Characterization of a latent cysteine proteinase from ascitic fluid as a high molecular weight form of cathepsin B

The latent cysteine proteinase present in ascitic fluid of patients with neoplasia and released from ascites cells in culture has been partially purified and the enzyme after pepsin activation was shown to be immunologically related to the lysosomal proteinase, cathepsin B. The latent form was characterized as a single chain of M_r 40 000 as determined by SDS-polyacrylamide gel electrophoresis under reducing conditions followed by Western blotting and immune staining with an antiserum to human cathepsin B. Using the same techniques the enzyme after pepsin activation gave a single band of M_r 33 000. Analysis by isoelectric focusing showed that the latent enzyme before and after pepsin treatment is composed of several acidic isoenzymes. These findings suggest that this latent proteinase represents a precursor form of cathepsin B which is released extracellularly rather than being processed and directed to the lysosome.     

Indole-3-acetic acid production by bacteroids from soybean root nodules

Purine nucleotide and RNA synthesis have been investigated at the different growth stages of carrot ( Daucus carota L.) cells grown in suspension cultures. At the early growth stages an increase in the content of RNA was observed, although at later stages RNA was degraded. The highest rates of incorporation of [¹⁴C]-labelled adenosine into ATP and GTP were observed at the late growth sttages. This indicated that purine slavage was more importnt at the late growth stages, while de novo synthesis was dominant during the initial growth stages. This pattern was also reflected by increased levels, in the cell dividison phase, of theenzymes glycinamide ribonucleotide synthetase (EC 6.3.1.3.) and phosphoribosylpyrophosphate amido-transferase (EC 2.4.2.14) involved in de novo purine synthesis. The activities of the purine salvage enzymes varied little during growth. Cells in the stationary phase, that were starved for sucrose and phosphate, showed a dramatic increase in cellular metabolism, as judged from a rapid uptake and incorporation of [³²P]-labelled phosphate into nucleotides and RNA, when incubated in fresh medium.     

Infrared-to-visible conversion in strontium sulphate through a defect-based infrared stimulated visible emission phenomenon

Strontium sulphate (SrSO₄) is a defect-based photoluminescence material, generally used in thermoluminescence applications, and has been studied for infrared (IR) stimulated visible emission. The SrSO₄ particles were synthesized using a precipitation method. The orthorhombic phase of SrSO₄ was confirmed from the X-ray diffraction pattern and the formation of micron-sized particles was authenticated from field emission scanning electron micrographs. The elemental composition of oxygen and strontium was determined using energy-dispersive X-ray analysis measurement that confirmed the presence of $V_O^{••}$ and $V_{\mathit{Sr}}^{\prime \prime }$ intrinsic defects in the material. Photoluminescence investigations showed the presence of various defect bands in the band gap giving rise to intrinsic luminescence in SrSO₄. The emission in the visible region was attributed to the defect band arising due to $V_O^{••}$. Photoluminescence lifetime measurement confirmed the presence of stable defect states with a lifetime in microseconds. The SrSO₄ sample was tested using IR lasers and a red–orange emission spot was observed from the powder sample when excited with IR lasers. The underlying principle for IR-to-visible conversion in the material is a defect-mediated phenomenon that has been described through the energy level diagram of the material.     

Two-dimensional separation of chloroplast membrane proteins by isoelectri focusing and electrophoresis in sodium dodecyl sulphate

Proteins of chloroplast subfragments enriched in Photosystem I and Photosystem II electron flow activity have been analyzed by two-dimensional polyacrylamide gel electrophoresis. In the first dimension, polyacrylamide gel isoelectric focusing (pH 5–7) was used in the presence of Triton X-100, followed at right angle by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Characteristic fingerprints were obtained for the Photosystem I and II fractions and a correlation between the major proteins separated by isoelectric focusing and the major polypeptides separated by undimensional SDS electrophoresis was established. Two dominant spots of 68 000 and 60 000 daltons appeared in the two-dimensional patterns of Photosystem I fractions $\text{p}\text{I}$ values about 5.6; two spots with molecular weights of 33 000 and 23 000 were characteristics for Photosystem II fractions $\text{p}\text{I}$ values about 5.3 and 6.3). Photosystem I fractions were furthermore characteristics by a series of spots in the 44 000–33 000 range $\text{p}\text{I}$ values from about 5.9 to 6.8). The two-dimensional system revealed that (a) several SDS-polypeptides have multiple forms differing in charge only, (b) some proteins separated by isoelectric focusing are resolved in the second dimensional into polypeptides of different size. The two-dimensional method combining Triton X-100 isoelectric focusing' and SDS electrophoresis provides a higher degree of resolution than either of the unidimensional methods thus allowing a detailed analysis of chloroplast membrane proteins.     

