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Thrombin and fibrinogen powders are the active components of advanced surgical hemostasis products including the EVARREST Fibrin Sealant Patch. Measuring the enzymatic activity of thrombin in the presence of fibrinogen is challenging, as hydration of the powders in a neutral aqueous environment will cause the enzyme to rapidly react with the fibrinogen to form a fibrin clot, which in turn binds and entraps the enzyme thus preventing subsequent measurement of thrombin activity. A novel approach has been developed to overcome this challenge. After isolation of the mixture of powders, an alkaline carbonate solution is used to solubilize the proteins, while reversibly inhibiting the activity of thrombin and preventing clot formation. Once the powders have been fully solubilized, thrombin activity can be restored by neutralization in a buffered fibrinogen solution resulting in fibrin clot formulation. The rate of clot formation can be quantified in a coagulometer to determine the thrombin activity of the original powder. Samples coated with powders containing fibrinogen and varying amounts of thrombin were tested using the method described herein. The results demonstrated that the method could consistently measure the activity of (alpha) thrombin in the presence of fibrinogen over a broad range of thrombin activity levels. The test was successfully validated according to International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use Guidelines and thus is suitable for use as part of a commercial manufacturing process. A method has been developed that enables thrombin activity to be measured in a mixture of fibrinogen and thrombin powders.

The objective of the present study was to compare the mechanical, kinetic, and biochemical properties of fibrin clots produced using EVICEL Fibrin Sealant (Human) and TISSEEL Fibrin Sealant. The stiffness/elasticity and strength of fibrin clots formed with EVICEL and TISSEEL were assessed using applied mechanical force and thromboelastography (TEG). The factor XIII content of the fibrin clots was also evaluated. Mean Young modulus and tensile strength of the fibrin clots produced by EVICEL were significantly higher than those of clots produced by TISSEEL (P < 0.05 for both). The mean time to initial clot formation and mean time to the predefined level of clot formation were numerically shorter for EVICEL compared with TISSEEL. Furthermore, mean maximal amplitude of the clots formed with EVICEL was significantly greater than that for the clots formed with TISSEEL. Mean concentration of factor XIII for the EVICEL fibrinogen samples tested was 9 IU/ml compared with undetectable concentrations of factor XIII for the TISSEEL fibrinogen samples. Fibrin clots formed with EVICEL have a much higher resistance to stretching and tensile strength and are more capable of maintaining their structure against applied force than those formed with TISSEEL. EVICEL also allows more rapid development of fibrin clots than TISSEEL. This superior clot strength and resilience obtained with EVICEL relative to TISSEEL may be due in large part to the presence of factor XIII.

Intravenous immunoglobulin (IVIg) is used successfully for therapy of inflammatory and autoimmune diseases, especially in cases of conventional therapy resistance. Within the broad spectrum of immunoregulatory activities of IVIg in vitro and in vivo, the anti-idiotypic activity neutralizing the related idiotypes is one of the main mechanisms. Furthermore, IVIg addresses integrins associated with inflammation and immune response thrombosis, such as the RGD (Arg-Gly-Asp) motif, expressed on a large number of cell surface and matrix proteins. In addition, during the last years, anti-Fas activity of IVIg was reported. We fractionated IVIg specific preparation (sIVIg) based on the multispecificities of the IVIg compound. We have generated an IVIg fraction that will show specific activity for lupus idiotypes in vitro. In NZBxW.F1 mice, results showed 200 times more beneficial effect. Using a peptide phage display library technology, we have identified a panel of lupus-related synthetic idiotypes that are mimetics of the idiotypes presented in patients with systemic lupus erythematosus. A column composed of these synthetic lupus-related idiotypes was used to prepare a large amount lupus-specific IVIg. Using the same approach, we prepared anti-anti-beta-2-glycoprotein-I (beta2GPI) specific IVIg for antiphospholipid syndrome (APS). This APS-specific IVIg reduced the fetal loss induced by anti-beta2GPI antibodies by improving the implantation process in a mouse model. Others prepared specific preparations of IVIg to RGD or for Fas. The molecular targeting with specific IVIg may be used for therapeutical purposes, using a smaller amount of IVIg, and targeting more specifically autoimmune diseases, thrombosis, or inflammatory condition.

