Simultaneous detection of different Rhizobium strains marked with either the Escherichia coli gusA gene or the Pyrococcus furiosus celB gene

A new marker system for gram-negative bacteria was developed on the basis of the celB gene from the hyperthermophilic archaeon Pyrococcus furiosus, which encodes a thermostable beta-glucosidase with a high level of beta-galactosidase activity. The celB gene is highly suitable as a marker for studying plant-bacterium interaction because endogenous background beta-glucosidase and beta-galactosidase enzyme activity can readily be inactivated by heat and because inexpensive substrates for detection are commercially available. Two celB-expressing transposons were constructed for use in ecological studies of a variety of gram-negative bacteria. The combined use of the gusA marker gene and celB allowed the simultaneous detection of several Rhizobium strains on a plant, and multiple-strain occupancy of individual modules also could be easily detected.     

Improved outcome in patients treated with postoperative radiation therapy for pathologic stage I/II endometrial cancer

OEE33, a component of the oxygen-evolving enzyme in chloroplasts, normally resides in the thylakoid lumen. In an attempt to study the fate of mistargeted proteins in chloroplasts, we substituted the bipartite transit peptide of OEE33 with that of CAB7, an integral thylakoid-membrane protein. As a result, when imported into isolated chloroplasts, the chimeric protein protein was targeted to the stroma instead of the thylakoid lumen. Whereas the wild-type OEE33 was totally stable for at least 2 h, the chimeric protein was rapidly degraded, with a half-life of 60 min. Degradation of the chimeric protein was stimulated by ATP supplementation. Degradation could also be observed in lysed chloroplasts, in an ATP-stimulated manner. When lysates were fractionated, the proteolytic activity was found to be associated mainly with the stromal fraction. This activity was very effectively inhibited by all tested inhibitors of serine proteases. Western blot analysis demonstrated that the stromal fraction active in degrading the chimeric OEE33 contains ClpC and ClpP, homologues of the regulatory and proteolytic subunits, respectively, of the bacterial, ATP-dependent, serine-type Clp protease.     

CTLA4Ig inhibits alloantibody responses to repeated blood transfusions

Allosensitization is a fundamental problem that limits the effectiveness of blood transfusions. Patients who receive multiple transfusions of blood or blood components frequently develop alloantibodies against donor alloantigens. Allosensitized patients are refractory to further transfusion and difficult to transplant successfully. CTLA4Ig fusion protein, which blocks the CD28-B7 costimulatory pathway in T-lymphocyte activation, was tested for its capacity to inhibit allosensitization to blood transfusions. Groups of LEW (RT1') rats were transfused with ACI blood (RT1a) together with L6 (a human immunoglobulin G1 [IgG1] antibody as isotype control) or CTLA4Ig in different doses (0.004, 0.02, 0.1, and 0.5 mg). Rats were retransfused with ACI blood after 28 and 84 days without any additional CTLA4Ig therapy. Weekly sera samples were tested for alloantibody against donor leukocytes using flow cytometry. CTLA4Ig caused a dose-dependent decrease in the IgM alloantibody response against donor major histocompatibility complex (MHC) class I antigens. In addition, 0.02-, 0.1-, and 0.50-mg doses of CTLA4Ig totally inhibited the IgG responses to the first transfusion, and this immunosuppressive effect persisted for the second and third transfusions. To study the capacity of CTLA4Ig to prevent a secondary immune response, three groups of LEW rats were transfused with ACI blood with no accompanying treatment. Animals were retransfused 28 days later with ACI blood together with L6 control antibody or 0.5 or 2.5 mg CTLA4Ig. CTLA4Ig, but not L6, prevented an increase in IgG alloantibody response despite repeated transfusions. The effects of CTLA4Ig treatment on helper T-lymphocyte proliferation was tested by limiting dilution analysis (LDA). Peripheral blood cells taken 30 days after blood transfusion and CTLA4Ig treatment contained significantly decreased donor-specific T-lymphocyte precursors compared with L6-treated rats. These data support the idea that blocking the B7/CD28 signal of T-lymphocyte activation by CTLA4Ig treatment at the time of transfusion may be an important therapeutic tool to inhibit alloantibody responses to blood transfusions.     

Use of naloxone in schizophrenic psychoses and manic syndromes

Since 1975, different morphinomimetic peptides have been isolated from hypophyseal-hypothalamic extracts: the pentapeptides methionine-enkephalin and leucine-enkephalin, and the longer peptides alpha-, beta- and gamma-endorphin. The primary structure of most of these peptides is also present in that of beta-lipotropin. The morphinomimetic properties of endorphins can be blocked with opiate-antagonists. In rats, moreover, the endorphins influence behavior which cannot be blocked with opiate antagonists. On the basis of the hypothesis that hyperactivity of endorphin systems may be involved in the pathogenesis of schizophrenia and manic syndromes, the effect of opiate antagonists on psychotic and manic symptoms has been examined in a number of clinical studies in the past few years. A transient therapeutic effect has been demonstrated in about 30% of the patients so treated. Our own double-blind controlled study of 5 schizophrenic and 5 manic patients in the context of a World Health Organization project failed to reveal any therapeutic effect after subcutaneous injection of 20 mg naloxone. The possible reasons of the negative results are discussed.     

Hepatotoxicity of anticholesterol, cardiovascular, and endocrine drugs and hormonal agents

Drugs in these categories may have effects on several organs in addition to the liver. For example, amiodarone may produce thyroid and corneal injury apart from or in addition to the phospholipidosis seen in the liver. Oral contraceptive steroids remain a rare but important concern for developing hepatic adenomas and possibly hepatocellular carcinoma, as well as a risk factor for developing Budd-Chiari syndrome in long-term users. Among the antihyperlipidemic agents, nicotinic acid, especially the sustained release formulations, may produce severe of even fatal hepatic injury. Increasing numbers of reports of hepatotoxicity from newer beta blockers and angiotensin-converting enzyme inhibitors require ongoing vigilance in patients receiving these medications.     

Highly sensitive and specific polymerase chain reaction assays for detection of baboon and pig cells following xenotransplantation in humans

Pig and baboon xenotransplants in humans require assays that discriminate source from human cells to investigate engraftment and identify true infection of recipients with xenogeneic endogenous retroviruses. We developed two polymerase chain reaction assays that target a porcine-specific sequence in the beta-globin gene and a baboon-specific sequence in the mitochondrial cytochrome oxidase subunit II gene. The sensitivity and specificity of both assays were evaluated on DNA lysates from baboon, pig, and human peripheral blood lymphocytes. Both assays detected single cells in backgrounds of human DNA. Additionally, both assays were highly specific and yielded negative results in reactions containing only human DNA. The two assays reliably detected peripheral blood lymphocyte samples from 14 baboons and 16 pigs. The baboon- and pig-specific target sequences are gender independent and nonpolymorphic and allow universal applicability. The high sensitivity and specificity is of particular importance in assessing low-level engraftment in human xenotransplant chimeras.