Phylogenetic distribution in the genus Mus of t-complex-specific DNA and protein markers: inferences on the origin of t-haplotypes

We have examined the phylogenetic distribution of two t-specific markers among representatives of various taxa belonging to the genus Mus. The centromeric TCP-1a marker (a testicular protein variant specific for all t-haplotypes so far studied) has also been apparently detected in several non-t representatives of the Mus IVA, Mus IVB, and probably M. cervicolor species. By contrast, a t-specific restriction- fragment-length polymorphism allele (RFLP) of the telomeric alpha- globin pseudogene DNA marker alpha-psi-4 was found only in animals belonging to the M. musculus-complex species either bearing genuine t- haplotypes or, like the M. m. bactrianus specimen studied here, likely to do so. This t-specific alpha-psi-4 RFLP allele was found to be as divergent from the RFLP alleles of the latter, non-t, taxonomical groups as it is from Mus 4A, Mus 4B, or M. spretus ones. These results suggest the presence of t-haplotypes and of t-specific markers in populations other than those belonging to the M. m. domesticus and M. m. musculus subspecies, implying a possible origin for t-haplotypes prior to the radiation of the most recent offshoot of the Mus genus (i.e., the spretus/domesticus divergence), some 1-3 Myr ago.      

Anisotropy decay associated fluorescence spectra and analysis of rotational heterogeneity. 2. 1,6-Diphenyl-1,3,5-hexatriene in lipid bilayers

The application of a new spectroscopic tool [Knutson, J. R., Davenport, L., & Brand, L. (1986) Biochemistry (preceding paper in this issue)] for studying rotational microheterogeneity of probe location in lipid bilayer systems is described. Anisotropy decay associated spectra are derived from experimentally obtained polarized emission components. "Early" difference spectra (IV - IH) contain contributions from both fast and slow rotors, while "late" difference spectra predominantly reflect the emission from slowly rotating fluorophores. Anisotropy decay associated spectra have been used to resolve the emission spectra of 1,6-diphenyl-1,3,5-hexatriene (DPH) imbedded within a known rotationally heterogeneous mixture of two vesicle types (L-alpha-dimyristoyllecithin and L-alpha-dipalmitoyllecithin). At 29 degrees C, diphenylhexatriene within pure dimyristoyllecithin vesicles rotates rapidly, with a small r infinity, while diphenylhexatriene in dipalmitoyllecithin vesicles exhibits a large r infinity. Spectra for diphenylhexatriene imbedded in the two vesicle types show small but significant spectral differences. A spectrum of a mixture of the two vesicle types with DPH lies between these characteristic component spectra. The spectrum extracted for "immobilized" probes in the mixture correctly overlays the dipalmitoyllecithin spectrum. Further studies have shown that diphenylhexatriene exhibits more than one emission anisotropy decay associated spectrum in vesicles of a single lipid type, when that lipid is near its phase transition temperature. Diphenylhexatriene apparently inhabits more than one rotational environment even in these "homogeneous" vesicle preparations.     

Decreased Fc receptor avidity and degradative function of monocytes from patients with systemic lupus erythematosus

We studied the binding and degradation of stable, soluble heat aggregates of 125I-IgG (A-IgG) by monocytes from 30 patients with systemic lupus erythematosus (SLE) and 30 normals. Relative avidities (KE) for Fc receptor (FcR) binding of A-IgG and maximal binding of A-IgG by monocytes were determined from Scatchard plots of binding data obtained at 4 degrees C. Rates of degradation (Vmax) of A-IgG at 37 degrees C were calculated from Lineweaver-Burke plots of the Michaelis-Menton equation. KE were decreased in SLE monocytes (15.5 X 10(-9) L/M) as compared with normals (20.1 X 10(-9) L/M, p less than 0.005) and Vmax were decreased for SLE (0.89 ng/hr) as compared with normals (1.11 ng/hr, p less than 0.005). The maximal FcR binding by SLE monocytes was not statistically different in SLE patients and normals, but monocytes from SLE patients with active disease showed a lower maximal binding capacity for A-IgG (4.9 ng/10(5) cells) than normals (5.4 ng/10(5) cells, p less than 0.05). KE and Vmax in SLE were also lower for patients with active disease than for normal subjects. KE in patients whose anti-ssDNA binding was greater than 20% were lower than for those with DNA binding of less than 20% (p less than 0.005). These data suggest that patients with active SLE have diminished numbers of available FcR on their circulating monocytes, possibly due to interiorization of FcR during endocytosis of endogenous circulating immune complexes.     