Azospirillum brasilense was attracted to capillaries containing either phosphate buffer, distilled water, or saline. The number of bacteria in these capillaries was 3-4×10(4), after 1 h of incubation. In the presence of phosphate buffer + attractants, the number of cells accumulated in the capillary increased only to 5×10(4)-1.1×10(5) cells. It was not possible, therefore, to measure chemotaxis inA. brasilense as distinct from aerotaxis by the capillary method. Chemotaxis was observed in semi-solid agar plates and was determined by a growth band oriented towards the attractant. Positive chemotactic response was obtained with peptone, tryptone, yeast extract, amino acids, organic acids, arabinose and galactose.

Laser tissue soldering is a method of repairing incisions. It involves the application of a biological solder to the approximated edges of the incision and heating it with a laser beam. A pilot clinical study was carried out on 10 patients who underwent laparoscopic cholecystectomy. Of the four abdominal incisions in each patient, two were sutured and two were laser soldered. Cicatrization, esthetical appearance, degree of pain, and pruritus in the incisions were examined on postoperative days 1, 7, and 30. The soldered wounds were watertight and healed well, with no discharge from these wounds or infection. The total closure time was equal in both methods, but the net soldering time was much shorter than suturing. There was no difference between the two types of wound closure with respect to the pain and pruritus on a follow-up of one month. Esthetically, the soldered incisions were estimated as good as the sutured ones. The present study confirmed that temperature-controlled laser soldering of human skin incisions is clinically feasible, and the results obtained were at least equivalent to those of standard suturing.

Laser tissue soldering is a method of repairing incisions. It involves the application of a biological solder to the approximated edges of the incision and heating it with a laser beam. A pilot clinical study was carried out on 10 patients who underwent laparoscopic cholecystectomy. Of the four abdominal incisions in each patient, two were sutured and two were laser soldered. Cicatrization, esthetical appearance, degree of pain, and pruritus in the incisions were examined on postoperative days 1, 7, and 30. The soldered wounds were watertight and healed well, with no discharge from these wounds or infection. The total closure time was equal in both methods, but the net soldering time was much shorter than suturing. There was no difference between the two types of wound closure with respect to the pain and pruritus on a follow-up of one month. Esthetically, the soldered incisions were estimated as good as the sutured ones. The present study confirmed that temperature-controlled laser soldering of human skin incisions is clinically feasible, and the results obtained were at least equivalent to those of standard suturing.

BACKGROUND Vascular access in children carries a significant risk of accidental extravasation of IV fluids and medications with the potential for tissue injury. In this prospective controlled study we assessed the diagnostic utility of using IV diluted sodium bicarbonate to confirm placement of IV catheters in ventilated children. Diluted sodium bicarbonate was created using undiluted standard 8.4% (1 mEq/mL) sodium bicarbonate mixed in a 1:3 and 1:5 ratio with sterile water to achieve a final diluted concentration of 2.1% (0. 25 mEq/mL) and 1.05% (0.125 mEq/mL) sodium bicarbonate, respectively. METHODS In 18 ASA I-II mechanically ventilated children ages 1 to 8 years, the effects of 1 mL/kg of dilute 2.1%, 1.05% sodium bicarbonate, or 0.9% normal saline, injected in a randomized order, were analyzed. All children had oxygen saturation, arterial blood pressure, electrocardiograph, and end-tidal carbon dioxide (ETCO(2)) monitoring. In addition, venous blood samples were taken before injection and 10 minutes after the final injection for analysis of venous blood pH and electrolytes. RESULTS In children, IV diluted 2.1% sodium bicarbonate resulted in significantly increased etco(2) (mean of 32.8 ± 3.4 mm Hg to 39.0 ± 3.5 mm Hg, P < 0.001), a mean increase of 6.2 mm Hg (95% prediction interval: 4.3 to 8.1 mm Hg) within 3 breaths. Intravenous diluted 1.05% sodium bicarbonate caused a less pronounced but still significant increase in etco(2) (33.4 ± 3.8 mm Hg to 36.3 ± 3.4 mm Hg, P < 0.001), a mean increase of 2.9 mm Hg (95% prediction interval: 1.8 to 4.1 mm Hg) within 3 breaths. Normal saline did not result in any significant changes, with a mean increase of 0.06 mm Hg (95% prediction interval: -1.3 to 1.4 mm Hg). Both concentrations of sodium bicarbonate were easily differentiated from normal saline injection by blinded anesthesiologists observing the change in etco(2) values immediately after injection. Analysis of pre- and postinjection venous pH, bicarbonate, and sodium levels could not detect clinically significant changes. A small but statistically significant increase in venous bicarbonate was noted. CONCLUSION The injection of 2.1% sodium bicarbonate in mechanically ventilated ASA I-II children identified intravascular placement and patency of an IV catheter by an increase in the exhaled CO(2) concentration. The injections did not have any clinically significant effects on blood pH, bicarbonate, or sodium concentration.

