A Two Step Stain for Normal and Leukemic Monocytes Using Two Different Dyes Applied in Sequence

A staining procedure for monocytes in specimens of blood and bone marrow was developed. The technique was a two step procedure in which unfixed cells were exposed first to a methanolic solution of C.I. basic blue 54. Next, an aqueous alkaline buffered solution of C.I. basic blue 141 was added to the first staining solution. After staining for 10 min in the solution with two stains, slides or coverslips were washed for 5 sec in pH 5.6 phosphate buffer and drained dry. The cytoplasm of monocytes stained intensely deep purple and frequently nuclei were stained red. Similar staining was not found in other types of normal or abnormal blood and bone marrow cells.     

Biochemical genetic markers of squirrel monkeys and their use for pedigree validation

Family data for 14 biochemical genetic markers of squirrel monkeys (genusSaimiri) were derived from 73 pedigreed progeny and both parents of each, as well as from 16 additional progeny and one parent of each. The data for each marker and the phenotypic patterns were consistent with autosomal codominant inheritance. It was concluded from the genetic marker data that the pedigree records of seven progeny were incorrect. Retrospective investigations of colony records followed by typing of animals that might possibly have been a parent enabled five of the pedigree records to be corrected. Although five of the pedigree errors were cases of mistaken paternity, the other two apparently were the consequence of infant swapping between dams shortly after birth. Because squirrel monkeys exhibit a high degree of allomaternal behavior, infant swapping between dams may occur more frequently than in many other nonhuman primate species.This research was supported in part by NIH Grant P40 RR01254.     

Amerindian mitochondrial DNAs have rare Asian mutations at high frequencies, suggesting they derived from four primary maternal lineages.

The mitochondrial DNA (mtDNA) sequence variation of the South American Ticuna, the Central American Maya, and the North American Pima was analyzed by restriction-endonuclease digestion and oligonucleotide hybridization. The analysis revealed that Amerindian populations have high frequencies of mtDNAs containing the rare Asian RFLP HincII morph 6, a rare HaeIII site gain, and a unique AluI site gain. In addition, the Asian-specific deletion between the cytochrome c oxidase subunit II (COII) and tRNA(Lys) genes was also prevalent in both the Pima and the Maya. These data suggest that Amerindian mtDNAs derived from at least four primary maternal lineages, that new tribal-specific variants accumulated as these mtDNAs became distributed throughout the Americas, and that some genetic variation may have been lost when the progenitors of the Ticuna separated from the North and Central American populations.     

The correlation between specific protein synthesis and tumoricidal function in murine peritoneal macrophages

Macrophages from the lipopolysaccharide (LPS)-responsive C3H/HeN mouse strain and the closely related LPS-nonresponsive C3H/HeJ strain were compared for tumoricidal activation and protein synthetic changes following in vivo and in vitro stimulation, utilizing two-dimensional polyacrylamide gel electrophoresis of [35S]methionine-labeled proteins. Peritoneal macrophages elicited from C3H/HeN mice with heat-killed Propionibacterium acnes exhibited tumoricidal activity in a 16-hr cytolytic assay and expressed cytoplasmic levels of a 23.5-kDa protein during 48 hr of culture. The inability to detect persistent expression of p23.5 in P. acnes-stimulated C3H/HeJ macrophages correlated with the cytolytic impotence of those cells in the 16-hr chromium release assay. C3H/HeN macrophage populations lacking tumoricidal capacity could be rendered lytic, as could P. acnes-elicited C3H/HeJ macrophages, following in vitro stimulation with bacterial lipopolysaccharide. Concomitant with the LPS-induced expression of new functional activity was the appearance of augmented levels of several macrophage-specific proteins, including p23.5. This effect was dependent upon the lipid A moiety of LPS as the effects of LPS could be blocked by inclusion of polymyxin B sulfate in the culture medium. However, neither tumoricidal function nor protein modulation could be readily induced in C3H/HeJ proteose peptone-elicited or resident macrophages. These results identify biochemical responses to stimuli which may be requisite to acquisition or execution of cytolytic activity.     

