Genetic transformation of Streptococcus thermophilus by electroporation

A rapid and convenient electroporation procedure was developed for the genetic transformation of intact cells of Streptococcus thermophilus with various species of plasmid DNA. Transformation frequency was influenced by the capacitance and voltage selected for electric pulsing, the pH and composition of the electroporation medium and the molecular mass of the transforming DNA. Electroporation is a simple and effective technique to introduce plasmid DNA into S. thermophilus and useful in the development of recombinant DNA technology for this important industrial microorganism.     

Evidence that Lyb-5 is a differentiation antigen in normal and xid mice

These studies addressed the question of whether the Lyb-5 determinant on mouse B cells might represent a marker for a unique lineage of B cells as has been thought, or rather, a differentiation antigen. Previous studies have addressed this question by treating splenic B cells with anti-Lyb-5 + complement and then culturing them with various antigens. In this study, we first immunized in vivo or in vitro and then determined susceptibility to anti-Lyb-5. These studies demonstrate that a substantial proportion of antibody-forming cells produced in response to immunization with sheep red blood cells, trinitrophenyl (TNP)-keyhole limpet hemocyanin, TNP-Brucella abortus, TNP-lipopolysaccharide, as well as TNP-Ficoll, are eliminated by anti-Lyb-5 + complement treatment at the end of culture. Some generation of Lyb-5+ antibody-forming cells occurred in the last 24 hr of culture. These studies suggest that the Lyb-5 marker represents a differentiation antigen on relatively mature B cells.     

Synchronized synthesis and intracellular transport of serum albumin and apolipoprotein B in cultured rat hepatocytes as studied by double immunofluorescence

Synthesis and intracellular transport of two secretory proteins, serum albumin (SA) and apolipoprotein B (apo B) have been synchronized in primary cultures of normal rat hepatocytes to make possible immunocytochemical study of the transport pathway. Under appropriate conditions of cycloheximide treatment, synthesis of new protein was inhibited and, by double immunofluorescent labeling, the cells were found to be largely depleted of the SA and apo B previously synthesized. Re-initiation of protein synthesis led to sequential appearance of SA and apo B, first in the endoplasmic reticulum, then in the Golgi complex, and finally at the cell surface. These results indicate that it should be feasible to use this cell system for high-resolution investigation of the sequence of structures involved in intracellular transport of SA and apo B by corresponding immunolabeling experiments as observed by electron microscopy.     

An autoradiographic study of the uptake of tritiated proline by osteoblasts during hibernation

Twenty-four LSH and LVG strain golden hamsters, Mesocricetus auratus, were used. Experimental animals were maintained at 5 C and allowed to hibernate. Control animals were kept at 27 C. Six animals (3 experimental, 3 control) were injected subcutaneously with 1 microCi of 3H-proline/gm body wt. (Spec. act. 3 Ci/mM) after hibernation lasting 12 hours, 1 day, 3 days, or 7 days. Animals were killed 1 hour after injection and autoradiographs were prepared from 5 microns thick decalcified sections of femurs. A greater number of endosteal cells were labeled than periosteal cells and also exhibited a greater magnitude of labeling throughout the study. Differences between endosteal and periosteal cells both in percentage of cells labeled and magnitude of labeling were maximum in control animals and progressively decreased with increasing periods of hibernation. A reduction in synthesis of matrix proteins during the early period of hibernation was seen and was attributed to a significant reduction both in average cell activity and in the number of active cells during hibernation. The latter phenomenon apparently made a large contribution to the reduced matrical synthesis. 3H-proline uptake by osteoblasts probably reflects the reduced requirements of matrical synthesis during hibernation.     