Objective To compare the efficacy of a fibrin preparation supplemented with tranexamic acid (Adhexil) with that of established devices, and to determine whether its effect is limited to the site of application. Design Rabbit uterine horns were abraded in nonbleeding and bleeding variants of an established adhesions model. In a separate study, a sidewall excision with approximation of the abraded cecum was added. Animals randomly received Adhexil at both, neither, or either loci. Setting Laboratory study. Animal(s) Seventy-two female New Zealand White rabbits (Oryctolagus cuniculus). Intervention(s) Adhexil, Seprafilm or SprayGel and Interceed. Main Outcome Measure(s) The extent of adhesions was evaluated 13 to 16 days after surgery. Result(s) Adhexil reduced adhesions (15 ± 7%; 15 ± 4%) compared with controls (74 ± 13%; 78 ± 9%) in the bleeding and nonbleeding models, respectively. The reductions resulting from the use of Seprafilm (39 ± 17%; 34 ± 14%) or SprayGel (61 ± 18%; 43 ± 14%) (n = 4) were not statistically significant. In the bleeding model, Interceed (48 ± 15%) reduced adhesions only modestly. Conclusion(s) In the combined uterine and sidewall model, Adhexil reduced selectively the extent and incidence of adhesions. The absolute and relative performance of Adhexil in an established adhesions model and in the presence of bleeding justifies its further investigation.

Two fibrinogen preparations, each an intermediate in the manufacture of the 'fibrinogen' component of a commercial human tissue sealant, were made from a common cryoprecipitate source. The first preparation, prepared according to the process described by Schwartz et al. had a higher ratio of clottable to total protein than the second preparation, prepared according to that by Martinowitz and Bal but a much lower ratio of fibronectin to fibrinogen. After clotting with thrombin and solubilization and reduction of the clots, sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a much higher content of high molecular weight polymers of fibrin(ogen) in the second preparation than in the first. The second preparation bound to collagen more strongly than did cryoprecipitate and much more strongly than did the first one. Experiments with highly purified proteins showed that fibronectin was essential in promoting progressive binding of fibrinogen to collagen under the action of activated factor XIII (transglutaminase). It was concluded that, because of their method of purification from cryoprecipitate, preparations of fibrinogen differ in their content of fibronectin and heteronectin. The binding of these proteins to collagen may improve the adhesion of tissue sealant clots to the extracellular matrix.

