A charge coupled device-based image cytophotometry system for quantitative histochemistry and cytochemistry

A rapid, semiautomated cytophotometry system for quantitative histochemistry and cytochemistry was constructed. The system consists of a Fairchild charge coupled device (CCD) image camera, a Zeiss Universal microscope, a Datacube analog to digital converter, and a digital Equipment Corporation LSI 11/23 computer operating under RT-11. Computer programs were written in FORTRAN and the MACRO assembly language for the acquisition of data from the CCD device. These data were then used for image segmentation, image display, and calculation of total optical density, perimeter, cell area, and several shape features. The reproducibility of measurement made with the CCD-based cytophotometry system was tested by repeated measurements. The coefficient of variation was estimated to be 1.7% for total optical density and 0.9% for cell area. The CCD-based cytophotometry system was further evaluated by comparing results with measurements made on the same cells with a scanning stage cytophotometer using the HIDACSYS computer programs. Correlation coefficients of 0.96 for total optical density and 0.91 for cell area were obtained between the two systems. We conclude that the high-speed, dimensional stability, small size, and linearity of the CCD-based cytophotometry system will make it useful for quantitative histochemistry and cytochemistry.     

Thymosin fraction 5 causes increased serum corticosterone in rodents in vivo

In these studies it was found that i.p. injection of thymosin fraction 5 (TF5) caused a dose-dependent increase in serum corticosterone in male Swiss Webster mice and in male Wistar rats. The maximum responses were seen at 1 and 2 hr, respectively. There was no effect on serum corticosterone in mice when Thymosin alpha 1 (a 28 amino acid peptide isolated from TF5) was injected i.p. at doses up to 100 micrograms. The steroidogenic effects of TF5 were seen only when the basal levels of serum corticosterone were low (less than 80 ng/ml). In studies in which the baseline levels in the animal colony were elevated (greater than 80 ng/ml), there were no steroidogenic effects, or they were minimal. These results suggest that some component of TF5 may influence pituitary adrenal function.     

Characterization of a T4+/Leu-8+ T cell clone that directly helps B cell Ig production by secreting B cell differentiation factor

A human T4+/Leu-8+ T cell clone (YA2) was established by phytohemagglutinin activation and interleukin 2 (IL 2) propagation. Functional characterization of this clone demonstrated that it provided potent help towards Ig production by pokeweed mitogen-stimulated B cells in the presence of small numbers of autologous T cells or by Staphylococcus aureus Cowan I (SAC)-activated B cells in the presence of B cell growth factor (BCGF). YA2 provided no help to resting B cells and minimal help to either unactivated B cells cultured with BCGF or SAC-activated B cells. Supernatant generated from clone YA2 by IL 2 stimulation had significant B cell differentiation activity but no BCGF or IL 2 activity. Thus, YA2 is a T4+/Leu-8+ potent direct helper only to B cells that are activated and proliferating due to its selective secretion of a differentiation factor, and not an activation and growth factor. The availability of phenotypically defined cloned populations of T cells with restricted functional helper activity related to the secretion of selected B cell tropic factors should prove useful in the dissection of the role of individual T cell subsets in the regulation of the human B cell cycle.     

