Allelic variants of Ly-5 in inbred and natural populations of mice

Allelic variants of Ly-5 in inbred commensal and other natural populations of mice were analyzed by patterns of restriction fragment length polymorphisms (RFLP) and Southern hybridization using an Ly-5 cDNA probe and by cell-surface staining with a panel of antibodies directed against polymorphic and nonpolymorphic Ly-5 determinants. New Ly-5 alleles were defined by RFLPs generated by both Eco RI and Bam HI restriction enzyme digests. The Mus musculus subspecies and other species within the genus Mus showed a strong correlation between allelic variants defined by restriction enzymes and serologic specificities. The data also suggest the conservation of the Ly-5 gene throughout the genus Mus.     

Demonstration of up-regulated IL 2 receptor expression on an in vitro cloned BCL1 subline

We have established BCL1 CL-3 cells capable of responding to B15-TRF and interleukin 2 (IL 2). This clone has both high affinity and low affinity receptors for IL 2 (IL 2R), but IL 2 by itself did not stimulate either proliferation or immunoglobulin (Ig) secretion. B15-TRF, which possesses both growth and differentiation activity, causes an increase in size of CL-3 cells and renders CL-3 cells responsive to IL 2, including an increased expression of IL 2R (eight-fold to 10-fold) and the differentiation of CL-3 cells into Ig secretion (60 to 80% of cultured cells). CL-3 cells pretreated with B15-TRF for 12 hr become competent to respond to IL 2 by up-regulation of IL 2R within 12 hr. In contrast CL-3 cells pretreated with IL 2 for 12 hr required 24 hr B15-TRF stimulation to result in IL 2R up-regulation. Thus the ordered action of B15-TRF and IL 2 is the most effective operational pathway for the up-regulation of IL 2R. This IL 2-mediated IL 2R up-regulation and induction of Ig synthesis depends upon the concentration of IL 2 in the culture. Both responses seem to be caused by IL 2 molecules bound to high affinity IL 2R. However, the possibility of involvement of low affinity IL 2R can not be vigorously excluded. In fact the level of IL 2 required for a response is far higher than that needed for activated T cell proliferation. This cloned BCL1 subline promises to be a useful tool for studying the regulation and mechanisms of B cell responses.     

Evolution of the 28S ribosomal RNA gene in anurans: regions of variability and their phylogenetic implications

Fifteen restriction sites were mapped to the 28S ribosomal RNA gene of individuals representing 54 species of frogs, two species of salamanders, a caecilian, and a lungfish. Eight of these sites were present in all species examined, and two were found in all but one species. Alignment of these conserved restriction sites revealed, among anuran 28S rRNA genes, five regions of major length variation that correspond to four of 12 previously identified divergent domains of this gene. One of the divergent domains (DD8) consists of two regions of length variation separated by a short segment that is conserved at least throughout tetrapods. Most of the insertions, deletions, and restriction-site variations identified in the 28S gene will require sequence-level analysis for a detailed reconstruction of their history. However, an insertion in DD9 that is coextensive with frogs in the suborder Neobatrachia, a BstEII site that is limited to representatives of two leptodactylid subfamilies, and a deletion in DD10 that is found only in three ranoid genera are probably synapomorphies.      

Pectate distribution and esterification in Dubautia leaves and soybean nodules,studied with a fluorescent hybridization probe

Carbohydrate-hybridization probes (Vreeland and Laetsch, 1989, Planta (177, 423–434) were used to localize the homogalacturonan (pectate) component of pectins in the cell walls of leaves and soybean root nodules. Leaves of two species of the dicotyledon Dubautia were compared; these species contain much pectin but differ in their tissue water relations with respect to their cell-wall properties. Maturation of the primary cell walls in nodules was studied in the Bradyrhizobium japonicum-Glycine max symbiosis. Probe labelling was based on the divalent-cation-mediated association between pectate in tissue sections and fluorescein-conjugated pectate fragments. Pectate was also labelled by mixed-dimer formation with fluorescent polyguluronate derived from alginate. The specificity of the probe for unesterified polygalacturonate was indicated by increased cell-wall labelling after chemical or enzymatic deesterification of tissue sections, in contrast to elimination of labelling by chemical esterification. Postfixation of tissue sections improved retention of soluble pectate. Pectate differences were found in the leaves among cell types, in degree of esterification, and between plant species. The cell walls of soybean nodules were strongly labelled by the pectate probe in nodules one week and three weeks after infection. Pectate was more highly esterified in the central infected zone than in the surrouding cortex. Within the infected zone, walls of uninfected cells and infected cells were similarly labelled by the pectate probe. The results indicate that the pectate molecular probe provides detailed information on pectate distribution at the cellular level for investigations of cell-wall structure, development and physiology.Abbreviations EDTA ethylenedinitrilotetraacetic acid (ethylenediaminetetraacetic acid) - NMR nuclear magnetic resonance spectroscopy - TTB 1,3,5-triazido-2,4,6-trinitrobenene     

