Description of a new species of Haemoproteus (Haemosporidia, Apicomplexa) from an Orange-tufted Sunbird Nectarinia osea

Haemoproteus osea sp. nov. is described from the blood of Nectarinia osea, sunbird species, endemic to Israel and the Red Sea zone of Arabia. Infection was found only in one out of 44 birds, in Eilat, at the north end of Gulf of Aqaba; level of parasitaemia was 0.07%. Young gametocytes adhere alongside the nucleus. The gametocytes extend alongside the erythrocyte nucleus, filling the entire space to the erythrocyte envelope and only slightly impose over the tip of the erythrocyte nucleus. Mean nuclear displacement ratio is 0.75. H. osea differs from H. sequeirae Tendeiro, 1947 in having the microgametocyte cytoplasm blue staining, similarly to the macrogametocyte, rather than reddish white, and also in causing less evident displacement of the erythrocyte nucleus.

Desseria zei sp. nov. (Adeleorina: Haemogregarinidae) infecting Zeus capensis from deep waters off the south and west coasts of South Africa

Blood films from a marine fish, the Cape Dory (Zeus capensis), trawled at depths between 149 and 389 m off the south and west coasts of South Africa, contained a new species of haemogregarine, Desseria zei sp. nov. The apicomplexan, found in 25/97 fish from the two regions, was generally intra-erythrocytic. Parasitaemia was usually low, but attained infection levels of 1/10 erythrocytes in one fish. The parasite stages were all gamonts and monomorphic, but likely varied in maturity; they were 3.3–4.5 μm wide by 14.7–18.3 μm long, curved with bluntly pointed ends, and existed singly within erythrocytes, or in pairs in the heavy infection. Their cytoplasm was deep blue, granular, and stained deeply at the anterior and sometimes, posterior extremities. Two or three vacuoles occurred in the cytoplasm between the anterior cap and the gamont nucleus, which was positioned in the posterior third of the parasite body. Gamonts lay close to the host cell nucleus, often curved around it. Distortion of host cells and host nucleus displacement occurred when single gamonts curved away from the host nucleus, or if they were paired. A few extracellular gamonts were observed, occasionally associated with the remnants of the erythrocyte nucleus.

Electron microscopic observations on the development and cytochemistry of the large granule complexes in chicken erythrocyte nuclei

Large granule complexes are structures found in a small percentage of chicken erythrocyte nuclei when observed in ultra-thin sections in the electron microscope. They consist of an amorphous region associated with a number of large (approximately 30 min) granules. We have shown, by a novel use of phenylhydrazine to synchronize populations of chicken erythrocytes in vivo, that large granule complexes do not occur in the nuclei until the cells have reached one-third to one-half of their normal intravascular lifespan. The mature large granule complexes are formed by aggregation of pre-existing fibrillar, granular and amorphous material, and their presence is correlated with the presence of another ultrastructural feature of the nucleus, the so-called “filled cavities' in the chromatin. Digestion of ultra-thin sections of erythrocytes embedded in the hydrophilic resin glycol methacrylate (GMA) has shown that the major component of the amorphous region is a rather acidic protein that is not haemoglobin, the most abundant protein in the erythrocyte. The large granules also contain protein and, almost certainly, RNA. The problems encountered in reaching this conclusion have emphasized the lack of unambiguous cytochemical tests for use on ultra-thin sections. We have shown that the large granule complex differs in many respects from the nucleolus in the erythrocyte series, even though the two organelles have certain superficial similarities such as their overall dimensions and the presence of granular and fibrillar regions. The most likely function of the large granule complex is as a repository for material, including RNA, the processing of which has ceased in the inactivated erythrocyte nucleus.

Transfer of mouse nuclear envelope specific proteins to nuclei of chick erythrocytes during reactivation in heterokaryons with mouse A9 cells

When chick erythrocyte nuclei are introduced into the cytoplasm of mouse A9 cells by cell fusion, proteins present in a fraction of the mouse nuclear envelope begin to appear in the envelope of the chick erythrocyte. The protein uptake was examined using antisera raised in chickens against the 3 major polypeptides of the nuclear pore complex-fibrous lamina fraction from rat liver nuclei. In indirect immunofluorescence studies these antisera give a strong envelope-specific staining with various mammalian but not chicken cells. Eighteen hours after cell fusion the first murine antigens can be observed in the erythrocyte nucleus. Two days after cell fusion the vast majority of the erythrocyte nuclei in cell hybrids contain some antigen and by 3 days the fluorescence of the reactivated erythrocyte nuclei reaches a level comparable to that of the mouse A9 nuclei. The rate of appearance of fluorescence in the chick nuclei depends upon the ratio of A9 cytoplasm to chick nuclei. Antigen uptake by the erythrocyte envelope is inhibited when protein synthesis is blocked suggesting that synthesis of mouse antigen, rather than a redistribution, determines the velocity or erythrocyte envelope reactivation. The early uptake of nucleospecific protein into the reactivating chick erythrocyte may not require any alteration in the nuclear envelope.

GLUTARALDEHYDE FIXATION OF ISOLATED EUCARYOTIC NUCLEI

Isolated chicken erythrocyte nuclei have been incubated with dilute concentrations of the bifunctional cross-linking agent glutaraldehyde (0–20 mM) in order to stabilize histone-histone interactions within the native nucleus. The kinetics of the disappearance of acid-soluble histones, free amino groups, and of individual histones have been observed to be pseudo first-order. Apparent first-order rate constants for the disappearance of individual histones correlate with the lysine mole percent of that fraction and follow the ranking, kapp: F1 > F2C > F2B ≥ F2A2, F2A1, F3. Histone polymers were observed to form very rapidly during the fixation reaction. Partial fractionation and amino acid analyses of these polymers support the view that they are composed principally of cross-linked (F2C)n molecules (where n = 2 to ∼8). The rate of glutaraldehyde reaction with free amino groups in histones is drastically reduced in solvents that promote chromatin decondensation (i.e., low ionic strengths in the absence of divalent cations) whereas the formation of cross-linked F2C polymers is less severely reduced. It is proposed that some F2C histones exist in close proximity within the isolated erythrocyte nucleus.

Further Experiments on the Role of the Nucleolus in the Expression of Structural Genes

When a chick erythrocyte nucleus is introduced into the cytoplasm of a mouse cell it undergoes reactivation and eventually determines the synthesis of chick-specific proteins in the cytoplasm of the heterokaryon. It has previously been shown that there is a close temporal correlation between the onset of synthesis of the chick-specific proteins and the appearance of nucleoli in the erythrocyte nuclei. We now show that the synthesis of chick-specific proteins in the heterokaryon decays if the nucleolus in the reactivated erythrocyte nucleus is irradiated with an ultraviolet microbeam. The synthesis of chick-specific proteins does not, however, decay if only one of two nucleoli in the erythrocyte nucleus or if extranucleolar areas of the nucleus are irradiated. These observations confirm that some function located at or near the nucleolus is essential for the full expression of structural genes.