Repair of deletions and double-strand gaps by homologous recombination in a mammalian in vitro system.

We have designed an in vitro system using mammalian nuclear extracts, or fractions derived from them, that can restore the sequences missing at double-strand breaks (gaps) or in deletions. The recombination substrates consist of (i) recipient DNA, pSV2neo with gaps or deletions ranging from 70 to 390 bp in the neo sequence, and (ii) donor DNAs with either complete homology to the recipient (pSV2neo) or plasmids whose homology with pSV2neo is limited to a 1.0- to 1.3-kbp neo segment spanning the gaps or deletions. Incubation of these substrates with various enzyme fractions results in repair of the recipient DNA's disrupted neo gene. The recombinational repair was monitored by transforming recA Escherichia coli to kanamycin resistance and by a new assay which measures the extent of DNA strand transfer from the donor substrate to the recipient DNA. Thus, either streptavidin- or antidigoxigenin-tagged beads are used to separate the biotinylated or digoxigeninylated recipient DNA, respectively, after incubation with the isotopically labeled donor DNA. In contrast to the transfection assay, the DNA strand transfer measurements are direct, quantitative, rapid, and easy, and they provide starting material for the characterization of the recombination products and intermediates. Accordingly, DNA bound to beads serves as a suitable template for the polymerase chain reaction. With appropriate pairs of oligonucleotide primers, we have confirmed that both gaps and deletions are fully repaired, that deletions can be transferred from the recipient DNA to the donor's intact neo sequence, and that cointegrant molecules containing donor and recipient DNA sequences are formed.     

Effect of the anthracycline group antibiotics, mitomycin C and bruneomycin, on the transduction of drug resistance in staphylococci]

The effect of various concentrations of antitumor antibiotics, such as carminomycin, rubomycin, adriamycin, mitomycin C and bruneomycin on transduction of erythromycin resistance from the donor strain 8325 P II/de of Staph, aureus to the recipient strain 8325-I in different transduction systems was studied. It was shown that the above antibiotics inhibited the transduction in the systems with constant presence of the drugs. Preliminary treatment of the recipient cells with the drugs in the subbacteriostatic doses did not decrease the transfer frequency. The preliminary treatment of the donor cells resulted in an increase in the phase titer and the transfer frequency in the "preliminary-treated donor + recipient" system.     

Protein synthesis in heterotopically transplanted rat hearts

This study examines protein synthesis in heterotopically transplanted rat hearts and several tissues of recipient rats. Donor hearts and recipient tissues synthesized many of the normally occurring proteins observed in tissues of unstressed rats. In addition, a stress-induced protein with a molecular mass of 71 kilodaltons was synthesized in donor heart, recipient heart and lung. Donor hearts incorporated more L-[35S]-methionine than did recipient hearts. Tissues of recipient rats also incorporated more label than the respective tissues of sham-recipient rats. These results suggest that ischemia, endured by the donor hearts during transplantation, induced these changes in protein synthesis.     

The rate of horizontal transmission of antibiotic resistance plasmids is increased in food preservation-stressed bacteria

AIM: The aim of this study was to investigate the possibility that sublethal food preservation stresses (high/low temperature, osmotic and pH stress) can alter the rate of horizontal transmission of antibiotic resistance (ABR) plasmids between Escherichia coli strains and between E. coli and Salmonella serotype Typhimurium. METHODS AND RESULTS: Escherichia coli donor cultures, carrying F1 plasmid R386 and Inc I1 plasmid TP307 and E. coli and Salm. Typhimurium recipient cultures were prestressed under a range of sublethal environmental conditions (high/low temperature, osmotic and pH stress). The prestressed donor and recipient cultures were then mated and the transmission rate calculated. The study found that the horizontal transmission rate of plasmids R386 and TP307 was significantly increased (P < 0.05) when prestressed donor and recipient cells are mated under conditions of environmental stress. CONCLUSION: The results from this study indicate that, the sublethal stresses that food pathogens encounter in modern food preservation systems increase the inter- and intra-specific horizontal transmission of selected ABR plasmids. SIGNIFICANCE AND IMPACT OF THE STUDY: Increased use of bacteriostatic (sublethal), rather than bacteriocidal (lethal) food preservation systems, may be contributing to the dissemination of ABR among important food borne pathogens.     

