Peak Cell Density Research Articles

The effects of oncogene transfection on growth and antibody production of human-mouse heterohybridomas

Human-mouse heterohybridomas producing human monoclonal antibodies show slower growth rates and lower peak cell densities than murine hybridomas. In order to improve the growth properties we transfected a heterohybridoma cell line with expression plasmids containing the oncogenes v-src, c-Ha-ras and SV40largeT. The plasmids were transferred by electroporation. Growth promoting activities of the plasmids were proven in NIH3T3 cells whereby a doubling of the maximum cell densities of this cell type was observed. The oncogene products were analyzed by means of northern blotting and immunofluorescence. After transfection of c-src and c-ras, a heterohybridoma cell line was derived which showed improved growth characteristics compared to the original cell line. Although specific antibody production was lower, antibody concentrations which accumulated in batch culture were higher due to increased maximum cell densities.

Neuropeptide Y-immunoreactive neurons in the retina of two Australian lizards.

Wholemounts and sectioned retina from adults of two lizard species, Pogona vitticeps and Varanus gouldii, were studied by immunohistochemistry for neuropeptide Y (NPY)-like immunoreactivity. In both species the morphology of two classes of amacrine cells (types A and B) were described. Cell somata were located mostly in the inner nuclear layer (INL) but were occasionally displaced into the ganglion cell layer (GCL). In the Pogona retina, type A cells had large somata and dendritic arbor that branched in sublamina (S) 1 and 2/3 of the inner plexiform layer (IPL). Type B amacrine cells had smaller somata and dendritic arbor branching mostly in S5 of the IPL. In the Varanus retina, the levels of dendritic branching of types A and B amacrine cells in the IPL were similar to those in Pogona although branching in the middle of the IPL occurred at S3. NPY-immunoreactive cells with small somata and narrow to medium sized dendritic fields were predominant. Unclassified cells also displayed NPY-like immunoreactivity; however, their dendritic morphology could not be determined due to the faint and inconsistent staining. In transverse retinal sections three bands of NPY-like immunoreactivity were evident in the IPL of both species, to which the unclassified cells also contributed. In both species type A cells were most numerous. Total NPY-immunoreactive cells were estimated to be 8,600 in Pogona and 32,860 in Varanus. In both species types A and B cells were non-uniformly distributed across the retina. The most apparent non-uniformity in distribution was observed in type A cells in Varanus. Peak cell density was found across the horizontal meridian of the retina from where cell density decreased towards the dorsal and ventral retinal margins. The results of this study provide evidence for the presence of NPY-immunoreactive amacrine cells in the lizard retina of which two types were morphologically characterized. Cross-species comparisons were also made among NPY-immunoreactive amacrine cells, and their possible function/s discussed.

Large serotonin-like immunoreactive amacrine cells in the retina of developing Xenopus laevis

The earliest appearance of serotonin-like immunoreactivity (SLI) in different cell types and the development of large SLI amacrine cells were studied in the retina of Xenopus laevis from stage 33/34 to adult. Intense SLI was first found in the somas of large amacrine cells at stage 39. The somas of small amacrine cells showed weak SLI at stage 41, followed by bipolar cells at stages 43. The number of large SLI amacrine cells in the inner nuclear layer of the retina increased from 57 at stage 40 to 774 in adult. Over the same period, retinal area increased from 0.19 mm 2 to 24.57 mm 2 with an accompanying decrease of cell density from 301/mm 2 to 32/mm 2. In adult animals large SLI amacrine cells were non-uniformly distributed. Peak cell density of 50–60/mm 2 was located in the center of the ventrotemporal quadrant and a trough of 8–15/mm 2 in the dorsal periphery of the retina. Peak cell density region of the adult retina correspond to part of the retina formed at early developmental stages where the rate of cell generation of large SLI amacrine cells was higher. These observations indicate that (1) SLI is expressed first by large amacrine cells, followed by small amacrine and bipolar cells; (2) large SLI amacrine cells are generated continuously throughout life, (3) the non-uniform retinal distribution of large cells results from a spatio-temporally differential cell generation at the ciliary margin.

Peak density, size and regional distribution of ganglion cells in the retina of the fur seal Callorhinus ursinus.

The total number, size, topographic distribution and peak density of ganglion cells were studied in retinal wholemounts of the fur seal, Callorhinus ursinus. The cell distribution showed a distinct zone of high ganglion cell density. It was located in the temporal retinal quadrant, near the horizontal meridian, 10-12 mm (25-31 degrees) from the optic disk. The peak cell density in this zone was 812-1332 cells/mm2 (mean 1053 cells/mm2), i.e. 125-205 cells/deg2 (mean 162 cells/deg2). These data predict a retinal resolution of 5.6-7.1 cycle/deg. The ganglion cell soma size ranged from 10 to 50 microns. Cell size histograms were bimodal in shape with modes below and above 30 microns.

