Influence of chain shortening on the inhibitor properties of hirudin and eglin c

To find out minimal sizes of the proteinase inhibitor proteins hirudin and eglin necessary for their biological activity the inhibitors were incubated with exopeptidases. From the incubation mixtures shortened derivatives were isolated and characterized. Eglin c can be N-terminally shortened by up to 6 amino-acid residues without any loss of affinity towards chymotrypsin. The complex of thrombin with hirudin lacking 3 C-terminal amino-acid residues showed a 15-20-fold increased Ki value as found previously for desulfato-hirudin and desulfato-hirudin shortened by 2 amino-acid residues. Obviously, the C-terminal part of the hirudin molecule has a positive influence on its affinity to thrombin.     

The effect of sucrose on the levels of abscisic acid, indoleacetic acid and zeatin/zeatin riboside in wheat ears growing in liquid culture

The influence of a varied sucrose supply on grain size and hormonal contents of detached wheat ears ( Triticwn aestivum L. cv. Schirokko) was investigated throughout grain development. In ears led limited amounts, or no sucrose, grain weights in both proximal and distal grain positions of the ear were reduced. Radioimmunoassay for abscisic acid, indoleacetic acid and zeatin/zeatin riboside showed that the changes in the levels of these hormones in grains and bracts were comparable to intact ears when detached ears were well supplied with sucrose. Under conditions of limited sucrose supply, higher abscisic acid levels in the distal and proximal grains of detached ears were found compared to ears supplied with adequate sucrose. Limiting sucrose supply to the ear did not alter the levels of indoleacetic acid or zeatin/zeatin riboside in either the grains or bracts of detached ears.     

Efficient oligonucleotide-directed construction of mutations in expression vectors by the gapped duplex DNA method using alternating selectable markers.

An efficient method for the construction of multiple mutations in a sequential manner is described. It is based on the gapped duplex DNA approach to oligonucleotide-directed mutagenesis (Kramer et al. 1984, Nucl. Acids Res. 12, 9441-9456) and a set of newly constructed phasmid vectors. These are characterized by the following features. Presence of the phage fl replication origin permits ready conversion to the single stranded DNA form. An amber mutation within, alternatively, the bla or cat gene provides a means for ready selection of the strand into which the mutagenic oligonucleotide has been incorporated. By means of the alternating antibiotic resistance markers any number of mutations can be constructed in consecutive rounds of mutagenesis. The optional presence of gene expression signals allows the direct overproduction of structurally altered proteins without re-cloning. Both the mutagenesis and expression aspects were tested using the lacZ gene as a model.     

Identification of two families of satellite-like repetitive DNA sequences from the zebrafish (Brachydanio rerio).

To further our understanding of the structure and organization of the zebrafish genome, we have undertaken the analysis of highly and middle-repetitive DNA sequences. We have cloned and sequenced two families of tandemly repeated DNA fragments. The monomer units of the Type I satellite-like sequence are 186 bp long, A+T-rich (65%), and exhibit a high degree of sequence conservation. The Type I satellite-like sequence constitutes 8% of the zebrafish genome, or approximately 8 x 10(5) copies per haploid genome. Southern analysis of genomic DNA, digested with several restriction endonucleases, shows a ladder of hybridizing bands, consistent with a tandem array, and suggests longer range periodic variations in the sequence of the tandem repeats. The Type II satellite has a monomer length of 165 bp, is also A+T-rich (68%), and constitutes 0.2% of the zebrafish genome (22,000 copies per haploid genome). Southern analysis reveals a complex pattern rather than a ladder of regularly spaced hybridizing bands.     

