Flow cytometric determination of atypical antigen expression in acute leukemia for the study of minimal residual disease.

The aim of this study was to investigate to which extent acute leukemias could be monitored for residual disease by using atypical antigen combinations as leukemia-related markers. Atypical antigenic features were determined by double color flow cytometry and included coexpression of lymphoid and myeloid related antigens, unphysiological coexpression of immature and mature antigens, and lack of an antigen that is normally expressed during maturation. Atypical immunophenotypes were detected in 35 of 68 patients with AML (51.5%) and 15 of 24 patients with ALL (62.5%). When 12 patients with leukemia-associated markers were again analyzed at relapse, the relevant antigen combinations were retained in 11 of them. The sensitivity of this two color flow cytometric assay as determined in dilution experiments was 1 in 10(3) to 10(4) cells. Follow-up studies of bone marrow samples revealed that, after induction chemotherapy cells with leukemia-associated markers were detectable in several patients at a frequency of 0.5 to 4%, but only patients in whom the cells with atypical antigens never disappeared suffered from relapse. In contrast, patients who became negative for the atypical cells remained in complete remission (median remission duration after the first negative bone marrow assessment by flow cytometry 52 weeks, range 20-102). We conclude that atypical antigen combinations, which are present in a meaningful number of acute leukemias, are a valuable means of monitoring acute leukemia patients during follow-up. This flow cytometric approach can complement other strategies to get a more accurate definition of remission in acute leukemia.     

Root Flooding of Muskmelon (Cucumis melo L.) Affects Fruit Sugar Concentration But Not Leaf Carbon Exchange Rate

Flooding the roots of greenhouse-grown muskmelon (Cucumis meloL. cv. Noy Yizreel) plants for 4 days reduced sucrose accumulation36% in the inner mesocarp and 88% in the outer mesocarp of developingfruit. Concentration of the translocated sugars raffinose andstachyose were also lower in fruit on flooded plants than inthose from nonflooded plants. In contrast, fruit hexose concentrationwas similar in both flooded and nonflooded plants. There wasno alteration in activities of enzymes associated with sucrosemetabolism in the fruit which could explain the decreased sucroseconcentration. Four days of root flooding caused no reductionin leaf carbon exchange rate or assimilate export rate, indicatingthat the reduction in fruit sucrose accumulation was not dueto source limitation. Root respiration, measured as CO₂ evolution,was approximately 30% lower in anaerobic roots than in aerobicroots. When viewed as carbohydrate consumed, a doubling of glycolyticactivity occurred in the anaerobic root mass. Increased demandfor carbohydrates by anaerobic roots may lead to a reductionin translocated carbohydrates available for sucrose biosynthesisin the developing fruit. (Received August 29, 1990; Accepted February 21, 1991)     

Determination for inflorescence development is a stable state,separable from determination for flower development in Pisum sativum L. buds

Terminal meristems of Pisum sativum (garden pea) transit from vegetative to inflorescence development, and begin producing floral axillary meristems. Determination for inflorescence development was assessed by culturing excised buds and meristems. The first node of floral initiation (NFI) for bud expiants developing in culture and for adventitious shoots forming on cultured meristems was compared with the NFI of intact control buds. When terminal buds having eight leaf primordia were excised from plants of different ages (i.e., number of unfolded leaves) and cultured on 6-benzylaminopurine and kinetin-supplemented medium, the NFI was a function of the age of the source plant. By age 3, all terminal buds were determined for inflorescence development. Determination occurred at least eight nodes before the first axillary flower was initiated. Thus, the axillary meristems contributing to the inflorescence had not formed at the time the bud was explanted. Similar results were obtained for cultured axillary buds. In addition, meristems excised without leaf primordia from axillary buds three nodes above the cotyledons of age-3 plants gave rise to adventitious buds with an NFI of 8.3 ±0.3 nodes. In contrast seed-derived plants had an NFI of 16.5 ±0.2. Thus cells within the meristem were determined for inflorescence development. These findings indicate that determination for inflorescence development in P. sativum is a stable developmental state, separable from determination for flower development, and occurring prior to initiation of the inflorescence at the level of meristems.     

