Retroviral-mediated gene transfer into mammalian cells

Retroviruses may be used as genetic vectors to transfer genes into mammalian cells with high efficiency. We have shown that the N2 vector will transfer a functional bacterial gene for neomycin resistance (NeoR) into more than 80% of mouse spleen foci. A derivative of the N2 vector was constructed to study transfer and expression of the human gene for adenosine deaminase (ADA) in mammalian lymphoid and hematopoietic stem cells. This vector, termed SAX, contains the human ADA cDNA with an SV40 promoter in addition to the NeoR gene. The SAX vector was found to efficiently transfer and express the ADA gene in an ADA-deficient human T-cell line. Gene transfer by SAX using an autologous nonhuman primate bone marrow transplant model resulted in expression of the human ADA gene in peripheral blood cells of treated animals. Human bone marrow treated with SAX produced 1%-2% of colonies in vitro that were expressing the vector genes. Transfer of genes into circulating hematopoietic stem cells of fetal sheep in utero was most efficient; vector gene expression was evident in 20%-40% of hematopoietic colonies. Therefore, retroviral vectors are capable of transferring functional genes into a wide variety of mammalian lymphoid and hematopoietic cells. Such vectors may be useful for clinical trials of gene therapy, that is, the correction of genetic diseases by insertion of a normal gene into a patient's defective cells.     

Transgenic HLA-DR alpha faithfully reconstitutes IE-controlled immune functions and induces cross-tolerance to E alpha in E alpha 0 mutant mice

We have constructed transgenic mice that express the human class II MHC molecule HLA-DR alpha on a genetic background in which the equivalent endogenous gene, H-2 IE alpha, is not expressed. In these mice, DR alpha complemented the E beta chain such that tissue-specific expression of an interspecies hybrid DR alpha-E beta heterodimer was obtained. Despite 25% amino acid differences between DR alpha and E alpha, immune responsiveness to IE-controlled antigens, clonal deletion of IE-reactive T cells, and alloantigenicity were quantitatively and qualitatively indistinguishable in IE-positive mice and in mice that had integrated at least four copies of the transgene. These results demonstrate a remarkable degree of structural, regulatory, and functional conservation. They also suggest that tolerance induction involves only discrete portions of MHC molecules.     

Lack of physiological stimulation induces decreased proteolytic activity in nerve terminals

The effect of optic nerve transsection on proteolytic degradation of axonally transported proteins in the superior colliculus of the rabbit was studied. Proteolysis of labeled proteins was determined in vitro in small pieces of the superior colliculus. Within 2 hours after sectioning the optic nerve there was a decreased degradation of slowly transported labeled proteins in the nerve terminals in the superior colliculus.Special Issue dedicated to Prof. Holger Hydén.     

Restriction fragment length polymorphism of the major histocompatibility complex of the dog

Human major histocompatibility complex (HLA) cDNA probes were used to analyze the restriction fragment length polymorphism (RFLP) of the DLA-D region in dogs. Genomic DNA from peripheral blood leucocytes of 23 unrelated DLA-D-homozygous dogs representing nine DLA-D types (defined by mixed leucocyte reaction) was digested with restriction enzymes (Bam HI, Eco RI, Hind III, Pvu II, Taq I, Rsa I, Msp I, Pst I, and Bgl II), separated by agarose gel electrophoresis, and transferred onto Biotrace membrane. The Southern blots were successively hybridized with radiolabeled HLA cDNA probes corresponding to DR, DQ, DP, and DO beta genes. The autoradiograms for all nine enzyme digests displayed multiple bands with the DRb, DQb, and DPb probes while the DOb probe hybridized with one to two bands. The RFLP patterns were highly polymorphic but consistent within each DLA-D type. Standard RFLP patterns were established for nine DLA-D types which could be discriminated from each other by using two enzymes (Rsa I and Pst I) and the HLA-DPb probe. Cluster analysis of the polymorphic restriction fragments detected by the DRb probe revealed four closely related supertypic groups or DLA-DR families: Dw3+Dw4+D1, Dw8+D10, D7+D16+D9, and Dw1. This study provides the basis for DLA-D genotyping at a population level by RFLP analysis. These results also suggest that the genetic organization of the DLA-D region may closely resemble that of the HLA complex.     

Proteolysis of tubulin and microtubule-associated proteins 1 and 2 by calpain I and II. Difference in sensitivity of assembled and disassembled microtubules

Calpain I and II (EC 3.4.22.17) are Ca2+-activated neutral thiol-proteases. Isolated brain tubulin and microtubule-associated proteins were found to be good substrates for proteolytic degradation by brain calpain I and II. The assembly of microtubules was totally inhibited when the calpains were allowed to act on microtubule proteins initially, and a complete disassembly was found after addition of calpain I to assembled microtubules. The high-molecular weight microtubule-associated proteins were degraded within a few minutes following incubation with calpain as shown by SDS-polyacrylamide gel electrophoresis and electron microscopy. When calpain was added to pre-formed microtubules, either in the presence or in the absence of microtubule-associated proteins, the proteolysis was significantly reduced. When tubulin was pre-assembled by taxol, the formation of proteolytic fragments was decreased indicating that assembly alters the availability of tubulin sites for proteolytic cleavage by calpain. Digested tubulin spontaneously formed aberrant polymers. No considerable change of apparent net charge was seen, thus indicating that calpain cleaves off fragments containing neutral amino acid residues and/or that the fragments of tubulin remain associated as an entity with the same charge as native tubulin. The results suggest that the calpains act as irreversible microtubule regulators.     

