Hypothesis: the target cell of GM-CSF is a macrophage precursor capable to produce cells with the property to secrete a G-CSF like activity.

The induction of granulocyte and macrophage colony formation by the granulocyte-macrophage colony stimulating factor (GM-CSF) on bone marrow cells (BMC) was evaluated as a function of time in agar cultures. We found that while macrophage cell clusters were very abundant on the first two days of culture, granulocytic cell clusters did not appear until the third day. We also found that macrophage colonies were present from the fourth day of culture, while granulocyte colonies did not appear until the fifth day. When two day cell clusters were transferred to cultures with GM-CSF we observed that only macrophage-colonies developed. On the other hand, when four day clusters were transferred, both granulocyte and macrophage colony formation was obtained in a similar way as the one obtained when using GM-CSF with fresh BMC. Two day clusters did not respond to granulocyte colony stimulating factor (G-CSF) while fourth day clusters generated granulocytic colonies in a similar way as when G-CSF was used with fresh BMC. In order to test the hypothesis that granulocyte colony formation in these assays could be a result of the secretion of G-CSF by the macrophages previously induced by GM-CSF, lysates from macrophage colonies were used to induce colony formation on BMC. We observed that colonies, mainly granulocytic, were induced in a similar way as when G-CSF was used. Finally, the possibility that GM-CSF is just a macrophage inducer with the property to produce cells that secrete G-CSF is discussed.     

Adhesive knob formation by conidia of the nematophagous fungusDrechmeria coniospora

We studied conidiogenesis and adhesive knob formation (maturation) by newly developed conidia of the nematophagous fungusDrechmeria coniospora. Upon conidiogenesis on infected nematodes or during saprophytic growth of the fungus in axenic cultures compact clusters of conidia developed. Less than 10% of such clustered conidia matured; mature conidia were invariably located on the periphery of the clusters.The kinetics and rate of maturation of conidia were studied inin vitro systems and in soil. In both cases adhesive knobs were formed; the rate at which knobs were formed appeared to be determined by the age of the conidia, the temperature and the soil moisture. In addition, knob formation was suppressed at increasing conidial densities. Under favorable conditions, however, over 90% of the conidia matured within a period of 3 days. The rate of knob formation was neither influenced by the presence of nematodes nor by that of exogenous nutrients, which suggests that maturation is an autonomous process. Electron-microscopical analysis indicated that budding of the conidia at the initial stage of maturation occurred simultaneously with the deposition of the sticky, adhesive layer around the wall of the developing knob.The ecological significance of the time- and spatially separated maturation of conidia after conidiogenesis is discussed with respect to survival of the conidia.     

To the intranucleolar translocation of AgNORs in leukemic early granulocytic and plasmacytic precursors

Early leukemic granulocytic and plasmacytic precursors were studied in vitro and in vivo to provide an information on the intranucleolar distribution of AgNORs (silver stained nucleolus organizer regions). In most of these cells AgNORs appeared as clusters of silver stained particles distributed in the whole nucleolar body. On the other hand, in some leukemic early granulocytic precursors, i.e., in myeloblasts and promyelocytes enlarged AgNORs were translocated in the nucleolar peripheral part. In addition, the number of translocated AgNORs at the nucleolar periphery was significantly smaller. Such translocation of a reduced number of AgNORs was easily produced by experimental aging, i.e., starving of cultured leukemic early granulocytic precursors (HL-60 and K562 cells) in vitro and seems to be reversible. Similar translocation of a reduced number of AgNORs was also produced by aging of leukemic plasmacytic precursors. Thus, the translocation of the reduced number of AgNORs to the nucleolar periphery in some blastic leukemic hematopoietic cells might be an useful marker of their aging at the single cell level. However, more studies in this direction are required in the future.     