The objective of the research was to determine the effect of the type, dose, and volume of anti-fibrinolytic agents (tranexamic acid, aprotinin) added to fibrin formulations, on adhesion development. Adhesions were induced in 228 male rats by creating apposing parietal and visceral peritoneal defects. Animals were randomized to receive no treatment or a fibrin formulation containing aprotinin or tranexamic acid. Seven days later the incidence of adhesions, and the force and energy required to detach them, were determined. Adhesions developed in 13/13 rats in the control and aprotinin groups. Treatment with fibrin (100 mg/ml tranexamic acid) resulted in adhesions in 4/14 rats (as strips, p < or = 0.0005), 4/10 rats (as spray, p < or = 0.0036), and 12/15 rats (by drip). The reduction of adhesions was dependent on the concentration of tranexamic acid with strip and spray application. Using commercial formulations, tranexamic-acid-containing fibrin (10/15, p = 0.042), but not aprotinin-containing fibrin (13/15), reduced the incidence of side-wall adhesions from 15/15 in controls. Fibrin containing either tranexamic or aprotinin reduced the incidence and severity of adhesions. This effect was greater when tranexamic acid was used and was dependent on the mode of administration, the volume, and to a degree, the concentration of tranexamic acid.

Conventional large (500-800 nm) multilamellar liposomes encapsulating DNA have been used in vivo as gene vectors into rat and pig liver. By using the intraportal vein route, high dose DNA (10 mg/kg) provided low efficiency and transient luciferase gene expression in the liver. This gene expression was, however, increased by liver resection (> 50%), ischemia (20 min) or orthotopic transplantation. As evidenced by histochemical analysis of beta-galactosidase expression, the gene transfection mainly ensued in Kupffer cells, but spleen and lung were contaminated. In comparison, injection into the bile duct of even 25-fold lower dose of liposome-encapsulated DNA (0.4 mg/kg) produced higher (100-fold) and long-lasting (during 6 days, at least) luciferase expression in rat liver. The gene expression was restricted to the liver and enhanced by liver resection. By this route, transgene-expressing cells were mainly hepatocytes. A treatment with colchicine prior to the administration of the vector allowed the persistence of relative high gene expression for at least 7 days. In pigs, qualitatively similar, but quantitatively less efficient gene expression was obtained by either the portal vein or the bile duct route. These results indicate that intrabile duct route might render large non-viral vectors applicable to gene transfer into the hepatocytes. The efficiency of liposome-mediated gene transfer into the liver can be increased by liver resection, ischemia or transplantation performed before DNA injection.

Hemophilia B is an X-chromosome-linked recessive disorder that is caused by a deficiency of biologically active clotting factor IX (FIX). In this work, liposomes (Lip) were used for non-viral, in vivo gene transfer of the human FIX gene into mouse organs. Plasmid DNA, containing the human FIX cDNA under the control of the Moloney murine leukemia virus (MoMLV) long terminal repeat (LTR), was encapsulated in 1-2-microns multilamellar Lip composed of egg phosphatidylcholine (EPC). The percentage of Lip-associated DNA was 47%, and 72% of the Lip DNA was protected from DNase I digestion. The Lip-encapsulated (Len) DNA was injected intravenously into Balb/c mice, and at various times post-injection, various tissues were examined for the presence of the exogenous DNA. Plasmid DNA was detected by Southern blot analysis mainly in the liver and spleen, but small amounts were also detected in the lungs, heart and kidneys. The plasmid DNA was retained in mouse liver cells for at least 7 days post-injection, and remained in an episomal state. The levels of human FIX protein in the mouse plasma were 190-650 pg per ml for 2 to 7 days post-injection. Treatment of mice with chloroquine (Cq) and colchicine (Cc) prior to Lip injection significantly increased the amount of plasmid DNA found in the liver cells, as well as the level of human FIX in the plasma. These results demonstrate the potential use of Len DNA for gene transfer into liver and spleen, and for gene therapy of inherited and acquired disorders.

