Synthesis of ribonucleic acid in normal bone in vitro

1. The incorporation of [2-(14)C]uridine into nucleic acids of bone cells was studied in rat and pig trabecular-bone fragments surviving in vitro. 2. The rapid uptake of uridine into trichloroacetic acid-soluble material, and its subsequent incorporation into a crude nucleic acid fraction of bone or purified RNA extracted from isolated bone cells, was proportional to uridine concentration in the incubation medium over a range 0.5-20.0mum. 3. During continued exposure to radioactive uridine, bulk RNA became labelled in a curvilinear fashion. Radioactivity rapidly entered nuclear RNA, which approached its maximum specific activity by 2hr. of incubation; cytoplasmic RNA, and particularly microsomal RNA, was more slowly labelled. The kinetics of labelling and rapid decline of the nuclear/microsomal specific activity ratio were consistent with a precursor-product relationship. 4. Bulk RNA preparations were resolved by zonal centrifugation in sucrose density gradients into components with approximate sedimentation coefficients 28s, 18s and 4s. 5. Rapidly labelled RNA, predominantly nuclear in location, demonstrated a polydisperse sedimentation pattern that did not conform to the major types of stable cellular RNA. Material of highest specific activity, sedimenting in the 4-18s region and insoluble in 10% (w/v) sodium chloride, rapidly achieved its maximum activity during continued exposure to radioactive precursor and decayed equally rapidly during ;chase' incubation, exhibiting an average half-life of 4.3hr. 6. Ribosomal 28s and 18s RNA were of lower specific activity, which increased linearly for at least 6hr. in the continued presence of radioactive uridine. There was persistent but variable incorporation into ribosomal RNA during ;chase' incubation despite rapid decline in total radioactivity of the acid-soluble pool containing RNA precursors.     

Acid hydrolases of the rat uterus in relation to pregnancy, post-partum involution and collagen breakdown

1. The rat uterus contains acid cathepsin, beta-glucuronidase, beta-galactosidase, acid phosphatase and deoxyribonuclease II at concentrations comparable with those found in liver. Two non-hydrolytic uterine enzymes, cytochrome c oxidase and aspartate aminotransferase, display only 2-6% of the activity found in liver. 2. The concentrations of acid cathepsin and beta-glucuronidase are significantly decreased in pregnancy and increase 3-4-fold during post-partum involution. 3. The concentrations of beta-galactosidase and acid phosphatase are not decreased in pregnancy and increase only 2-3-fold during involution. 4. The concentrations of these four acid hydrolases increase linearly during the first 4 days post partum and reach their peak values at the same time that wet weight and collagen content fall to their lowest point. 5. The concentration of deoxyribonuclease is depressed in pregnancy but does not rise above normal in the post-partum period. 6. Only a small proportion of each hydrolytic activity can be isolated in the mitochondrial-lysosomal fraction of sucrose homogenates of the rat uterus. This proportion increases during involution. However, the extensive mitochondrial rupture occurring during homogenization indicates that the technique is probably too harsh to obtain a true measure of the proportion of lysosomes present in the intact tissue. 7. There are no significant changes in either the concentration or subcellular distribution of the five acid hydrolases in the livers of the experimental rats during pregnancy or involution. In each case the largest proportion of the activity is found in the mitochondrial-lysosomal fraction of liver homogenates. 8. The results are interpreted in terms of the lysosomal theory of intracellular digestion.     

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.     

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.     

Characterization of human alpha fetoprotein charge microheterogeneity during fetal development.

Aliquotes of human amniotic fluid (AF), fetal serum (FS), and cord blood (CB) were obtained as by-products of routine clinical diagnostic procedures at term or in the second trimester of pregnancy. When samples of CB were applied to a pH 5.5-4 chromatofocusing gradient, three isoforms of AFP could be resolved; a pl 4.57 form (isoform IA, 52% AFP), a pl 4.27 form (isoform IB, 43% AFP), and one species that was bound to the column but could be eluted with 1.0 M NaCl (isoform II, pl less than 4.00, 5% AFP). Term AF displayed a profile similar to that observed in term CB. When samples of 15-20-week gestation AF were chromatofocused, the immunoreactive AFP recovered was distributed between isoform IA and IB (60%) and isoform II (40%). FS and AF obtained from same pregnancy (23-26 weeks) displayed an identical chromatofocusing profile. Aliquotes of AF subjected to conA revealed 83% reactive variants compared with greater than 95% reactive variants for CB. FS displayed a conA profile identical to CB. When individual CB charge isoforms were isolated and subjected to conA analysis, greater than 97% of the AFP bound to conA. In contrast, when AFP isoform IA and IB were isolated from midgestation AF, approximately 22% of the AFP did not bind to the lectin while 100% of isolated AFP isoform II eluted as the reactive variant. These data suggest that human AFP exists as at least three charge and two lectin variants and that the charge profile may change during fetal development.     

Restriction site mapping for three or more enzymes

Restriction site mapping requires a generator to put forwardpossible maps and a constraint checker to reject false maps.Ideally these combine to give an algorithm which calculatesa sound and complete solution set. Three algorithms for generationare presented and compared. Two decompose a multi-enzyme problem(3) into subproblems. The constraint checker is based on separationtheory. Some insights into the extent of constraint checkinginvolved in and the feasibility of more checking for three ormore enzymes are discussed. The trade-off between computationtime and the soundness of the solution set is examined. Received on July 30, 1989; accepted on April 4, 1990     

Characterization of the functional properties of env genes from long-term survivors of human immunodeficiency virus type 1 infection.

A small number of persons infected with human immunodeficiency virus type 1 (HIV-1) remain clinically and immunologically healthy for more than a decade after infection. Recent reports suggest that these individuals may be infected with an attenuated strain of HIV-1; however, a common genetic basis for viral attenuation has not been found in all cases. In the present study, we examined the functional properties of the HIV-1 env genes from six long-term survivors. env clones were generated by PCR amplification of proviral env sequences, followed by cloning of the amplified regions into expression vectors. Eight to ten clones from each subject were screened by transient transfection for expression of the envelope precursor glycoprotein, gp160. Those clones expressing gp160 were then cotransfected with an HIV-1 luciferase reporter vector, pNL4-3Env(-)LUC(+) and evaluated for their ability to mediate infection of phytohemagglutinin-activated peripheral blood mononuclear cells in single-cycle infectivity assays. Clones expressing gp160 were identified for all six long-term survivors, indicating the presence of proviral env genes with intact open reading frames. For two subjects, D and DH, the encoded envelope glycoproteins yielded high levels of luciferase activity when pseudotyped onto HIV-1 virions and tested in single-cycle infectivity assays. In contrast, envelope glycoproteins cloned from four other long-term survivors were poorly processed and failed to mediate infection. Sequencing of the gp120/41 cleavage site and conserved gp41 cysteine residues of these clones did not reveal any obvious mutations to explain the functional defects. The functional activity of env clones from long-term survivors D and DH was comparable to that seen with several primary HIV-1 env genes cloned from individuals with disease progression and AIDS. These results suggest that the long-term survival of subjects D and DH is not associated with overt functional defects in env; however, functional abnormalities in env may contribute to maintaining a long-term asymptomatic state in the other four cases we studied.