture of 'high-mannose' chains containing either five or six mannose residues (approx. 50% of each). Amino acid analysis of the glycopeptides showed that the predominant oligosaccharide at glycosylation-site Asn-23 was of 'high-mannose' type, whereas the other two sites (Asn-75 and Asn-99) were glycosylated with 'complex-type' chains. Both these sites were shown to be variably glycosylated. The major glycans linked to Asn-75 were of structures I and IIB, whereas all three 'complex-type' chains were represented at Asn-99. The results presented explain the previously reported carbohydrate heterogeneity of thymocyte Thy-1 glycoprotein.     

Oligosaccharide structure of a functional unit RvH1-b of Rapana venosa hemocyanin using HPLC/electrospray ionization mass spectrometry

In the present study the structures of two glycopeptides (G1 and G1'), isolated from FU RvH(1)-b and two glycopeptides (G2 and G3), isolated from the structural subunit RvH(1) of Rapana venosa hemocyanin, were determined. To structurally characterize the site-specific carbohydrate heterogeneity and binding site of the N-linked glycopeptide(s), a combination of capillary reversed-phase chromatography and ion trap mass spectrometry was used. The amino acid sequences of glycopeptides G1 and G1' determined by Edman degradation and MS/MS sequencing demonstrated that the oligosaccharides are linked to N-glycosylation sites. Two peptides (a glycosylated (G1) and non-glycosylated one) were identified in this fraction and no linkage sites were observed in the latter one. Based on the sequencing of the glycosylated fractions G1, G1', G2 and G3, the carbohydrate structure Man(alpha1-6)Man(alpha1-3)Man(beta1-4)GlcNAc(beta1-4)[Fuc(alpha1-6)]GlcNAc-R could be identified for glycopeptides G1 and G3, and only the typical core structure Man(alpha1-6)Man(alpha1-3)Man(beta1-4)GlcNAc(beta1-4)GlcNAc-R was found for G1' and G2. The Fuc residue found in glycopeptides G1 and G3 is attached to N-acetyl-glucosamine of the carbohydrate core, as often found in other glycoproteins.     

Growth-promoting effects of lactoferrin on L. acidophilus and Bifidobacterium spp.

We investigated the effects of lactoferrin on the growth of L. acidophilus CH-2, Bifidobacterium breve ATCC 15700, B. longum ATCC 15707, B. infantis ATCC 15697, and B. bifidum ATCC 15696. The growth of L. acidophilus was stimulated by bovine holo-lactoferrin but not by apo-lactoferrin. With bifidobacteria, bovine lactoferrin stimulated growth of three strains: B. breve, B. infantis and B. bifidum under certain conditions. Both apoprotein and holoprotein had similar effects. However, B. longum growth was not affected by lactoferrin. Thus, the mechanism of stimulating growth of bifidobacteria may be different from that of L. acidophilus. By far-western blotting using biotinylated lactoferrin and horseradish peroxidase-conjugated streptavidin, lactoferrin-binding proteins were detected in the membrane protein fraction of L. acidophilus, B. bifidum, B. infantis and B. breve. The molecular weights of lactoferrin-binding proteins of L. acidophilus were estimated from SDS-polyacrylamide gel electrophoresis to be 27, 41 and 67 kDa, and those of the three bifidobacterial strains were estimated to be 67-69 kDa. However, no such lactoferrin-binding components were detected in the membrane fraction of B. longum. It is interesting that the appearance of lactoferrin-binding proteins in the membrane fraction of these species corresponds to their growth stimulation by lactoferrin.     