Muscle cells, when transduced with a factor IX (FIX) expression vector, are capable of producing FIX protein and secreting it into the blood. These cells can thus serve as appropriate targets for gene transfer in hemophilia B patients. In this work, we studied the potential of retrovirus-mediated in vivo gene transfer of the FIX gene into muscle cells. Producer cells, which produce recombinant retroviruses containing the human FIX cDNA under the transcriptional control of the chicken beta-actin promoter, were treated with the cytostatic drug mitomycin C (MC), and injected into the rectus femoris muscle of 6-week-old CB-17/1cr severe combined immune-deficient (SCID) mice. As a control, SCID mice were injected with NIH 3T3 cells, which were stably transformed by the retroviral vector described above and therefore secreted FIX, but did not produce retroviruses. The injected MC-treated cells did not cause any tumours to develop in the mice, and the amount of human FIX in the plasma of mice injected with the producer cells was significantly higher, at any of the time points measured beyond 10 days postinjection, than the amount of human FIX in the plasma of mice injected with FIX-secreting 3T3 cells. These results indicate that muscle cells transduced by retroviruses produced human FIX and secreted it into the blood. In vivo gene transfer to muscle cells by injection of retrovirus-producing cells, or by direct injection of recombinant retroviruses, could prove to be important for the gene therapy of hemophilia B.

The novel use of nonradioactive sulfone labeling of DNA has been utilized in the highly sensitive ChemiProbe system, enabling the detection of 0.1 pg of DNA in less than 16h in dot, slot, and Southern blot hybridization techniques (Herzberg et al., 1987; Nur et al., 1989).

Human papillomavirus (HPV) was detected by using two sets of deoxyribonucleotide primers for differentiating between 'low-risk' types (HPV11 and HPV6) and 'high-risk' (hri) types (HPV16, HPV18 and HPV33). A new application of the Chemiprobe method for labeling DNA was used to detect products of the polymerase chain reaction (PCR) from 36 cervical biopsies. This method, first demonstrated by Uchimura et al. (submitted), is based on the sulfonation of a polycytidylic acid tail of 5-20 monomers attached to the 5' end of either one or both of the PCR primers. This procedure can increase the sensitivity of detection of PCR products more than 100-fold with respect to ethidium bromide (EtdBr) staining. Various methods were used to detect hri HPV DNA in the 36 clinical samples. The number of positive results obtained was as follows, two by Southern-blot hybridization; five by PCR amplification followed by electrophoresis and detection of products by EtdBr staining; six by PCR amplification using one or two sulfonated C-tailed primers followed by electroblotting and immunoenzymatic visualization; and five by hybridization of sulfonated genomic viral recombinant with a PCR product immobilized on a membrane. The yield of the PCR product was significantly greater when one of the primers was C-tailed than when both or neither of the primers were C-tailed. PCR employing sulfonated C-tailed oligo primers is very specific and sensitive, and the entire procedure can be employed as a nonradioactive substitute for radioactive dot-blot or Southern-blot hybridization procedures, routinely used for detection of HPV in clinical samples.

The association between nitrogen-fixing bacteria from the genus Azospirillum and the grasses Zea mays and Setaria italica was investigated in sterilized Leonard-jar assemblies. Nitrogen-fixing bacteria isolated from Cynodon dactylon roots in Israel and Azospirillum brasilense (Sp-7, Sp-80, and Cd) were examined. C(2)H(2) reduction activity was detected in systems containing 0.0 to 0.08 but not in those containing 0.16 gram per liter NH(4)NO(3). The organisms tested significantly increased plant dry weight (50-100%), total N content of leaves (50-100%) and C(2)H(4) production (300-1000 nanomoles C(2)H(4) per plant per hour). Highest C(2)H(2) reduction activities were obtained above 30 C and with high light intensities. Significant increases in S. italica dry weight (DW) and nitrogen (N) content were observed in sand (DW = 80%, N = 150%), sandy loam soil (DW = 80%, N = 75%) and loess (DW = 37%, N = 25%). The results obtained in this work clearly demonstrate the potential benefit of inoculating grasses with Azospirillum.

Two cultivars of Triticum aestivum and T. turgidum were inoculated with bacteria of the genus Azospirillum in field experiments carried out in the Northern Negev and Bet Shean Valley. The results showed significant increases in yield of grain and foliage. There were significant increases in the number of fertile tillers per unit area and in the nitrogen yield. The possible mechanisms by which Azospirillum inoculation benefited wheat and other crops are discussed.