Anti-c-myc DNA increases differentiation and decreases colony formation by HL-60 cells

Summary The proto-oncogene c-myc, whose gene product has a role in replication, is overexpressed in the human promyelocytic leukemia HL-60 cell line. Treatment of HL-60 cells with an antisense oligodeoxyribonucleotide complementary to the start codon and the next four codons of c-myc mRNA has previously been observed to inhibit c-myc protein expression and cell proliferation in a sequence-specific, dose-dependent manner. Comparable effects are seen upon treatment of HL-60 cells with dimethylsulfoxide (Me₂SO), which is also know to induce granulocytic differentiation of HL-60 cells. Hence, the effects of antisense oligomers on cellular differentiation were examine and compared with Me₂SO. Differentiation of HL-60 cells into forms with granulocytic characteristics was found to be enhanced in a sequence-specific manner by the anti-c-myc oligomer. No synergism was observed between the anti-c-myc oligomer and Me₂SO in stimulating cellular differentiation. In contrast, synergism did appear in the inhibition of cell proliferation. Finally, the anti-c-myc oligomer uniformly inhibited colony formation in semisolid medium. It is possible that further reduction in the level of c-myc expression by antisense oligomer inhibition may be sufficient to allow terminal granulocytic differentiation and reverse transformation. This work was supported by grants to E. W. from the National Institutes of Health, Bethesda, MD (CA 42960), and the Leukemia Society of America.     

Kinetics of recA function in conjugational recombinant formation.

Summary Genetic recombination was studied in F^- strains of E. coli carrying a mutation (recA200) that confers a thermosensitive Rec^- phenotype. Recombination during Hfr matings at 35C was monitored by raising the temperature of incubation to 42C at various intervals so that only merozygotes that had completed those functions dependent on the activity of the recA gene product could form recombinant progeny. The results indicated that no more than 1–2% of the merozygotes present while mating was in progress were able to form recombinant colonies at 42C. Separation of mating pairs reduced the yield of recombinants obtained at 35C by 50 to 200-fold if plating on agar medium was delayed for 15–30min by continuing incubation in broth medium. recA200 merozygotes that were also recB21 sbcB15 proved relatively stable when plating was delayed in this manner, which suggested that Hfr DNA is prone to exonuclease inactivation in recA200 merozygotes after mating pairs have separated. Post-mating incubation in high salt medium or on agar plates promoted the recovery of recombinants at 35C. However, the majority of recA200 merozygotes did not acquire the ability to form recombinant colonies at 42C under these more stable conditions until mating pairs had been separated and incubation continued at 35C for 40–60 min. It was concluded that recA200 strains are partially defective for recombination even at low temperature but that terminating mating promotes the recovery of recombinants. A mechanism involving the stimulation of RecA activity by mating pair separation is postulated to account for the efficient recovery of recombinants from HfrxF^- recA200 crosses at 35C.     

Studies on colony formation in vitro by mouse bone marrow cells. II. Action of colony stimulating factor

An analysis was made of some of the processes involved in the stimulation by colony stimulating factor (CSF) of cluster and colony formation by mouse bone marrow cells in agar cultures in vitro. Colony formation was shown to be related to the concentration and not the total amount of CSF. The concentration of CSF determined the rate of new cluster initiation in cultures and the rate of growth of individual clusters. Colony growth depleted the medium of CSF suggesting that colony cells may utilise CSF during proliferation. Bone marrow cells incubated in agar in the absence of CSF rapidly died or lost their capacity to proliferate and form clusters or colonies. CSF appears (a) to be necessary for survival of cluster-and colony-forming cells or for survival of their proliferative potential, (b) to shorten the lag period before individual cells commence proliferation and (c) to increase the growth rate of individual clusters and colonies.     

Differences in cluster-root formation and carboxylate exudation in Lupinus albusL. under different nutrient deficiencies