Biology ofPterolonche inspersa (Lep.: Pterolonchidae), a biological control agent forCentaurea diffusa andC. maculosa in the United States

The moth,Pterolonche inspersa (Staudinger) (Lepidoptera: Pterolonchidae), is widely distributed in southern Europe, north Africa, Turkey and the former Soviet Union. It occurs in both thick and scattered stands of knapweeds in disturbed sites, usually on sandy and/or stony soil. Larvae bore in the roots of diffuse and spotted knapweeds (Centaurea diffusa De Lamarck andC. maculosa De Lamarck). There is one generation per year in northern Greece, and larvae feed in the roots for about 11 months during the growing season (August–September, to the following July–August). In the laboratory garden, emergence took place between the second half of July and the end of August, with peak emergence during mid August. In the field, adults were observed from early to late July. Female moths oviposited on rosettes during the first ten days of July and continued through the end of July. Eggs were laid singly or in groups of five or six, firmly attached to the leaves of the host plant. In the laboratory, females mated within 24 hours of emergence and the preoviposition period lasted 2.6±0.8 days. The oviposition period lasted 7.4±2.2 days and the average number of eggs per female was 142.2±59.2. The incubation period was 12±4.7 days; the pupal stage lasted 14.7±2.4 days; and females lived 15.8±2.4 days, while males lived 10.7±1.4 days. First instar larvae failed to survive on economically important Compositae in the generaCynara L.,Helianthus L.,Zinnia L. andCalendula L. (Dunnet al., 1989).     

Preparation of monoclonal antibody-ferritin conjugates of high specific activity

125I-monoclonal IgG anti-gamma chain antibodies were conjugated to ferritin using glutaraldehyde as a bifunctional reagent. The molar ratio of IgG:ferritin:glutaraldehyde resulting in the highest yield was determined. Free IgG was separated from IgG bound to ferritin by sucrose density gradient ultracentrifugation; free ferritin was separated from antibody-ferritin conjugates by differential salt precipitation. The IgF:ferritin molar ratio of the resulting product was 1:1.4, containing over 90% ferritin-IgG "monomers"; 70-90% of the 125I activity bound immunospecifically to sepharose-IgG or aggregated human globulin (AHG). The product was used as an immunologic EM marker for AHG. Monoclonal antibody-ferritin conjugates prepared by this method should prove useful for quantitative ultrastructural analysis of surface antigens.     

The contractile basis of amoeboid movement: V. The control of gelation, solation, and contraction in extracts from dictyostelium discoideum

Motile extracts have been prepared from Dictyostelium discoideum by homogenization and differential centrifugation at 4 degrees C in a stabilization solution (60). These extracts gelled on warming to 25 degrees Celsius and contracted in response to micromolar Ca++ or a pH in excess of 7.0. Optimal gelation occurred in a solution containing 2.5 mM ethylene glycol-bis (β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA), 2.5 mM piperazine-N-N'-bis [2-ethane sulfonic acid] (PIPES), 1 mM MgC1(2), 1 mM ATP, and 20 mM KCI at ph 7.0 (relaxation solution), while micromolar levels of Ca++ inhibited gelation. Conditions that solated the gel elicited contraction of extracts containing myosin. This was true regardless of whether chemical (micromolar Ca++, pH >7.0, cytochalasin B, elevated concentrations of KCI, MgC1(2), and sucrose) or physical (pressure, mechanical stress, and cold) means were used to induce solation. Myosin was definitely required for contraction. During Ca++-or pH-elicited contraction: (a) actin, myosin, and a 95,000-dalton polypeptide were concentrated in the contracted extract; (b) the gelation activity was recovered in the material sqeezed out the contracting extract;(c) electron microscopy demonstrated that the number of free, recognizable F-actin filaments increased; (d) the actomyosin MgATPase activity was stimulated by 4- to 10-fold. In the absense of myosin the Dictyostelium extract did not contract, while gelation proceeded normally. During solation of the gel in the absense of myosin: (a) electron microscopy demonstrated that the number of free, recognizable F- actin filaments increased; (b) solation-dependent contraction of the extract and the Ca++-stimulated MgATPase activity were reconstituted by adding puried Dictyostelium myosin. Actin purified from the Dictyostelium extract did not gel (at 2 mg/ml), while low concentrations of actin (0.7-2 mg/ml) that contained several contaminating components underwent rapid Ca++ regulated gelation. These results indicated : (a) gelation in Dictyostelium extracts involves a specific Ca++-sensitive interaction between actin and several other components; (b) myosin is an absolute requirement for contraction of the extract; (c) actin-myosin interactions capable of producing force for movement are prevented in the gel, while solation of the gel by either physical or chemical means results in the release of F-actin capable of interaction with myosin and subsequent contraction. The effectiveness of physical agents in producting contraction suggests that the regulation of contraction by the gel is structural in nature.     