The effects of carbon monoxide and hypoxic hypoxia on amine-containing cells in the tracheal epithelium of young rabbits

Summary Endogenously fluorescent, singly occurring, amine-containing cells in tracheal epithelium were examined in 3-, 10-, and 28-day-old rabbits. These cells are pyramidal in shape with the apex projected toward the tracheal lumen. The cytoplasm exhibits a yellow fluorescence which is predominantly supranuclear. Occasional, infranuclear, fluorescent cytoplasmic processes project from the cells. The numbers of fluorescent cells per unit length of trachea increase with age. Acute exposure of 10-day-old rabbits to 13% O₂ decreases the number of detectable fluorescent cells in the trachea compared to controls exposed to room air. Similarly, exposure to 750 ppm carbon monoxide decreases the number of fluorescent epithelial cells appearing in tracheas of 10- and 28-day-old rabbits. These results suggest that the amine-containing epithelial cells of the trachea respond to tissue hypoxia and that decreased airway pO₂ is not necessary to elicit a response.Supported by a grant from The Council for Tobacco Research, U.S.A., Inc. We are grateful to Margaret Hogan and Scott Pine for technical assistance     

IF-3 crosslinking to Escherichia coli ribosomal 30 S subunits by three different light-dependent procedures: Identification of 30 S proteins crosslinked to IF-3—Utilization of a new two-stage crosslinking reagent, p-nitrobenzylmaleimide

We here report the results of using three light-dependent procedures for crosslinking IF-3 to 30 S proteins within an IF-3·30 S complex. In the first procedure, employing FMN as a photosensitizer, protein S12 is found to be the only major crosslinked protein. In the second procedure, IF-3 is first reacted with the new two-stage crosslinking reagent, p-nitrobenzylmaleimide (PNBM), and the PNBM—IF-3·30 S complex is irradiated. The major crosslinked proteins are S3 > S2, S12, S18. Small amounts of crosslinked S11 and S21 are also found. In the third procedure, the IF-3·30 S complex is reacted with PNBM and then irradiated. The major crosslinked proteins are S12 > S3 > S11 and small amounts of crosslinked S1, S13, and S21 are also found. These results are compared with results obtained by others using different crosslinking procedures and are used to discuss the Lake and Kahan model (J. A. Lake and L. Kahan, 1975, J. Mol. Biol., 99, 631–644, and J. A. Lake, 1978, in Advanced Techniques in Biological Electron Microscopy II, Koehler, J. K., ed., pp. 173–211, Springer-Verlag, Berlin) for IF-3 binding to 30 S subunits.     

XY female mice express H-Y antigen

Karyotypically XY individuals of the C57BL/6J-Y^POS mouse stock develop as females or hermaphrodites, but never as normal males. The aberrant sexual development results from the interaction of the C57BL/6J genetic background with the M. poschiavinus-derived Y chromosome. XY females from this stock were assayed for H-Y antigen. By the criteria of skin-grafting, the cell-mediated lympholysis test, and the popliteal lymph node assay, these XY females are antigenically indistinguishable from normal C57BL/6 males. Implications for the hypothesis that H-Y antigen induces formation of the mammalian testis are discussed.     

Anhydrobiosis in Nematodes II: Carbohydrate and Lipid Analysis in Undesiccated and Desiccate Nematodes

Glycogen, trehalose, glucose, and total lipid contents of six nematode species were studied. Anhydrobiotic Anguina tritici and Ditylencbus dipsaci stored trehalose in preference to glycogen and only small amounts of glucose were detected. Glycogen content was also reduced in anhydrobiotic Aphelenchus avenae. Conversely, Panagrellus redivivus and Turbatrix aceti contained large amounts of glycogen, appreciable amounts of glucose, and minimal amounts of trehalose. Ditylenchus myceliophagous "curds" contained low amounts of glycogen and very little trehalose; total lipid was 60% of that in fresh samples. The lipid contents of fresh samples of P. redivivus, T. aceti, and A. avenae were high (23.1, 21.9, and 36.7% dry weight, respectively), but in anhydrobiotic A. avenae larvae the level was reduced by over 60%. In contrast, lipid levels remained high in anhydrobiotic A. tritici and D. dipsaci larvae (40.6 and 38.3%, respectively). Analysis of lipid composition in anhydrobiotic A. tritici and A. avenae did not indicate any specific metabolic adaptations to desiccation survival.