Identification and characterization of alkali-tolerant clostridia isolated from biodeteriorated casein-containing building materials

Summary Reports on malodour in buildings constructed in the late 1970s gave rise to thorough investigations on the possible role of vapours of chemical compounds emitted by building materials. The odour could be related to the use of casein as an additive to improve the fluidity of concrete materials used as a self-levelling floor topping compound. Casein was suggested to be degraded by microorganisms, resulting in an accumulation of malodorous substances in the topping compounds.Bacteria isolated from biodeteriorated concrete materials containing caseins exhibited unusual tolerance towards high pH. Two dominant species were found among a total of 80 sporeforming, anaerobic isolates from concrete and raw products of caseins, namely Clostridium bifermentans and Clostridium sporogenes. C. bifermentans had a maximal pH tolerance of 12.2 while C. sporogenes could reproduce up to pH 11.7. The study includes the identification of the clostridia with API multitest as well as an investigation of the volatile organic acid and monoamine patterns. About 100 cfu clostridia/g material could be obtained during the isolation procedures.     

Correction of chlorophyll-defective male-sterile winter oilseed rape (Brassica napus) through organelle exchange: Characterization of the chlorophyll deficiency

Jarl, C. I., Ljungberg, U. K. and Bornman, C. H. 1988. Correction of chlorophyll-defective male-sterile winter oilseed rape ( Brassica napus ) through organelle exchange: Characterization of the chlorophyll deficiency. - Physiol. Plant. 72: 505–510.
As is known, the introduction of male-sterile Raphanus sativus L. cytoplasm into Brassica napus L. results in male-sterile oilseed rape plants, which display a temperature-related chlorophyll defect. The influences of temperature and irradiance on this defect were investigated. Compared to a line of normal (green phenotype) male-fertile oilseed rape, the male-sterile line had reduced chlorophyll content, fewer chloroplasts per cell, an altered ultrastructure of the chloroplasts and reduced activities of both photosystems, although the relative amounts of the photosystems and the chlorophyll a/b ratio were similar. The lower activity of the photosystems is explained by a decreased functional antennae size and a reduced efficiency in the interactions between the nuclear-encoded light-harvesting proteins and the reaction centres coded for by the plastome. Some thylakoid polypeptides differed in proportion between the male-fertile line with green phenotype and the male-sterile line with chlorotic phenotype. Characters, in which the two lines exhibited differences, are ascribed to difficulties in molecular communication between the oilseed rape nucleus and the radish cytoplasm, which are combined in the deficient male-sterile line.     

Phytochrome is not involved in the red-light-enhancement of the stomatal blue-light-response in wheat seedlings

The stomatal response to blue light (BL) in wheat seedlings ( Triticum aestivum L. cv. Starke II, Weibull) was enhanced by background red light (R). This enhancement was only slightly affected by the addition of background far-red light (FR). Under similar light treatments, the addition of FR induced a 43% transformation from the far-red-absorbing form towards the red-absorbing form of phytochrome from etiolated oat ( Avena sativa L. cv. Sol II), immobilized on phenyl-sepharose. Furthermore, the enhancement of the stomatal BL-response by 15 min R was not reversed by a subsequent irradiation with 5 min FR. It is concluded that the red-light-enhancement of the stomatal blue-light-response in wheat seedlings does not involve a change in the photostationary state of phytochrome.     

A chicken beta-actin gene can complement a disruption of the Saccharomyces cerevisiae ACT1 gene.

Recently it was demonstrated that beta-actin can be produced in Saccharomyces cerevisiae by using the expression plasmid pY beta actin (R. Karlsson, Gene 68:249-258, 1988), and several site-specific mutants are now being produced in a protein engineering study. To establish a system with which recombinant actin mutants can be tested in vivo and thus enable a correlation to be made with functional effects observed in vitro, a yeast strain lacking endogenous yeast actin and expressing exclusively beta-actin was constructed. This strain is viable but has an altered morphology and a slow-growth phenotype and is temperature sensitive to the point of lethality at 37 degrees C.     

Glycosphingolipid patterns of the gastrointestinal tract and feces of germ-free and conventional rats

Acid and non-acid glycosphingolipids of stomach, small and large intestine, and stimulated feces of germ-free and conventional rats of the same stain have been isolated and characterized. The glycosphingolipid patterns of the intestinal organs were chemically and immunologically very similar between the two groups of rats and relatively unaffected by the presence of an intestinal microbial flora. The major exception was the presence of hematoside with N-glycoloylneuraminic acid (NeuGc) (NeuGc alpha 2----3Gal beta 1----4Glc beta 1----1Cer) in the stomach of conventional rats not found in the stomach of germ-free animals. Glycosphingolipids of stimulated feces of germ-free animals were derived from epithelial cells mainly of the small intestine and showed no signs of degradation. Glycosphingolipids of feces of conventional rats completely retained the pattern of blood group A-, B-, and H-active glycolipids as found in sterile feces but contained less of hematoside and more of lactosylceramide. This effect was probably due to degradation by bacteria, as demonstrated in vitro with the production of lactosylceramide after treatment of the isolated acid glycolipids of sterile feces with neuraminidase from Clostridium perfringens. The amount of total non-acid glycosphingolipids per dry weight was similar for stomach, was 50% higher for small intestine, and 300% higher for large intestine of germ-free animals compared to conventional animals. Due to the presence of large amounts of mucins the dry sterile feces contained 12% less non-acid glycolipids than conventional feces. However, calculated per rat per day the germ-free animal excreted more of non-acid glycosphingolipids (1.8 and 1.2 mg, respectively).