Isolation, characterization and immunological determination of basement membrane-associated heparan sulfate proteoglycan

Basement membrane-associated heparan sulfate proteoglycan (HSPG) was extracted from isolated porcine glomerular basement membranes and purified by ion-exchange chromatography. The proteogycan was characterized by specific enzymatic digestions, by amino-acid analysis, by SDS-polyacrylamide gel electrophoresis and by density gradient centrifugation. Polyclonal antibodies were raised against the purified HSPG in rabbits. Antibodies were characterized by enzyme immunoassays, immunoprecipitation and immunohistological methods. They were shown to recognize specifically the core protein of HSPG from porcine, human and rat glomerular basement membrane but did not recognize HSPG from guinea pig or rabbit kidney. The affinity-purified antibodies did not cross-react with other basement membrane proteins like laminin, fibronectin or collagen type IV nor with chondroitin sulfate-rich or keratan sulfate-rich proteoglycans from human or bovine tissue. Using these antibodies an enzyme immunoassay was developed for determination of HSPG in the range of 1-100 ng/ml. Studies with cultured porcine endothelial cells showed that subendothelial basement membrane-associated HSPG may be determined with the enzyme immunoassay.     

A specific point mutation in the mitochondrial genome of Caucasians with MELAS

Summary The mitochondrial DNA (mtDNA) of Japanese patients suffering from the syndrome of mitochondrial myopathy, encephalopathy, lactic acidosis and strokelike episodes (MELAS) exhibits a specific heteroplasmic AG transition in the tRNA^Leu at position 3243. In this study, we investigated mtDNA from skeletal muscle, cardiac muscle, brain, liver, diaphragm, fibroblasts and blood cells of four Caucasians with MELAS, one younger healthy sister of two MELAS patients, and eleven controls. We found that 1) the mutation was present in all investigated tissues of Caucasians with MELAS but not in controls, 2) within a single patient, the tissue-specific variation of the copy number of mutated mtDNA covered the same range as in the skeletal muscle of different patients, 3) the mutation was also present in the blood cells of the healthy sister of two MELAS siblings.     

Sequence homologies of glucose-dehydrogenases of Bacillus megaterium and Bacillus subtilis

The sequence homologies of the glucose dehydrogenase subunits of B. megaterium and B. subtilis are compared. From the known B. megaterium aminoacid sequence and the base sequence of the cloned B. subtilis structural gene we predict the B. megaterium structural glucose dehydrogenase gene. Assuming the minimal mutational changes to convert one gene into the other 23 transitions, 30 transversions, 1 inversion, 3 insertion-deletions, but no frameshifts are postulated necessary to interconvert the structural genes. The homology of both enzyme subunits of 85% reflects the close evolutionary distance between B. subtilis and B. megaterium.     

CO2 sensitive receptors on labial palps ofRhodogastria moths (Lepidoptera: Arctiidae): physiology,fine structure and central projection

Summary The tips of the labial palps ofRhodogastria possess a pit housing uniform sensilla (Fig. 1), histologically characterized by wall-pores and receptor cells with lamellated outer dendrites (Fig. 2). The receptor cell axons project to glomeruli in the deutocerebrum (cf. Fig. 3) which are not innervated by antennal receptors. From their histology as well as from their central projection these sense organs are identical with palpal pit organs of other Lepidoptera (Lee et al. 1985; Kent et al. 1986; Lee and Altner 1986).Physiologically, the palp-pit receptors respond uniformly; they are most excitable by stimulation with carbon dioxide (Fig. 6) while they exhibit relatively moderate responses to various odorants (Fig. 4). The responses to CO₂ (Fig. 7) show a steep dose-response characteristic. In ambient atmosphere (i.e., ca. 0.03% CO₂) the cells are in an excited condition already; the seeming spontaneous activity exhibited in air is decreased if the preparation is kept under N₂ or O₂ or CO₂-free air (Figs. 7, 10). There is hardly any adaptation of the responses to continuous or repeated stimulation (Fig. 8). Perhaps CO₂ sensitivity is correlated with sensilla characterized by both wall-pores and lamellated dendrites. Pilot tests indicate that CO₂ perception might be widespread in the Lepidoptera (cf. Fig. 12), but the biological significance remains obscure.     

Demonstration of cytoplasmic receptors for the molting hormones in crayfish

Summary From in vitro experiments using different binding assays it is in crayfish demonstrated that the cytosol of target tissues is able to bind both ecdysone and ecdysterone. The ability to bind ecdysteroids is destroyed by heating and by treatment with -chymotrypsin and N-ethyl-maleinimide (NEM) (Figs. 4, 5). In target tissues there is a strong positive correlation between protein content and binding (Fig. 6). The association of the hormone-protein-complex is rapid, taking only a few min even at 5° C (Fig. 3). The binding of the two hormones to the cytosol is both specific and saturable. The association constants for the cytoplasmic receptors from hypodermis, hindgut and midgut gland are in the range of 3–6×10⁷ M^–1 for ecdysone and 5–7×10⁸ M^–1 for ecdysterone (Fig. 8). The data suggest the existence of cytoplasmic ecdysteroid receptors.     