Studies on glycopeptides derived from acidic glycoproteins of guinea-pig serum

1. Fraction I, a fraction containing acidic glycoproteins, isolated from guinea-pig serum, was digested with Pronase after removal of sialic acid and a major and a minor glycopeptide fraction were isolated by chromatography with Sephadex G-25 and G-50. 2. The major fraction was examined by various methods and shown to contain several glycopeptides. Estimates of molecular weight of the glycopeptide fractions were obtained. Although some variation appeared to occur, the glycopeptides were not grossly heterogeneous with respect to size. An average prosthetic group was estimated to contain about 15 sugar residues. 3. Aspartic acid was the principal amino acid present in the fractions and in all subfractions of the major fraction investigated. Where examined, ammonia was liberated on acid hydrolysis in approximately equimolar amounts to the aspartic acid present. The carbohydrate composition of the fractions was also determined. 4. The glycopeptides showed relatively little degradation in alkaline solution. 5. These results suggest that an N-acylglycosylamine bond involving aspartic acid forms the major type of linkage between carbohydrate and polypeptide. The isolation of a compound with the composition and chromatographic properties of 2-acetamido-1-(l-beta-aspartamido)-1,2-dideoxy-beta-d-glucose supports this view, and indicates that N-acetylglucosamine is the sugar involved in at least many linkages. 6. Fraction I contains some glycoproteins that are susceptible to Pronase and one or more others that resist digestion before the removal of sialic acid. A brief examination revealed some similarities between prosthetic groups derived from both kinds of glycoprotein.     

Immunomodulating effect of protein fractions isolated from bifidobacteria

The screening of the immunomodulating activity (IMA) of different protein fractions isolated from bifidobacteria was carried out and the capacity of these fractions for changing the proliferative activity of immunocompetent cells was evaluated. Soluble proteins were extracted from lyophilized and sonicated bacterial mass of B. bifidum strain 1 in Na2HPO4 (pH 8) in a water bath at 65 degrees C for 30 minutes. After the formation and removal of nucleic acid sediment the resulting supernatant fluid was dialyzed, its adsorption spectra were analyzed and the fluid was fractionated in a specially proposed device for preparative electrophoresis. Protein fractions were tested for IMA on spleen cells of CBA mice in the reaction of lymphocyte blast-transformation by the level of the inclusion 3H thymidine. The analysis of IMA of protein fractions revealed that their high-molecular components produced a pronounced dose-dependent effect on the proliferative activity of spleen cells. The fractions containing low-molecular components were either inactive (fraction 4) or active only in the maximum dose (fraction 5).     

Glycosylation of LDL decreases its ability to interact with high-affinity receptors of human fibroblasts in vitro and decreases its clearance from rabbit plasma in vivo

Incubation of human LDL in vitro at 37 degrees C for 48 h with [14C]glucose at concentrations from 5 to 200 mM resulted in a glycosylated LDL, containing 0.4-20 mol of glucose incorporated per apolipoprotein B of 250 000 daltons. The extent of glucose incorporated was proportional to the time of incubation and concentration of glucose. Glycosylation of LDL abolished its uptake and degradation by the high-affinity process for LDL in normal human skin fibroblasts. 125I-labeled glycosylated LDL was bound, internalized and degraded by the fibroblasts via a nonspecific low-affinity process. The 125I-labeled glycosylated LDL and 125I-labeled LDL were taken up and degraded at similar rates in a non-saturable, low-affinity process by peritoneal macrophages isolated from mice. When 125I-labeled glycosylated LDL or 125I-labeled LDL were injected into rabbits, the glycosylated LDL had a delayed plasma clearance in comparison to the LDL. The mean fractional catabolic rates were 0.67 day-1 and 1.70 day-1 for 125I-labeled glycosylated LDL and 125I-labeled LDL, respectively. The uptake and degradation of 125I-labeled LDL by human skin fibroblasts was decreased as the concentration of free carbohydrate, glucose, sucrose or sorbitol, in the medium was increased from 10 mM to 1 M. It is speculated that pathologic levels of plasma glucose in vivo could result in a decrease in LDL uptake as a result of glycosylation of LDL. A decrease in uptake of native or modified LDL in vivo could contribute to hypercholesterolemia and its pathophysiology.     