Evidence for natural horizontal transfer of tetO gene between Campylobacter jejuni strains in chickens

AIMS: The transfer of tetO gene conferring resistance to tetracycline was studied between Campylobacter jejuni strains, in the digestive tract of chickens. METHODS AND RESULTS: In vitro conjugation experiments were first performed in order to select donor/recipient couples for further in vivo assay. Then, chickens were inoculated with a donor/recipient couple of C. jejuni strains displaying spontaneous in vitro tetracycline resistance gene transfer. The donor was a tetracycline-resistant ampicillin-susceptible strain, and the recipient was a tetracycline-susceptible ampicillin-resistant strain. Chicken droppings were streaked on antimicrobial selective media and bi-resistant Campylobacter isolates were further characterized according to their donor or recipient flaA gene RFLP profile. The acquisition of tetracycline-resistance gene by the recipient C. jejuni strain from the donor C. jejuni strain was confirmed by tetO PCR. CONCLUSIONS: The study showed that transfer of tetO gene occurs rapidly and without antimicrobial selection pressure between C. jejuni strains in the digestive tract of chickens. SIGNIFICANCE AND IMPACT OF THE STUDY: The rapid and spontaneous transfer of tetO gene may explain the high prevalence of tetracycline resistance in chicken Campylobacter strains.     

Plasmid-associated transfer of tetracycline resistance in Bacteroides ruminicola

Tetracycline resistance was transferred at frequencies between 10(-7) and 10(-6) per recipient cell in anaerobic matings between two strains of the strictly anaerobic rumen bacterium Bacteroides ruminicola. The donor strain, 223/M2/7, was a multiple-plasmid-bearing tetracycline-resistant strain from the ovine rumen, and the recipient, F101, was a rifampin-resistant mutant of B14, a bovine strain belonging to B. ruminicola subsp. brevis. Resistance transfer could occur in the presence of DNase, but not in dummy mating mixtures in which filtrate from a donor culture replaced donor cells. Acquisition of tetracycline resistance by the recipient was accompanied by the appearance of a 19.5-kilobase pair plasmid (designated pRRI4) which was homologous with a plasmid of similar size and restriction pattern present in the donor strain. A transconjugant (F115) carrying pRRI4 was also able to act as a donor of tetracycline resistance and plasmid DNA in matings with another recipient. Derivatives of F115 that had spontaneously lost tetracycline resistance lacked detectable plasmid DNA. It is concluded that pRRI4 mediated the transfer of tetracycline resistance. Transfer of resistance was not detectably enhanced by pregrowth of the donor in medium containing tetracycline. Transfer of tetracycline resistance was not detected from 223/M2/7 to a strain, 23 belonging to B. ruminicola subsp. ruminicola.     

Plasmid-associated transfer of tetracycline resistance in Bacteroides ruminicola.

Tetracycline resistance was transferred at frequencies between 10(-7) and 10(-6) per recipient cell in anaerobic matings between two strains of the strictly anaerobic rumen bacterium Bacteroides ruminicola. The donor strain, 223/M2/7, was a multiple-plasmid-bearing tetracycline-resistant strain from the ovine rumen, and the recipient, F101, was a rifampin-resistant mutant of B14, a bovine strain belonging to B. ruminicola subsp. brevis. Resistance transfer could occur in the presence of DNase, but not in dummy mating mixtures in which filtrate from a donor culture replaced donor cells. Acquisition of tetracycline resistance by the recipient was accompanied by the appearance of a 19.5-kilobase pair plasmid (designated pRRI4) which was homologous with a plasmid of similar size and restriction pattern present in the donor strain. A transconjugant (F115) carrying pRRI4 was also able to act as a donor of tetracycline resistance and plasmid DNA in matings with another recipient. Derivatives of F115 that had spontaneously lost tetracycline resistance lacked detectable plasmid DNA. It is concluded that pRRI4 mediated the transfer of tetracycline resistance. Transfer of resistance was not detectably enhanced by pregrowth of the donor in medium containing tetracycline. Transfer of tetracycline resistance was not detected from 223/M2/7 to a strain, 23 belonging to B. ruminicola subsp. ruminicola.     