In contrast with the well document role of proteoid root formation and carboxylate exudation in acclimation to P deficiency in white lupin (Lupinus albus L.), their role under other nutrient deficiencies and their ecological significance are still poorly understood. In the present work, differences in proteoid root formation, exudation of carboxylates by root clusters, non-proteoid and proteoid root tips by using a non-destructive method, and concentrations of organic acids in the tissues of plants grown in the absence of P, Fe or K were studied. Proton release from roots increased soon after withdrawing Fe from the medium; within three days the solution pH decreased from 6 to about 4, and this increased release in protons continued until the end of the experiment. Acidification appeared much later, on the 10th day and the 14th day after withdrawal of P and K, respectively; the extent of the acidification was also weaker than under –Fe (5.2 for –P and 5.7 for control on the 10th day; 6.0 for –K and 6.1 for control on the 14th day). Root clusters formed when plants were grown under –P and –Fe, but not under –K conditions. The root clusters developed sooner under –Fe conditions, but the number of clusters was far less than under –P. Under P deficiency, root clusters released mainly citrate, but also some malate; while the major organic acid released by root tips of both non-proteoid and proteoid roots was malate. However, under Fe deficiency, the majority of the organic acids exuded both by the root clusters and root tips was malate, whereas only a small amount of citrate was detected. The release rate of citrate by – P root clusters was greater than that by – Fe root clusters. Moreover, the release rate of malate was greater in –Fe root clusters than in –P root clusters, but the opposite was found in proteoid root tips, i.e. faster in –P than in –Fe proteoid root tips. The significances of proteoid root formation and release of organic acids in acclimation to different nutrient deficiencies for white lupin plants are discussed.     

4. CLIMPSES OF THE NARINA TROGON

Bertram, B. C. R. &; Burger, A. E. 1981. Aspects of incubation in Ostriches. Ostrich 52:36-43.

We studied incubation in domesticated Ostriches Struthio camelus in South Africa and wild Ostriches in Kenya. Although the eggs were large, with relatively high thermal capacities, unattended eggs exposed to the sun reached dangerously high temperatures (40,5°C). Experimental exposure of fresh eggs to the sun for seven days prior to incubation greatly reduced the percentage of embryos which developed, and no embryos survived 15 days of exposure. In the wild. Ostriches frequently shade their eggs in the pre-incubation period to prevent overheating.

During natural incubation, temperatures in the eggs (range 30,8-33,8°C) and of nest-air (31,9-34,6°C) were remarkably constant, despite the daily ambient fluctuations of air temperatures (17,8-38,9°C). Similarly the humidity of the nest-air (39–52%) was lower and less variable than the ambient air (39–72%). Water loss during 42 days of incubation was 11–12% of initial egg weight and, in addition, early laid eggs lost 3–4% during the 2½-3 week pre-incubation period. The water vapour conductivity and the daily water loss of Ostrich eggs were similar to those of other birds, in proportion to epg size, despite the arid environment inhabited by Ostriches. Some of the constraints on the feeding and breeding behaviour of Ostriches imposed by the physical requirements of their eggs are discussed.     

The mechanism of aggregate formation by Selenomonas ruminantium

Summary The mechanism of granule formation by Selenomonas ruminantium was investigated. A basic protein has been isolated from the lysate of S. ruminantium which triggers cluster formation (small aggregates of 20–100 cells) of suspended cells. Evidence is presented that these basic proteins were of ribosomal origin. It is suggested that ribosomes are released into the culture broth by lysis and that the associated basic proteins are subsequently dissociated by high monovalent cation concentrations. It was found that these positively charged basic proteins interact with the negatively charged lipopolysaccharide of the organism to form the clusters. Adding lysate to suspended cells, followed by lowering of the pH from 5.8 to 4.5 also induced clustering. At dilution rates exceeding the maximum growth rate clusters were retained in anaerobic gas-lift reactors and grew into granules (1–3 mm). It is postulated that granules evolve from clusters. Within the clusters, lysis and a low pH are induced due to diffusion limitations. As a consequence dividing cells are entrapped within the clusters, resulting in growth.     

Communal roosting and formation of sleeping clusters in barbary macaques (Macaca sylvanus)

Sleeping cluster composition and distribution were studied in a semi-free-ranging population of 174 Barbary macaques (Macaca sylvanus) in Rocamadour/France. Over a period of 2 months 341 sleeping clusters comprising 754 animals were recorded as animals left the sleeping trees. To control for nocturnal activities five observation periods were conducted, each of which covered a complete night. Sleeping partners were selected from a particular set of individuals. From night to night there was considerable fluctuation among the individuals, which finally formed a sleeping cluster. Preferred size of sleeping clusters was two and three individuals per cluster. The majority of females spent the nights in body contact to infants or female juveniles; whereas males either slept alone or with older male juveniles. Adult males and females never shared a sleeping cluster. During the night the animals showed hardly any locomotive and vocal activity. Sleeping clusters remained stable until dissociation the next morning. © 1992 Wiley-Liss, Inc.     