SUMMARY: Azospirillum brasilense strain Cd was grown in a medium containing NH4 + in a chemostat at a range of constant dissolved oxygen tensions (d.o.t.) (0·007--0·18 atm). Poly-β-hydroxybutyrate (up to 12% of the cell dry weight) increased under oxygen limitation and moderate dilution rate (D = 0·14 h-1). The highest carotenoid content was observed at high d.o.t. and dilution rates up to 0·12 h-1. The amount of protein varied with d.o.t. from 0·29 mg protein (mg dry wt)-1 at 0·007 atm to 0·54 mg at 0·18 atm. The yield efficiency and respiration rate were highest at low d.o.t. and decreased significantly at a d.o.t. of 0·18 atm. Succinate dehydrogenase and malate dehydrogenase activities increased 2·5-fold at 0·10-0·18 atm O2, whereas succinate oxidase and NADH oxidase activities increased consistently with increasing d.o.t. Azospirillum brasilense showed a low specific activity for catalase; the specific activity of superoxide dismutase increased sharply above 0·16 atm O2.

Small fragments of DNA from an 8-kbp plasmid, pRA1, from a plant pathogenic strain of Spiroplasma citri were shown previously to be present in the chromosomal DNA of at least two species of Spiroplasma. We describe here the shot-gun cloning of chromosomal DNA from S. citri Maroc and the identification of two distinct sequences exhibiting homology to pRA1. Further subcloning experiments provided specific molecular probes for the identification of these two sequences in chromosomal DNA from three distinct plant pathogenic species of Spiroplasma. The results of Southern blot hybridization indicated that each of the pRA1-associated sequences is present as multiple copies in short, dispersed, and repetitive sequences in the chromosomes of these three strains. None of the sequences was detectable in chromosomal DNA from an additional nine Spiroplasma strains examined.

Bacteria belonging to the genus Azospirillum isolated from Cynodon dactylon roots in Israel were compared with Azospirillum brasilense from Brazil and California for their ability to fix nitrogen in association with grasses under greenhouse conditions. The plants were grown in a system which avoided cross inoculation from the inoculated soil to the control, while maintaining the natural soil microflora and humidity level in the soil close to field capacity. The organisms tested significantly increased the dry weight of Zea mays and Setaria italica leaves, the total nitrogen content of these leaves (as measured by the Kjeldahl method), and supported acetylene reduction in intact nonsterile systems as compared with the noninoculated controls. Ethylene production in intact systems could be detected after 6 h and was linear for 72 h, providing a constant soil temperature (28–32 °C) was maintained.

Five strains of Azospirillum brasilense and two of Azospirillum spp., from Israel, responded to self-created and preformed oxygen gradients by forming aerotactic bands in capillary tubes and actively moving toward a specific zone with low dissolved oxygen. Increasing the oxygen concentration in capillaries containing phosphate buffer increased the number of attracted bacteria and decreased band velocity. High O2 concentrations and H2O2 temporarily repulsed the bacteria, causing the formation of a bacterial arc around the capillary mouth. There was no band formation under anaerobic conditions, although the bacteria remained highly motile. Exogenous energy sources were unnecessary for aerotaxis in Azospirillum spp. The addition of oxidizable substrates to the capillary slightly enhanced aerotaxis, possibly by accelerating O2 consumption. Aerotactic band formation was affected by pH, bacterial concentration and age, incubation time, and respiratory inhibitors, but not by the lack of combined nitrogen in the growth medium. It is proposed that aerotaxis plays a role in the capacity of Azospirillum spp. to reach an environment suitable for N2 fixation.