Exosomes communicate protective messages during oxidative stress; possible role of exosomal shuttle RNA

Background

Exosomes are small extracellular nanovesicles of endocytic origin that mediate different signals between cells, by surface interactions and by shuttling functional RNA from one cell to another. Exosomes are released by many cells including mast cells, dendritic cells, macrophages, epithelial cells and tumour cells. Exosomes differ compared to their donor cells, not only in size, but also in their RNA, protein and lipid composition.

Methodology/Principal Findings

In this study, we show that exosomes, released by mouse mast cells exposed to oxidative stress, differ in their mRNA content. Also, we show that these exosomes can influence the response of other cells to oxidative stress by providing recipient cells with a resistance against oxidative stress, observed as an attenuated loss of cell viability. Furthermore, Affymetrix microarray analysis revealed that the exosomal mRNA content not only differs between exosomes and donor cells, but also between exosomes derived from cells grown under different conditions; oxidative stress and normal conditions. Finally, we also show that exposure to UV-light affects the biological functions associated with exosomes released under oxidative stress.

Conclusions/Significance

These results argue that the exosomal shuttle of RNA is involved in cell-to-cell communication, by influencing the response of recipient cells to an external stress stimulus.     

A mathematical model of twin-twin transfusion syndrome with pulsatile arterial circulations

The twin-twin transfusion syndrome (TTTS) is a severe complication of monochorionic twin pregnancies caused by a net transfusion of blood from one twin (the donor) to the other (the recipient) through placental anastomoses. To examine the pathophysiology of TTTS evolving through clinical stages I to IV, we extended our mathematical model to include pulsating circulations propagating along the arterial tree as well as placental and cerebral vascular resistances, and arterial wall thickness and stiffness. The model demonstrates that abnormal umbilical arterial flow (TTTS stage III) in the donor twin results from increased placental resistance as well as reduced resistance in the cerebral arteries. In contrast, recipient twin abnormal umbilical arterial flow requires a significantly greater increase in placental resistance, resulting from the compressive effects of high amniotic fluid pressure. Thus simulated abnormalities of donor umbilical arterial pulsations occur in the donor more commonly and earlier than in the recipient. The "normal" staging sequence (I, II, III, IV) correlates with the presence of compensating placental anastomoses, constituting the majority of monochorionic twin placentas. However, TTTS stage III may occur before manifestations of stage II (lack of donor bladder filling), in our model correlating with severe TTTS from a single arteriovenous anastomosis, an infrequent occurring placental angioarchitecture. In conclusion, this mathematical model describes the onset and development of the four stages of TTTS, reproduces a variety of clinical manifestations, and may contribute to identifying the underlying pathophysiology of the staging sequence in TTTS.     

Transferability of antibiotic resistance determinants in strains of Staphylococcus aureus belonging to different phage groups

A total of 107 donor strains of Staphylococcus aureus isolated from clinical material, with a high incidence of multiresistant strains belonging predominantly to phage group III, were tested for transmission of determinants of resistance to 6 antibiotics using mixed cultures of donor strains and the recipient Staphylococcus aureus strain 5849-fur-r, rif-r. The capability of strains to transfer resistance markers to the recipient was found to depend neither on phage group nor phage type to which the donor strain belonged, but strains possessing multiple resistance to antibiotics effectuated transfers at comparatively higher frequencies.     

