Monoclonal antibody against K562 cell line accelerates killing of the target cells by large granular lymphocytes

A monoclonal antibody (MoAb 11-4) was raised against K562, a human erythroleukemia cell line sensitive to natural killer cell-mediated cytotoxicity (NK-CMC). Immunological analysis revealed MoAb to be IgG_2b. Alone, the MoAb was not cytotoxic for K562 and did not bind to the effector cells, but the addition of this antibody to macrophage-depleted human peripheral blood lymphocytes increased killing of K562 in a 4-hr NK-CMC assay. The maximum increase in NK-CMC was observed when MoAb 11-4 was added to target cells prior to the formation of effector/target cell conjugates. This effect was dose dependent, was specific for K562, and, contrary to conventional antisera, occurred at very low concentrations of MoAb. When MoAb was added either to Percoll-purified large granular lymphocytes (LGL) or to LGL-depleted lymphocytes, only the latter demonstrated a significant increase in the killing of K562 in a 4-hr chromium release assay. Kinetics studies revealed that although the overall LGL-mediated lysis was only slightly increased at 4 hr, the maximum lytic activity was reached within 2 hr. These studies suggest that (1) human LGL and LGL-depleted cell populations bear Fc receptors for mouse IgG_2b and (2) although the cytotoxic activities of both cell populations are increased by treatment with MoAb 11-4, the kinetics of this increase are different.     

Molecular cloning and sequence analysis of cDNA for human transferrin

A cDNA clone for human transferrin was identified from a human liver cDNA library by pre-screening with different ss-cDNA probes against length-fractionated liver mRNAs, positive hybridization-selection and nucleotide sequence analysis. The insert was of 1 kb, encoding human transferrin from aminoacid 403 through the COOH terminus, with a 3' non coding region of 166 nucleotides. This insert hybridized with a single major mRNA species of about 2.4 kb and several genomic DNA restriction fragments. Hybridization of the Southern blots with different parts of the transferrin insert and at different stringences suggest that the various bands observed correspond to splice sites inside one gene rather than to hybridization to several related genes. Finally, a single or a low number of transferrin gene copies seem to exist in the human genome.     

An antibody probe to determine the native species of glycinamide ribonucleotide transformylase in chicken liver

Antibody probes of Western blots [Renart, J., Reiser, J., & Stark, G. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 3116] of chicken liver homogenates under various conditions revealed that glycinamide ribonucleotide transformylase can be rapidly proteolyzed in such homogenates. These findings, along with molecular weight measurements by ultracentrifugation, identify the true form of glycinamide ribonucleotide transformylase as a monomeric protein of 117000 daltons. This protein has been purified 400-fold in 44% yield from chicken liver in one step on an affinity column of 10-formyl-5,8-dideazafolate-Sepharose. Native glycinamide ribonucleotide transformylase retains full activity after proteolytic cleavage to a form (Mr 55000) similar to fragments seen in the Western blot of the homogenates. This phenomenon may be responsible for the previous identification of glycinamide ribonucleotide (GAR) transformylase as a dimer of 55000-dalton subunits. Similar analyses using antibodies to 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase [Mueller, W. T., & Benkovic, S. J. (1981) Biochemistry 20, 337] and trifunctional enzyme [Smith, G. K., Mueller, W. T., Wasserman, G. F., Taylor, W. D., & Benkovic, S. J. (1980) Biochemistry 19, 4313] confirm that these two proteins were isolated in their native forms.     

New affinity adsorbents containing deoxycytidine, deoxyadenosine, or deoxyguanosine and their interactions with deoxynucleoside-metabolizing enzymes

3'-(4-Aminophenyl phosphate) derivatives of deoxycytidine (dCyd), deoxyadenosine (dAdo), and deoxyguanosine ( dGuo ) were synthesized. The inhibitory effects of these compounds on mammalian and bacterial deoxynucleoside kinases and several other deoxynucleoside-metabolizing enzymes were examined. The same derivatives were coupled to carboxyl-terminal Sepharose CL-6B (3-8 mumol of ligand/mL of gel), and each of the resulting affinity adsorbents was tested with various partially purified enzymes. Reasonable correlation between the inhibitory effect of a soluble deoxynucleoside 3'-phosphate diester and affinity of the corresponding Sepharose adsorbent for the enzyme was observed. Among the three dCyd kinases examined, only the bovine mitochondrial enzyme was adsorbed onto the dCyd-Sepharose column and eluted biospecifically by 1 mM dCyd (1400-fold purification). Its Ki toward the dCyd derivative was relatively low (1.1 mM), whereas no measurable inhibition was seen with mammalian cytosol or bacterial enzymes that did not stick to the column. The Ki of the dAdo derivative toward three dAdo kinases was more than 5 mM in each case, and none of these were retained by dAdo-Sepharose. Among the other dAdo-metabolizing enzymes examined, nucleoside phosphotransferase from barley (Ki = 1.2 mM) was adsorbed to dAdo-Sepharose at pH 5.0 and was biospecifically eluted with dAdo or AMP after suppressing ionic binding by adjusting the pH to 6.0 (480-fold purification to homogeneity). Mammalian mitochondrial dGuo kinase (beef liver) showed the lowest Ki (0.16 mM) among the enzymes tested and was biospecifically purified with dGuo -Sepharose (2800-fold purification).(ABSTRACT TRUNCATED AT 250 WORDS)     