PMA induces the ligand-independent internalization of CR1 on human neutrophils

Phorbol myristate acetate (PMA) has been reported to confer on the C3b receptor (CR1) of neutrophils a capacity for phagocytosis of particles bearing C3b without the involvement of other membrane receptors. In the present study, we employed a monoclonal antibody, YZ-1, that is specific for CR1 to assess the effect of PMA on plasma membrane expression of CR1, total cellular CR1, and internalization of CR1 by neutrophils. PMA had a biphasic effect on the membrane expression of CR1 by purified neutrophils, with 4 ng/ml inducing a 60% increment in receptor expression, and higher concentrations causing up to a 70% decrement. PMA-dependent increases in CR1 expression were not accompanied by corresponding changes in total cellular CR1 and were preempted by treatment of cells with formyl-methionyl-leucyl-phenylalanine (FMLP). PMA-induced decreases in CR1 expression by neutrophils, as measured by binding of indirectly fluoresceinated or radiolabeled YZ-1, or of 125I-labeled dimeric C3b, were maximal with 20 to 30 ng/ml PMA, and occurred within 30 min of incubation at 37 degrees C. The PMA-dependent down-regulation of CR1 by neutrophils was not associated with a comparable decrease in total cellular CR1, and this response was observed to occur also with monocytes but not with peripheral blood lymphocytes. By tagging neutrophil CR1 with 125I-YZ-1 Fab and monitoring accessibility to Protease, intracellular CR1 (inaccessible) was discriminated from receptor on plasma membrane (accessible). Internalization of CR1 occurred within 5 min after addition of PMA to neutrophils, was dose dependent, and involved up to two-thirds of the tagged receptors. Therefore, PMA caused internalization of CR1 by neutrophils in the absence of ligand, indicating that this response was independent of a transmembrane signal generated by a C3b-CR1 interaction.     

Tobacco single-copy DNA is highly homologous to sequences present in the genomes of its diploid progenitors

Summary We compared the single-copy DNA sequences of the tetraploid tobacco plant, Nicotiana tabacum, with those of its diploid progenitors N. sylvestris and N. tomentosiformis. We observed that 65% of N. sylvestris and N. tomentosiformis single-copy DNA fragments reacted with each other using moderately stringent hybridization conditions (60° C, 0.18 M Na⁺). An additional 10% sequence homology was detected when the hybridization temperature was reduced by 10° C. The thermal stability of interspecific single-copy DNA duplexes indicated that they were approximately 6% more mispaired than homologous single-copy DNA duplexes. In contrast, we observed almost no single-copy DNA divergence between N. tabacum and its diploid progenitors. Greater than 99% of N. sylvestris and N. tomentosiformis single-copy DNAs reacted with N. tabacum DNA using moderately stringent hybridization conditions. The thermal stability of these duplexes indicated that they contained no more sequence mismatch than homologous single-copy duplexes. Together, our results show that significant single-copy DNA sequence divergence has occurred between the diploid N. sylvestris and N. tomentosiformis genomes. However, by applying our experimental criteria these single-copy DNAs are indistinguishable from their counterparts in the hybrid N. tabacum nucleus.     

10 nm RecA protein filaments formed in the presence of Mg2+ and ATP gamma S may contain RNA

Summary Filaments formed by the polymerization of RecA protein along DNA in the presence of Mg²⁺ and adenosine 5-0-(3-thiotriphosphate) (ATPS) are seen by electron microscopy to have a 10 nm diameter with a 9 nm helical repeat. When certain preparations of apparently pure RecA protein are incubated with Mg²⁺ and ATPS in the absence of nucleic acid for extended times, very long filaments with the same 10 nm diameter and 9 nm axial repeat are seen. We show here that these long 10 nm filaments can contain RNA which is present as a contaminant of the RecA protein and poly(A) which is synthesized during the incubations by an activity that is apparently polynucleotide phosphorylase. RecA protein purified by a procedure developed in this laboratory did not contain RNA and did not form these very long 10 nm filaments. However, when exogenous RNA was added to this protein, 10 nm filament formation was observed.     

Long-term infertility and dominant lethal mutations in male mice treated with adriamycin

