A computer program for enzyme kinetics that combines model discrimination, parameter refinement and sequential experimental design.

A method of model discrimination and parameter estimation in enzyme kinetics is proposed. The experimental design and analysis of the model are carried out simultaneously and the stopping rule for experimentation is deduced by the experimenter when the probabilities a posteriori indicate that one model is clearly superior to the rest. A FORTRAN77 program specifically developed for joint designs is given. The method is very powerful, as indicated by its usefulness in the discrimination between models. For example, it has been successfully applied to three cases of enzyme kinetics (a single-substrate Michaelian reaction with product inhibition, a single-substrate complex reaction and a two-substrate reaction). By using this method the most probable model and the estimates of the parameters can be obtained in one experimental session. The FORTRAN77 program is deposited as Supplementary Publication SUP 50134 (19 pages) at the British Library (Lending Division), Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1986) 233, 5.     

H1° histones of normal and cancer human cells

Summary H1° histones were purified by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis from human lung carcinoma (line DMS79), human hepatoblastoma (HepG2), human adult lung and human adult and fetal liver. The purified human H1° histones were analyzed for their amino acid composition and terminal residues. The comparative analysis of the amino acid compositions of the different human H1° histones showed that: (a) all the H1° preparations have the characteristically high lysine content associated with a low arginine content, which distinguishes outer histones from core histones; (b) H1° is distinguishable from other H1 histones by the presence of methionine and histidine; (c) H1° histones from human adult, fetal and cancer cells are very similar in amino acid composition, and in cancer cells the level of the H1° histone is not inversely related with cell growth rate nor with the expression of the -fetoprotein gene.     

Kinetics of the 5′-nucleotidase and the adenosine deaminase in subcellular fractions of rat brain

Suspensions of rat brain microsomes, synaptosomes, and synaptic vesicles were able to convert adenosine to inosine by means of adenosine deaminase. Isosbestic points of this transformation, at 222, 250 and 281 nm, remained unchanged with time-course. This fact suggests that adenosine deaminase (ADA, E.C. 3.5.4.4) is located on the surface of the vesicles whereas purine nucleoside phosphorylase (PNP, E.C. 2.1.2.4) is located inside the vesicles. Kinetic parameters of the particulate 5-nucleotidase (5N, E.C. 3.1.3.5) and adenosine deaminase were analogous to those of the cytosolic enzymes. These results suggest that soluble and particulate enzymes represent different pools of the same molecular species.     

Immune reactivity in cattle with ocular squamous cell carcinoma after intralesional BCG immunotherapy

Summary Lymphocyte stimulation with Con A and specific immune reactivity to BCG (antibody formation to BCG and DTH reaction to PPD) were determined in BCG-treated, surgically treated and untreated cows with ocular squamous cell carcinoma. In tumor-bearing cows the Con A-induced proliferation of lymphocytes was reduced when compared to healthy controls. This suppression consisted of a reduced blastogenic response to Con A of lymphocytes from tumor-bearing cows, and the presence of a factor in the sera of these animals, as these sera suppressed the blastogenic response of lymphocytes from healthy cows. BCG had only a minor influence on the suppressive activity. Antibodies to BCG were demonstrated in 50% of the BCG-treated animals. The formation of antibodies was not influenced by intradermal injection of PPD of Mycobacterium bovis. Absorption of a BCG antibody containing serum with BOSCC tumor extracts did not reveal the existence of cross reacting antigens between BCG and BOSCC. Pretherapeutic and posttherapeutic Con A reactivity could not be correlated with clinical response. Of the 30 BCG treated cows 29 developed a positive DTH reaction to PPD. Correlation between clinical response and immune reactivity was seen only with regard to the DTH reaction to PPD: this reaction remained positive for a longer period after treatment in animals with a favorable clinical outcome than in nonresponding animals.Animals were maintained under the guidelines laid down by the Faculty of Veterinary Medicine, State University, Utrecht, The NetherlandsGrant recipient of the Koningin Wilhelmina Fonds (Netherlands Cancer Foundation) Abbreviations used: BCG, Bacillus Calmette-Guerin; BOSCC, bovine ocular squamous cell carcinoma PBL peripheral blood leukocytes; PPD, purified protein derivative of Mycobacteria; DTH, delayed type hypersensitivity Con A, concanavalin A; PHA, phytohemagglutinin; PWM, pokeweed mitogen     

Heterogeneous localization of some purine enzymes in subcellular fractions of rat brain and cerebellum

The activity of guanine deaminase (GAH, E.C. 3.5.4.3) was lower in rat cerebellum soluble and microsomal fractions than in rat brain subfractions. Adenosine deaminase (ADA, E.C. 3.5.4.4) activity was released in higher proportion than guanine deaminase, purine nucleoside phosphorylase (PNP, E.C. 2.1.2.4), 5-nucleotidase (5N, E.C. 3.1.3.5), and lactate (LDH, E.C. 1.1.1.27) and malate (MDH, E.C. 1.1.1.37) dehydrogenase in press-juices of rat brain. Furthermore, nerve ending-derived fractions (synaptosomes and synaptic vesicles) showed an enrichment of adenosine deaminase and also of 5-nucleotidase. The action of deoxycholate over the subfractions did not increase the activity of either enzyme. The contrary occurred with the remaining enzymes studied. Thus, it is possible that one set of enzymes are located on the surface of the particulate vesicles, whereas another set are located inside these vesicles, suggesting a compartmentation of purine catabolic enzymes in different areas of the central nervous system.     