Internalization of cationized ferritin receptors by rat hepatoma ascites cells.

Cationized ferritin (CF) was used to label the cell surface anionic sites of Chang rat hepatoma ascites cells. If the hepatoma cells were fixed with glutaraldehyde and treated with CF, the label was distributed evenly over the external surface of the plasma membrane. Treatment of unfixed ascites cells with CF yielded clusters of ferritin particles separated by label-free areas of the plasma membrane. Some unfixed ascites cells were treated firstly with CF, then incubated in veronal buffered saline at 37 °C for 10, 20, 30 and 45 min, subsequently fixed in glutaraldehyde and re-exposed to CF. After 10 min of incubation, the label was arranged into large clusters with the remaining areas of the plasma membrane lightly labelled with CF. At 20 min, only clusters of ferritin were present on the plasma membrane; the remaining area of the cell surface was totally free of label. The ability of the plasma membrane to bind additional CF was completely restored after 45 min of incubation. These results suggest that for some period of time after internalization of CF label on cell surface the plasma membrane is devoid of any detectable negative charge.     

Lactate formation by rat small intestine in vitro.

The formation of lactic acid by mucosal slices, rings and muscle from rat jejunum has been studied for periods of up to 8 min. Lactate output by mucosal slices incubated in the absence of glucose was characterised by two phases: a rapid, initial phase of release lasting about 1 min, followed by a much slower phase extending over the remainder of the incubation period. Glucose addition at 30 s initiated a second rapid phase of lactate release into the medium which was again followed by a slower rate of lactate output up to 8 min. The time course of lactate output suggested that there was a negative Pasteur effect in mucosal slices, which could not be reversed by the addition of ADP or glucose 6-phosphate. By contrast, the rate of lactate formation by rings and muscle from rat jejunum increased steadily over the incubation period, indicating a positive Pasteur effect. When Na+ in the incubating medium were replaced by K+, lactate formation by mucosal slices and rings was considerably reduced. Measurements of tissue lactate content before and during incubation revealed that about three-quarters of the lactate released by mucosal slices during the first 30 s of incubation was present initially in the tissue. After the first 30 s the tissue lactate remained constant both in the presence and absence of glucose so that the lactate released into the incubation medium is equivalent to the lactate formed by the slices. The role of the various tissue components of the small intestine in lactate formation is discussed in relation to sites of glucose entry.     

Biological removal of explosive 2,4,6-trinitrotoluene byStenotrophomonas sp. OK-5 in bench-scale bioreactors

The biological removal of 2,4,6-trinitrotoluene (TNT) was studied in a bench-scale bioreactor using a bacterial culture of strain OK-5 originally isolated from soil samples contaminated with TNT. The TNT was completely removed within 4 days of incubation in a 2.5 L benchscale bioreactor containing a newly developed medium. The TNT was catabolized in the presence of different supplemented carbons. Only minimal growth was observed in the killed controls and cultures that only received TNT during the incubation period. This catabolism was affected by the concentration ratio of the substrate to the biomass. The addition of various nitrogen sources produced a delayed effect for the TNT degradation. Tween 80 enhanced the degradation of TNT under these conditions. Two metabolic intermediates were detected and identified as 2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene based on HPLC and GC-MS analyses, respectively. Strain OK-5 was characterized using the BIOLOG system and fatty acid profile produced by a microbial identification system equipped with a Hewlett packard HP 5890 II gas chromatograph. As such, the bacterium was identified as aStenotrophomonas species and designated asStenotrophomonas sp. OK-5.     

In vitro propagation of caper (Capparis spinosa L.)