Autumn stomatal closure in six conifer species of the Central Rocky Mountains

Summary Environmental and water relations parameters during fall were monitored for six conifer tree species common to the central Rocky Mountains growing naturally at the same location (Pinus contorta, Pinus ponderosa, Pinus flexilus, Pseudotsuga menziesii, Abies lasiocarpa, Picea engelmannii). Subsequent to what appeared to be the beginning of seasonal stomatal closure, leaf conductance to water vapor declined sharply following the onset of freezing air temperatures at night. A coincident rapid decline in morning xylem pressure potentials (_p) also occurred which resulted in values that were considerably below afternoon _p. Continuing decreases in maximum leaf conductance during the day were highly correlated with corresponding decreases in minimum nocturnal air temperatures of the preceding night. By mid-December, morning _p returned to values very near afternoon _p and were only slightly lower than before the onset of subfreezing nights. A preliminary model is proposed which interprets the qualitative interaction between air and soil temperatures, soil and plant water potentials, and leaf conductance during seasonal stomatal closure in fall.     

Extreme temperatures and thermal tolerances for seedlings of desert succulents

Summary Extreme temperatures near the soil surface, which can reach 70°C at the main study site in the northwestern Sonoran Desert, markedly affect seedling survival. Computer simulations indicated that for the rather spherical barrel cactus Ferocactus acanthodes (Lem.) Britt. & Rose the maximum surface temperature decreased 8°C and the minimum temperature increased 3°C as the seedling height was increased from 1 mm up to 50 mm. Simulated changes in shortwave and longwave irradiation alone showed that shading could decrease the maximum temperature by about 5°C for the common desert agave, Agave deserti Engelm., and raise the minimum 1°C. Actual field measurements on seedlings of both species, where shading would affect local air temperatures and wind speeds in addition to irradiation, indicated that shading decreased the average maximum surface temperature by 11°C in the summer and raised the minimum temperature by 3°C in winter.Seedlings grown at day/iight air temperatures of 30°C/20°C tolerated low temperatures of about -7°C and high temperatures of about 56°C, as measured by the temperature where stain uptake by chlorenchyma cells was reduced 50%. Seedling tolerance to high temperatures increased slightly with age, and F. acanthodes was more tolerant than A. deserti. Even taking the acclimation of high temperature tolerance into account (2.7°C increase per 10°C increase in temperature), seedlings of A. deserti would not be expected to withstand the high temperatures at exposed sites, consistent with previous observations that these seedlings occur only in protected microhabitats. Based primarily on greater high temperature acclimation (4.3°C per 10°C), seedlings of F. acanthodes have a greater high temperature tolerance and can just barely survive in exposed sites. Wide ranges in photoperiod had little effect on the thermal sensitivities of either species. When drought increased the chlorenchyma osmotic pressure from about 0.5 MPa to 1.3 MPa, seedlings of both species became about 2°C less tolerant of high temperatures, which would be nonadaptive in a desert environment, and 2°C more tolerant of low temperatures, which also occurs for other species.In conclusion, seedlings of A. deserti and F. acanthodes could tolerate tissue temperatures over 60°C when acclimated to high temperatures and below -8°C when acclimated to low temperatures. However, the extreme environment adjacent to desert soil requires sheltered microhabitats to protect the plants from high temperature damage and also to protect them from low temperature damage at their upper elevational limits.     

