Development of 7--11-week-old human embryonic gonads in organ culture]

The author suggests a simplified method of organic culture of the human gonads. Gonads (160) from 7--11-week embryos were cultivated on millipore filters in penicillin bottles. During the first week of cultivation the central necroses were observed both in the male and the female gonads, but later they disappeared in some of the explants. In the ovaries from the 9--11-week embryos the germ cells underwent development which continued to the pachytene stage, while in the ovaries from the 7--9-week embryos the development of the germ cells failed to reach the leptotene stage. In the case of testis germ cells they remained at the stage of spermatogonia.     

Bone formation in organ cultures of bone marrow

Summary Bone formation in organ cultures of intact marrow fragments from mouse is described. Marrow explants were cultured on the top surface of a millipore filter at a gas-liquid interface. Observations with both light- and electron microscopes demonstrated the formation of a well-organised trabecular matrix lined with osteoblast-like cells. The tissue and cells were positive for alkaline-phosphatase activity. Large amounts of thick, well-banded collagen fibrils and matrix vesicles typical of those found in bone were present. The tissue became mineralised in the presence of 10 mM Na--glycerophosphate; in its absence a similar trabecular matrix developed but mineralisation did not take place.     

Genetic factors influencing bystander signaling in murine bladder epithelium after low-dose irradiation in vivo

Radiation-induced bystander effects occur in cells that are not directly hit by radiation tracks but that receive signals from hit cells. They are well-documented in vitro consequences of low-dose exposure, but their relevance to in vivo radiobiology is not established. To investigate the in vivo production of bystander signals, bladder explants were established from two strains of mice known to differ significantly in both short-term and long-term radiation responses. These were investigated for the ability of 0.5 Gy total-body irradiation in vivo to induce production of bystander signals in bladder epithelium. The studies demonstrate that irradiated C57BL/6 mice, but not CBA/Ca mice, produce bystander signals that induce apoptosis and reduce clonogenic survival in reporter HPV-G-transfected keratinocytes. Transfer of medium from explants established from irradiated animals to explants established from unirradiated animals confirmed these differences in bladder epithelium. The responses to the in vivo-generated bystander signal exhibit genotypic differences in calcium signaling and also in signaling pathways indicative of a major role for the balance of pro-apoptosis and anti-apoptosis proteins in determining the overall response. The results clearly demonstrate the in vivo induction of bystander signals that are strongly influenced by genetic factors and have implications for radiation protection, medical imaging, and radiotherapy.     

Role of distant lymphoid cell interactions for the development of in vitro antibody formation

In order to clarify the nature of the recently described intercellular local interactions which were shown to inhibit the antibody-forming cells (AFC) proliferation in vitro, a possibility of realization of this effect at a distance was studied. It was shown that the population with a great number of cells could inhibit in vitro at a distance an increase of the AFC in the population of cells separated from the former by a millipore filter, impermeable for the cells. The results were the same with the use of polymethylmetacrylate film (5 to 10 micrometer in thickness) impermeable for proteins with a molecular weight of 150000 daltons (125I-IgG-antibodies) and some ions (51CrO4), but permeable for other substances with low molecular weight.     

The role of external tensions in differentiation of Xenopus laevis embryonic tissues

Explants extirpated from Xenopus laevis embryos at the early gastrula stage were placed on pieces of hydrophilized latex film which were then either stretched or remained intact. In explants cultivated on the intact films most cells emigrated out of the explants and remained undifferentiated, whereas the explants on the films stretched for 10 min or more developed a normal set of rudiments. In the explants of suprablastoporal zone stretched perpendicularly to the cranio-caudal direction, the axial organs were oriented in the direction of stretching. In the stretched explants, unlike the intact ones, a system of microfilament-associated intercellular contacts was formed within a few minutes.     

