Superior resolution of gamma-crystallins from microdissected eye lens by cation-exchange high-performance liquid chromatography

A novel procedure is presented for the rapid quantitative analysis of eye lens gamma-crystallins and beta s-crystallin by ion-exchange high-performance liquid chromatography on Synchropak CM300. At least six different gamma-crystallin gene products can be resolved from the soluble fraction of calf lens extract. This method is applicable to the analysis of microsections from individual lenses, and can be used to rapidly characterize spatial variations in gamma-crystallin composition which occur with aging and cataractogenesis.     

Tensile strength of bovine trabecular bone

Data on the tensile and compressive properties of trabecular bone are needed to define input parameters and failure criteria for modeling total joint replacements. To help resolve differences in reports comparing tensile and compressive properties of trabecular bone, we have developed new methods, based on porous foam technology, for tensile testing of fresh/frozen trabecular bone specimens. Using bovine trabecular bone from an isotropic region from the proximal humerus as a model material, we measured ultimate strengths in tension and compression for two groups of 24 specimens each. The average ultimate strength in tension was 7.6 +/- 2.2 (95% C.I.) MPa and in compression was 12.4 +/- 3.2 MPa. This difference was statistically significant (p = 0.013) and was not related to density differences between the test groups (p = 0.28). Strength was related by a power-law function of the local apparent density, but, even accounting for density influences, isotropic bovine trabecular bone exhibits significantly lower strengths in tension than in compression.     

A histamine-releasing factor from activated human mononuclear cells

Human mononuclear cells activated by streptokinase-streptodornase have been shown to elaborate a factor capable of releasing histamine from human basophils. We have developed reproducible methods for its production in large quantities by using cells obtained from leukapheresis packs, by detection utilizing donor basophils known to release well with anti IgE, and by quantitation of histamine by the radioenzyme method. Human histamine-releasing factor (HRF) gave a single peak upon gel filtration with an estimated m.w. of 32,000; SDS gel electrophoresis revealed a single major band as seen at m.w. 30,000. HRF can be resolved into at least two forms separable by ion-exchange chromatography on QAE Sephadex, and two peaks of activity were obtained by chromatofocusing or isoelectric focusing in gels at pH 6.9 and between 7.4 and 8.3. This factor represents an important potential link between cellular immunity and immediate hypersensitivity.     

Antigen presentation in the rat. II. An Ia+ radiosensitive T cell can present antigen to primed Ia- T cells

We demonstrated previously the presence of an Ia+ (OX-6+) antigen-presenting cell within the rat T cell fraction that is capable of presenting antigen to antigen-primed OX-6-T cells. This antigen-presenting cell (T-APC) reacted with the monoclonal antibodies W3/25 and W3/13, which is known to react mainly with rat T cells. Further characterization of the T-APC indicated that the cell also reacted with the monoclonal antibody OX-19, which is highly specific for rat T cells. Moreover, the antigen-presenting function of the T-APC was sensitive to treatment with mitomycin C or gamma-irradiation (2000 rad). Under similar conditions, antigen presentation by partially purified dendritic cells or macrophages was totally resistant to these treatments. The antigen-presenting activity of gamma-irradiated T-APC was not reconstituted by the addition of the lymphokines IL 1, IL 2, or Con A supernatants. Although unirradiated T-APC were able to stimulate an MLR response, this function was also sensitive to gamma-irradiation, whereas the MLR-stimulating ability of macrophages and dendritic cells was resistant to gamma-irradiation. These data indicate that Ia+ T cells from the rat are capable of presenting antigen to antigen-primed T lymphocytes and that, in contrast to antigen presentation by macrophages and dendritic cells, the function of T-APC is gamma-radiation sensitive.     

Induction of functional Fc receptors in P388 leukemia cells : Requirement for multiple differentiation signals

The development of functional Fc receptors (FcR) during induced differentiation with the tumor promoter, phorbol myristate acetate (PMA), was studied in the murine tumor cell line, P388. PMA induced the appearance of FcR on the membranes of P388 cells as indicated by the binding of IgG-coated sheep red blood cells (IgG-SRBC). Concentrations of PMA as low as 1 ng/ml were sufficient to induce the expression of FcR as well as to inhibit cellular division and to induce adherence in the P388 tumor cell line; however, optimal FcR induction occurred at PMA concentrations of 10-100 ng/ml. Immunofluorescent analysis with heat-aggregated myeloma proteins indicated that PMA induced FcR which were capable of binding IgG2a and IgG2b immunoglobulins, but not IgG1. Adherence to a substratum was determined to be a second required signal for expression of FcR, since PMA induction of P388 tumor cells in teflon dishes failed to fully develop FcR and adherence of P388 cells to poly-L-lysine-coated culture dishes in the absence of PMA was insufficient for FcR expression. FcR which appeared after PMA induction were non-functional in the sense that membrane-bound IgG-SRBC were not ingested to any significant extent by the tumor cells. However, if FcR induction occurred in the presence conA-induced rat spleen cell culture supernatants, phagocytosis of membrane-bound erythrocytes occurred. These findings suggest that for the expression of FcR which are capable of particle internalization, at least three identifiable membrane-transmitted signals are required during differentiation.     

Diffusion of autoinducer is involved in regulation of the Vibrio fischeri luminescence system.