Lectin affinity fractionation of asparagine-linked oligosaccharides from normal human and chronic leukemic leukocytes

Asparagine-linked oligosaccharides were isolated from normal and chronic leukemic leukocytes (normal neutrophils, normal lymphocytes, chronic myeloid, chronic lymphoid and hairy cell leukemic leukocytes) and analyzed by sequential lectin affinity column chromatography. The neutral and sialylated glycopeptides ranged in size from 1,800 to 4,000 da. on gel filtration. Sequential lectin affinity analysis was then used to fractionate the Asn-oligosaccharides into major structural classes of high mannose, hybrid, and bi-, tri- and tetraantennary complex structures. Using lectins of well defined specificity, the sequential chromatography provided a satisfactory means of assessing the overall glycopeptide profiles of the different leukocyte types. Results from 10 patient samples show that alterations in leukocyte Asn-oligosaccharides occur during leukemogenesis. Most notable was an average twofold increase in the relative amount of high mannose glycopeptides compared to complex glycopeptides for the leukemic cells. High mannose glycopeptides comprised 8.6 percent of the total lectin-adherent glycopeptides from leukemics, and 4.2 percent in the normals. In addition, carbohydrate analysis has revealed that the total amount of neutral hexose was markedly decreased in all leukemic samples. Leukemics ranged from 10.5 to 18.8, while normals ranged from 24.2 to 49.2 nanomole of hexose per 100 micrograms protein. The sialic acid content of the leukemic glycopeptides was relatively unchanged from that of normals, resulting in an apparent increase in the sialic acid: hexose ratio for all leukemic glycopeptides. The results suggest that in the leukemic cells, high mannose structures constitute a larger proportion of the total Asn-linked oligosaccharides, while the overall level of protein glycosylation is decreased. Complex multiantennary glycopeptides, when synthesized, tended to be more fully sialylated than their normal counterparts.     

Proteolytic fragmentation and peptide mapping of human carboxyamidomethylated tracheobronchial mucin

Human tracheobronchial mucin was isolated from lung mucosal gel by chromatography on Sepharose 4B in the presence of dissociating and reducing agents, and its thiol residues were carboxyamidomethylated with iodo[1(-14)C]acetamide. The 14C-carboxyamido-methylated mucin was purified by chromatography on Sepharose 2B. No low molecular weight components were detected by molecular sieve chromatography or polyacrylamide gel electrophoresis in the presence of dissociating and reducing agents or by analytical density centrifugation in CsCl/guanidinium chloride. After digestion of the purified 14C-mucin with trypsin-L-1-tosylamido-2-phenylethyl chloromethyl ketone, three fractions (TR-1, TR-2, and TR-3) were observed by chromatography on Sepharose 4B. TR-1, a 260-kDa mucin glycopeptide fragment, contained all of the neutral hexose and blood group activity and 20% of the radioactivity in the undigested mucin. TR-1 was refractory to a second incubation with trypsin but could be digested by papain or Pronase to a smaller mucin glycopeptide fraction, as judged by the slight decrease in apparent molecular weight on Sepharose CL-4B. These mucin glycopeptides contained approximately 50% of the radioactivity in the TR-1 fraction, indicating that the glycosylated domains of carboxyamidomethylated tracheobronchial mucin contained thiol residues. The remainder of the radioactivity from papain or Pronase digests of TR-1 eluted, like the TR-3 fractions, in the salt fraction on Sepharose CL-4B. Peptide mapping of the nonglycosylated TR-3 fraction by TLC and high voltage electrophoresis yielded six principal and several less intensely stained ninhydrin reactive components, with the radiolabel concentrated in one of the latter peptides. Peptide purification of the TR-3 fraction by high pressure liquid chromatography on a C18 reverse phase column demonstrated the presence of four major peptides, with TR-3A being the dominant component. The TR-3D peptide contained S-carboxy-aminomethylcysteine and had 69% sequence similarity to the sgs-7 salivary glue protein of Drosophila.     