Mechanisms of extracellular vesicle uptake in stressed retinal pigment epithelial cell monolayers

PurposeExtracellular vesicles (EVs) can mediate long-distance communication in polarized RPE monolayers. Specifically, EVs from oxidatively stressed donor cells (stress EVs) rapidly reduced barrier function (transepithelial resistance, TER) in naïve recipient monolayers, when compared to control EVs. This effect on TER was dependent on dynamin-mediated EV uptake, which occurred rapidly with EVs from oxidatively stressed donor cells. Here, we further determined molecular mechanisms involved in uptake of EVs by naïve RPE cells.MethodsRPE cells were grown as monolayers in media supplemented with 1% FBS followed by transfer to FBS-free media. Cultures were used to collect control or stress EVs upon treatment with H₂O₂, others served as naïve recipient cells. In recipient monolayers, TER was used to monitor EV-uptake-based activity, live-cell imaging confirmed uptake. EV surface proteins were quantified by protein chemistry.ResultsClathrin-independent, lipid raft-mediated internalization was excluded as an uptake mechanism. Known ligand-receptor interactions involved in clathrin-dependent endocytosis include integrins and proteoglycans. Desialylated glycans and integrin-receptors on recipient cells were necessary for EV uptake and subsequent reduction of TER in recipient cells. Protein quantifications confirmed elevated levels of ligands and neuraminidase on stress EVs. However, control EVs could confer activity in the TER assay if exogenous neuraminidase or additional ligand was provided.ConclusionsIn summary, while EVs from both stressed cells and control contain cargo to communicate stress messages to naive RPE cells, stress EVs contain surface ligands that confer rapid uptake by recipient cells. We propose that EVs potentially contribute to RPE dysfunction in aging and disease.     

Transformation of Chloroplast Ribosomal RNA Genes in Chlamydomonas: Molecular and Genetic Characterization of Integration Events

Transformation of chloroplast ribosomal RNA (rRNA) genes in Chlamydomonas has been achieved by the biolistic process using cloned chloroplast DNA fragments carrying mutations that confer antibiotic resistance. The sites of exchange employed during the integration of the donor DNA into the recipient genome have been localized using a combination of antibiotic resistance mutations in the 16S and 23S rRNA genes and restriction fragment length polymorphisms that flank these genes. Complete or nearly complete replacement of a region of the chloroplast genome in the recipient cell by the corresponding sequence from the donor plasmid was the most common integration event. Exchange events between the homologous donor and recipient sequences occurred preferentially near the vector:insert junctions. Insertion of the donor rRNA genes and flanking sequences into one inverted repeat of the recipient genome was followed by intramolecular copy correction so that both copies of the inverted repeat acquired identical sequences. Increased frequencies of rRNA gene transformants were achieved by reducing the copy number of the chloroplast genome in the recipient cells and by decreasing the heterology between donor and recipient DNA sequences flanking the selectable markers. In addition to producing bona fide chloroplast rRNA transformants, the biolistic process induced mutants resistant to low levels of streptomycin, typical of nuclear mutations in Chlamydomonas.     

Biological aspects of limb transplantation: I. Migration of transplanted bone marrow cells into recipient

The transplanted limb contains bone marrow tissue. The hematopoietic cells contained in the bone of the graft normally differentiate after transplantation and can be released to the recipient. The cells migrate to the recipient bone marrow cavities and lymphoid organs. This causes the immune reaction between the donor and the recipient, which develops not only in the graft itself but also in the recipient immune organs where donor bone marrow cells home. The purpose of this study was to investigate the process of migration of the hematopoietic cells from the donor limb to the recipient bone marrow cavities and lymphoid tissues. The questions the authors asked were: what is the rate of release of bone marrow cells from the transplanted bone, where do the released bone marrow cells home in the recipient, how fast are donor bone marrow cells rejected by the recipient, and can some bone marrow cells homing in the recipient tissues survive and create a state of microchimerism. Experiments were performed on Brown Norway and Lewis inbred rat strains (n = 30). Limb donors received intravenous chromium-51-labeled bone marrow cells. Twenty-four hours later, the limb with homing labeled bone marrow cells was transplanted to an allogeneic or syngeneic recipient. The rate of radioactivity of bone marrow cells released from the graft and homing in recipient tissues was measured after another 24 hours. To eliminate factors adversely affecting homing such as the "crowding effect" and allogeneic elimination of bone marrow cells by natural killer cells, total body irradiation and antiasialo-GM1 antiserum were applied to recipients before limb transplantation. In rats surviving with the limb grafts for 7 and 30 days, homing of donor bone marrow cells was studied by specific labeling of donor cells and flow cytometry as well as by detecting donor male Y chromosome. The authors found that transplantation of the limb with bone marrow in its natural spatial relationship with stromal cells and blood perfusion brings about immediate but low-rate release of bone marrow cells and their migration to recipient bone marrow and lymphoid tissues. Large portions of allogeneic bone marrow cells are rapidly destroyed in the mechanism of allogeneic elimination by radioresistant but antiasialo-GM1-sensitive natural killer cells. Some transplanted bone marrow cells remain in the recipient's tissues and create a state of cellular and DNA microchimerism. A low number of physiologically released donor bone marrow cells do not seem to adversely affect the clinical outcome of limb grafting. Quite the opposite, a slight prolongation of the graft survival time was observed.     