Terbium as a solid-state probe for RNA

This paper continues previous work on the analysis of nucleic acid-terbium complexes in the solid state. The fluorescence excitation and emission spectra of the RNA-terbium(III) complex is reported. The fluorescence excitation and emission spectra of both the RNA-terbium(III) and DNA-terbium(III) complexes as trapped on millipore filters is reported. One hundred percent of the DNA combined with terbium was trapped on millipore filters. Deoxyribonucleic acid was recovered from DNA-terbium(III) complexes trapped on millipore filters using SDS-extraction. Energy transfer was shown to occur from the bases in nucleic acids to the terbium ion, whereas the actual binding of terbium to nucleic acids was due to phosphate groups. The relative fluorescence of homopolyribonucleotide-terbium complexes showed that the guanine moiety was responsible for most of the observed fluorescence. Binding studies showed an equal affinity of radioactive terbium for all the homopolyribonucleotides. The fluorescence of solid-state DNA and RNA terbium complexes was used to measure picomole quantities of DNA or RNA.     

Two-stage carcinogenesis induced by foreign bodies

Replacement of non-perforated polyvinylchloride films 3.5 months after implantation to CBA mice by perforated polyvinylchloride films or by millipore filters with 0.45 micron pores led to the development of sarcoma at the site of implantation in 37.0 and 21.4% of cases, respectively. The implantation of only perforated films led to the development of sarcoma in 6.9% of cases, while the implantation of filters failed to induce tumour growth. The data obtained show that the latent period of tumour development can be divided into two stages. The first stage occurs in the presence of non-perforated film, while the second stage ensues in the presence of non-cancerogenic films. Complete removal of films 6 months after implantation prevented tumour development. It is suggested that non-perforated films serve as initiating agents, while non-cancerogenic films act as protectors.     

Osteoblast-osteoclast interaction in bone resorption. Preliminary results

Osteoclastic bone resorption has been evaluated in vitro by release of tritiated collagen fragments from 3H-proline prelabeled bone particles incubated for 48 hours in presence of avian isolated osteoclasts. Cells were co-incubated with periosteum-free chick calvarial fragments by interposition of 0.4 micron millipore membrane transwells, in presence or absence of 10(-8) M 1.34 bovine parathyroid hormone (PTH). Results demonstrated that i) calvaria exert a stimulating effect over osteoclastic bone resorption which was 1.8 fold enhanced with respect to controls (p less than 0.001). ii) the stimulating effect is exerted by calvarium-derived soluble molecules capable of crossing the 0.4 micron millipore membrane interposed between calvarial fragments and osteoclasts, iii) in this experimental system no further enhancement of calvarial stimulating effect is operated by PTH treatment.     

A bicellular mechanism in the immune response to chemically defined antigens. 3. Interaction of thymus and bone marrow-derived cells

The role of thymus and bone marrow-derived cells in the in vitro response to the dinitrophenyl (DNP) determinant was studied using the millipore filter well technique for spleen organ cultures. Antibodies to DNP were assayed by the technique of inactivation of DNP-coupled T-4 bacteriophage. It was found that spleens of mice total-body irradiated at 750 R, treated with bone marrow and thymus cells after exposure and immunized against rabbit serum albumin (RSA) were able to produce antibodies to DNP when challenged in vitro with DNP-RSA. Such a response was not produced by spleen explants from x-irradiated mice treated with either thymus or bone marrow cells. Neither were antibodies to DNP produced by spleens of animals repopulated with thymus and bone marrow cells, but not immunized with the carrier. This carrier effect was manifested when the irradiated mice were treated with RSA and thymus cells 6–8 days before administration of the bone marrow cells. Yet, such an effect was not observed when the RSA and bone marrow cells were given 6–8 days before injection of the thymus cells. Thus, the thymus-derived cells appear to play the role of cells sensitive to the carrier (RSA), whereas the bone marrow seems to be involved in the production of antibodies.     