Plant protoplasts and genetic engineering II

Book Review

Plant protoplasts and genetic engineering IIY.P.S. Bajaj (Ed.), (Biotechnology in Agriculture and Forestry, Vol. 9). Berlin: Springer-Verlag, 1989. 510 pages. DM398.00. ISBN 3-540-50789-2     

The refined 2.15 A X-ray crystal structure of human liver cathepsin B: the structural basis for its specificity.

From the lysosomal cysteine proteinase cathepsin B, isolated from human liver in its two-chain form, monoclinic crystals were obtained which contain two molecules per asymmetric unit. The molecular structure was solved by a combination of Patterson search and heavy atom replacement methods (simultaneously with rat cathepsin B) and refined to a crystallographic R value of 0.164 using X-ray data to 2.15 A resolution. The overall folding pattern of cathepsin B and the arrangement of the active site residues are similar to the related cysteine proteinases papain, actinidin and calotropin DI. 166 alpha-carbon atoms out of 248 defined cathepsin B residues are topologically equivalent (with an r.m.s. deviation of 1.04 A) with alpha-carbon atoms of papain. However, several large insertion loops are accommodated on the molecular surface and modify its properties. The disulphide connectivities recently determined for bovine cathepsin B by chemical means were shown to be correct. Some of the primed subsites are occluded by a novel insertion loop, which seems to favour binding of peptide substrates with two residues carboxy-terminal to the scissile peptide bond; two histidine residues (His110 and His111) in this "occluding loop' provide positively charged anchors for the C-terminal carboxylate group of such polypeptide substrates. These structural features explain the well-known dipeptidyl carboxypeptidase activity of cathepsin B. The other subsites adjacent to the reactive site Cys29 are relatively similar to papain; Glu245 in the S2 subsite favours basic P2-side chains. The above mentioned histidine residues, but also the buried Glu171 might represent the group with a pKa of approximately 5.5 near the active site, which governs endo- and exopeptidase activity. The "occluding loop' does not allow cystatin-like protein inhibitors to bind to cathepsin B as they do to papain, consistent with the reduced affinity of these protein inhibitors for cathepsin B compared with the related plant enzymes.     

Defect of sperm assembly in a neurological mutant of the mouse, wobbler (WR)

In the wobbler (WR) mouse, a neuromuscular mutant characterized by a motoneuron degeneration and male infertility, the cellular basis of the defect in spermiogenesis was studied by light and electron microscopy as well as by lectin binding. Spermatozoa of the wobbler mutant had rounded heads, and their motility was reduced. In histological sections of WR testes, spermatogenesis appeared normal up to the stage of round spermatids, but the elongation and flattening of the nucleus during late spermiogenesis did not occur. Numbers of spermatid nuclei in WR testes were reduced to 70%-80% of controls. The acrosomal marker glycoprotein, peanut agglutinin receptor, was synthesized, but the acrosomal membrane did not attach to the nucleus. The disturbance in spermiogenesis of the wobbler mouse is not due to impaired descent of the testis, nor to a lack of testosterone, and is distinct from that observed in other mouse mutants (quaking, QK; Purkinje cell degeneration, PCD) with combined neurological and spermiogenesis defects.     

Differential expression and localization of brain-type and mitochondrial creatine kinase isoenzymes during development of the chicken retina: Mi-CK as a marker for differentiation of photoreceptor cells