Biochemical and tissue print analyses of hydroxyproline-rich glycoproteins in cell walls of sporophytic maize tissues

In an effort to understand the role of hydroxyproline-rich glycoproteins (HRGPs) in plant cell wall structure, we studied the distribution and physical properties of PC-1-like proteins (PC-1 being the major pericarp HRGP) throughout sporophytic tissues of two maize (Zea mays L.) varieties. We determined total amounts of hydroxyproline, an indicator of HRGPs, and did tissue print and Western blot analysis. We found hydroxyproline in cell walls of stems, leaves, roots, tassels, and silks. We also observed reactivity of anti-PC-1 monoclonal antibodies with anatomical prints of these tissues on nitrocellulose paper. Stem nodes and silks contained the most hydroxyproline and exhibited the strongest reaction with the antibody. PC-1 was localized in vascular bundles and the epidermis of stem tissue. However, localization to a specific cell type in the silk could not be determined at the resolution of the tissue print. The stem node protein had the same electrophoretic mobility as the pericarp protein as determined on Western blots prepared from cationic neutral gels. Protein extracts from silk tissues of both varieties studied contained one protein of the same size/charge as that found in pericarp, as well as some minor variant bands. The data presented here document that cell wall proteins are present in many tissues of the maize plant, although they are primarily in cell types contributing to support.     

Influence of the H-2u haplotype on immune function in F1 hybrid mice. I. Antigen presentation

Previously, we reported that myelin basic protein (MBP) peptide 1-37 contains an encephalitogenic epitope for PL/J mice, and MBP peptide 89-169 is encephalitogenic for SJL/J mice. (SJPL)F1 hybrid mice do not respond to immunization with these peptides in a co-dominant manner because the encephalitogenic response to peptide 1-37 dominates. To examine this phenomenon more closely, we tested the ability of MBP-primed parental or F1 T cells to respond to MBP or MBP peptides in the context of PL, SJL, or F1 antigen-presenting cells (APC). It was found that the F1 T cells responded to either the protein or the peptides when these were presented in the context of F1 or PL APC. However, F1 T cells would not respond to MBP in the context of SJL APC, although the latter cells were functionally intact. This effect was not antigen-specific because SJL APC would not present ovalbumin or PPD to primed F1 T cells. F1 T cells from mice immune to the strongly antigenic bacterium Listeria monocytogenes responded to bacterial antigens presented by SJL APC, although at a significantly lower level compared to the results obtained when these antigens were presented by F1 or PL APC. This finding implied that unbalanced antigen presentation was a quantitative rather than a qualitative phenomenon. When F1 hybrid mice from other strain combinations were tested, a similar effect was observed whenever one of the parental strains was PL/J. This effect was mapped to the MHC in MHC-congenic B10 mice.     

Fluorescence methods for localizing proteinases and proteinase inhibitors in skeletal muscle

Summary Proteinases and proteinase inhibitors have become suspect in a wide variety of muscle wasting conditions that might be treatable if knowledge of the cellular locale and function of these molecules were known. Fluorescent probes have been useful in the localization of proteinases in muscle samples from human and animal specimens. These include the histochemical localization of proteinases based on the specific fluorescence of hydrolysis product derivatives, but this approach has been limited to the lysosomal proteinases because of the acidic requirements of the trapping reaction of the primary reaction product. Immunohistochemical techniques do not have the same restrictions and a number of lysosomal and nonlysosomal proteinases have been identified in muscle by this means. Unfortunately, they do not yield any information as to the activity of the enzymes. This is an important consideration since the extracellular environment contains a number of proteinase inhibitors, some of which may be internalized by the cell.     

Decreased lysosomal protease content of skeletal muscles from streptozotocin-induced diabetic rats: a biochemical and histochemical study

Summary The activity of four lysosomal proteases in soleus and extensor digitorum longus muscles was studied in streptozotocin-induced diabetic rats using newly developed fluorescence histochemical and biochemical techniques. The results indicate that the content of lysosomal protease in skeletal muscle cells was decreased three weeks after the induction of diabetes. The reduction was most pronounced in the extensor digitorum longus for all the proteases tested, but in the soleus only cathepsin B and dipeptidyl peptidase II showed a decrease. Biochemical assays on total muscle homogenates and muscle extracts confirmed the histochemical observations that protease activity was significantly lower in diabetic muscles. This decrease in activity varied with the duration of diabetes beginning as early as 48 h for the soleus. In conclusion, myofibre-specific decreases in lysosomal proteases occur following diabetes.