A new neuraminic acid derivative and three types of glycopeptides isolated from the Cuvierian tubules of the sea cucumber Holothuria forskali

The Cuvierian tubules of Holothuria forskali Della Chiaje, a sea cucumber found in the Adriatic Sea, were investigated with regard to their carbohydrate moieties. From a Pronase digest of these tubules three types of carbohydrate units were isolated and characterized. 1. A high-molecular-weight glycopeptide fraction was shown to contain sulphated polyfucose, galactosamine, a uronic acid and a previously unknown neuraminic acid derivative. The sulphate was shown by i.r. analysis to be present as an O-ester. The carbohydrate unit was linked O-glycosidically to threonine and serine residues in the polypeptide chain. The hitherto unknown neuraminic acid derivative (Hf-neuraminic acid) was resistant to enzymic cleavage by neuraminidase, even after mild alkaline hydrolysis for the removal of O-acyl residues. However, the glycosidic linkage of this compound to the other part of the carbohydrate moiety was readily cleaved by mild acid hydrolysis. Its chromatographic properties distinguished Hf-neuraminic acid from other known neuraminic acid derivatives (N-acetyl-, NO-diacetyl-, NOO-triacetyl- and N-glycollyl-neuraminic acid). Further, this acidic sugar was shown to possess neuraminic acid as its basic structure. Thus, an as yet unknown substituent lends the distinct properties to Hf-neuraminic acid. 2. The carbohydrate composition of a second glycopeptide fraction consisting of a derivative of neuraminic acid, galactose, mannose and glucosamine was similar to that of the well-known carbohydrate groups of the globular glycoproteins. 3. The third fraction contained two glycopeptides containing the disaccharide, glucosylgalactose, which was shown to be linked to the hydroxyl group of hydroxylysine residues of a collagen-like protein. Approximately half of these residues were glycosylated. In addition to these glycopeptides, a small amount of a third glycopeptide that carried only a galactosyl residue was detected. The amino acid sequence of the two major compounds were found to be Gly-Ala-Hyl*-Gly-Ser and Gly-Pro-Hyl*-Gly-Asp, where Hyl* represents a glycosylated amino acid residue.     

The vaginal Bifidobacterium flora in women of reproductive age

The composition of vaginal bifidoflora in 56 clinically healthy women of reproductive age was studied. The study revealed that four species of bifidobacteria, viz. Bifidobacterium bifidum, B. breve, B. adolescentis 2 and B. longum, dominated in the composition of this bifidobacterial population. Nine out of 11 isolated strains were found to be capable of inhibiting indicator microorganisms Staphylococcus aureus and Enterococcus faecalis when tested in vitro; in addition, strains B. adolescentis 2 F1, B. bifidum G1, B. breve P2 and B. longum Z4 inhibited Klebsiella ozaenae, Pseudomonas aeruginosa, Escherichia coli and were also active acid producers. Three of these 4 bifidobacterial strains were capable of adhesion to vaginal epitheliocytes, while B. bifidum G1 was practically incapable of adherence to these cells, similarly to B. bifidum strain 791 of intestinal origin. In addition, the spectra of antibiotic susceptibility varied from strain to strain, but all bifidobacterial strains were susceptible to benzylpenicillin and resistant to lomefloxacin, most of them being also resistant to cyprofloxacin and gentamicin. Thus the data presented in this work are indicative of the possibility and advantages of using bifidobacterial strains belonging to this ecological niche as probiotics for the correction of the microflora of the urogenital tract in females.     

Determination of antifungal activities in serum samples from mice treated with different antifungal drugs allows detection of an active metabolite of itraconazole

