Quantitative analysis of protein mixtures by second derivative absorption spectroscopy

A new method for the quantitative analysis of protein mixtures based on multicomponent analysis of the second derivative near uv spectra is described. Using bovine eye lens crystallins, we demonstrate that the technique can provide precise concentrations of closely related proteins within mixtures, under both native and denaturing conditions. We have also successfully used the method to analyze the subunit composition of a heteromultimeric protein aggregate. The method is more rapid and precise than alternative approaches and offers the advantage of substantially reduced interference from many extraneous solution components and light scattering. It is also nondestructive and extremely sensitive, requiring only small volumes of sample at low total protein concentrations. Prospective applications are proposed for the study of eye lens crystallins, as well as for other protein/protein and protein/nonprotein mixtures.     

Phytochemical and Cytogenetic Study of Two Centaurea Species from Croatia: the Particular Case of Diploid and Tetraploid C. salonitana

Natural wild populations of C. rupestris and C. salonitana were studied to determine possible relationships between the volatile oil (VO) composition and ploidy level. The chemical composition of the volatile oil was investigated using the GC/MS technique. The predominant components of the VO of diploid and tetraploid C. salonitana were hexadecanoic acid and α-linoleic acids, while in C. rupestris they were germacrene D and β-caryophyllene in one population and heptacosane and germacrene D, in another. The nuclear DNA amounts (2 C DNA), determined by flow cytometry, were 3.54 pg for C. rupestris, 3.39 pg for the diploid and 6.79 pg for the tetraploid population of C. salonitana. Evidence that the degree of ploidy solely influences the chemical composition of the essential oil of C. salonitana was not found. The results presented are the first data to be reported on the DNA content of the studied Centaurea populations from Croatia, as well as on the chemical composition of C. salonitana volatile oil.     

Karyotype morphology in Hyperiella dilatata Stebbing 1888 (Amphipoda: Hyperiidae) from the Ross Sea (Antarctica)

Summary The chromosome complement and some karyological features were investigated in the pelagic amphipod Hyperiella dilatata Stebbing 1888 from the Ross Sea (Antarctica). The diploid karyotype consists of 48 metacentric and 10 submetacentric elements (2n = 58). The presence of secondary constrictions and supernumerary chromosomes is described. Available chromosome numbers of Hyperiidea exhibit a wide range of distribution, among which Hyperiella dilatata is the closest to the modal number of other amphipods.     

Structural and biochemical remodelling in catecholamine-induced cardiomyopathy: comparative and ontogenetic aspects

Excessive release or administration of beta-mimetic catecholamines may induce cardiomegaly, necrotic lesions and accumulation of connective tissue in the heart of adult homoiotherms. It was examined here whether similar changes can also be observed at different stages of evolution of the cardiovascular system, i.e. in poikilotherms and in homoiotherms during embryonic life.Sensitivity of the poikilothermic hearts (carp, frog, turtle) to isoproterenol (IPRO) was significantly lower than in the homoiotherms. Necrotic lesions, if present, were localized in the inner spongious musculature which has no vascular supply but which exhibits higher activities of enzymes connected with aerobic oxidation. Moreover, the IPRO-induced decrease of the phospholipid content was also significantly more expressed in the spongious layer. IPRO treatment did not influence the total weight of the fish heart but the proportion of the outer compact layer was significantly higher. These changes were accompanied by an increase of collagen, higher water content and an increase of isomyosin with a lower ATPase activity. The response of the poikilothermic heart to IPRO-induced overload thus differs significantly from that in the homoiotherms.The administration of IPRO during embryonic life of homoiotherms (chick) induces serious cardiovascular disturbances, including cardiomegaly and cellular oedema. Necroses of myofibrils, characteristic of IPRO-induced lesions of adults, were, however, rather exceptional. IPRO did not elevate the concentration of⁸⁵Sr (as a calcium homologue) in the immature myocardium; it seems, therefore, that IPRO-induced changes of the embryonic heart are not necessarily due to an intracellular calcium overload.It may be concluded that the character of catecholamine-induced cardiomyopathy is not uniform and depends strictly on the stage of cardiac development.     

Glucose-induced secretion of Trichoderma reesei xylanases.

To produce two xylanases with Trichoderma reesei grown on glucose, recombinant strains which carry either the xyn1 or the xyn2 (xylanase I and II [XYN I and XYN II]-encoding) structural genes under the expression signals of the homologous pki1 (pyruvate kinase-encoding) gene were constructed. The two types of transformants secreted XYN I or II, respectively, during growth on glucose, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunostaining. The corresponding specific xylanase activities of the best transformants on glucose were 76 and 145 U/mg of protein for XYN I and XYN II, respectively, as opposed to that obtained by the parent strain (26 U/mg of protein). When related to the amount of biomass formed, however, they produced only about 4 to 5 U/g, in contrast to much higher activities (10 to 12 U/g) during growth on xylan. The ultrastructural location of XYN II in the transformant strain producing the highest constitutive XYN II formation (ATX2-12) was investigated by immunoelectron microscopy and compared with that in the wild-type strain growing on xylan. Cell extracts from both types of transformants grown on glucose exhibited a higher intracellular xylanase activity than did the parent strain grown on xylan. By using electron microscopy and immunogold labelling, XYN II was detected in the endoplasmic reticulum, Golgi-like vesicles, secretory vesicles, vacuoles, and cell walls. The immunolabel in the vacuoles was detected preferentially in subapical cells. When a recombinant strain which expressed xyn2 from the pki1 promoter was compared with the parent strain during growth on xylan, the former exhibited a less proliferated endoplasmic reticulum and a smaller number of secretory vesicles; however, a higher density of labelling was observed. The relationship of these findings to the efficacy of protein secretion during growth on glucose is discussed.     

