Some epidemiological models with nonlinear incidence

Epidemiological models with nonlinear incidence rates can have very different dynamic behaviors than those with the usual bilinear incidence rate. The first model considered here includes vital dynamics and a disease process where susceptibles become exposed, then infectious, then removed with temporary immunity and then susceptible again. When the equilibria and stability are investigated, it is found that multiple equilibria exist for some parameter values and periodic solutions can arise by Hopf bifurcation from the larger endemic equilibrium. Many results analogous to those in the first model are obtained for the second model which has a delay in the removed class but no exposed class.Research supported in part by Centers for Disease Control Contract 200-87-0515. Support services provided at University House Research Center at the University of IowaResearch supported in part by NSERC A-8965 and the University of Victoria President's Committee on Faculty Research and Travel     

Molecular characterization of a patient with del(1)(q23–q25)

Summary We report a patient (S.T.) with multiple congenital anomalies and developmental delay associated with an interstitial deletion of 1q23–1q25. Molecular analysis of the deletion was performed using DNA markers that map to 1q. Five DNA markers, MLAJ-1 (D1S61), CRI-L1054 (D1S42), HBI40 (D1S66), OS-6 (D1S75), and BH516 (D1S110), were demonstrated to be deleted. Informative polymorphisms demonstrated this to be a de novo deletion of the maternally derived chromosome. Deletion status was determined using restriction fragment length polymorphism (RFLP) analysis supplemented with densitometry in the experiments where RFLP analysis was not fully informative. Deletions were confirmed by Southern analysis using genomic DNA from a somatic cell hybrid retaining the del(1)(q23–q25) chromosome that was constructed from patient S.T. Flow karyotyping confirmed the deletion and estimated that the deletion encompassed 11,000–16,000 kb. The clinical and cytogenetic characteristics of S.T. are compared with those of ten previously described patients with monosomy 1q21–1q25.     

Application of flow karyotyping in prenatal detection of chromosome aberrations

This paper describes the application of bivariate flow karyotyping to (1) classification of chromosomes isolated from cultures of cells taken by amniocentesis and (2) detection of numerical and structural aberrations. Chromosomes were isolated from primary cultures 2-5 wk after amniocentesis, stained with Hoechst 33258 and chromomycin A3, and analyzed using dual beam flow cytometry. Information about chromosome DNA content and DNA base composition was derived from the locations of the peaks in the flow karyotypes, each peak being produced by one or more chromosome types with similar DNA content and DNA base composition. Information about the relative frequency of each chromosome type was determined on the basis of the relative volume of the peak for that chromosome type. Cytogenetic information determined on the basis of flow karyotypes was compared with that obtained by visual analysis following G-banding. Variability among the peak means and volumes in flow karyotypes was determined from analyses of 50 normal amniocyte cultures. Numerical aberrations involving chromosomes 21, 18, and Y were detected correctly in all of 28 analyses, including eight in a blind study. Structural aberrations involving chromosomes 1, 2, 3, 6, 9-12, 13, 14, 15, 21, and 22 were detected in all of seven cultures in a blind study. Flow karyotypes proved to be insensitive to small, normally occurring chromosome polymorphisms detected by banding analysis. In addition, a few samples were erroneously scored as having numerical aberrations.     

Large-scale preparation and reconstitution of apo-flavoproteins with special reference to butyryl-CoA dehydrogenase from Megasphaera elsdenii. Hydrophobic-interaction chromatography