To establish an in vitro method of predicting in vivo efficacy of antifungal drugs against Candida albicans and Aspergillus fumigatus, the antifungal activities of fluconazole, itraconazole, and amphotericin B were determined in mouse serum. The minimum inhibitory concentration (MIC) of each drug was measured using mouse serum as a diluent. For C. albicans, the assay endpoint of azoles was defined as inhibition of mycelial extension (mMIC) and for A. fumigatus, as no growth (MIC). The MICs of amphotericin B for both pathogens were defined as the MIC at which no mycelial growth occurred. Serum MIC or mMIC determinations were then used to estimate the concentration of the drugs in serum of mice treated with antifungal drugs by multiplying the antifungal titer of the serum samples by the serum (m)MIC. The serum drug concentrations were also determined by HPLC. The serum concentrations estimated microbiologically showed good agreement with those determined by HPLC, except for itraconazole. Analysis of the serum samples from itraconazole-treated mice by a sensitive bioautography revealed the presence of additional spots, not seen in control samples of itraconazole. The bioautography assay demonstrated that the additional material detected in serum from mice treated with itraconazole was an active metabolite of itraconazole. The data showed that the apparent reduction in the itraconazole serum concentration as determined by HPLC was the result of the formation of an active metabolite, and that the use of a microbiological method to measure serum concentrations of drugs can provide a method for prediction of in vivo efficacy of antifungal drugs.     

Feruloyl oligosaccharides stimulate the growth of Bifidobacterium bifidum

Insoluble dietary fiber from wheat bran contains some feruloyl groups linked to the arabinose residues in the cell wall arabinoxylan. Treatment of wheat bran insoluble dietary fiber with xylanase from Bacillus subtilis yielded feruloyl oligosacchairdes, which were purified with Amberlite XAD-2. Saponification of the feruloyl oligosaccharides released ferulic acid and arabinoxylan oligosaccharides which consist of arabinose and xylose. The effect of the feruloyl oligosacchairdes on the growth of Bifidobacterium bifidum F-35 was investigated in vitro. The B. bifidum produced acid when cultivated anaerobically in TPY broth with 0.5% feruloyl oligosacchairdes as the carbohydrate source. The biomass yield of the B. bifidum increased with increasing the concentration of feruloyl oligosaccharides in TPY broth. The maximum cell growth was increased by 50% in TPY broth supplemented with 0.1% feruloyl oligosaccharides compared to TPY broth. These results indicated that the growth of B. bifidum F-35 was promoted by the feruloyl oligosaccharides from wheat bran insoluble dietary fiber, and not suppressed by the ferulic acid moiety of them.     

Evidence for the in vivo degradation of human respiratory mucins during Pseudomonas aeruginosa infection

The comparison of distribution of glycopeptides of sputa from patients suffering from various chronic hypersecretions has already shown an increased acidity with a decreased proportion of neutral glycopeptides in the respiratory secretions of patients suffering from cystic fibrosis, as compared to those of patients with chronic bronchitis. In order to find out whether this decrease is specific to cystic fibrosis mucins or whether it is due to a degradation of mucus by Pseudomonas aeruginosa, which infects most of the sputa from patients with this disease, mucus glycopeptides from patients with different chronic bronchial disorders, infected by Pseudomonas or not, were prepared and fractionated by ion-exchange chromatography. The neutral fraction, which has never been studied in detail, was gel-filtered, and provided two fractions, one containing true mucin glycopeptides and the other containing a mixture of peptides and glycopeptides with a lower molecular mass. In the Pseudomonas-infected samples, the true mucin glycopeptide fraction was greatly diminished as compared to this same fraction in non-Pseudomonas-infected samples; this was not specific to cystic fibrosis secretions. In contrast, the glycopeptide fraction with a lower molecular mass was greatly increased in all the Pseudomonas-infected samples. Polyacrylamide gel electrophoresis of this second fraction showed unique glycopeptide bands between 40-50 kDa in the Pseudomonas-infected samples, regardless of the origin of the samples. These bands were revealed by an antibody directed against whole cystic fibrosis mucin. Infected chronic bronchitis sputa and cystic fibrosis samples without P. aeruginosa did not show these bands. These studies therefore suggest that there are P. aeruginosa-associated changes in mucins which may result from degradation of mucins.     