Uptake and early fate of metaphase chromosomes ingested by the Wi-L2 human lymphoid cell line

Aspects of the ingestion and early intracellular fate of homologous. [3H]-thymidine-labeled chromosomes (donor) were studied in recipient Wi-L2 cells in the absence of reutilized radioactivity. As much as 67% of the cell-associated radioactivity was resistant to hydrolysis by DNase I after 4 h of incubation. Cell fractionation and electron microscope autoradiography indicated that chromosome uptake was rapid, into both cytoplasmic and nuclear fractions and was facilitator and dose dependent. Sedimentation analysis demonstrated that at 4 h donor DNA of approximate single-strand mol wt of 1--6 X 10(6), as compared to 6--12 X 10(6) for chromosomal DNA, was recoverable in cell fractions. By 6 h, a significant portion of the nucleus-associated donor DNA was converted into material of higher mol wt, although no evidence was found for integration into recipient DNA. Cytoplasmic donor DNA continued to be degraded. An average number of chromosome equivalents of nucleus-associated donor DNA to recipient cell nuclei of 1--4 was obtained and its relationship to the lower frequency of chromosome-mediated gene transfer is discussed.     

Temporal changes in the distribution of thoracic duct lymphoblasts to synovium and other tissues of rats with adjuvant-induced arthritis

The distribution of lymphoblasts(lymphocytes in cell cycle) obtained from the central lymph of donor rats and transferred adoptively to syngeneic recipients has been shown previously to be influenced by the presence of arthritis in either donor or recipient rats. The intent of the present study was to examine patterns of distribution of lymphoblasts in the early period after transfer, when extravasation of donor lymphoblasts was expected to occur. Thoracic duct lymphoblasts labelled in vitro with [125I]-iododeoxyuridine were detected in recipient rats by external radiometry and autoradiography.Irrespective of donor status, fewer donor lymphoblasts accumulated in the feet of normal recipients when compared to arthritic recipients at 15 min, 2 h and 24 h after cell transfer.When recipients of similar disease status were compared, the percentages of injected lymphoblasts from normal and arthritic donors recovered in the feet were similar at 15 min and 2 h after transfer. The proportions of lymphoblasts recovered in the feetat 24 h after injection declined in normal recipients and arthritic recipients of cells from normal donor rats. Importantly,this decline did not occur when both the donor and the recipient were arthritic. In the hindpaws, donor lymphoblasts were located predominantly in the bone marrow, except in transfers between arthriticrats, when at 24 h they were predominantly in the synovium.At 15 min, lymphoblasts were detected within the lumen of vessels within synovium, whereas by 2 h extravasation of these cells was evident. In conclusion, lymphoblasts accumulate more readily in hindfeet that are inflamed. In the early hours after injection, lymphoblasts from normal and arthritic donors are recruited equally, but these early levels are only maintained for 24 hin the combination of arthritic donor and arthritic recipient. Adramatic change in the proportion of lymphoblasts located in synoviumat this later time suggests that a dynamic process of relocation,retention and/or local cell division maintains the numbers of arthritic donor cells in the latter combination.     