Development and characterization of cell culture systems from Puntius (Tor) chelynoides (McClelland)

Puntius (Tor) chelynoides, commonly known as dark mahseer, is a commercially important coldwater fish species which inhabits fast-flowing hill-streams of India and Nepal. Cell culture systems were developed from eye, fin, heart and swim bladder tissues of P. chelynoides using explant method. The cell culture system developed from eye has been maintained towards a continuous cell line designated as PCE. The cells were grown in 25cm(2) tissue culture flasks with Leibovitz' L-15 media supplemented with 20 % fetal bovine serum (FBS) at 24°C. The PCE cell line consists of predominantly fibroblast-like cells and showed high plating efficiency. The monolayer formed from the fin and heart explants were comprised of epithelial as well as fibroblast-like cells, a prominent and rhythmic heartbeat was also observed in heart explants. Monolayer formed from swim bladder explants showed the morphology of fibroblast-like cells. All the cells from different tissues are able to grow at an optimum temperature of 24°C and growth rate increased as the FBS concentration increased. The PCE cell line was characterized using amplification of mitochondrial cytochrome oxidase subunit I (COI) & 16S rRNA genes which confirmed that the cell line originated from P. chelynoides. Cytogenetic analysis of PCE cell line and cells from fin revealed a diploid count of 100 chromosomes. Upon transfection with pEGFP-C1 plasmid, bright fluorescent signals were observed, suggesting that this cell line can be used for transgenic and genetic manipulation studies. Further, genotoxicity assessment of PCE cells illustrated the utility of this cell line as an in vitro model for aquatic toxicological studies. The PCE cell line was successfully cryopreserved and revived at different passage levels. The cell line and culture systems are being maintained to develop continuous cell lines for further studies.     

Grouping of RNA Phages by a Millipore Filtration Method

Twenty-five strains of RNA phages were tested for their filtration and elution patterns (F-E patterns) by a millipore filtration method. These strains, including MS2, f2 and R17, were separated in three groups in this aspect. We named these groups as group I, II and III. According to this grouping, MS2, f2 and R17 belonged to group I and Qβ belonged to group III. Furthermore, it was shown that group III was further divided in two sub-groups (IIIa and IIIb) by this method. Grouping based on the F-E patterns was in extremely good accordance with the grouping based on the serological properties. This grouping was also supported by the results of chemical and physical analyses of these RNA phages, that is, RNA phages which belonged to the same group had several common properties. Basic Information on the millipore filtration method was also presented in this paper.     

Diffusable growth factors induce bladder smooth muscle differentiation

Bladder smooth muscle differentiation is dependent on the presence of bladder epithelium. Previously, we have shown that direct contact between the epithelium and bladder mesenchyme (BLM) is necessary for this interaction. In this study, we tested the hypothesis that bladder smooth muscle can be induced via diffusable growth factors. Fourteen-day embryonic rat bladders were separated into bladder mesenchyme (prior to smooth muscle differentiation) and epithelium by enzymatic digestion and microdissection. Six in vitro experiments were performed with either direct cellular contact or no contact (1) 14-d embryonic bladder mesenchyme (BLM) alone (control), (Contact) (2) 14-d embryonic bladders intact (control), (3) 14-d embryonic bladder mesenchyme combined with BPH-1 cells (an epithelial prostate cell line) in direct contact, (4) 14-d embryonic bladder mesenchyme with recombined bladder epithelium (BLE) in direct contact, (No Contact) (5) 14-d embryonic bladder mesenchyme with BPH-1 prostatic epithelial cells cocultured in type 1 collagen gel on the bottom of the well, and (6) 14-d embryonic bladder mesenchyme with BPH-1 epithelium cultured in a monolayer on a transwell filter. In each case the bladder tissue was cultured on Millicell-CM 0.4-microm membranes for 7 d in plastic wells using serum free medium. Growth was assessed by observing the size of the bladder organoids in histologic cross section as well as the vertical height obtained in vitro. Immunohistochemical analysis of the tissue explants was performed to assess cellular differentiation with markers for smooth muscle alpha-actin and pancytokeratin to detect epithelial cells. Control (1) bladder mesenchyme grown alone did not exhibit growth or smooth muscle and epithelial differentiation. Contact experiments (2) intact embryonic bladder, (3) embryonic bladder mesenchyme recombined with BPH-1 cells, and (4) embryonic bladder mesenchyme recombined with urothelium each exhibited excellent growth and bladder smooth muscle and epithelial differentiation. Both noncontact experiments (5) and (6) exhibited growth as well as bladder smooth muscle and epithelial differentiation but to a subjectively lesser degree than the contact experiments. Direct contact of the epithelium with bladder mesenchyme provides the optimal environment for growth and smooth muscle differentiation. Smooth muscle growth and differentiation can also occur without direct cell to cell contact and is not specific to urothelium. This data supports the hypothesis that epithelium produces diffusable growth factors that induce bladder smooth muscle.     

