Comparison of the DNA content of megakaryocytes identified immunologically with that identified morphologically

 We devised a new microfluorometric method for determining the ploidy of megakaryocytes identified immunologically in bone marrow smears. The smears were immunostained by incubation with mouse monoclonal anti-glycoproteins (GP) IIb antibodies, followed by fluorescein isothiocyanate-conjugated goat anti-mouse IgG antibodies. They were then stained with 4′,6-diamidino-2-phenylindole (DAPI). Megakaryocytes were identified by their GPIIb immunofluorescence using a microfluorometer and, after the filters were changed, their DNA content was assayed by measuring the intensity of DAPI fluorescence. This intensity was shown to be proportional to the DNA content when the aperture of the objective lens was reduced. We compared these results with those obtained when megakaryocytes were identified morphologically, using DAPI staining after Wright-Giemsa destaining. In all 12 normal controls, the ploidy peaks were shown to be 16N by both methods, and the mean ploidy detected by the immunological method was only reduced 0.961 times relative to the estimate from the morphological method. In contrast, in eight myelodysplastic syndrome (MDS) patients, the ploidy peaks were either 8N or 4N and the mean was reduced by 0.906 times (P=0.018). Thus we could immunologically identify small megakaryocytes which we could not identify morphologically. Therefore, this method is useful for measuring megakaryocytic ploidy, especially in the pathological megakaryocytes of MDS patients. Accepted: 29 April 1997     

Deactivation of macrophage oxidative burstin vitro by different strains ofHistoplasma capsulatum

It was previously reported thatHistoplasma capsulatum (Hc) yeast not only failed to stimulate a murine macrophage oxidative burst (OB), but they also blunted or abolished OB stimulation by a subsequent encounter with potent stimuli such as zymosan or phorbol 12-myristate 13-acetate (PMA). The present studies show that macrophage deactivation is proportional to the time of incubation and the dose of Hc yeast that induce the deactivated state. Hc yeast derived from a virulent strain (G217B) are more efficient inducers of macrophage deactivation than similar preparations derived from the avirulent Downs Hc strain. Yeast cells of two other pathogenic fungi,Candida albicans andCryptococcus neoformans are shown to stimulate rather than deactivate a macrophage OB.     

Effect of Methionine Sulfoximine on Photosynthetic Carbon Metabolism in Wheat Leaves

Methionine sulfoximine (MSO) greatly reduced the carbon dioxideexchange rate (CER) of detached wheat (Triticum aestivvm L.cv Roland) leaves in 21% O₂, but only slightly reduced it in2% O₂. A supply of 50 mM NH₄Cl had little effect on the CERirrespective of the O₂ concentration. A simultaneous additionof glutamine and MSO protected against the inhibition of photosynthesisto a considerable extent and caused the accumulation of moreNH₃ than did the addition of MSO alone. Fixation of ¹⁴CO₂ in wheat leaves was inhibited by MSO treatmentin 22% O₂, and there was decreased incorporation of ¹⁴G intoamino acids and sugars and increased label into acid fractions.The addition of MSO and glutamine together eliminated the effectof MSO on the photosynthetic ¹⁴CO₂ fixation pattern. NH₄Cl stimulatedthe synthesis of amino acids from ¹⁴CO₂, especially the synthesisof serine in 22% O₂. Our observations show that factors other than the uncouplingof photophosphorylation by accumulated NH₃ may be responsiblefor the early stage of photosynthesis inhibition by MSO underphotorespiratory conditions. ¹Present address: Department of Agricultural Chemistry, KyushuUniversity, Fukuoka 812 Japan. ²Also at U.S. Department of Agriculture, Agricultural ResearchService, Urbana, Illionois 61801, U.S.A. (Received September 13, 1983; Accepted February 2, 1984)     

Immunohistochemical study of a large molecular fragment of thyroglobulin in parafollicular cells

Thyroglobulin-like immunoreactivity of the parafollicular cells was studied by an immunoperoxidase bridge technique using antisera against dog thyroglobulin fragments. 1. The dog parafollicular cells were specifically stained by anti-peak I (27S and larger components fraction) antiserum absorbed with peak II (19S fraction). By this method, they were easily distinguishable from the non-reactive follicular cells and colloid droplets. More sensitive staining of the parafollicular cells was possible with anti-peak I' (larger components fraction) antiserum. The staining reactions indicated that the antigenic material responsible for immunoreactivity of the parafollicular cells was due to larger molecular components of thyroglobulin corresponding to 32S, 37S or greater than 37S, and was not due to either the 19S thyroglobulin or to the 27S iodoprotein. 2. A conspicuous decrease of the immunoreactive material in the parafollicular cells occurred in the dog after both chronically induced hypercalcemia and antithyroid drug treatment. This coincided with movement of secretory granules containing calcitonin as shown by staining with silver impregnation, HCl-basic dye, and lead-hematoxylin. 3. The antisera against larger molecular components of dog thyroglobulin showed a high degree of cross-reactivity to the parafollicular cells of most of the mammalian species investigated; rats, rabbits, hamsters, mice, cats, lions, goats, cows, and human.     

Insuppressibility of plasma glucagon by orally or intravenously administered glucose in diabetes mellitus

In order to study the response of pancreatic alpha cells to the change blood glucose, plasma pancreatic glucagon levels were measured after glucose loading given orally (50g) or intravenously (25g) in twenty-two normal controls and eighty untreated diabetics. Basal plasma pancreatic glucagon levels did not differ significantly in the two groups. However, oral or intravenous glucose administration caused a decrease in plasma pancreatic glucagon in normal subjects but not in diabetics. In "moderate" or "severe" diabetics, plasma pancreatic glucagon tended to increase paradoxically following oral glucose loading. To evaluate the sensitivity of pancreatic alpha cells to glucose, we calculated the index, -sigma delta IRG/sigma delta BS, after oral glucose loading. It was 1.96 +/- 0.57 in normal subjects, and significantly higher than in "mild" (0.11 +/- 0.05), "moderate" (-0.002 +/- 0.06) and "severe" (-0.09 +/- 0.07) diabetics. These results demonstrate the insensitivity of alpha cells to hyperglycemia in patients with diabetes mellitus as compared with normal subjects.     

Evidence for "twisted plane" undulations in golden hamster sperm tails

Motile spermatozoa from the golden hamster have been arrested by rapid freezing and then fixed with glutaraldehyde at low temperature after substitution with ethylene glycol. As far as can be judged, the flagellar waveforms thus stabilized are similar to those seen in living sperm; in contrast, fixation in glutaraldehyde, without prior freezing, induces agonal changes in flagellar conformation. The characteristics waveform after freeze substitution contains three bends. Approx. half of these flagella are entirely planar. The rest are three dimensional, with the third bend displaced in a regular way from the plane containing the proximal two bends. From the geometry of these flagella, it is concluded that the plane of action of a given bending cycle undergoes a clockwise twist (from a forward viewpoint) as the cycle is succeeded by new bending cycles. This "twisted plane" undulation is quite different from helical movement. The twisting seems to occur abruptly, between cycles, as if each bending cycle has a preferred plane of action. The mechanism underlying the twisting is uncertain. However, on the basis of the angular displacements between the preferred planes, and the findings from electron microscopy, the following idea is presented as a working hypothesis: that, if the most proximal plane of bending is topographically determined by peripheral doublet 1, then successive distal planes of action are influenced predominantly by doublets 2, 3, etc., in clockwise sequence. The merits and weaknesses of this hypothesis are discussed.