Summary A twenty fold multiplication per twenty days of caper was achieved by culturing nodal shoot segments in the presence of BAP (4 μM) plus IAA (0.3 μM) and GA₃ (0.3 μM). The use of a modified MS medium facilitated this response. Plantlet regeneration was induced on single shoots taken from proliferating clusters subcultured for 20 days on a reduced BAP (2 μM) without auxin and gibberellin Higher rooting responses (70%) were obtained after a 20-day incubation period in darkness on solid half-strength MS1 medium plus IAA (30 μM), followed by a subsequent 20 day culture period on half-strength MSI basal medium. Proliferation was mainly due to axillary shoot-bud development as revealed by histological studies. The extensive meristematic activities observed indicated the enormous morphological potential of this species.     

Aspergillus flavus colonization and aflatoxin B1 formation in barley grain during interaction with other fungi

Colonization of barley grain by Aspergillus flavus and formation of aflatoxin B₁ in the presence of Penicillium verrucosum, Fusarium sporotrichioides, and Hyphopichia burtonii were studied over a three-week period in all combinations of 20 or 30 °C and 0.97, 0.95 or 0.90 a_w. Grain colonization was assessed initially by observing hyphal extension on the grain surface, using scanning electron microscopy, and then from the proportion of seeds infected and numbers of colony forming units (cfu) formed. Aflatoxin b¹ concentrations were determined by enzyme linked immunosorbent assay using a monoclonal antibody. These studies showed that interaction between A. flavus and other fungi in paired culture had different effects on both colonization and aflatoxin formation depending on the species involved and environmental conditions. Germination of A. flavus spores was unaffected by the presence of other species on the grain surface. Subsequently, three principal patterns of A. flavus colonization of barley grain were observed through the incubation period in the presence of other fungal species: (a) colonization unaffected by the presence of other species; (b) colonization initially slower in the presence of other species but later differing little from pure cultures; and (c) colonization adversely affected by the presence of other species. Five main patterns of aflatoxin B₁ production were observed relative to pure culture but with no consistent relationship with species, a_w, temperature or incubation period; (a) little changed; (b) increased slowly; (c) decreased; (d) enhanced; and (e, f) increased initially but later decreased to (e) the same level as in pure culture or (f) to less than in pure culture. Generally, production of aflatoxin B₁ by A. flavus was less than in pure culture but sometimes was changed only slightly by the presence of P. verrucosum, F. sporotrichioides or H. burtonii or was temporarily enhanced.     

The infection of apples, cv. Bramley's Seedling, by Nectria galligena Bres

In laborator experiments germinating conidia penetrated leticels and wounds but not the intact surfaces of apples. Date of harvest had no significant effect on the numbers of apples infected with Nectria galligena but the earliest picks rotted first in barn store. Inoculations of unpicked apples resulted in small arrested lesions which only developed into progressive rots after a considerable period in store. Rots developed mosy quickly from inoculations made between mid-August and mid-September. The size of rot increased with spore number and many inoculations with 10–100 conidia remained as arrested lesions. Arrested lesions developed 10–15 days after unripe apples were inoculated and consisted of a zone of fungal colonization surrounded by suberized, necrotic cells in which compounds toxic to both N. galligena and Penicillium expansum were detected. No antifungal compounds were found in progressive rots to mature apples or in healthy apples of any age. Antifungal activity, measured by inhibition of P. expansum, was greatest 15–20 days from inoculation of unripe apples with N. galligena but decreased after a total of 35 days incubation at 20 d? C. Much less antifungal activity was produced in ripe or desert apples.     

Analysis of colonies developing in vitro from mouse bone marrow cells stimulated by kidney feeder layers or leukemic serum

An analysis has been made of cell colonies developing in agar cultures from mouse bone marrow cells following stimulation either by neonatal kidney cell feeder layers or AKR lymphoid leukemia serum. Colonies arose by cell proliferation and were mixtures of granulocytic and mononuclear cells. Colonies stimulated by kidney feeder layers reached a mean size of 2000 cells by day 10 of incubation and remained predominantly granulocytic in nature. When bovine serum was substituted for fetal calf serum, cell colonies grew to a smaller size and lost their granulocytic nature, finally becoming almost pure populations of mononuclear cells. Colonies stimulated by AKR leukemic serum reached a mean size of 350 cells by day 10 of incubation. Although these colonies initially were granulocytic in nature, they finally became almost pure populations of mononuclear cells. The colony mononuclear cells actively phagocytosed carbon, and contained metachromatic granules probably derived from ingestion of agar. The mononuclear cells in these colonies may not have been members of the original colony, but may have been incorporated in the colony as it expanded in size, subsequently proliferating in the favourable environment of the colony.     

