Qualitative and quantitative immunofluorescence studies of chloroplast ferredoxin

Antibodies were raised in rabbits against 2Fe–2S ferredoxin from N. tabacum L. The antibodies showed partial cross-reactivity in the double diffusion test with ferredoxins from Spinacia oleracea L., Petunia inflata Fries., P. axillaris Lam., Phaseolus vulgaris L., Chlamydomonas remhardii Dang. A complete cross-reaction was observed with ferredoxins from five other Nicotiana species, thus with this test it was impossible to discriminate between these ferredoxins. Therefore the following test was performed. Heterologous ferredoxin (i.e., ferredoxin other than from N. tabacum) was coupled covalently to Sepharose beads. Rabbit anti-N. tabacum-serum was then pre-incubated with this ferredoxin which resulted in complete abolition of cross-reactivity with free heterologous ferredoxin. However, the serum retained antibody activity against specific antigenic determinants of N. tabacum ferredoxin. When this serum was tested against ferredoxin purified from the hybrid: N. tabacum ()xN. glutinosa () it gave a positive reaction. The relative content of maternal N. tabacum ferredoxin in the hybrid was estimated by using a fluorescent derivative of this specific antibody and estimating the cross-reactivity compared with that of artificial mixtures of pure N. tabacum and N. glutinosa ferredoxins. The hybrid contained 50% of maternal ferredoxin. This technique was also applied to ferredoxins of other species of Nicotiana and to the ferredoxin from the hybrid N. clevelandii ()xN. glutinosa (). We conclude that it provides a good test system for the study of the expression of chloroplast ferredoxin in Nicotiana hybrids in general.Abbreviations PBS phosphate buffered saline - FITC fluorescein isothiocyanate - S.E.M. standard error of means     

Conditions for a high plating efficiency of free cell suspensions of Haplopappus gracilis (Nutt) gray

Summary Suspensions of Haplopappus gracilis cells, containing about 80% free cells, were obt ained from log-phase cultures by filtration through 3 nylon sieves having decreasing mesh widths from 297, 210 and 88 m. From the free cell suspensions, 75 to 90% of the cells developed into visible colonies when the plating procedure was divided into two steps: a) plating the cells at high concentration in soft agar on feeder agar; b) replating the resulting aggregations at appropriate concentrations on fresh feeder agar. From the results, it is inferred that, in the replating step, the volume of the inoculum is the deciding factor which influences the resulting plating efficiency.     

Angiotensin-like activity in resistance vessels

Summary Angiotensin II (ANG II) was localized immunocytochemically in kidney and various other organs of the chinese hamster. In the kidney ANG II-like activity was found in the epitheloid cells of the juxtaglomerular apparatus as well as in the media i.e. the smooth muscle cells of arcuate and interlobular arteries and afferent arterioles. ANG II-like activity was also observed in the medial muscle cells of resistance vessels in other organs and tissues such as submandibular gland and brown adipose tissue. The site of synthesis of ANG II needs to be investigated but the data point to the possibility of an intracellular function of ANG II in smooth muscle cells of blood vessels.These studies were supported by the Deutsche Forschungsgemeinschaft within the SFB 90 Cardiovasculäres System     

Heterogeneity in 5′-nucleotidase activity of mouse peritoneal macrophages

Summary After stimulation of the mouse peritoneal cavity with newborn calf serum (NBCS), four types of monocyte and macrophage were distinguished on the basis of peroxidase (PO) patterns. Cytochemically, these cells showed strong heterogeneity in 5-nucleotidase (5N) activity. Monocytes and monocyte-derived macrophages with PO activity in granules lacked 5N activity. Resident macrophages (with PO activity in RER and nuclear envelope) generally had significant 5N activity on the plasma membrane, the pattern showing close correlation with the biochemical findings. The group of PO-negative macrophages comprised both 5N-negative and 5N-positive cells. These findings suggest two possibilities, i.e., that monocytes (5N-)transform via PO-negative cells (5N-/+) into resident macrophages (5N+), or that the monocytes and monocyte-derived macrophages and the resident macrophages represent separate lineages. The fourth type of macrophage, the exudate-resident cell (with PO activity both in granules and in the RER and nuclear envelope), occurred only in low numbers and very late after NBCS stimulation, and is therefore considered not to be a transitional cell between monocytes and resident macrophages.     