Field evaluation of starter N and delayed inoculation ofLespedeza cuneata grown in minesoil

A field study was conducted on freshly reclaimed surface-mined area to determine response of sericea lespedeza (Lespedeza cuneata [Dumont] G. Don.) to delayed rhizobial inoculation. Soybeans (Glycine max L.) were used as a control legume. Plots were inoculated with spray applications of rhizobial suspensions at seeding, cotyledon stage or second trifoliate leaf stage, or not inoculated. Starter N at 0, 10 or 20 kg ha^?1 was applied preplant in a factorial arrangement with inoculation timings.G. max. was grown for 92 days andL. cuneata for 121 days. Starter N increased plant growth and total shoot N in both species. However, % shoot N was found to increase only inL. cuneata. Delaying inoculation had no significant effect upon total shoot N or % shoot N accumulation inL. cuneata. Inoculation ofG. max at planting produced greater plant growth and N accumulation than delayed inoculation treatments. Application of inoculum as a surface spray appeared to be an effective method for delayed inoculation as evidenced by nodule formation. Lack of increased plant growth, regardless of time of inoculation, suggests that delayed inoculation does not improve establishment and growth ofL. cuneata in minesoil.     

Characterization of cross-bridge elasticity and kinetics of cross-bridge cycling during force development in single smooth muscle cells

Force development in smooth muscle, as in skeletal muscle, is believed to reflect recruitment of force-generating myosin cross-bridges. However, little is known about the events underlying cross-bridge recruitment as the muscle cell approaches peak isometric force and then enters a period of tension maintenance. In the present studies on single smooth muscle cells isolated from the toad (Bufo marinus) stomach muscularis, active muscle stiffness, calculated from the force response to small sinusoidal length changes (0.5% cell length, 250 Hz), was utilized to estimate the relative number of attached cross-bridges. By comparing stiffness during initial force development to stiffness during force redevelopment immediately after a quick release imposed at peak force, we propose that the instantaneous active stiffness of the cell reflects both a linearly elastic cross-bridge element having 1.5 times the compliance of the cross-bridge in frog skeletal muscle and a series elastic component having an exponential length-force relationship. At the onset of force development, the ratio of stiffness to force was 2.5 times greater than at peak isometric force. These data suggest that, upon activation, cross-bridges attach in at least two states (i.e., low-force-producing and high-force-producing) and redistribute to a steady state distribution at peak isometric force. The possibility that the cross-bridge cycling rate was modulated with time was also investigated by analyzing the time course of tension recovery to small, rapid step length changes (0.5% cell length in 2.5 ms) imposed during initial force development, at peak force, and after 15 s of tension maintenance. The rate of tension recovery slowed continuously throughout force development following activation and slowed further as force was maintained. Our results suggest that the kinetics of force production in smooth muscle may involve a redistribution of cross-bridge populations between two attached states and that the average cycling rate of these cross-bridges becomes slower with time during contraction.     

Sequences encoding the protein and RNA components of ribonuclease P from Streptomyces bikiniensis var. zorbonensis.

The genes encoding the RNA (rnpB) and protein (rnpA) subunits of ribonuclease P (RNase P) of Streptomyces bikiniensis var. zorbonensis have been cloned by complementing the temperature-sensitive growth phenotype of Escherichia coli strains that carry mutations in these genes. The rnpB sequence of S. bikiniensis includes new covariations that lead to refinement of the previous secondary structure models for RNase P RNAs. The deduced amino acid sequence of S. bikiniensis RNase P is conserved with that of other known RNase P proteins only to a limited extent. Immediately upstream from rnpA is an open reading frame that codes for the highly conserved ribosomal protein, L34. This same gene arrangement occurs in all bacteria studied to date.     