Chalone inhibition of granulocyte colony growth in agar: kinetic quantitation by capillary tube scanning.

Mouse bone marrow cells were seeded into capillary tubes containing agar with colony stimulating factor. The development of myelomonocytic clusters and colonies was followed by daily tube scanning using their light scattering properties. Three kinetic scanning parameters were determined and the significance of different threshold settings was evaluated; viz. the number of signals, the mean signal height and the signal integrals. The inhibitory effect of two extracts with known granulocyte chalone activity which had been prepared from human peripheral leukocytes and rat bone marrow cells, was followed with the scanning method. A continuous reduction of clusters and colony formation and their growth throughout the incubation period was observed which suggested a sustained retardation of proliferation of both the stem cells committed for myelomonopoiesis and their progeny.     

Effects of temperature, cultivar and isolate on the incubation period of white leaf spot (Mycosphaerella capsellae) on oilseed rape {Brassica napus)

When leaves of oilseed rape (cv. Cobra) were inoculated with conidial suspensions of Mycosphaerella capsellae (white leaf spot) and incubated in controlled environments, the lag period from inoculation to the appearance of the first lesions decreased, and the total number of lesions produced increased, as temperature increased from 5^oC to 20^oC, although differences between 15^oC and 20^oC were small. With incubation period estimated as the time from inoculation until 5%, 50% or 95% of the lesions were produced, there was a linear relationship between l/(incubation period in days) and temperature over the range 5^oC to 20^oC, from which values at intermediate temperatures could be estimated. Summed mean daily temperatures from inoculation to the production of 5% of the lesions were estimated as 115–130 degree-days in the controlled environment experiments at 5^oC to 20^oC. When pods or leaves of plants in oilseed rape crops (cv. Cobra or cv. Libravo) were inoculated with conidial suspensions of M. capsellae on five occasions from January to October, with variable temperatures during the incubation period, degree-days until the first appearance of lesions were in the range 115–230. The numbers of white leaf spot lesions cm^-2 which developed on inoculated leaves differed greatly between nine oilseed rape cultivars, with most on cv. Tapidor and fewest on cv. Libravo, but the incubation period differed little between cultivars. Similarly, the number of lesions which developed differed between four M. capsellae isolates from different regions but the incubation period did not.     

Immunohistochemical study of the postnatal development of pituitary thyrotrophs in the rat,with special reference to cluster formation

Summary The postnatal development of rat pituitary thyrotrophs was investigated immunohistochemically on days 1, 3, 5, 10, 15 and 25. Fetal thyrotrophs are strongly immunoreactive. In the postnatal period, however, weakly immunoreactive thyrotrophs increase in number to constitute clusters on days 3–5. The numbers and dimensions of the clusters reach a maximum on day 10. Thereafter the clusters break down to give rise to single, scattered neogenic thyrotrophs. Thyrotrophs in clusters on day 10 were investigated by electron microscopy in adjacent sections. They can be characterized as an immature type of basophil, according to the classification of Yoshimura et al. (1977): 1) Type I basophils, which are irregularly shaped with elongate processes, and characterized by rows of secretory granules about 100 nm in diameter. 2) Type I/II basophils, i.e., forms intermediate between Types I and II, containing less numerous secretory granules about 100–150 nm in diameter. Type II basophils which correspond to the classical thyrotrophs are not fully developed on day 10. Thus, most thyrotrophs develop from the clusters in the neonatal period. Such neogenic thyrotrophs retain the immature characteristics of Type I and I/II cells and may develop into Type II cells during subsequent maturation.