A new method is described for the large-scale reversible dissociation of flavoproteins into apoprotein and prosthetic group using hydrophobic-interaction chromatography. Lipoamide dehydrogenase from Azotobacter vinelandii and butyryl-CoA dehydrogenase from Megasphaera elsdenii are selected to demonstrate the usefulness of the method. In contrast to conventional methods, homogeneous preparations of apoproteins in high yields are obtained. The apoproteins show high reconstitutability. The holoenzymes are bound to phenyl-Sepharose CL-4B at neutral pH in the presence of ammonium sulfate. FAD is subsequently removed at pH 3.5-4.0 by addition of high concentrations of KBr. Large amounts of apoenzymes (200-500 mg), showing negligible residual activity, are eluted at neutral pH in the presence of 50% ethylene glycol. The holoenzyme of lipoamide dehydrogenase can be reconstituted while the apoprotein is still bound to the column or the apoenzyme can be isolated in the free state. In both cases the yield and degree of reconstitution of holoenzyme is more than 90% of starting material. Apo-lipoamide-dehydrogenase exists mainly as a monomer in solution and reassociates to the native dimeric structure in the presence of FAD. The apoenzyme is stable for a long period of time when kept in 50% ethylene glycol at -18 degrees C. Steady-state fluorescence-polarization measurements of protein-bound FAD indicate that reconstituted lipoamide dehydrogenase possesses a high stability which is governed by the low dissociation rate constant of the apoenzyme-FAD complex. The holoenzyme of butyryl-CoA dehydrogenase cannot be reconstituted when the apoenzyme is bound to the column. However, stable apoprotein can be isolated in the free state yielding 50-80% of starting material, depending on the immobilization conditions. The coenzyme A ligand present in native holoenzyme is removed during apoprotein preparation. The apoenzyme is relatively stable when kept in 50% ethylene glycol at -18 degrees C. From kinetic and gel filtration experiments it is concluded that the reconstitution reaction of butyryl-CoA dehydrogenase is governed by both the pH-dependent hydrodynamic properties of apoenzyme and the pH-dependent stability of reconstituted enzyme. At pH 7, the apoenzyme is in equilibrium between dimeric and tetrameric forms and reassociates to a native-like tetrameric structure in the presence of FAD. The stability of reconstituted enzyme is strongly influenced by the presence of CoA ligands as shown by fluorescence-polarization measurements. The degree of reconstitution of butyryl-CoA dehydrogenase is more than 80% of the original specific activity under certain conditions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Post-translational modification of the beta-subunit of the human fibronectin receptor

Monoclonal antibody DH12, directed against the beta-subunit of the fibronectin receptor recognizes a doublet of proteins (100 and 110 kDa) in Western blots of solubilized whole fibroblasts. Pulse-chase experiments with [35S]methionine in human skin fibroblasts suggested that the two proteins might be metabolically related as precursor (100 kDa) and product (110 kDa). Endo H digestion and [3H]fucose labeling suggested that maturation converted the high-mannose oligosaccharides (100 kDa) to the endoglycosidase H resistant complex type (110 kDa). This was supported by N-glycanase digestion and by chemical deglycosylation which showed a single polypeptide. Surface iodination of intact cells labeled only the presumed mature beta-subunit.     

Regulatory aspects of mitochondrial phospholipase A2: correlation of hydrolysis rates with substrate configuration as evidenced by 31P-NMR

The influence of variation of the phospholipid composition in model membranes composed of phosphatidylcholine and phosphatidylethanolamine on the hydrolysis of these phospholipids by rat liver mitochondrial phospholipase A2 was investigated. With the pure phospholipids, phosphatidylethanolamine was hydrolyzed over 30-times faster than phosphatidylcholine. Upon increasing the mole percentage of phosphatidylethanolamine in mixtures, a gradual, though non-linear, increase in the initial rate of hydrolysis of this phospholipid was observed. By contrast, phosphatidylcholine hydrolysis remained constant up to about 50 mol% phosphatidylethanolamine, whereafter a sudden fall-off of activity was observed. This drop in the hydrolysis rate coincided with a transition of the phospholipid structure from bilayer to an as yet unidentified organization characterized by an isotropic signal in the 31P-NMR spectra recorded in the presence of Ca2+. The occurrence of this phase was clearly dependent on Ca2+, since mixtures with identical composition in the absence of Ca2+ remained largely in bilayer configuration. That the structure adopted by phospholipids is of importance for their susceptibility to attack by this intracellular phospholipase A2 became evident also in studies with the single phospholipids in the absence or presence of Triton X-100 above the critical micellar concentration. While phosphatidylcholine hydrolysis was inhibited in mixed micelles as compared to its bilayer organization, the hydrolysis of phosphatidylethanolamine in mixed micelles was 3-fold that in the hexagonal HII phase.     

Mineralization of adult mouse bone marrow in vitro

Murine adult bone marrow exhibits mineralizing capacity in vitro as is demonstrated by the new in vitro assay we report here. In less than 2 weeks after the onset of the cultures, mineralization is obtained in more than 80% of the marrow cultures. Moreover, morphological studies reveal that during incubation phenotypic changes related to osteogenic differentiation occur at the extracellular matrix as well within cell populations. Well banded collagen is synthesized. Matrix vesicles and needles of hydroxy-apatite crystals are observed via transmission electron microscopy. Osteoblast-like cells are present with membrane-associated alkaline phosphatase activity. The mineralization is specific for cultured bone marrow and is not observed in cultured spleen fragments as is shown via 85Sr uptake, calcein uptake and histomorphology. No inducing agent is added to the tissue culture medium except for 10% fetal calf serum, beta-glycerophosphate (10(-2) M) and ascorbic acid. However, the prerequisite for obtaining mineralization is the three-dimensional structure of the marrow in culture. The in vitro organ culture we developed may provide the opportunity to identify which marrow cells have osteogenic potential and to investigate the mechanisms triggering differentiation towards osteogenesis.     