Retinal topography in the koala (Phascolarctos cinereus).

Nissl-stained retinal wholemounts were used to investigate the topographical organization of the ganglion cell layer of the koala (Phascolarctos cinereus); the visual resolution limit of this animal was subsequently estimated from retinal ganglion cell density data. Two types of cells could be differentiated on the basis of their size and staining characteristics: a subpopulation of presumed ganglion cells, consisting of medium to large cells with Nissl substance in the cytoplasm and pale uniformly staining nuclei, and a further subpopulation of small, densely staining cells. The latter group were presumed to be neuroglia and displaced amacrine cells. Iso-density contour maps were prepared from total cell counts and also counts of presumed ganglion cells; in all cases, the density of cells was greatest in the inferior retina where there was an area of peak density occurring as a poorly developed, horizontal streak that extended across the inferior retina. The inferior position of the streak in the koala contrasts with reports of the superior position of streaks in other marsupials. Peak cell densities of 2370 cells/mm2 and 1480 cells/mm2 were recorded for the total cell population and the presumed ganglion cell subpopulation, respectively. The latter value is equivalent to a visual resolution of 2.4 cycles/degree, based on sampling theory and a square packing paradigm, placing the koala close in visual performance to two other marsupials, the Australian Northern native cat and the American Virginia opossum.

Human urine stimulates growth of Leishmania in vitro

Enhancement of trypanosomatid metacyclogenesis by insect urine components led us to test the effect of human urine as a culture additive. The addition of 1–5% urine to Schneider's Drosophila medium containing 10% foetal calf serum enhanced the growth of 11 Leishmania strains representing 8 different taxonomic groups. Cell division was stimulated in cultures with non-dividing organisms. Peak cell density was increased, as was the efficiency with which L. donovani could be isolated from infected hamsters. Preliminary work suggested that the modified medium would be useful for field isolation of L. donovani and L. braziliensis. The active nutrients or growth factors are not known.

Enhanced growth characteristics of hybridoma cells infected with myc- and ras-containing retroviruses

The introduction of retrovirally encoded myc or ras genes into the mouse hybridoma cell line PQXB 1 2 , in which antibody production is unstable, resulted in altered growth characteristics: infected cells showed increased growth rates, higher peak cell densities, and slower decline of viability following maximum density than did control cultures. In addition, some oncogeneinfected cultures maintained initial levels of antibody production during 16 weeks in culture, while antibody levels fell by 90% in uninfected PQXB cells. Both myc and ras oncogenes were expressed at only low levels in control and infected cells. These results suggest that oncogene expression in hybridomas can lead to enhanced growth characteristics and can stabilize antibody production.

Distribution and size of ganglion cells in the retinae of large Amazon rodents

The topographical distribution of density and soma size of the retinal ganglion cells were studied in three species of hystricomorph rodents. Flat-mounted retinae were stained by the Nissl method and the ganglion cells counted on a matrix covering the whole retinae. Soma size was determined for samples at different retinal regions. The agouti, a diurnal rodent, shows a well-developed visual streak, reaching a peak density of 6250 ganglion cells/mm2. The total number of ganglion cells ranged from 477,427-548,205 in eight retinae. The ganglion-cell-size histogram of the visual streak region exhibits a marked shift towards smaller values when compared to retinal periphery. Upper and lower regions differ in both cell density and cell size. The crepuscular capybara shows a less-developed visual streak with a peak ganglion cell density of 2250/mm2. The shift towards small-sized cells in the visual streak is less marked. Total ganglion cell population is 368,840. In the nocturnal paca, the upper half of the fundus oculi includes a tapetum lucidum. The retina of this species shows the least-developed visual streak of this group, with the lowest peak ganglion cell density reaching 925/mm2. The total ganglion cell number (230,804) is also smaller than in the two other species. Soma-size spectra of this species are characterized by the presence, in the lower hemi-retina, of very large perikarya comparable in size to the cat's alpha ganglion cells.

Quantitative analysis of the retinal ganglion cell layer and optic nerve of the barn owl Tyto alba.