Differentiation under the control of insulin of rat preadipocytes in primary culture: Isolation of homogeneous cellular fractions by gradient centrifugation

Using a density gradient medium (Percoll) we succeeded in isolating homogeneous cell populations from the stromal-vascular fraction of the inguinal tissue of 3-day-old rats. In primary culture, in medium 199 supplemented with 10% fetal calf serum and 5.5 mM glucose, almost complete differentiation (90%) of these fractions was obtained for the first time in presence of a physiological concentration of insulin (10^?9 M). During the adipose conversion, insulin markedly enhanced the activities of glycerol-3-phosphate dehydrogenase and acid:CoA ligase. When VLDL and heparin were added with insulin to the medium, this effect was not potentiated. On the contrary, VLDL and heparin in presence of insulin increased the triglyceride content of the cells. With VLDL and heparin only, the biochemical and morphological characteristics of the cells were very similar to those observed in control culture. The heavier fraction was morphologically heterogeneous and did not undergo the adipose conversion to the same extent as the two lighter fractions. It was concluded that this model could be helpful in studying the proliferation and the differentiation of preadipocytes at an early stage of development.     

Biochemical and functional comparison of DNA polymerases alpha, delta, and epsilon from calf thymus.

DNA polymerase alpha, delta and epsilon can be isolated simultaneously from calf thymus. DNA polymerase delta was purified to apparent homogeneity by a four-column procedure including DEAE-Sephacel, phenyl-Sepharose, phosphocellulose, and hydroxylapatite, yielding two polypeptides of 125 and 48 kDa, respectively. On hydroxylapatite DNA polymerase delta can completely be separated from DNA polymerase epsilon. By KCl DNA polymerase delta is eluted first, while addition of potassium phosphate elutes DNA polymerase epsilon. DNA polymerases delta and epsilon could be distinguished from DNA polymerase alpha by their (i) resistance to the monoclonal antibody SJK 132-20, (ii) relative resistance to N2-[p-(n-butyl)phenyl]-2-deoxyguanosine triphosphate and 2-[p-(n-butyl)anilino]-2-deoxyadenosine triphosphate, (iii) presence of a 3'----5' exonuclease, (iv) polypeptide composition, (v) template requirements, (vi) processivities on the homopolymer poly(dA)/oligo(dT12-18), and (vii) lack of primase. The following differences of DNA polymerase delta to DNA polymerase epsilon were evident: (i) the independence of DNA polymerase epsilon to proliferating cell nuclear antigen for processivity, (ii) utilization of deoxy- and ribonucleotide primers, (iii) template requirements in the absence of proliferating cell nuclear antigen, (iv) mode of elution from hydroxylapatite, and (v) sensitivity to d2TTP and to dimethyl sulfoxide. Both enzymes contain a 3'----5' exonuclease, but are devoid of endonuclease, RNase H, DNA helicase, DNA dependent ATPase, DNA primase, and poly(ADP-ribose) polymerase. DNA polymerase delta is 100-150 fold dependent on proliferating cell nuclear antigen for activity and processivity on poly(dA)/oligo(dT12-18) at base ratios between 1:1 to 100:1. The activity of DNA polymerase delta requires an acidic pH of 6.5 and is also found on poly(dT)/oligo(dA12-18) and on poly(dT)/oligo(A12-18) but not on 10 other templates tested. All three DNA polymerases can be classified according to the revised nomenclature for eukaryotic DNA polymerases (Burgers, P.M. J., Bambara, R. A., Campbell, J. L., Chang, L. M. S., Downey, K. M., Hübscher, U., Lee, M. Y. W. T., Linn, S. M., So, A. G., and Spadari, S. (1990) Eur. J. Biochem. 191, 617-618).     

Influence of the nontarget mollusc Marisa cornuarietis on the hourly cercarial production of Schistosoma mansoni from Biomphalaria glabrata

A comparative study of hourly cercarial productivities of Schistosoma mansoni from infected Biomphalaria glabrata was carried out in the presence of either healthy B. glabrata (control) or healthy Marisa cornuarietis (experimental). The results showed that, with M. cornuarietis, almost all the hourly cercarial productivities increased by a factor varying from 1.3 to 2.5 without modification of the shedding period.