SUMMARY: Four different carotenoids were isolated and purified from membranes of the N2-fixing bacterium Azospirillum brasilense strain Cd, grown under aerobic conditions, and one from strain Cd-1. Carotenoid synthesis did not occur under microaerobic conditions. Cells of these strains formed aggregates (>0-1 mm in diameter) when subjected to aerobic conditions. Neither carotenoids nor cell aggregation were observed in A. brasilense strains Sp 7, Sp81 and Sp51e. All of the differently pigmented Azospirillum strains tested contained similar amounts of soluble cytochrome c. In the presence of diphenylamine, which specifically inhibits carotenoid synthesis, the rate of acetylene reduction (N2 fixation) in strain Cd decreased to 50% of the control. Carotenoid synthesis was inhibited in cells grown in the presence of NH4Cl at concentrations higher than 10 mM. Carotenoid synthesis started in liquid cultures of suain Cd only after the concentration of NH4Cl in the medium decreased, and N2 fixation became evident at the same time. In comparison, strain Sp81 did not grow or fix nitrogen after NH4Cl was depleted. Carotenoids appear to protect the nitrogenase of A. brasilense strains Cd and Cd-1 from oxidative damage.

The potential of the nitrogen fixing bacteriumAzospirillum brasilense to enhance development and increase growth of several gramineae was investigated. In both sterilized and non-sterilized systems heading and flowering occurred earlier in the inoculated plants as compared to the noninoculated ones. Total shoot and root weights, total-N content, plant height and leaf length were significantly increased by inoculation.

Spiroplasma citri was found to carry an 8.0 kb plasmid that differed from previously describedS. citri plasmids in its restriction map. It was also clonable in pBR322. The plasmid, named pRA1, was found in large quantities as free plasmid inS. citri (R8A2, Maroc) subclones of low passage level. In subclones of higher passage levels, free plasmid was replaced by plasmid sequences integrated into the spiroplasma chromosome. Significant quantities of integrated plasmid sequences were also observed in the corn stunt spiroplasma,S. kunkelii, while small quantities of free and/or integrated plasmid DNA could be detected in some spiroplasmas serologically and genotypically remote fromS. citri. Integrated plasmid sequences were cloned into theEscherichia coli plasmid pUC13. Hybridization tests and restriction maps of these clones indicated that the integrated plasmid sequences consisted of fragments, rather than entire plasmid DNA, inserted into specific sites in the spiroplasma chromosome. Although the biological role of the pRA1 plasmid remains unclear, theS. citri subclones containing large quantities of free plasmid exhibited slower growth rates and a tendency to lyse.

Azospirillum brasilense strain Cd responded chemotactically to amino acids, sugars and organic acids. Chemotactic rings were observed in semisolid agar plates containing oxidizable substrates. Increasing sodium succinate concentration decreased the velocity of ring expansion. Chemotactic activity of Azospirillum was also examined by a newly developed technique using a channelled chamber. Varying the concentrations of aspartic and glutamic acids affected the chemotactic response of the bacteria. In both assays chemotaxis was obtained under conditions that prevented aerotaxis.

The association between the nitrogen-fixing bacterium Azospirillum brasilense (strain cd) and the grass Setaria italica was studied under different environmental and soil conditions. Highest acetylene reduction rates in intact plants were observed at the booting stage of Setaria (2350 nmol ethylene produced hour−1 plant−1) at 27 C. Higher temperatures, up to 32 C, enhanced ethylene reduction. Significant increases in shoot dry weight, panicle weight, and length were obtained in inoculated plants fertilized with suboptimal NH4NO3 levels. The increase in nitrogen content of plants inoculated with A. brasilense was shown to be due to N2 fixation. This was demonstrated by growing plants in washed quartz sand with no combined nitrogen. The bacteria also increased branching and development of roots. It was concluded that inoculation of Setaria with A. brasilense may lead both to increases in plant yield and saving of nitrogen fertilizer.

Free-living, dinitrogen-fixing bacteria associated with roots of grasses were isolated from several locations in Israel. Bacteria with characteristics similar to those of Azospirillum were isolated from Cynodon dactylon roots and were compared with Azospirillum brasilense from Brasil (Sp-7) and from California (Cd). Colonies of the Israeli isolates were yellow and consisted of curved rods, 0.5–0.6 μm in diameter with polar flagella, whereas colonies of A. brasilense were pink (Sp-7) and red (Cd) and the cells were 1.0–1.1 μm in diameter with polar flagella. Ultraviolet absorption spectra of soluble c and membrane-bound c and b cytochromes were similar in all isolates. When grown in semisolid agar medium with or without ammonium chloride all isolates formed a growth zone below the surface. However, they grew best under aerobic conditions in liquid medium containing NH4Cl. All isolates could use salts of malate and lactate, arabinose, and galactose, but not mannitol, as sole carbon sources; they did not need biotin to shorten their lag phase. One Israeli isolate was capable of growing and fixing nitrogen with glucose as a sole carbon source. The Israeli isolates formed aggregates above pH7.6 in liquid or semisolid medium and were capable of reducing nitrate to nitrogen gas under anaerobic conditions.