Abnormal binding of soluble IgG immune complexes to hepatic nonparenchymal cells of autoimmune mice

Because the liver is the major organ responsible for removal of soluble immune complexes (IC), the surface binding characteristics of preformed model IC to unstimulated mouse liver nonparenchymal cells (NPC) in suspension were studied. NPC of non-autoimmune C3H/FeJ, C3H/HeJ, A/J, DBA/2 and the autoimmune NZB/W F1 and MRL/lpr female mice of various ages were isolated by perfusion of the portal vein with collagenase followed by separation of NPC from hepatocytes with a metrizamide gradient. Thirty-five percent of NPC of all mouse strains were nonspecific esterase-positive and phagocytosed latex beads. Radiolabeled mouse IgG anti-DNP covalently cross-linked stable IC were separated by gel filtration and bound to NPC under various conditions. Marked differences were noted in maximal number of IC bound per cell between the autoimmune and non-autoimmune mouse strains: 3.3 to 4.0 X 10(5) in the non-autoimmune strains vs 0.3 to 1.4 X 10(5) molecules of IC bound per cell in the autoimmune strains at 1 to 6 mo. Insignificant differences were noted in Ka by Scatchard plot analysis (3.5 to 5.0 X 10(8) M-1) and rate of reversibility of binding as determined by dissociation of surface-bound IC with an excess of heat-aggregated gamma-globulin (T 1/2:1.5 to 2 min). These data demonstrate a decreased number of available binding sites for IC in unstimulated NPC from NZB/W F1 and MRL/lpr female mice throughout their life spans. Although the findings are consistent with saturation of binding sites of the NPC with native IC, the abnormality found in the 1-mo-old autoimmune mice (who do not have detectable autoantibodies) suggests a primary defect in FC receptor expression or an altered state of activation of NPC that may contribute to the disease process.     

COMPARISON OF DAYLENGTH AND TEMPERATURE RESPONSES IN NICOTIANA AND ITS TAXONOMIC SECTIONS

Steinberg , Robert A. (U.S.D.A., Beltsville, Md.) Comparison of daylength and temperature responses in Nicotiana and its taxonomic sections. Amer. Jour. Bot. 46(4): 261–268. Illus. 1959.—Fifty-seven of the sixty species of Nicotiana were grown in the greenhouse under long- and short-day regimes. Supplemental tungsten light of about 30 ft.-c. (bench) was used to extend natural illumination to 16 hr. daily. Short-day controls received natural illumination for 9.5–12 hr. daily from about September to March. Two temperature levels were also employed—one with temperature held uniformly at about 25°C. and the other with a day temperature of about 20°C. and a night temperature of about 10°C. Daylength behavior of the species ranged from long-day to day-neutral to short-day. All species were brought into flower and all, except N. acaulis and N. ameghinoi, formed viable seed in at least 1 of the 4 environments. A modified classification of photoperiodic flowering responses based on rapidity and not ability to flower was adopted to permit quantitative comparison of species responses to both daylength and temperature. Very few species flowered equally rapidly (day-neutral) in both the 10- and 16-hr. day-lengths. Temperature level caused modifications in response from long-day to day-neutral and vice versa, and from short-day to day-neutral and vice versa. Data for N. glauca and some other species would indicate that a greater spread between temperature levels could possibly lead to opposite classifications at upper and lower temperatures. Excellent agreement was found between daylength responses of the species and the 14 taxonomic sections of Goodspeed for the genus Nicotiana. Only 2 of the sections (Paniculatae and Undulatae) were heterogeneous in that both included short- and long-day species in the same section. The native habitat of all short-day species was South America. Certain of the species gave a compensatory response to variations in light duration and low temperature similar to that given by sugarbeets and other biennials. This phenomenon may therefore be of general occurrence. Use of a quantitative expression for photoperiodic flowering responses is proposed to avoid ambiguity. It is the quotient of days from sowing to first blossom on short-days divided by that on long-days. The value 0.6₂₀°C. (9–12) would read short-day at 20°C. with 9–12 hr. daylengths. Close agreement was found in daylength flowering ratios in successive tests in the greenhouse. The ratios alter under cold treatment with species susceptible to low-temperature stimulation or inhibition of blossoming.