Cloning, sequence analysis, and expression of a cDNA encoding a plastid-localized heat shock protein in maize

We have cloned and characterized a cDNA encoding a maize (Zea mays L.) heat shock protein (HSP), HSP26. The mRNA of HSP26 is present as a single mRNA species of 1.1 kilobase pairs in size and is detectable when maize seedlings are treated at 40°C but not at 28°C. Accumulation of HSP26 mRNA was detected after 10 minutes of incubation at 40°C, reaching the maximum level after 1 hour. Comparison of the deduced amino acid sequence of maize HSP26 to other HSPs indicated a strong homology to the sequences of two nuclear encoded HSPs that are transported into the chloroplasts during heat shock: pea HSP21 and soybean HSP22. Maize HSP26 was also found to cross-react with anti-pea chloroplast HSP21 antibodies. Because of the sequence homology between maize HSP26, soybean HSP22, and pea HSP21, in vitro chloroplast protein import experiments were conducted. The in vitro synthesized maize HSP26 is specifically imported to the soluble fraction of the chloroplast and processed to a smaller polypeptide. The sequence homology and antibody cross-reactivity between maize HSP26 and pea HSP21 have allowed us to conclude that maize HSP26 is a nuclear-encoded, plastid-localized protein in maize.     

A major gibberellic Acid-induced barley aleurone cysteine proteinase which digests hordein : purification and characterization

We previously described the purification and characterization of a 37,000 M_r cysteine proteinase, designated EP-A, from gibberellic acid (GA₃)-induced barley (Hordeum vulgare L.) aleurone layers (S Koehler, T-HD Ho [1988] Plant Physiol 87: 95-103). A second, more abundant protease has now been purified from this tissue. This protease, designated EP-B, has an apparent M_r of 30,000 on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). It resolves into two bands during native isoelectric focusing with pl of 4.6 to 4.7. The analysis of hemoglobin digestion products by both gradient SDS-PAGE and Bio-Gel P2 chromatography, the inhibition of protease activity by E-64, leupeptin, iodoacetate, and p-hydroxymercuribenzoate, and N-terminal amino acid sequence analysis all indicate that EP-B is a cysteine proteinase. The first 22 amino acids at the N terminus of EP-B have been determined, and their sequence is 90% similar to that of EP-A. EP-B has properties similar to EP-A; however, EP-B is much more sensitive to high pH during gel electrophoresis and therefore is not detectable on native activity gels used to detect EP-A. Its pH optimum against azocasein and hemoglobin is 4.5 to 4.6. Both of these proteinases digest hordeins enriched for the B and D fractions into similar peptides of 25,000 to 2,000 M_r as determined by gradient SDS-PAGE.     

Prognostic usefulness of certain defined indicators for achieving complete remission and survival of ANLL in adults: proposal of a prognostic scale

Retrospective analysis has included 323 patients with acute nonlymphocytic leukaemia. The comparable patient groups were treated since 1981, according to protocols used by the Polish Acute Leukaemia Group. The prognostic value for achieving complete remission and survival of 67 pre-treatment factors (42 quantitative and 25 qualitative) was evaluated. The most important 9 parameters were scored according to their prognostic value as follows: age, percent of blasts in bone marrow, peripheral blood blast count, morphological subtype, percent of granulocytes in bone marrow, percent of blasts with CD-15 antigen, thrombocyte count, spleen/liver enlargement, CSF protein levels. Proposed scoring system enables classification of ANLL patients to a standard and high risk groups.     

Inward current generated by Na-Ca exchange during the action potential in single atrial cells of the rabbit.

To investigate the underlying ionic mechanism of the late plateau phase of the action potential in rabbit atrium the whole-cell patch-clamp technique with intracellular perfusion was used. We recorded the inward current during repolarizations following a brief 2 ms depolarizing pulse to +40 mV from a holding potential of between -70 and -80 mV. The development of this current coincides with the onset of the late plateau phase of the action potential. Peak activation of the current occurs about 10 ms from the beginning of the depolarizing pulse, and it decays spontaneously with a slow timecourse. Its voltage dependency from -40 mV to +40 mV shows very steep activation (-40 to -20 mV) and shows almost the same maximum magnitude between -10 mV and +40 mV. This behaviour is quite different from that of the calcium current. The inward current and the late plateau phase of the action potential were both abolished by the application of 5 mM EGTA, 1 microM ryanodine and by reducing the Na+ gradient. The fully activated current-voltage relation of the inward current was plotted as the difference current before and after treatment with Ryanodine, Diltiazem, 20 mM Na+ inside or 30% Na+ outside and shows an exponential voltage dependence with the largest magnitude of the current occurring at negative potentials. The current-voltage (I-V) curve was well fitted by the Na-Ca exchange equation, i = A exp (-(1 - r)EF/RT). The results suggest that the inward current contributes to the generation of the late plateau phase of the rabbit atrial action potential, and is activated by intracellular calcium released from the sarcoplasmic reticulum. Sarcoplasmic reticulum calcium release appears to be triggered both by the membrane voltage and by the calcium current. It is concluded that the inward current is generated by Na-Ca exchange.     

Purification, characterization, and amino terminal sequence of xylose reductase from Candida shehatae.