A high affinity,calmodulin-responsive (Ca2+ + Mg2+)-ATPase in isolated bone cells

Although acute alterations in Ca²⁺ fluxes may mediate the skeletal responses to certain humoral agents, the processes subserving those fluxes are not well understood. We have sought evidence for Ca²⁺-dependent ATPase activity in isolated osteoblast-like cells maintained in primary culture. Two Ca²⁺-dependent ATPase components were found in a plasma membrane fraction: a high affinity component (half-saturation constant for Ca²⁺ of 280 nM, $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of 13.5 nmol/mg per min) and a low affinity component, which was in reality a divalent cation ATPase, since Mg²⁺ could replace Ca²⁺ without loss of activity. The high affinity component exhibited a pH optimum of 7.2 and required Mg²⁺ for full activity. It was unaffected by potassium or sodium chloride, ouabain or sodium azide, but was inhibited by lanthanum and by the calmodulin antagonist trifluoperazine. This component was prevalent in a subcellular fraction which was also enriched in 5′-nucleotidase and adenylate cyclase activities, suggesting the plasma membrane as its principal location. Osteosarcoma cells, known to resemble osteoblasts in their biological characteristics and responses to bone-seeking hormones, contained similar ATPase activities. Inclusion of purified calmodulin in the assay system caused small non-reproducible increases in the Ca²⁺-dependent ATPase activity of EGTA-washed membranes. Marked, consistent calmodulin stimulation was demonstrated in membranes exposed previously to trifluoperazine and then washed in trifluoperazine-free buffer. These results indicate the presence of a high affinity, calmodulin-sensitive Ca²⁺-dependent ATPase in osteoblast-like bone cells. As one determinant of Ca²⁺ fluxes in bone cells, this enzyme may participate in the hormonal regulation of bone cell function.     

The Formation of Otoliths in the Frog Rana esculenta. Scanning Electron Microscopic and X-Ray Diffraction Studies

Two crystal forms of calcium carbonate were observed: calcite (utricle) and aragonite (saccule, lagena, endolymphatic sac). The first step in otolith formation is the appearance of organic structures in the macula. The subsequent step is characterized by fast growing primitive crystals with a prismatic habitus that successively transform into adult or mature crystals. With the metamorphosis, the aragonite crystals of the endolymphatic organ show clear signs of erosion that can be related to a process of CaCO₃ mobilization from such deposits.     

Dopamine Uptake by Rat Striatal Synaptosomes: A Compartmental Analysis

Abstract: Dopamine (DA) uptake into synaptosomes from rat corpus striatum was studied in the presence of a monoamine oxidase (MAO) inhibitor and dithiothreitol, by means of a filtration technique. Under these conditions a steady state develops rapidly in which the synaptosomal DA content remains constant while the continuing DA uptake is counterbalanced by DA efflux from the synaptosome. Exchange of synaptosomal [³H]DA and [¹⁴C]DA was measured under these conditions. In timecourse experiments it was found that exchange could be described significantly better by a three-compartment model than by a two-compartment model. However, if synaptosomes from reserpine-pretreated animals were used, analysis according to a three-compartment model did not result in a significantly better fit compared with a two-compartment model. Subsequently, kinetic transfer parameters describing DA fluxes between compartments at different DA concentrations were calculated from the fitted exchange curves. A Michaelis-Menten kinetic analysis indicated that only the in-series three-compartment configuration, in which DA is taken up from the medium into one synaptosomal compartment, from which it can subsequently be transferred to a second compartment without direct access to the medium, gave kinetically acceptable results. Transfer parameters in synaptosomes from reserpine-treated rats were comparable to those parameters describing DA transport between the medium and the first intrasynaptosomal compartment as measured under control conditions. Morover, it was found that potassium depolarization of synaptosomes resulted in a release of DA in a quantity similar to that found in the second intrasynaptosomal compartment. It is suggested that the two intrasynaptosomal compartments found correspond to a cytoplasmatic and vesicular DA pool, respectively. The functional significance of these findings is discussed in terms of the regulation of DA levels within the nerve terminal.     

Description of Thecavermiculatus andinus n.sp. (Meloidoderidae), a Round Cystoid Nematode from the Andes Mountains of Peru

Thecavermiculatus andinus n.sp. is described and illustrated from Oxalis tuberosa originally collected in the vicinity of Lake Titicaca high in the Andes mountains of southern Peru. This new species differs markedly front the other two species in the genus, especially in having a much greater female vulval-anal distance and annules with lined punctation on most of the female body with a lacelike pattern restricted to the posterior portion, particularly at the vulva and anus which do not protrude. Females are essentially spherical with protruding neck, white to yellowish in color, and can easily be mistaken for potato cyst nematodes. Among the dozen or more known weed and crop host plants are potato and eggplant. In order to accommodate this new species, the genus Thecavermieulatus is emended. A key to the species of this genus is presented.