Removal of endotoxin from antibody preparations for clinical use

Cell Biochemistry and Biophysics - Despite attempts to maintain asepsis, good manufacturing practices, and the use of terminal sterilization by millipore filtration, the nuclear practitioner is...     

Scanning Electron Microscopy of Cell-Surface Changes in Methylnitrosurea (MNU)-Treated Rat Bladders in vivo and in vitro

Scanning electron microscopy has been used (1) to characterize epithelial cells of bladders from normal rats and from rats treated with a single initiating but non-carcinogenic dose of 2 mg methylnitrosurea (MNU), 24 h and 6 weeks after treatment; and (2) to compare morphological aspects of epithelial differentiation in organ culture of bladder explants taken from untreated and MNU-treated rats at these time intervals.
There are marked differences in vivo between the surface organization of normal urothelium and urothelium undergoing reversible hyperplasia following MNU treatment. Maturation of the normal rat bladder epithelium in vivo is shown to be related to a series of well-defined cell-surface changes readily identified by SEM. By contrast the maturation response is perturbed in the hyperplastic epithelium; the cells lose their ability to differentiate normally and form instead an excess of stubby globular microvilli which project from the cell surface.
In organ culture, maturation of normal bladder epithelium (both in re-epithelialized areas of the explant and in areas of epithelial outgrowth over cellulose acetate substrates) can be also related to a series of cell surface changes showing close similarities to those in vivo. However, epithelial maturation remains defective in organ cultures of bladders from MNU-treated animals. The closely parallel behaviour of the bladder epithelium in vivo and in vitro in both normal and treated tissues underlines the potential value of the bladder organ culture system for studying the comparative biology of hyperplastic development produced by a single initiating dose of MNU and suggests it will be useful with which to study carcinogenesis following multiple doses of MNU.     

3-dimensional growth (spheroid formation) of tumor cells in diffusion chambers]

The three-dimension growth -- spheroidoformation of tumor cells of the primary MX-induced sarcoma in mice BALB/c, C57BL, and C3H/Sn, was studied. The tumor cells were cultured on millipore filters of the diffusion chambers implanted into the peritoneal cavity of normal syngeneic mice.     

A millipore--blue dextran affinity binding system.

Blue dextran has been adsorbed to millipore filter discs in a linkage stable under a variety of conditions. These discs have been to bind lactic dehydrogenase, which may then be specifically eluted with NADH, one of its substrates. In contrast, another enzyme, alkaline phosphatase, not expected to bind to blue dextran, was indeed completely recovered in the filtrate.     

TRANSLOCATION OF 14C METABOLITES IN CARROT ROOT

Four-month-old carrot plants exposed to ¹⁴CO₂ for 1 hr in light were harvested successively at 1, 5, 10, 26, and 78 hr after initial exposure. Half-mm thick transverse slices from 3 and 10 cm below the crown were frozen quickly, freeze-dried, and autoradiographed on film. Radioactivity was first localized in a ring surrounding the cambium. The radioactive region extended centrifugally along radii as discrete loci to half the phloem thickness in 10 hr, in a pattern similar to radial rows of callose-stained sieve elements. Median longitudinal sections stained for callose demonstrated the presence of anastomosing sieve tube strands between the more vertical sieve tubes of differing age. Radioactive materials did not move across the cambium for 5–10 hr. These data fit with the decreased growth, in earlier studies, of uniform phloem explants removed from increasing distances from the cambium and of the lesser growth than phloem of similar xylem explants.     

Spheroid formation in explants of human breast tumor and fibroadenomatous tissues and lymphoid infiltration of explants during cultivation in diffusion chambers

An extensive formation of epithelioid cell spheroids was shown during tumour tissue growth on millipore filters. In addition, the appearance of such multicellular structures as an early sign of malignant transformation was demonstrated in some fibroadenomatous tissue cultures. The increased fibroadenomatous tissue proliferative activity was accompanied by the rising level of lymphoid cell infiltration.