Isolation and Characterization of Pyrophosphate:D-Fructose-6-phosphate 1-Phosphotransferase from Cucumber Seeds

Pyrophosphate:D-fructose-6-phosphate 1-phosphotransferase waspurified over 700-fold from germinating cucumber (Cucumis sativuscv. Fletcher) seeds. The purified enzyme has a specific activityof 5.2 µmol.min^–1.mg protein^–1 in the presenceof 1 µM fru-2,6-P₂. The pH optima is similar for boththe forward and reverse reactions (pH 7.5–7.8). Magnesium,manganese and cobalt activate the enzyme, with the highest affinitybeing for magnesium. The enzyme exhibits normal Michaelis-Mentenkinetics in both the presence and absence of fru-2,6-P₂. Half-maximumactivation of the enzyme was obtained with 35 nM fru-2,6-P2.Fru-2,6-P₂ stimulates activity by increasing V_max and increasingthe affinity for fru-6-P, fru-1,6-P₂ and PPi. Phosphate causesnoncompetitive inhibition with respect to both fru-6-P and PPi.On the basis of the steadystate substrate interaction and Piinhibition data a sequential ternary complex mechanism is proposed. (Received April 28, 1986; Accepted July 9, 1986)     

Extracellular Fluid Proteins of Goldfish Brain: Evidence for the Presence of Proteases and Esterases

Preparations of enriched fractions of extracellular fluid (ECF) proteins from goldfish brain were found to contain protease(s) and esterase(s). The N-substituted furanacryloyl (FA) peptides FA-Phe-Gly-Gly and FA-Phe-OMe were used as model substrates for determining protease and esterase activity, respectively, in a spectrophotometric assay. Studies of the profile of substrate specificity and identification of the types of compounds that were effective as inhibitors showed that these ECF enzymes have some distinctive properties. GSH, but not GSSG, and EDTA inhibited the protease(s) without influencing the esterase(s), whereas L-1-tosylamide-2-phenylethylchloromethyl ketone blocked both protease and esterase activities of ECF. Most of the protease and esterase properties of ECF could be bound to concanavalin A-Sepharose affinity chromatographic columns in association with ependymin--a brain extracellular protein. These observations indicate that ECF may contain a metalloprotease(s) and raise the possibility that the ependymins might be a substrate for these ECF enzymes.     

Phenotypic heterogeneity of Gross virus-induced thymic lymphomas in the rat: cellular origins and migratory properties

Neoplastic thymocytes from rat thymic lymphoma-leukemias induced by the rat-adapted Gross leukemia virus (RAGV) were analyzed for a variety of differentiation markers. The neoplasms from individual rats all expressed the antigenic phenotype MP+, W3/13+, Thy-1+, RT-1+, RT-7+, W3/25-. However, approximately two-thirds of the neoplasms were positive for the OX 8 antigen, and one-third were negative. The OX 8- neoplasms only involved the thymus, whereas approximately 40% of the OX 8+ neoplasms involved the spleen as well as the thymus. Virtually all OX 8+ and OX 8- neoplastic cells contained terminal deoxynucleotidyl transferase (TdT), and both OX 8+ and OX 8- lymphomas expressed the lactate dehydrogenase (LDH)-5' isozyme and the primary, but not the secondary, ADA isozyme. This enzymatic phenotype is characteristic of thymocyte precursors, but not thymocytes. Our results therefore indicate that RAGV-induced lymphomas arise from transformed prethymic TdT+ cells which contain the LDH-5' and the primary ADA isozymes. These preleukemic cells presumably migrate to the thymus where they express the RT-7 pan-T-cell antigen and, in some instances, the OX 8 antigen during the development of overt leukemia. The OX 8+ neoplasms, being more differentiated than their OX 8- counterparts, then migrate to peripheral lymphoid tissues.     

Sex ratio and intrasexual kin competition in mammals

Summary Asymmetries in both intrasexual competition and generation overlap occur in Antechinus (Dasyuridae; Marsupialia). We show that the range of interpopulation variation in the sex ratio of pouch young spans and exceeds the range of sex ratios at birth hitherto recorded from eutherians (Clutton-Brock and Albon 1983). Although postweaning dispersal and male mortality are similar among all Antechinus populations, interpopulation variance in female longevity leads to variable duration of mother/daughter interaction. As this duration increases, parental investment is increasingly biased toward males, supporting the view that local competition among female kin for resources may influence mammalian sex ratios.     

Restriction fragment polymorphisms as probes for plant diversity and their development as tools for applied plant breeding

Summary Maize and tomato cDNA clones have been hybridized in Southern blotting experiments to plant genomic DNA prepared from different lines to detect restriction fragment polymorphisms (RFPs). In maize we have found that a high degree of genetic variability is present, even among domestic inbred lines. Most randomly chosen maize cDNA clones can be used to detect elements of this variability. Similar levels of polymorphism are observed when genomic DNA is digested with any of a number of different restriction enzymes and probed with individual clones. When a clone is hybridized to genomic DNAs prepared from several different maize lines, a number of different alleles are often detected at a single locus. At the same time one clone can often detect more than one independently segregating locus by cross hybridization to related sequences at other loci. As expected these markers are inherited as simple codominant Mendelian alleles from one generation to the next and colinkage of these markers can be demonstrated in the progeny from a heterozygous parent. In similar studies with tomato, remarkably different results were found. Few RFPs were demonstrable among domestic Lycopersicon esculentum lines although a higher level of variability could be detected when comparing esculentum with its wild Lycopersicon relatives. These results are discussed in relation to the applied uses of RFPs in plant breeding as well as the inherent variability of different plant genomes.This work was supported in part by funds from Sandoz Ltd. (Basel, Switzerland) and its subsidiary company, Northrup King Co. (Minneapolis, Minn., U.S.A.) as well as by NSF SBIR grant #BSR-8360870.     