Productivity of Agave deserti: measurement by dry weight and monthly prediction using physiological responses to environmental parameters

Summary An environmental productivity index based on physiological responses to three environmental variables was used to predict the net productivity of a common succulent perennial of the Sonoran Desert, Agave deserti, on a monthly basis. Productivity was also independently measured in the field from dry weight changes. The index was based on soil water availability, day/night air temperatures, and photosynthetically active radiation (PAR), which were individually varied in the laboratory and the effect on net CO₂ uptake by the leaves determined. From monthly precipitation, temperature, and PAR at the field site together with the responses measured in the laboratory, an index (maximum value of unity) was assigned to each of these three environmental variables and their product was termed the environmental productivity index. This index indicates the fraction of maximal CO₂ uptake expected in the field for each month (well-watered A. deserti assimilated 285 mmol CO₂ m^-2 leaf area day^-1 at PAR saturation and optimal day/night temperatures of 25° C/15° C). The dry weight analysis was based on the monthly unfolding of new leaves from the central spike of the rosette and their seasonal increase in dry weight, which were determined in the field. The production of new leaves was highly correlated with the environmental productivity index (r²=0.93), which in turn was highly correlated with the water status index (r²=0.97). After correction for respiration by folded leaves, stem, and roots, plant productivity predicted by the average environmental productivity index (0.36) over a wet June-to-October period agreed within 4% with the productivity based on the conventional dry weight analysis. The net productivity of A. deserti over this 5-month period was 0.57 kg m^-2 ground area (5.7 Mg ha^-1), a large value for a desert CAM plant. The environmental productivity index proposed here may provide a reliable means for predicting net productivity on a monthly basis, which may be particularly useful for species in relatively variable environments such as deserts.     

Discriminative stimulus properties of the serotonin agonist 1-(3-trifluoromethylphenyl)piperazine (TFMPP)

Using a standard two-lever drug discrimination procedure, twelve rats were trained to discriminate 1.0 mg/kg of the serotonin (5-HT) agonist TFMPP from saline. Once trained, the animals displayed a dose-related decrease in discriminative performance upon administration of lower doses of TFMPP. Tests of stimulus generalization were performed using the purported 5-HT agonist RU-24, 969 and 1-(2,5-dimethoxy-4-methylphenyl)-2-aminopropane (DOM). While TFMPP produced stimulus effects similar to those of RU-24,969, these effects seem to be dissimilar to those of DOM. The results of the present study suggest that the discriminative stimulus effects of TFMPP may involve a 5-HT1-related mechanism.     

Gene amplification in Bacillus subtilis

A strain of Bacillus subtilis that carries in its genome a staphylococcal chloramphenicol acetyltransferase gene (from pC194) responds to growth at different concentrations of chloramphenicol by an alteration in the number of copies per genome of the sequences encoding the gene. Growth at 20 micrograms chloramphenicol ml-1 results in a 15-fold amplification of the sequences, whereas growth in the absence of chloramphenicol results in their loss. The mechanism of in situ amplification probably has much in common with that involved in 'R factor transitioning'. The hybridization procedures that have been used for accurately determining the number of copies of the amplified DNA sequences are potentially useful for plasmid copy number determination. The findings reported here also provide a potentially useful alternative to more conventional cloning strategies that are based on autonomous plasmids in B. subtilis. The particular advantages that can be envisaged include enhanced stability of the cloned sequences and control of the number of copies that are present.     

Some factors involved in the dissolution of Autographa californica nuclear polyhedrosis virus polyhedra by digestive fluids of Trichoplusia ni larvae

Factors involved in the dissolution of polyhedra of Autographa californica nuclear polyhedrosis virus (AcNPV) by digestive fluid collected from fifth stage Trichoplusia ni larvae were studied in vitro. Observations were made at timed intervals using phase contrast microscopy and transmission electron microscopy. When digestive fluid was heated at 50°C proteases retained activity. Exposure of polyhedra to digestive fluid previously heated to 50°C resulted in polyhedral matrix dissolution and envelope disruption in a manner similar to that of unheated digestive fluid, only delayed slightly. After exposure of polyhedra for 3 min, only enveloped virons were observed. Heating the digestive fluid to 60° or higher inactivated the proteases and altered the effect on polyhedra. Dissolution of the occlusion body matrix occurred but the polyhedral envelope remained and only a few weakened areas were observed in its structure. Within the polyhedral envelope, enveloped virons were not observed, only nucleocapsids and capsids. Exposure of polyhedra to 0.1 m sodium carbonate buffer at pHs of 9.5 or higher had effects similar to those of the digestive fluid with heat (60°C)-inactivated proteases. The addition of trypsin and chymotrypsin to the 0.1 m sodium carbonate buffer had no effect, while the addition of a bacterial protease (Streptomyces griseus) at pHs of 9.5 or higher resulted in dissolution of the matrix and disruption of the polyhedral envelope like the digestive fluid. Material infectious to TN-368 cells was obtained by exposure of AcNPV to T. ni digestive fluid. Maximum infectivity resulted from a 5-min exposure to unheated digestive fluid, with a dramatic decrease in infectivity with longer exposure. Exposure to digestive fluid with heat (60°C)-inactivated proteases resulted in a slower release of infectious material from the occlusion body, with a steady increase in the level of infectivity throughout the 30-min digestion period.