Role of V1V2 and other human immunodeficiency virus type 1 envelope domains in resistance to autologous neutralization during clade C infection

Biologically functional clade C envelope (Env) glycoproteins from the chronically (donor) and newly (recipient) infected partners of four heterosexual transmission pairs in Zambia were cloned and characterized previously. In each case, the donor viral quasispecies contained Envs that were resistant to autologous neutralization by contemporaneous plasma, while the recipient Envs were sensitive to neutralizing antibodies in this donor plasma sample. The donor Envs also varied in length, glycosylation, and amino acid sequence of the V1V2 hypervariable domain of gp120, while the recipient Envs were much more homogeneous. To assess the contribution of V1V2 to the neutralization phenotype of the donor Envs, V1V2 domains from neutralization-sensitive recipient Envs were replaced with donor V1V2 domains, and the autologous neutralization sensitivities of the chimeric Envs were evaluated using a virus-pseudotyping assay. Long donor V1V2 domains regulated sensitivity to autologous neutralization, although the effect was dependent on the Env background. Short donor V1V2 domains did not confer neutralization resistance. Primary sequence differences in V2 were also found to influence neutralization sensitivity in one set of recipient Envs. The results demonstrate that expansion of the V1V2 domain is one pathway to escape from autologous neutralization in subtype C Envs. However, V1V2-independent mechanisms of resistance also exist, suggesting that escape is multifaceted in chronic subtype C infection.     

Recipient preparation is critical for spermatogonial transplantation in the rat

Testis cell transplantation from mice or rats into recipient mouse seminiferous tubules results in donor cell-derived spermatogenesis in nearly all host testes. Normal spermatozoa are produced and, in the most successful mouse transplantations, the donor haplotype is transmitted to progeny of the recipient. However, few studies have been performed in other species. In this report, we demonstrate that rat and mouse testis cells will generate donor cell-derived spermatogenesis in recipient rat seminiferous tubules. Depletion of endogenous spermatogenesis before donor cell transplantation was more difficult in rat than reported for mouse recipients. A protocol employing treatment of neonatal rats with busulfan was most effective in preparing recipients and allowed more than 90% of testes to be colonized by donor cells. Transplantation of mouse testis cells into rat seminiferous tubules was most successful in recipients made cryptorchid and treated with busulfan. In the best experiments, about 55% of rat testes were colonized by mouse cells. Both rat and mouse donor cell-derived spermatogenesis were improved by treatment of rat recipients with leuprolide, a gonadotropin-releasing hormone agonist. The studies indicated that recipient preparation for spermatogonial stem cell transplantation was critical in the rat and differs from the mouse. However, modification of currently used techniques should allow male germ line stem cell transplantation in many species.     

Transferable tetracycline resistance in Clostridium perfringens strains of porcine origin

These studies represent the first systematic survey of the incidence of conjugative antibiotic resistance in Clostridium perfringens. Ninety-two antibiotic-resistant porcine strains were examined to see if they could donate their antibiotic-resistance determinants to sensitive recipient strains. Fifteen of the 89 tetracycline-resistant strains transferred their tetracycline resistance in mixed-plate mating experiments but no transfer of macrolide-lincosamide resistance was detected. The efficiencies of transfer of tetracycline resistance varied from 1.3 X 10(-3) to 1.9 X 10(-6) transconjugants per donor cell. Significantly higher transfer efficiencies were observed when both the donor and recipient strains were derivatives of strain CW 362. These values ranged from 3.7 X 10(-1) to 4.6 X 10(-2) transconjugants per donor cell. This high frequency transfer system should prove invaluable for further genetic studies on this microorganism.     

Transduction transmission of the extrachromosomal markers of antibiotic resistance in staphylococcal populations]

Transduction of extrachromosomal markers of resistance to penicillin and erythromycin in staphylococcal strains isolated from patients was studied. Two transduction methods were compared, i. e. transduction with a phage filtrate of the donor culture resistant to erythromycin and transduction on mixed cultivation of the donor and recipient. A higher transduction rate was observed with the latter method. Mixed cultivation of the donor cultures resistant to penicillin or erythromycin and the recipient strains of the wild type sensitive to these antibiotic resulted in transduction of the respective markers. Transductants which acquired prophage 6 simultaneously with marker Egg became donors of erythromycin resistance.