No genetic differences between affected and unaffected members of a German family with Leber's hereditary optic neuropathy (LHON) with respect to ten mtDNA point mutations associated with LHON.

In order to investigate possible synergistic influences of different mtDNA mutations on penetrance and severity of Leber's hereditary optic neuropathy (LHON), a large German LHON pedigree is characterized with respect to 10 different mutations associated with LHON. All members of the family carry three different mtDNA mutations (at nucleotide 4,216, 11,778 and 13,708) in a homoplasmic form, regardless of whether or not they are clinically affected. Testing for another 7 mutations reveals negative results in all family members. Hence, the variable disease expression of the family members cannot be explained by varying combinations of LHON-associated mtDNA mutations.     

Regulation of heavy metal concentrations in cereal grains from uranium mine soils

Aims

Along a gradient of diminishing heavy metal (HM) concentrations formed by local inclusions of uranium mine soils into non-contaminated cropland, duplicate 1-m² plots of 3 winter wheat cvs. (Akteur E, Brilliant A, and Bussard E) were established at 3 positions within a winter rye (cv. Visello) culture. It was the goal to determine permissible soil HM concentrations tolerated by cereal cvs. with variable excluder properties, and regulatory mechanisms which optimize the concentrations of essential minerals and radionuclide analogues in viable seeds from geologically related soils with diverging HM content.

Methods

Total metal concentrations / nitrogen species in soils, shoots, and mature grains were determined by ICP-MS / spectrophotometry, and Kjeldahl analyses.

Results

No non-permissible concentrations in grains of the 4 cereal cvs. were caused by elevated but aged total soil resources (mg kg^-1 DW) in As (156); Cu (283); Mn (2,130); Pb (150); and in Zn (3,005) in the case of Bussard although CdCuZn elicited phytotoxicity symptoms. Uranium (41) contaminated grains of Akteur and Brilliant but not of Bussard and Visello due to their excluder properties. The concentration in Cd (41) had to be reduced to 20/2 mg kg^-1 for the production by excluder cvs. of fodder/food grains. Cultivars excluding both HM and radionuclide analogues such as BaCsSr synchronously were not identified. Whereas plant tissue concentrations in the metalloprotein-associated elements CdCoCuMnNiZn rise and fall generally with N_org, grains of the wheat cvs. differed too little in N_org to designate variations in their metal acquisition rates solely as protein-regulated. Wheat grains confined nevertheless the concentrations in Cu to 11–14 mg kg^-1 although the respective soil concentrations varied by factor 19. Grain deposition in CaFeMn(Zn) and in nuclides followed the same rules.

Conclusions

It is hypothesized that cereals down-/up-regulate grain:soil transfer rates from soils with excessive/deficient trace metal resources to equip viable seeds with an optimum but not maximum in essential minerals. Positive correlations between metal concentrations in planta to those in soil can thereby be lost.     

Reconstitution of EBV-directed T cell immunity by adoptive transfer of peptide-stimulated T cells in a patient after allogeneic stem cell transplantation for AITL

Reconstitution of the T cell repertoire after allogeneic stem cell transplantation is a long and often incomplete process. As a result, reactivation of Epstein-Barr virus (EBV) is a frequent complication that may be treated by adoptive transfer of donor-derived EBV-specific T cells. We generated donor-derived EBV-specific T cells by stimulation with peptides representing defined epitopes covering multiple HLA restrictions. T cells were adoptively transferred to a patient who had developed persisting high titers of EBV after allogeneic stem cell transplantation for angioimmunoblastic T-cell lymphoma (AITL). T cell receptor beta (TCRβ) deep sequencing showed that the T cell repertoire of the patient early after transplantation (day 60) was strongly reduced and only very low numbers of EBV-specific T cells were detectable. Manufacturing and in vitro expansion of donor-derived EBV-specific T cells resulted in enrichment of EBV epitope-specific, HLA-restricted T cells. Monitoring of T cell clonotypes at a molecular level after adoptive transfer revealed that the dominant TCR sequences from peptide-stimulated T cells persisted long-term and established an EBV-specific TCR clonotype repertoire in the host, with many of the EBV-specific TCRs present in the donor. This reconstituted repertoire was associated with immunological control of EBV and with lack of further AITL relapse.