The expression and the cellular- as well as subcellular-distribution of brain-type B-CK and mitochondrial Mi-CK during development of the chicken retina was studied by immunoblotting, immunofluorescence and immunogold methods. B-CK expression and accumulation in retina was high from early stages of embryonic development on, decreased slightly around hatching and remained high again during adulthood. At early stages of development (days 2-5), B-CK was more or less evenly distributed over the entire retina with the exception of ganglion cells, which were stained more strongly for B-CK than other retinal precursor cells. Then, at around day 10, the beginning of stratified immunostaining by anti-B-CK antibody was noted concomitant with progressing differentiation. Finally, a dramatic increase in staining of the differentiating photoreceptor cells was seen before hatching (day 18) with weaker staining of other cell types. At hatching, as in the adult state, most of the B-CK was localized within rods and cones. Thus, during retinal development marked changes in the immunostaining pattern for B-CK were evident. By contrast, Mi-CK expression was low during development in ovo and rose just before hatching with a predominant accumulation of this isoenzyme within the ellipsoid portion of the inner photoreceptor cell segments. Mi-CK accumulation in the retina coincided with functional maturation of photoreceptors and therefore represents a good marker for terminal differentiation of these cells. B-CK, present from early stages of retina development, seems to be relevant for the energetics of retinal cell proliferation, migration and differentiation, whereas the simultaneous expression of both B- and Mi-CK around the time of hatching indicates a coordinated function of the two CK isoforms as constituents of a PCr-circuit involved in the energetics of vision, which, in autophagous birds, has to be operational at this point in time.     

Site-directed mutagenesis of beta-galactosidase (E. coli) reveals that tyr-503 is essential for activity

By using the technique of site-directed mutagenesis we have succeeded in replacing tyr-503 of beta-galactosidase (E. coli) with a phe. A study of the kinetic and stability properties of this mutant enzyme (F-503 beta-galactosidase) showed that the loss in activity upon this change is due to the loss of a catalytic group (rather than a detrimental change in the enzyme's overall structure or a change in the enzyme's binding capacity). This confirms previous suggestions that this tyr residue is involved in catalysis.     

Identification of two histidines as copper ligands in Streptomyces glaucescens tyrosinase

The physiochemical properties of wild type and two mutants of Streptomyces glaucescens tyrosinase are reported. The native enzyme contains two coppers at the active site which are EPR nondetectable. The two coppers react stoichiometrically with one hydrogen peroxide molecule giving rise to oxytyrosinase. Its optical features are similar to those reported earlier for a molluscan hemocyanin. The two mutants in which histidine-62 and -189 were changed to asparagine by site-directed mutagenesis have lost their enzymatic activity and their ability to bind oxygen and contain only one copper ion which is fully EPR detectable. The EPR parameters indicate that the remaining copper is in a tetragonally distorted ligand environment. These data are in agreement with His-62 and His-189 serving as copper ligands in S. glaucescens tyrosinase.     

The use of limited proteolysis to probe interdomain and active site regions of beta-galactosidase (Escherichia coli)

Limited proteolysis by pancreatic elastase (EC 3.4.21.36) and chymotrypsin (EC 3.4.21.1) was used to study the domain structure and active site of beta-galactosidase (EC 3.2.1.23) (Escherichia coli). Treatment with elastase resulted in a rapid cleavage between residues Ala-732 and Ala-733. No inactivation accompanied this cleavage suggesting that this bond is in a hinge region of the protein. Some slow cleavages beyond the initial one were observed to occur and were accompanied by inactivation. Treatment of beta-galactosidase with chymotrypsin resulted in cleavages first between Trp-585 and Ser-586 and then between Phe-601 and Cys-602. The first of these cleavages resulted in total inactivation of beta-galactosidase. The presence of monovalent ions or isopropyl-beta-D-thiogalactopyranoside protected against the cleavages but when Mg2+ or Mn2+ was present in the reaction mixture, the bond between Trp-585 and Ser-586 was more susceptible to the action of chymotrypsin. These data demonstrate that the conformation of beta-galactosidase around Trp-585 and Ser-586 is dramatically affected by the binding of ions and isopropyl-beta-D-thiogalactopyranoside. The mutant M15 beta-galactosidase, which is missing residues 11 through 41 and is an inactive dimer rather than an active tetramer, was found to be much more labile to proteases than native beta-galactosidase, but the same initial cleavages were found to occur. In addition, trypsin cleaved the M15 protein between Arg-431 and Trp-432 while native beta-galactosidase was stable to trypsin.