The enzymes for luminescence in Vibrio fischeri are induced by the accumulation of a species-specific metabolite (autoinducer) in the culture medium. Tritium-labeled autoinducer was used to study the mechanism of autoinduction. When 3H-autoinducer was added to suspensions of V. fischeri or Escherichia coli, cellular concentrations equaled external concentrations. For V. fischeri, equilibration of 3H-autoinducer was rapid (within 20 s), and greater than 90% of the cellular tritium remained in unmodified autoinducer. When V. fischeri or E. coli cells containing 3H-autoinducer were transferred to autoinducer-free buffer, 85 to 99.5% of the radiotracer escaped from the cells, depending on the strain. Concentrations of autoinducer as low as 10 nM, which is equivalent to 1 or 2 molecules per cell, were sufficient for induction, and the maximal response to autoinducer occurred at about 200 nM. If external autoinducer concentrations were decreased to below 10 nM after induction had commenced, the induction response did not continue. Based on this study, a model for autoinduction is described wherein autoinducer association with cells is by simple diffusion and binding of autoinducer to its active site is reversible.     

Decreased Incorporation of [3H]Inositol and [3H]Glycerol into Glycerolipids of Sciatic Nerve from the Streptozotocin Diabetic Rat

The incorporation of [3H]myo-inositol into individual phosphoinositides and of [3H]glycerol into glycerolipids was determined in sciatic nerve obtained from normal and streptozotocin diabetic rats and incubated in vitro. The uptake of inositol into lipid was approximately linear with time. More than 80% of the label was present in phosphatidylinositol with the remainder divided about equally between phosphatidylinositol phosphate and phosphatidylinositol-4,5-bisphosphate. Labeling was unchanged 2 weeks after induction of diabetes, but was reduced by 32% after 20 weeks of the disease. Glycerol incorporation occurred primarily into phosphatidylcholine and triacylglycerol and was depressed up to 45% into major phosphoglycerides in nerves from both 2- and 20-week diabetic animals. Triacylglycerol labeling was also substantially decreased, and the reduction was comparable in intact and epineurium free nerve, suggesting that a metabolically active pool of this compound, which is sensitive to hyperglycemia and/or insulin deficiency, is located in or immediately adjacent to the nerve fibers. The considerable decline in incorporation of these lipid precursors in diabetic nerve may be related to impaired inositol transport and to decrease overall energy utilization by the tissue.     

The nitrogen cycle in lodgepole pine forests,southeastern Wyoming

Storage and flux of nitrogen were studied in several contrasting lodgepole pine (Pinus contorta spp.latifolia) forests in southeastern Wyoming. The mineral soil contained most of the N in these ecosystems (range of 315–860 g · m^–2), with aboveground detritus (37.5–48.8g · m^–2) and living biomass (19.5–24.0 g · m^–2) storing much smaller amounts. About 60–70% of the total N in vegetation was aboveground, and N concentrations in plant tissues were unusually low (foliage = 0.7% N), as were N input via wet precipitation (0.25 g · m^–2 · yr^–1), and biological fixation of atmospheric N (<0.03 g · m^–2 · yr^–1, except locally in some stands at low elevations where symbiotic fixation by the leguminous herbLupinus argenteus probably exceeded 0.1 g · m^–2 · yr^–1).Because of low concentrations in litterfall and limited opportunity for leaching, N accumulated in decaying leaves for 6–7 yr following leaf fall. This process represented an annual flux of about 0.5g · m^–2 to the 01 horizon. Only 20% of this flux was provided by throughfall, with the remaining 0.4g · m^–2 · yr^–1 apparently added from layers below. Low mineralization and small amounts of N uptake from the 02 are likely because of minimal rooting in the forest floor (as defined herein) and negligible mineral N (< 0.05 mg · L^–1) in 02 leachate. A critical transport process was solubilization of organic N, mostly fulvic acids. Most of the organic N from the forest floor was retained within the major tree rooting zone (0–40 cm), and mineralization of soil organic N provided NH₄ for tree uptake. Nitrate was at trace levels in soil solutions, and a long lag in nitrification was always observed under disturbed conditions. Total root nitrogen uptake was calculated to be 1.25 gN · m^–2 · yr^–1 with estimated root turnover of 0.37-gN · m^–2 · yr^–1, and the soil horizons appeared to be nearly in balance with respect to N. The high demand for mineralized N and the precipitation of fulvic acid in the mineral soil resulted in minimal deep leaching in most stands (< 0.02 g · m^–2 · yr^–1). These forests provide an extreme example of nitrogen behavior in dry, infertile forests.     

Commercial freezing of bovine embryos in straws

Bovine embryos were frozen commercially in clear double length $\text{1}\text{2}$ cc French straws with the wick and powder plug in the center of the straw. One-half of the double length straw serves as a handle and contains a color coded $\text{1}\text{4}$ cc straw around which an adhesive backed label has been applied. After plunging into liquid nitrogen, straws are transferred into goblets on canes while under liquid nitrogen. The straws are stored in the liquid phase of a nitrogen tank and canes containing straws are not transferred from one container to another unless the goblet containing the straws is full of liquid nitrogen.Embryos held for longer than 4 hours after collection prior to freezing showed a steady decline in pregnancy rate related to the length of time held prior to freezing. The percentage of embryos thawed and then evaluated as being transferrable was related to the quality of the embryos prior to freeze (Grade 1–93.6%, Grade 2–87.0%, Grade 3–63.8%). There was no statistical difference in pregnancy rates obtained from prefreeze Grade 1 embryos when comparing advanced blastocysts (45.2%), blastocysts (38.7%), early blastoclyst (43.1%) and advanced morula (41.6%).