Sperm production and fertility were studied in male mice treated with adriamycin (ADR) at 6 or 8 mg/kg. Testicular sperm production and epididymal sperm counts were markedly reduced after ADR treatment. Gradual recovery of counts occurred, but sperm counts had not reached control levels even more than 1 year after treatment. Epididymal sperm showed treatment-induced morphological abnormalities throughout the experiment; the frequencies of sperm with detached tails and the frequencies of sperm with morphologically abnormal heads remained elevated about 2-3-fold above control. According to the frequency of vaginal plugs, treated male mice mated at control rates with untreated females during the post-treatment sterile period. However, after some fertility was regained the fertilization rate (calculated as the fraction of eggs, flushed from the oviduct 2 days after mating, that had been fertilized and had cleaved) was markedly reduced and remained depressed for the remainder of the experiment. The fertilization rate reached only 0.29 at 23-32 weeks after 8 mg/kg ADR and 0.76 at 16-23 weeks after 6 mg/kg ADR; both values were significantly below the control value of 0.94. Dominant lethal mutations in the zygotes flushed from the oviduct were measured in culture by the loss of the zygote's ability to develop to a stage characterized by trophectoderm outgrowths and formation of an inner cell mass. The frequencies of dominant lethal mutations detected in vitro were 1.7 or 7.4% after 6 mg/kg, and 32 or 40% after 8 mg/kg ADR; each value was calculated in two different ways, with 3 of these 4 values significantly different from zero. We conclude that even after mice regain fertility following ADR exposure, the level of fertility remains permanently subnormal as evidenced by a lack of fertilization of eggs that is probably due to the decreased quantity and quality of spermatozoa produced. Furthermore, ADR can induce genetic damage in stem spermatogonia, which can be transmitted through fertile spermatozoa. Thus, there may be a genetic risk to the offspring of cancer patients treated with ADR chemotherapy, but at present we are unable to quantitate that risk.     

Morphological changes along an altitude gradient and their consequences for an andean giant rosette plant

Summary Selected morphological features were measured in five populations of the giant rosette plant Espeletia schultzii occurring along an elevation gradient from 2600 to 4200 m in the Venezuelan Andes. Pith volume per amount of leaf area increases with elevation resulting in significantly larger water storage capacity at higher elevations. Thickness of leaf pubescence and, therefore, leaf boundary layer resistance, also increases with elevation resulting in both potentially higher leaf temperatures relative to air temperature and higher leaf to air vapor pressure gradients. The net effect on transpiration rate would depend on ratios of stomatal to boundary layer resistance and leaf energy balance. At higher elevations the central rosette leaves are more vertically oriented and the leaf bases show a pronounced curvature as the intersection with the main axis is approached. This gives these rosettes a distinctly paraboloid appearance and probably enhances capture and retention of incident long and shortwave radiation by the apical bud and expanding leaves. Features which result in enhanced water storage capacity and higher plant temperatures relative to air temperature without greatly increasing water loss are adaptive in high altitude paramo habitats where water availability and growth are limited by year round low temperatures (mean 2–3° C).     

Phosphate Transport across the Plasma Membrane of Wheat Leaf Protoplasts: Characteristics and Inhibitor Specificities

The kinetics and inhibitor specificities of phosphate transport across the plasma membrane of wheat leaf mesophyll protoplasts have been examined. Studies were also carried out on the effects of light and pH on phosphate transport and the plasma membrane electropotential. At pH 5.8 (30°C), protoplasts accumulated phosphate at the rate of 3.9 ± 0.2 nanomoles per milligram protein per hour. Phosphate uptake rates and inhibitor specificities for the leaf cell plasma membrane phosphate transporter were qualitatively similar to those observed with root protoplasts. Neither picrylsulfonic acid, or p-chloromercuribenzene sulfonate affected phosphate uptake significantly at 0.1 millimolar. Of all compounds tested, carbonyl cyanide-p-trifluoromethoxy phenylhydrazone was the most effective inhibitor of phosphate uptake (60% at 0.1 millimolar). Tribenzylphosphate inhibited uptake by 34% while dibenzylphosphate had no effect. The plasma membrane electropotential was found to be −37 ± 3 millivolts. Initiation of photosynthesis lowered the membrane potential to −39 ± 3 millivolts. Inhibition of phosphate uptake by 34% with the substrate analog tribenzylphosphate resulted in a measured membrane potential of −33 ± 3 millivolts. These changes in potential were not significant at the 5% probability level. Phosphate uptake rates remained constant under photosynthetic and nonphotosynthetic conditions. The utility of tribenzylphosphate as an inhibitor in plant systems is demonstrated.