Induced plasmon mutations affecting the growth habit of peanuts, A. hypogaea L.

The effectiveness of the acridines ethidium bromide (EB) and acriflavine in inducing plasmon mutations was compared with the alkylating agents ethyl methanesulphonate (EMS) and diethyl sulphate and to γ-rays. The growth habit (trailing versus bunch) of peanuts (A. hypogaea), controlled by geniccytoplasmic interactions, was utilized. Breeding tests distinguishing nuclear from plasmon mutations were developed and are described in detail. Plasmon mutations were induced, but there were differences in mutation yields between the cultivars and the mutagens.In the trailing line, TBR[V4], 135 independent bunch mutations (in 1804 M₂ families) were recovered: 28 bred true while 97 continued to segregate into M₃ and M₄. Of the 28, 14 were nuclear from an Hb to an hb allele while 14 were in the plasmon. Of the latter, 6 were induced by EMS, 7 by γ-rays and 1 by acriflavine. Somatic segregation of heteroplasmons, i.e. more plasmon mutations, could be responsible for many of the mutations that continued to segregate, but in some cases chromosomal aberrations might be involved.In the bunch cultivars there were 32 independent trailing mutations (in 3895 M₂ families), one bred true for trailing, while the others continued to segregate into M₃ and M₄. Plasmon mutations could not be ascertained because of the continuing segregations, but these mutations manifested sorting out of heteroplasmons.     

Evidence for the occurrence of calmodulin in the yeasts Candida albicans and Saccharomyces cerevisiae

The lectin extracted from Vicia graminea seeds has been purified by conventional techniques but such procedures did not give a satisfactory yield. We describe a new purification which involves 3 steps after obtention of the crude extract. The first step is based on affinity chromatography on con A—Sepharose. Further purification steps were performed on DEAE-Sephacel chromatography and ultrogel AcA₄₄ gel filtration. The homogeneity of the lectin was demonstrated by polyacrylamide gel electrophoresis. Purification of the lectin by this new method was less time consuming, the yield was higher and the specific activity increased.     

Isolation and biochemical characterization of leukemia-associated inhibitory activity that suppresses colony and cluster formation of cells

Leukimia-associated inhibitory activity suppresses colony and cluster formation in vitro of cells derived from granulocyte-macrophage progenitor cells of normal donors. It does not inhibit these same progenitor cells from patients with leukemia and it may contribute to the proliferative advantage leukemia cells appear to possess over normal hematopoietic cells during acute leukemia. The inhibitory activity was isolated by a combination of procedures including: ultracentrifugation, Sephadex G-200, carboxymethylcellulose, SDS-polyacrylamide gel electrophoresis, thin-layer and preparative isoelectric focusing and concanavalin A-Sepharose. Leukemia-associated inhibitory activity was characterized as a glycoprotein. It was inactivated by trypsin, chymotrypsin, pronase and periodate treatment. It bound to and was eluted by α-methylmannose from concanavalin A-Sepharose columns and had an apparent M_r range of 450–550 000 and an isoelectric focus value between pH 4.6 and 4.9. Crude leukemia associated inhibitory activity was temperature sensitive but the more purified preparations were heat stable.     

Protein degradation in mammary gland explants evidence for limited heterogeneity of protein degradation rates

Mammary gland explants in organ culture were subjected to hormonal manipulation, and rates of protein degradation during 1 and 2 day periods were measured by a double-isotope method. Isotope ratios for protein subunits in subcellular fractions were measured after resolution by two-dimensional polyacrylamide gel electrophoresis. Frequency distribution analysis shows that the isotope ratios for each fraction are coupled predominantly in exponential distributions corresponding to populations of protein subunits with different mean degradation rates. The result also suggest that heterogeneity of protein degradation rates within each population is limited. There is no consistent correlation of degradation rate with protein isoelectric point or subunit molecular weight either overall or within any population of degradation rates. Therefore, the similarity of protein degradation rates within each population is clearly not related to these molecular properties of the proteins.     

Concentration of Babesia bigemina-infected erythrocytes using a dextran sulphate affinity column

Babesia bigemina-infacted erythrocytes preferentially bind to dextran sulphate affinity columns. Subsequent elution yields suspensions containing up to 95% infected erythrocytes. Preliminary immunoblotting studies indicate a parasite antigen of 35,000 mol. wt might be implicated in the binding.