Differences in the type of base methylated (cytosine or adenine) and in the extent of methylation were detected by high-pressure liquid chromatography in the DNAs of five spiroplasmas. Nearest neighbor analysis and digestion by restriction enzyme isoschizomers also revealed differences in methylation sequence specificity. Whereas in Spiroplasma floricola and Spiroplasma sp. strain PPS-1 5-methylcytosine was found on the 5' side of each of the four major bases, the cytosine in Spiroplasma apis DNA was methylated only when its 3' neighboring base was adenine or thymine. In Spiroplasma sp. strain MQ-1 over 95% of the methylated cytosine was in C-G sequences. Essentially all of the C-G sequences in the MQ-1 DNA were methylated. Partially purified extracts of S. apis and Spiroplasma sp. strain MQ-1 were used to study substrate and sequence specificity of the methylase activity. Methylation by the MQ-1 enzyme was exclusively at C-G sequences, resembling in this respect eucaryotic DNA methylases. However, the MQ-1 methylase differed from eucaryotic methylases by showing high activity on nonmethylated DNA duplexes, low activity with hemimethylated DNA duplexes, and no activity on single-stranded DNA.

Here we report that the 600 bp promoter-like region at the left end of a newly isolated and characterized rat L1 DNA element can activate the prokaryotic chloramphenicol acyltransferase gene in a rat cell line. Activation only occurs when the promoter region is oriented to the transferase gene as it is to the L1 protein encoding sequences and is 75% inhibited by methylation of just 5 of the 22 CpGs present in the promoter. The G + C rich promoter contains enough CpGs to qualify it as a CpG island, but in contrast to other CpG islands, genomic L1 promoters are fully methylated in both somatic cell and sperm DNA as judged by restriction enzyme analysis. Partial demethylation of the genomic promoters by treatment with 5-azacytidine failed to produce discrete L1 transcripts. The relationship of methylation to the evolutionary history and fate of the rat L1 promoter is discussed.

Background West Nile Virus (WNV) is endemic in Israel and a significant level of antibodies is present in the population due to natural exposure. Anecdotal cases suggested that the presence of anti-WNV antibodies in intravenous immunoglobulin (IVIG) from Israeli donors (IVIG-IL) assisted the recovery of patients with severe WNV infection. Methods To enhance the therapeutic efficacy of IVIG-IL against WNV infection, OMRIX Biopharmaceuticals, Israel, have developed a strategy for selection of plasma units from a 10% fraction of Israeli blood donors with anti-WNV antibodies. Positive units were processed into pharmaceutical grade WNV IVIG (WNIG). Following inoculation with WNV, mice received i.p. injections of different doses (0.01–8 mg/mouse) of IVIG-IL or WNIG, according to the specific experimental protocol. Results WNIG was about 10 times more potent (per gr of IgG) than was regular IVIG-IL when tested by ELISA and neutralization assays. In a mouse lethal WNV infection model, prophylactic treatment with WNIG was at least 5–10-fold more potent as compared to treatment with IVIG-IL. Treatment with WNIG during active encephalitis, three or four days following WNV infection, had a significant protective effect. WNIG was also very effective in protecting immunosuppressed mice. Indeed, treatment of dexamethasone-immunosuppressed mice with 0.2 or 1.0 mg WNIG 4 h after virus infection, led to 100% survival. Conclusion IVIG produced from selected plasma donated in WNV endemic regions can be used to produce WNV IVIG with superior activity for therapeutic and prophylactic measures.