Effects of non-MHC loci on resistance to retrovirus-induced immunodeficiency in mice

Mice of certain strains are highly sensitive to development of a severe immunodeficiency disease following inoculation as adults with LP-BM5 murine leukemia viruses (MuLV) whereas others are extremely resistant. These strain-dependent differences in response to infection have been shown to be genetically determined with resistance to disease being, in general, associated with homozygosity for Fv-1 ⁿand H-2 haplotypes a and d and sensitivity with homozygosity for Fv-1 ^band other H-2 haplotypes including b, s, and q. The Fv-1 ^b, H-2 ^rstrain RIIIS/J (RIIIS) was found to be highly resistant to disease even though B10.RIII(71NS)/J (B10.RIII), also H-2 ^r, was very sensitive, thus excluding a role for H-2 in the resistance of RIIIS. The characteristics of RIIIS resistance were evaluated in studies of infected (B10.RIII×RIIIS) F₁, F₂ and reciprocal backcross mice. Resistance to disease was shown to be semidominant and determined by more than one gene, although a preponderant influence of a single gene was suggested. Studies of segregating populations showed that resistance was not associated with or linked to polymorphisms of the V _\complex or genes in proximity to the Emv-2 locus on chromosome 8. However, there was almost complete concordance between absence of disease in infected mice and inhibition of ecotropic virus spread. These results demonstrate that genes other than Fv-1 or H-2 can profoundly influence the development of retrovirus-induced immunodeficiency and replication of ecotropic viruses.Abbreviations MuLV murine leukemia virus - MCF mink cell focus-inducing MuLV - B6 C57BL/6 - BM5d the defective virus in LP-BM5 MuLV - MAIDS murine acquired immunodeficiency syndrome - RIIIS RIIIS/J - B10.RIII B10.RIII (71NS)/J - MLR mixed lymphocyte reaction - FACS fluorescence activated cell sorter     

Purification and characterization of sulfatases from Haliotis rufescens: evidence for changes in synthesis and heterogeneity during development

Summary The digestive glands of many marine molluscs are rich sources of arylsufatase enzymes which may function in the catabolism of sulfated polysaccharides in the diets of herbivorous species. Arylsulfatases, partially purified from the hepatopancreas of the red abalone, Haliotis rufescens, were investigated with respect to heterogeneity, catalytic requirements, and timing of induction during development. Four hepatopancreatic enzymes were purified from adult animals using a combination of hydrophobic interaction and anion-exchange chromatography. Zymograms of the four partially-purified enzymes produced by electrophoresis under nondenaturing conditions revealed a fifth, relatively more basic isozyme. All four partially-purified enzymes appear to be monomeric, with molecular weights of approximately 43 000 Da each, as measured by gel filtration. The affinities for p-nitrocatechol sulfate, pH optima, and strengths of inhibition by anions displayed by these enzymes are similar to the values reported for other molluscan arylsulfatases. Three of the four enzymes have K _m values between 0.8 and 2.0 mM for p-nitrocatechol sulfate; the remaining enzyme (A₂) has a K _m of 6.7 mM. All four enzymes have pH and temperature optima of 5.5 and 45°C, respectively. Three of the four enzymes have-t_1/2(50°C) values of 3.5 min; the enzyme A₄ has a t_1/2 has a t_1/2(50°C) of 8.5 min. A monoclonal antibody directed against form A_1b does not cross react with any of the other hepatopancreatic arylsulfatases when assayed by Western blot, confirming the structural heterogeneity of the adult enzymes.Total arylsulfatase activity increases in a biphasic manner during early abalone development, with the first increase occurring early in larval maturation. The secoad phase of enzyme expression is dependent upon the induction of settlement and metamorphosis of the competent veliger larvae, strongly suggesting that the expression of arylsulfatase synthesis (and the maturation of the digestive gland, the hepatopancreas) is controlled by genetic events which occur as a result of metamorphosis. Competent veliger larvae express only two arylsulfatase forms, which share many physicochemical and kinetic characteristics with the adult hepatopancreatic enzymes. However, neither of the larval arylsulfatases is recognized by the monoclonal antibody to form A_1b from adult hepatopancreas. Endogenous enzyme inhibitor levels in larvae remain constant throughout the period of arylsulfatase induction, and therefore do not contribute to the control of arylsulfatase activity levels during development.These results are the first documentation of the developmental induction of a specific protein(s) in abalone as a result of metamorphosis. The significance of the timing of arylsulfatase expression is discussed in relation to potential physiological substrates and the dietary switching which occurs at metamorphosis. Possible genetic events which are consistent with the observed patterns of expression of these enzymes also are considered.Abbreviations BSA bovine serum albumin - C centigrade - Da daltons - DEAE diethylaminoethyl - ELISA enzyme-linked immunosorbent assay - FPLC fast protein and polynucleotide liquid chromatography - GABA -aminobutyric acid - HAT hypoxanthine, aminopterin, thymidine - Hepes N-(2-Hydroxythyl)piperazine-N'-(2-ethanesulfonic acid) - HPLC high pressure liquid chromatography - KBS Kantor's balanced salt solution - K _m Michaelis constant - PBS phosphate buffered saline - R _m relative mobility - RPMI Roswell Park Memorial Institute - SDS sodium dodecyl sulfate - T time - TBS TRIS buffered saline - V _max maximal velocity