Gramicidin-induced hexagonal HII phase formation in negatively charged phospholipids and the effect of N- and C-terminal modification of gramicidin on its interaction with zwitterionic phospholipids

The effect of gramicidin on macroscopic structure of the negatively charged membrane phospholipids cardiolipin, dioleoylphosphatidylglycerol and dioleoylphosphatidylserine in aqueous dispersions was investigated and compared with the effect of gramicidin on dioleoylphosphatidylcholine. It was shown by small-angle X-ray diffraction, 31P nuclear magnetic resonance and freeze-fracture electron microscopy that in all these lipid systems gramicidin is able to induce the formation of a hexagonal HII phase. 31P-NMR measurements indicated that the extent of HII phase formation in the various lipids ranged from about 40% to 60% upon gramicidin incorporation in a molar ratio of peptide to lipid of 1 : 10. Next, the following charged analogues of gramicidin were prepared: desformylgramicidin, N-succinylgramicidin and O-succinylgramicidin. The synthesis was verified with 13C-NMR and the effect of these analogues on lipid structure was investigated. It was shown that, as with gramicidin itself, the analogues induce HII phase formation in dioleoylphosphatidylcholine, lower and broaden the bilayer-to-HII phase transition in dielaidoylphosphatidylethanolamine and form lamellar structures upon codispersion with palmitoyllysophosphatidylcholine. Differential scanning calorimetry measurements indicated that, again like gramicidin, in phosphatidylethanolamine the energy content of the gel-to-liquid-crystalline phase transition is not affected by incorporation of the analogues, whereas in phosphatidylcholine a reduction of the transition enthalpy is found. These observations were explained in terms of a similar tendency to self-associate for gramicidin and its charged analogues. The results are discussed in the light of the various factors which have been suggested to be of importance for the modulation of lipid structure by gramicidin.     

Localization of lysine residues in the binding domain of the K99 fibrillar subunit of enterotoxigenic Escherichia coli

Modification of lysine residues with 4-chloro-3,5-dinitrobenzoate results in the loss of the binding capacity of K99 fibrillae to horse erythrocytes (Jacobs, A.A.C., van Mechelen, J.R. and de Graaf, F.K. (1985) Biochim. Biophys. Acta 832, 148-155). In the present study we used dinitrobenzoate as a spectral probe to map the modified residues. After the incorporation of 0.7 mol CDNB per mol subunit, 90% of the binding activity disappeared and the lysine residues at positions 87, 132 and 133 incorporated 20%, 27.5% and 52.2% of the totally incorporated label, respectively. In the presence of the glycolipid receptor, Lys-132 and Lys-133 were partially protected against modification, while Lys-87 was not protected. The results suggest that Lys-132 and Lys-133 are part of the receptor-binding domain of the K99 fibrillar subunit and that the positive charges on these residues are important for the interaction of the fibrillae with the negatively charged sialic acid residue of the glycolipid receptor. A striking homology was found between a six-amino-acid residue segment of K99, containing Lys-132 and Lys-133, and segments of three other sialic-acid-specific lectins; cholera toxin B subunit, heat-labile toxin B subunit of Escherichia coli and CFA1 fimbrial subunit, suggesting that these segments might also be part of the receptor-binding domain in these three proteins.     

Effect of membrane cholesterol on dimyristoylphosphatidylcholine-induced vesiculation of human red blood cells

During incubation of intact human erythrocytes with sonicated dimyristoylphosphatidylcholine (DMPC) vesicles, the cells change their discoid morphology to form echinocytes and finally give rise to the release of membrane vesicles. In this process, the red cell membrane accumulates DMPC and loses up to 15% of its cholesterol. On the other hand, replacement of 25% of the endogenous phosphatidylcholine species by DMPC without affecting the cholesterol level of the erythrocytes can be achieved by incubation with DMPC/cholesterol (1:1, mol/mol) sonicated vesicles in the presence of the phosphatidylcholine-specific phospholipid-transfer protein from bovine liver. This replacement also gives rise to an echinocytic cell morphology, but no membrane vesiculation can be observed. However, the vesiculation process can as yet be initiated upon a subsequent decrease of the cholesterol level, by incubation of those modified cells in the presence of sonicated vesicles of pure egg phosphatidylcholine. Incubation of native erythrocytes with pure egg phosphatidylcholine vesicles, on the other hand, results in cholesterol depletion, but does neither induce the formation of echinocytes nor the release of membrane vesicles. Cellular ATP levels are not affected during these incubations. From these results, it can be concluded that a decrease in cholesterol content of the erythrocyte membrane is essential for the DMPC-induced vesiculation of those cells.