Complete sequence of an HLA-dR beta chain deduced from a cDNA clone and identification of multiple non-allelic DR beta chain genes

At least three polymorphic class II antigens are encoded in the human major histocompatibility complex (HLA): DR, DC and SB. cDNA clones encoding beta chains of HLA-DR antigen, derived from mRNA of a heterozygous B-cell line, were isolated and could be divided into four subsets, clearly distinct from cDNA clones encoding DC beta chains. Therefore, at least two non-allelic DR beta chain genes exist. The complete sequence of one of the DR beta chain cDNA clones is presented. It defines a putative signal sequence, two extracellular domains, a trans-membrane region and a cytoplasmic tail. Comparison with a DC beta chain cDNA clone revealed a homology of 70% between the two beta chains and that the two genes diverged under relatively little selective pressure. A set of amino acids conserved in immunoglobulin molecules was found to be identical in both DR and DC beta chains. Comparison of the DR beta chain sequence with the amino acid sequence of another DR beta chain revealed a homology of 87% and that most differences are single amino acid substitutions. Allelic polymorphism in DR beta chains has probably not arisen by changes in long blocks of sequence.     

Transgenic Tobacco plants with T-DNA Phytohormone synthesis genes

Agrobacterium tumefaciens binary vectors carrying kanamycin resistance gene and either C58 T-DNA gene 4 for cytokinin synthesis or genes 1 and 2 for auxin synthesis were constructed and used for transformation of a short-day tobacco Maryland Mammoth. Kanamycin resistant plants were regenerated from a small fraction of transformed tissue and the presence of T-DNA in their genome was verified by Southern blotting. The level of endogenous cytokinin in plants transgenic for gene 4 and the level of endogenous IAA in those transgenic for genes 1 and 2 increased by more than 100 %. A number of morphological characteristics distinguish them from untransformed controls.     

Detection of proteins and phenol in DNA samples with second-derivative absorption spectroscopy.

We have employed near-uv second-derivative spectra of DNA, N-acetyl-L-tryptophanamide, N-acetyl-L-tyrosinamide, N-acetyl-L-phenylalanine ethyl ester, and phenol in a matrix least-squares multicomponent analysis algorithm to detect the presence of tryptophan, tyrosine, phenylalanine, and/or phenol in DNA preparations. With this method, each of these compounds can be detected in a DNA sample (absorbance, 0.1) at absorbance levels of less than 0.002. In practice, the presence of proteins can be detected at absorbance levels of less than 0.003. Using second-derivative spectra of proteins, contents of mixtures of proteins and DNA can be determined with less than 1% error. Mixtures of DNA and RNA can also be quantitatively analyzed with an error of approximately 2%. This technique can be easily implemented with computer-controlled spectrophotometers equipped with standard spectral analysis software. With prerecorded standard spectra, the time of analysis does not exceed a few seconds.     

Biosynthesis and endocytosis of lysosomal enzymes in human colon carcinoma SW 1116 cells: impaired internalization of plasma membrane-associated cation-independent mannose 6-phosphate receptor.

The human colon adenocarcinoma cell lines SW 948, SW 1116, and SW 1222 were tested for their ability to sort and internalize lysosomal enzymes. The biosynthesis of the lysosomal enzymes cathepsin B, arylsulfatase A, and beta-hexosaminidase in these cell lines exhibits no significant differences to that in human fibroblasts. The intracellular targeting of newly synthesized hydrolases to the lysosomes relies in colon carcinoma cells on the mannose 6-phosphate receptor system. Both the cation-independent mannose 6-phosphate receptor (CI-MPR) and the cation-dependent mannose 6-phosphate receptor are expressed in all colon carcinoma cell lines investigated. Endocytosis of lysosomal enzymes via mannose 6-phosphate receptors is reduced in colon carcinoma cells as compared with human fibroblasts. SW 1116 cells were shown to be deficient in receptor-mediated endocytosis of mannose 6-phosphate containing ligands. Ligands of other endocytic receptors as well as the fluid-phase marker horseradish peroxidase were internalized at normal rates. While antibodies against CI-MPR bind to the surface of SW 1116 cells, these antibodies cannot be internalized. These data suggest that the cycling of CI-MPR is specifically impaired in SW 1116 cells.