Primitive erythropoiesis in early chick embryogenesis. II. Correlation between hemoglobin synthesis and the mitotic history

Primitive erythroblasts in the circulating blood of the chick embryo continue to divide while synthesizing hemoglobin (Hb). Hb measurements on successive generations of erythroblasts show that there is a progressive increase in the Hb content of both interphase and metaphase cells. Furthermore, for any given embryo the Hb content of metaphase cells is always significantly greater than that of interphase cells. The distribution of Hb values for metaphase cells suggests that there are six Hb classes corresponding to the number of cell cycles in the proliferative phase. The location of erythroblasts in the cell cycle was determined by combining Feulgen cytophotometry with thymidine radioautography on the same cells. Measurements of the Hb content for erythroblasts in different compartments of the cell cycle (G1, S, G2, and M) show a progressive increase through the cycle. Thus, the amount of Hb per cell is a function of the number of cell divisions since the initiation of Hb synthesis and, to a lesser degree, the stage of the cell cycle. Earlier generations of erythroblasts synthesize Hb at a faster rate than the terminal generation. Several models have been proposed to explain these findings.     

Thorotrast uptake and transit in embryonic glia, heart fibroblasts and neurons in vitro

Thorotrast (colloidal ThO₂) is incorporated into coated vesicles, various agranular vesicles and sacs, and a surface-associated system of membranous channels in times as short as 1 min by single cultured glial and heart cells. Thorotrast appears in ‘C’-shaped bodies and in small, dense bodies of the lysosomal series within ca. 25 min. With longer chase periods, thorotrast ‘clears’ from all cytoplasmic organelles except the lysosomal series. The technique of applying thorotrast and using varying chase periods fails to distinguish a class of membranous organelles, located close to the cell periphery, that might serve as a source of new cell surface during locomotory activity. Similarly, thorotrast (colloidal ThO₂) is incorporated into almost all classes of membrane-bounded organelles of growth cones and axons of single nerve cells in vitro in times as short as 1 min. This includes elements of the smooth endoplasmic reticulum. No thorotrast enters the lysosomal granules in this short time. During various chase periods, the tracer disappears from the initial sites of incorporation and accumulates in dense bodies of the lysosome series within growth cones and axons. ‘C’-shaped bodies may be an intermediate in that process. No unique sites of endocytotic activity or of a complete absence of endocytosis were observed that could be correlated with growth cone function and axonal elongation, though the presence of the tracer in agranular sacs of the smooth endoplasmic reticulum in growth cones could reflect hypothesized cycling of cell surface (Bray, 1973).     

Observations on freeze-fractured membranes of a Trypanosome

Pure preparations of Trypanosoma brucei, free from plasma and cellular components were isolated from rat blood, and concentrated into loose pellets by low-speed centrifugation. Pellets were either processed for thin sectioning as a control for general morphology, or glycerol-treated after glutaraldehyde fixation for preparation of freeze-fracture replicas. Concentration of cells of 50,000–100,000/mm² of sectioned or fractured surface facilitated identification of fracture faces of the cell body, invaginated flagellar pocket and flagellum. Particle distribution and A and B faces of these regions of the cell are described. A collar of B face particles occurs around the neck of the flagellar pocket, possibly associated with a junction controlling ingress of ingested materials to coated vesicles formed along the membrane defining the pocket. A and B faces of the flagellum and adjoining surface of the cell body have shown that the only intra-membrane specialization corresponding to the miniature ‘maculae adherentes’ described previously in thin sections is probably an uninterrupted series of small clusters (3–6) of 80 Å particles on the A face of the flagellar membrane. It is proposed that these arrays represent attachment points for strands linking the axoneme and paraxial rod to the flagellar surface, and are not directly concerned with the physical adhesion of the flagellum to the cell body surface—a linkage that appears to be established within the extracellular gap between these apposed surfaces of the cell. The potential use of freeze-etching in further study of the external antigens of the infective cell is discussed.     

Observations on the mechanism of indirect induction by mating with ultraviolet-irradiated col I donors

Summary Irradiation of the colI donor itself is required to initiate indirect induction of phage following mating with a lysogenic recipient. Attempts to demonstrate that some other cytoplasmic inducer is responsible for induction have been negative. However the level of transfer of a viable (colicin-producing) colI factor (to a non-lysogenic recipient) is not correlated with the transfer of indirect induction (to a lysogenic recipient) either with respect to relative UV sensivities, or relative kinetics. Transfer of viable colI factor from the irradiated donor is delayed for up to 40 minutes whereas indirect induction is initiated early after contact. There is some lethality and inhibition of division of recipient cells mated with the irradiated donor, these effects similarly being initiated extremely early after cell contact. The question as to whether these effects of mating with an irradiated donor on the recipient follow transfer of inviable colI, or reflect a disturbance of transfer itself remains unresolved.     

DEVELOPMENT AND GERMINATION OF THE AZOTOBACTER CYST

The fine structure of Azotobacter vinelandii has been studied by means of electron microscopy of ultrathin sections made of the encysting and germinating cells. The organisms were fixed with KMnO₄ and embedded in epoxy resin. On an encystment medium the rod-shaped bacteria begin to assume an almost spherical form and then bark-like exine appears in 1½ to 2 days. The exine thickens and an electron permeable intine forms between it and the shrinking cell body. In 5 days the intine makes up more than half of the cyst volume and begins to show a definite two-layered structure. Meanwhile the peripheral bodies, which may be extensions of the cell membrane of the vegetative cell, disappear as the encystment progresses. The cell wall and membrane of the vegetative cell remain demonstrable as the confining structure of the shrinking central body of the mature cyst. In this central body lipoidal globules appear together with aggregations of nuclear material. Cyst germination begins with an increase in the size of the central body at the expense of the intine. The nuclear aggregations become more diffuse and the lipoidal globules disappear. The exine may be pushed outward and the bark-like fragments separate as the emerging vegetative cell develops. Invagination of the cell wall and membrane may occur at this stage leading to cell division. Empty exines remain as horseshoe-shaped structures.     

A PLUCHEA HYBRID FROM THE PACIFIC

Two species of Pluchea have been introduced into the Hawaiian Islands in the present century. These are P. indica, native to southeastern Asia, and P. odorata, native to the western hemisphere. Both are erect shrubs. Within the past 30 years a third Pluchea taxon, a sprawling shrub morphologically distinct from the two species, has appeared in the Hawaiian Islands and on other islands in the tropical Pacific. On the basis of morphology, fertility, meiotic behavior, and other evidence, it is concluded that the third taxon is a hybrid between the two species. The hybrid is highly sterile; it has a limited capacity for asexual reproduction. Metaphase I configurations for both species were found to be 10_II; the most frequent configuration in the hybrid was 8_II + 4_I. The hybrid is given a binomial.     

A practical method for rescuing desired hybridomas during monoclonal antibody production

Summary We present a practical method for the rescue of previosly stable hybridoma clones which increases the proportion of desired cells in the population before cloning by limiting dilution. When the antibody activity of a culture supernatant was lower than that previously obtained, a precloning distribution at a density of 10 cells per microtiter well greatly improved the chances of obtaining a single active clone by subsequent limiting dilution. The Poisson distribution model was used to evaluate the method. Probabilities calculated clearly demonstrate the advantage of this precloning distribution step when attempting to isolate a hydridoma cell line that is relatively rare in a population. This work was supported in part by grants EY 06225 and EY 06226 from the National Eye Institute of the National Institutes of Health, Bethesda, MD and by an unrestricted departmental award from Research to Prevent Blindness, Inc.