The visual capacity of the common barn owl (Tyto alba) was studied by quantitative analysis of the retina and optic nerve. Cell counts in the ganglion cell layer of the whole-mounted retina revealed a temporal area centralis with peak cell density of 12,500 cells/mm2 and a horizontal streak of high cell density extending from the area centralis into the nasal retina. Integration of the ganglion cell density map gave an estimated total of 1.4 million cells for the ganglion cell layer. Electron microscopy of a single, complete section of the optic nerve revealed a bimodal fiber diameter spectrum (modes at 0.3 and 0.9 microns; bin width = 0.2 microns), with diameters ranging from 0.15 microns (unmyelinated) to 6.05 microns (myelinated, sheath included). The total axon count for the optic nerve was estimated from sample counts to be about 680,000 axons (25% unmyelinated). Therefore, roughly half of the cells in the retinal ganglion cell layer do not send axons into the optic nerve. With certain assumptions, the data predict a visual spatial acuity for barn owls on the order of 8 cycles/degree, a value similar to the known behaviorally measured acuities of masked owls (10 cycles/degree) and domestic cats (6 cycles/degree).

Visual acuity of the northern native cat ( Dasyurus hallucatus) - Behavioural and anatomical estimates

Behavioural estimates of the visual acuity of the Northern Native Cat (or Northern Quoll) - Dasyurus hallucatus - were made using the Mitchell jumping stand technique. A value of 2.3–2.8 cycles per degree was obtained. This functional acuity compared well with predictions based on the peak ganglion cell density (2600 cells/mm 2) determined from the retinal ganglion cell density map.

Hybridoma growth and antibody secretion in serum-supplemented and low protein serum-free media

Hybridoma cell growth and monoclonal antibody secretion were studied with three murine hybridomas, SS1.1, NS6.3 and 455, propagated in four low protein serum-free media and various serum-supplemented media. Cell metabolism, as indicated by cell growth and glucose uptake, and antibody production rates were measured. The analysis focused on the secretion of monoclonal antibodies as a function of the medium makeup. Although cell densities achieved were generally higher for serum-supplemented media, glucose uptake rates did not vary significantly, and antibody secretion rates measured at peak cell density were higher for serum-free media with all three hybridomas. Decreasing the serum concentration had opposite effects for the IgM and IgG secretors, NS6.3 and SS1.1; secretion rates measured at peak cell density were higher for the former and lower for the latter.

Distribution of ganglion cells in the retina of adult pigmented ferret

The retinal ganglion cell distribution in adult pigmented ferret was mapped in Nissl-stained retinas and in retinas back-filled with HRP after large bilateral injections of the enzyme into the brain. In common with other carnivores the ferret has an area of peak cell density equivalent to the area centralis and a prominent visual streak of high cell density extending horizontally across the retina. The maximum ganglion cell densities for the retinas were estimated to be 3500–5200 cells/mm 2 in Nissl-stained, dehydrated retinas and 3300–4300 cells/mm 2 in HRP-labelled, undehydrated retinas. Three cell types were distinguished in the HRP-labelled retinas and they appear to correspond to alpha-, beta- and gamma-cell types of cat retina. However, unlike in cat, the retinal ganglion cells of the ferret do not consistently fall into 3 distinct groups with respect to cell size, nor is there a tendency for the cells in the area centralis to be smaller than those in the peripheral retina. Estimates for the total number of ganglion cells of 82,000 and 88,000 were obtained from Nissl-stained retinas, and of 74,000, 75,000 and 78,000 from HRP-labelled retinas.

Density, soma size, and regional distribution of rabbit retinal ganglion cells

Cell soma area or diameter distributions are generally very skewed and present some unusual problems in characterization and description. In this study of ganglion cell soma size in rabbit retina, our conclusions are based on a statistical method which not only characterizes this particular neuronal population but which also may be of considerable value in other species and other parts of the nervous system. To facilitate comparisons between retinas, we used ganglion cell density as the measure of retinal location. The rabbit retina has a horizontally extended area centralis, the visual streak, which we show to have a uniformly high peak cell density along most of its length. Cell density maps were used to estimate the total number of ganglion cells in the retina; the mean for three retinas was about 406,000 cells, which corresponds well to an earlier count of optic nerve axons (394,000 +/- 20,000; Vaney, D.I., and A. Hughes (1976) J. Comp. Neurol. 170: 241-252). Contrary to other reports, we could not find any large differences in ganglion cell size distributions between the inferior peripheral retina and the visual streak nor could we confirm the report of a large cell area temporalis (Provis, J. M. (1979) J. Comp. Neurol. 185: 121-138). Cell size distributions in the superior and inferior retina were very different, however, and, within the inferior retina, there was a small but systematic change in cell size between the periphery and the visual streak. In general, small and medium size cells were present in nearly constant proportions throughout the inferior retina, while the large ganglion cells showed a small decrease in proportion from the periphery to the visual streak; the decline in large cells was a linear function of cell density. In terms of ganglion cell soma size, there was no sharp distinction between the visual streak and the peripheral retina.