Rapid measurement of creatine kinase activity in a coronary care unit using a portable benchtop reflectance photometer.

Rapid measurements of plasma creatine kinase activity using an inexpensive benchtop reflectance photometer (Ames Seralyzer) and disposable reagent strips were evaluated in the laboratory and coronary care unit. The system proved simple to use and capable of yielding rapid (four minutes per analysis), precise (coefficient of variation less than 9%), and accurate results (correlation with routine method 0.995) when used by medical staff. Creatine kinase values were available 6.5-102 hours earlier than routine laboratory data, depending on the time and day of sampling, thereby facilitating appropriate and economic patient management. This instrument might be used to supplement the routine enzyme service for selected admissions, resulting in greatly improved availability of results and hence contributing to the early discharge of patients from intensive care facilities.     

Binding of human lymphocyte-specific monoclonal antibodies to common marmoset lymphoid cells

Commercially available monoclonal antibodies which bind to human lymphocyte subsets were screened for their ability to bind to lymphoid cells from the common marmoset Callithrix jacchus. Anti-Leu-5 and T11 were the only pan T-cell antibodies which reacted strongly. None of the antibodies which bind human lymphocytes of the helper/inducer subpopulation reacted with C. jacchus cells and only one antibody, T8, specific for the cytotoxic/suppressor subset, bound to the marmoset cells. The two antibodies tested which bind human B cells, B1 and anti-HLA-DR, were also reactive with marmoset cells. The cellular specificity of the T11, T8, and B1 antibodies was determined by dual binding studies on the fluorescence-activated cell sorter. The B1 antibody bound only Ig+ cells and all Ig+ cells were B1+. The T11 and T8 antibodies bound only to Ig- marmoset lymphoid cells and, as in the human, all T8+ marmoset cells were also T11+. Thus, using these monoclonal antibodies in the common marmoset one can identify three populations of lymphoid cells: (1) T11+, T8+ cells; (2) T11+, T8- cells; (3) B1+ cells.     

General method for the derivation and numerical solution of epithelial transport models

Summary A general method is presented for the formulation and numerical evaluation of mathematical models describing epithelial transport. The method is based on the principles of conservation of mass, and maintenance of electroneutrality within the cells and bathing solutions. It is therefore independent of the specific membrane transport mechanisms, and can be used to evaluate different models describing arbitrary transport processes (including passive, active and cotransport processes). Detailed numerical methods are presented that allow computation of steady-state and transient responses under open-circuit, current-clamp and voltage-clamp conditions, using a general-purpose laboratory minicomputer. To evaluate the utility of this approach, a specific model is presented that is consistent with the Koefoed-Johnson and Ussing hypothesis of sodium transport in tight epithelia (Acta Physiol. Scand. 42:298–308, 1958). This model considers passive transport of an arbitrary number of permeant solutes, active transport of sodium and potassium, and osmotically induced water transport across the apical and basolateral membranes. Results of the model are compared to published experimental measurements in rabbit urinary bladder epithelium.     

Reproductive strategies of the orang-utan: New data and a reconsideration of existing sociosexual models

Recent field data indicate that MacKinnon’s model of the orang-utan’s sexual and agonistic activity needs to be revised. In this model, male reproductive activity is concentrated in an extended phase of subadulthood and in early adulthood. According to this model, the role of older adult males is primarily that of range guardian, and in that role they would ensure that the offspring they had generated earlier would have safe access to food resources. This study presents cases suggesting that subadult males, even though sexually active, may have low reproductive success. In previous studies adult males were shown to display less sexual initiative than subadult males. In this study an adult male was at times involved infrequent mating activity in response to proceptive activity of females in the course of consortship. This adult male proved to be a successful breeder, thus refuting the hypothesis of adult male sterility. The female is most likely to conceive through cooperative mating in lengthy consortships with the dominant resident adult male. We hypothesize that the extended subadult phase represents a submissive strategy, allowing subadult males to remain in the home range of adult males but with minimal reproductive success.     

The interaction of cadmium with calcium and cisplatin with chloride

Changes in the locomotor rate of the ciliateTetrahymena pyriformis were used to quantitatively evaluate chemical interactions produced by: cadmium in combination with varying amounts of calcium, andcis-dichlorodiammineplatinum (II) (cisplatin) with varying amounts of sodium chloride. Cadmium (as CdCl₂) produces a measurable decline in the locomotor rate of the cells. Cadmium's detrimental effect can be reduced by the addition of calcium (as CaCl₂) in combination with cadmium. At a ratio of 30∶1 (calcium: cadmium), cadmium's negative effect upon motility is essentially nullified. It is suggested that the “protective” action afforded by calcium stems from the chemical similarity of the two cations and their involvement/competition for molecular sites responsible for the energy release and/or delivery of ciliary activity. Cisplatin will also effect a reduction in ciliary activity. However, the interaction between cisplatin, sodium chloride, and the cell appears more complex than that found with cadmium-calcium. At the lower range of chloride (as NaCl) used in this study, increased chloride concentration produces an increase in cisplatin's action against ciliary activity. At the higher levels, the chloride reduced cisplatin's negative effects. It is suggested that the increases in cisplatin's effects are caused by mass chemical action of increased chloride, which increases the concentration of the nonpolar cisplatin. The reduced effects found with the higher concentrations of sodium chloride may be because of the presence and action of elevated NaCl in/on the cell. This study clearly demonstrates differences in biologically relevant chemical interactions occurring with the two sets: cadmium-calcium and cisplatin-chloride.