Calcium transients during Fc receptor-mediated and nonspecific phagocytosis by murine peritoneal macrophages

Studies with populations of macrophages have produced conflicting results concerning the possibility that the concentration of intracellular ionized calcium [( Ca2+]i) may act as an important mediator for phagocytosis. Since asynchronous changes in [Ca2+]i in individual cells undergoing phagocytosis may be averaged to undetectability in population studies, we studied single adhering murine macrophages using fura-2 and our previously described digital imaging system. The proportion of macrophages phagocytosing IgG-coated latex beads was greater than for uncoated beads (percent phagocytosing cells: 71 +/- 7 vs. 27 +/- 7, P less than 0.01). Phagocytosis of IgG-coated and uncoated beads was always associated with a calcium transient that preceded the initiation of phagocytosis. No calcium transients were detected in cells that bound but did not phagocytose beads. Four major differences between Fc receptor-mediated and nonspecific phagocytosis were detected: (a) the duration of calcium transients was longer for nonspecific phagocytosis compared with Fc receptor-mediated phagocytosis (69.9 +/- 10.2 vs. 48.7 +/- 4.7 s, P less than 0.05) and the magnitude of calcium transients was less for nonspecific phagocytosis (178 +/- 43 vs. 349 +/- 53 nM, P less than 0.05); (b) removal of extracellular calcium abolished the calcium transients associated with nonspecific phagocytosis but had no effect on those associated with receptor-mediated phagocytosis; (c) in the absence of extracellular calcium, buffering intracellular calcium with a chelator reduced Fc receptor-mediated phagocytosis but had no additive inhibitory effect on nonspecific phagocytosis; and (d) inhibition of protein kinase C (PKC) with staurosporine inhibited nonspecific phagocytosis but had no effect on receptor-mediated phagocytosis. Our observations suggest that despite both types of phagocytosis being associated with intracellular calcium transients, the role played by intracellular calcium in the signaling pathways may differ for Fc receptor-mediated and nonspecific phagocytosis by elicited murine macrophages.     

Ligand-independent internalization and recycling of the insulin receptor. Effects of chronic treatment of 3T3-C2 fibroblasts with insulin and dexamethasone.

Upon binding insulin at the plasma membrane, the insulin receptor internalizes into the endosomal compartment of the cell with a half-time of approximately 10 min. Our earlier work demonstrated that receptor inactivation (loss of insulin binding capacity) is a regulated process. Long term treatment of cultured cells with insulin or the glucocorticoid dexamethasone increases or decreases, respectively, the rate constant for insulin receptor inactivation (Knutson, V. P., Ronnett, G. V., and Lane, M. D. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2822-2826). In these studies, monolayer cultures of 3T3-C2 fibroblasts were chronically treated with insulin or dexamethasone. Subsequently, the surface receptors were labeled with the photoactivatable cross-linking agent 125I-labeled 2-(p-azidosalicylamido)ethyl-1,3'- dithiopropionate -insulin. Following equilibration of the radiolabeled receptor between the plasma membrane and internal pools, the steady-state rate constant for receptor recycling was determined by quantitating the rate at which internal radiolabeled receptor was inserted into the plasma membrane. The steady-state rate constant for this recycling process was the same in control, insulin-treated, or steroid-treated cells (t1/2 = 2h). In contrast, the rate constant for receptor internalization was regulated; the half-times were 10 h for control cells, 5 h for insulin-treated cells, and 19 h for dexamethasone-treated cells. These changes in rate constants for internalization and inactivation lead to changes in the relative numbers of receptor molecules undergoing recycling versus inactivation. Therefore, whereas the recycling of the insulin receptor is not a regulated process, the internalization of surface receptor in the absence of bound ligand is a metabolically controlled step in receptor processing.     

Sugar transport by the bacterial phosphotransferase system. Fluorescence studies of subunit interactions of enzyme I

Enzyme I of the bacterial phosphoenolpyruvate:glycose phosphotransferase system (PTS) exhibits a temperature-dependent monomer/dimer equilibrium. The accompanying paper (Han, M. K., Roseman, S., and Brand, L. (1990) J. Biol. Chem. 265, 1985-1995) shows that the C-terminal -SH residue (Cys-575) can be modified specifically with fluorescent probes such as pyrene maleimide. The derivative retains full enzyme activity, and is capable of forming dimers at room temperature. In the present studies, Enzyme I labeled in this way is found to exhibit a temperature-, concentration-, and pH-dependent monomer/dimer association. The kinetics of dimer formation of Enzyme I is measured in the following way. A derivatized Enzyme I sample is prepared with a pyrene moiety irreversibly attached to the C-terminal -SH residue and 5,5'-dithiobis-2-nitrobenzoic acid reversibly attached to the other 3 -SH residues. This modified enzyme does not form dimers at room temperature. Addition of dithiothreitol results in total release of the thionitrobenzoate anion within 2 min. After the three -SH groups are unblocked, steady-state and nanosecond time-resolved emission anisotropy measurements indicate the dimer is formed over a period of 30 min. In a similar experiment, little dimer formation is observed at 3 degrees C, at temperature at which the native enzyme also does not form dimers. Tryptophan fluorescence is also examined during the release of the thionitrobenzoate. After the completion of thionitrobenzoate release, additional slow steady-state tryptophan fluorescence changes are observed. These results suggest that dimer formation may be preceded by a conformational change following thionitrobenzoate release.