Polymyxin B is an inhibitor of insulin-induced hypoglycemia in the whole animal model. Studies on the mode of inhibitory action

The cyclic decapeptide, polymyxin B (PMXB), was found to inhibit hypoglycemia in mice receiving exogenous insulin (Amir, S., and Shechter, Y. (1985) Eur. J. Pharmacol. 110, 283-285). In this study, we have extended this observation to rats. Insulin-dependent hypoglycemia in rats is efficiently blocked at a 12:1 molar ratio of PMXB to insulin. This effect is highly specific, as it could not be mimicked by a variety of antibiotics or positively charged substances. Chemical modifications of PMXB have revealed that the ring structure, rather than the tail structure, is important for anti-insulin-like activity. Colistin A, which differs from PMXB by one conservative amino acid substitution in the ring structure, is devoid of this activity. Polymyxin B does not interact with insulin, nor does it alter the rate of insulin absorption and/or degradation, or the ability of insulin to bind to target tissues. This peptide inhibits hypoglycemia by blocking insulin-dependent activation of the hexose transport mechanism, as deduced by in vitro studies. The effect of insulin in stimulating hexose uptake (and subsequent glucose metabolism) in both isolated muscle tissue and adipocytes is blocked with little or no effect on the basal activities of these processes. Colistin A has no significant inhibiting effect. Other insulin-dependent activities, such as inhibition of lipolysis in adipocytes or synthesis of DNA in muscle cells, are not inhibited. It is concluded that PMXB inhibits, in a highly specific manner, the action of insulin in stimulating hexose transport and subsequent glucose metabolism, both in vitro and in the whole animal model.     

以双歧杆菌为载体实现量子点在体靶向肿瘤的可行性研究

应用双歧双歧杆菌作为量子点输送载体,为小动物在体生物肿瘤成像提供依据. 采用电穿孔方法,将
二棕榈酰磷脂酰胆碱（DPPC）包被的硫硒镉水溶性量子点转入细菌内,得到“量子点-细菌”复合探针,在
“量子点-细菌”表面通过1-(3-二甲氨基丙基)-3-乙基碳二亚胺 (EDC)活化法进一步偶联叶酸分子,制得
“量子点-细菌-叶酸”复合纳米生物探针. 将纳米生物探针经尾静脉注入Lewis肺癌小鼠体内,采用冰冻组
织切片,考察探针在小鼠体内脏器及肿瘤的分布情况. 结果表明,在肿瘤部位检测到较强的量子点光致发光
信号,而在肺、肝、脾等脏器中只检测到微弱的量子点发光信号. “量子点-细菌-叶酸”复合纳米生物探
针小动物在体肿瘤靶向成像是可行的.     

Inhibitory impact of bifidobacteria on the transfer of beta-lactam resistance among Enterobacteriaceae in the gnotobiotic mouse digestive tract

While looking for new means to limit the dissemination of antibiotic resistance, we evaluated the role of potentially probiotic bifidobacteria on the transfer of resistance genes between enterobacteria. Transfers of bla genes encoding extended-spectrum beta-lactamases (SHV-5 and CTX-M-15) were studied in the absence or presence of bifidobacteria. In vitro, transfer frequencies of these bla genes decreased significantly in the presence of three of five tested strains, i.e., Bifidobacterium longum CUETM-89-215, Bifidobacterium bifidum CIP-56.7T, and Bifidobacterium pseudocatenulatum CIP-104168T. Four transfer experiments were conducted in the digestive tract of gnotobiotic mice, the first three observing the effect of B. longum CUETM-89-215, B. bifidum CIP-56.7T, and B. pseudocatenulatum CIP-104168T on blaSHV-5 transfer and the fourth experiment studying the effect of B. bifidum CIP-56.7T on blaCTX-M-15 transfer. These experiments revealed significant decreases in the transconjugant levels (up to 3 logs) in mice having received B. bifidum CIP-56.7T or B. pseudocatenulatum CIP-104168T compared to control mice. Bifidobacteria appear to have an inhibitory impact on the transfer of antibiotic resistance genes. The inhibitory effect is associated to specific bifidobacterial strains and may be related to the production of thermostable metabolites by these strains.