D-Xylose is a major component of the carbohydrates derived from agricultural residues and forest products. Among more than two hundred known xylose-utilizing yeasts, only a few species are known to be able to ferment xylose anaerobically. Candida shehatae is one of such xylose-fermenting yeasts. Xylose reductase (E.C. 1.1.1.21) is a key enzyme responsible for xylose metabolism in xylose-utilizing as well as xylose-fermenting yeasts. In this paper, we report the development of a convenient and reliable procedure for the purification of xylose reductase from C. shehatae to near homogeneity. The amino acid composition and N-terminal sequence of the enzyme have also been analyzed. C. shehatae seems to contain only a single xylose reductase, but the enzyme has a dual coenzyme specificity for both NADPH and NADH. The enzyme is remarkably stable at room temperature and 4 degrees C.     

Fine mapping of the McLeod locus (XK) to a 150-380-kb region in Xp21.

McLeod syndrome, characterized by acanthocytosis and the absence of a red-blood-cell Kell antigen (Kx), is a multisystem disorder involving a late-onset myopathy, splenomegaly, and neurological defects. The locus for this syndrome has been mapped, by deletion analysis, to a region between the loci for Duchenne muscular dystrophy (DMD) and chronic granulomatous disease (CGD). In this study, we describe a new marker, 3BH/R 0.3 (DXS 709), isolated by cloning the deletion breakpoint of a DMD patient. A long-range restriction map of Xp21, encompassing the gene loci for McLeod and CGD, was constructed, and multiple CpG islands were found clustered in a 700-kb region. Using the new marker, we have limited the McLeod syndrome critical region to 150-380-kb. Within this interval, two CpG-rich islands which may represent candidate sites for the McLeod gene were identified.     

Extracellular lipase from Pseudomonas aeruginosa is an amphiphilic protein.

Lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) secreted by Pseudomonas aeruginosa PAC1R was purified from cell-free growth medium by preparative isoelectric focusing. After blotting the N-terminal amino acid sequence and the amino acid composition were determined and compared to P. fragi and P. cepacia lipases yielding significant homology between all three species. Additionally, a consensus sequence K-Y-P-i-v-l-V-H-G was identified residing at the N-terminus of Pseudomonas lipases and in the central part of Staphylococcus lipases. Treatment of lipase with the serine-specific inhibitor diethyl p-nitrophenyl phosphate caused a rapid and complete inhibition of enzyme activity indicating the presence of a serine at the catalytic site as expected from lipase consensus sequences. Upon charge-shift electrophoresis the electrophoretic mobility of purified lipase was shifted either anodally or cathodally in the presence of sodium deoxycholate and cetyltrimethylammoniumbromide, respectively. This result demonstrates that extracellular lipase of P. aeruginosa exhibits an amphiphilic character like intrinsic membrane proteins.     

The influence of various physical parameters on anther culture of broccoli (Brassica oleracea var. italica)

Embryo formation by cultured broccoli (Brassica oleracea L. var. italica) anthers was best in the pH range of 5.5 to 5.8. Manipulation of the initial medium pH showed, however, that embryos could be recovered throughout the entire pH range tested. Experiments designed to test the influence of anther density on embryo production exhibited an apparent population effect. Comparison of anthers cultured with and without filaments showed a significantly lower level of embryo formation with filaments attached. The importance of anther orientation with the adaxial surface up was also demonstrated. Detailed studies of the effect of temperature on anther response showed the importance of 35°C treatments. Other temperatures and a variety of temperature manipulations were either comparatively ineffective or inhibitory. The duration of 35°C exposure required for optimal response varied widely between 18 and 48 h. Wide variation in plant to plant response was observed despite attempts to optimize the manipulation of physical parameters. Individual plants were identified that reliably formed many thousands of embryos, whereas other plants failed to form embryos under all tested conditions.     

Hormonal regulation, processing, and secretion of cysteine proteinases in barley aleurone layers.

Barley aleurone layers synthesize and secrete several proteases in response to gibberellic acid (GA3). Two major cysteine proteinases designated EP-A (37,000 M(r)) and EP-B (30,000 M(r)) have been described [Koehler and Ho (1988). Plant Physiol. 87, 95-103]. We now report the cDNA cloning of EP-B and describe the post-translational processing and hormonal regulation of both cysteine proteinases. Three cDNAs for cysteine proteinases were cloned from GA3-induced barley aleurone layers. Genomic DNA gel blot analysis indicated that these are members of a small gene family with no more than four to five different genes. The proteins encoded by two of these clones, pHVEP1 and 4, are 98% similar to each other and are isozymes of EP-B. The proteins contain large preprosequences followed by the amino acid sequence described as the mature N terminus of purified EP-B, and are antigenic to EP-B antiserum. The results of pulse-chase experiments indicated that the post-translational processing of large prosequences proceeds in a multistep fashion to produce the mature enzymes. Processing intermediates for EP-B are observed both in the aleurone layers and surrounding incubation medium, but only mature EP-A is secreted. The regulation of synthesis of EP-A, EP-B, and other aleurone cysteine proteinases was compared at the protein and mRNA levels. We conclude that barley aleurone cysteine proteinases are differentially regulated with respect to their temporal and hormonally induced expression.