Petri dish

Am J Physiol, 1991 Oct, 261(4 Pt 2), H1329 - 34
Simultaneous measurement of contraction and oxygen consumption in cardiac myocytes; Rose H et al.; A setup has been developed that simultaneously measures the mechanics and the energetics of electrically induced contractions at physiological frequencies of isolated cardiac myocytes . The core of the setup is a self-manufactured stimulation chamber in which most of the myocytes are in suspension while some are attached to a plastic cover slip prepared from culture Petri dishes . The analysis of the contractile behavior of the attached myocytes is based on an image-processing system with digitized frames of a charge-coupled device camera . Thirty-six frames illuminated by a stroboscope are taken at increasing time intervals between stimulus and flash (snap), allowing one to resolve the contraction cycle with a very high time resolution (down to 1 ms) . The number of pixels that differ between each of these frames and a "reference" frame of the cells in the relaxed state (slack cell length) are used to quantify the contractions . An oxygen electrode in the chamber registers the drop of oxygen tension resulting from the consumption by the myocytes, which exhibit a strictly aerobic metabolism . The resulting data are also stored and analyzed in an IBM-AT-compatible computer.

Infect Immun, 1991 Oct, 59(10), 3387 - 92
A role for gamma interferon, tumor necrosis factors, and soluble T-cell receptors in the depressed blastogenic response of spleen cells of Mycobacterium lepraemurium-infected mice; Richard L et al.; Spleen cells of Mycobacterium lepraemurium-infected mice were cultured on petri dishes coated with mycobacterial antigens, and antigen-reactive cells were isolated . Upon incubation in mitogen- or antigen-free culture medium, these cells released mediators capable of depressing the in vitro proliferative response of normal splenocytes to specific antigen and to concanavalin A and lipopolysaccharide . One of these mediators was identified with gamma interferon (IFN-gamma), mainly on the basis that treatment of supernatants with monoclonal anti-IFN-gamma antibodies markedly reduced the suppressive activity contained therein . Detectable levels of tumor necrosis factor alpha (TNF-alpha) and TNF-beta were present in spleen cell culture supernatants of infected mice . Moreover, low doses of recombinant TNF-alpha and TNF-beta were found to potentiate the suppressive activity of exogenous IFN-gamma . Soluble T-cell receptors beta were also detected in the culture supernatants . The elimination of these molecules with monoclonal anti-T-cell receptor beta (F23.1) antibodies immobilized on a plastic surface partially reversed the depression of the response to mycobacterial antigen but did not affect the response to mitogens . These results revealed the complex nature of suppressor mediators that are produced by mycobacterial antigen-reactive cells and that regulate the in vitro proliferative response.

J Neurol Sci, 1991 Oct, 105(2), 211 - 6
Enhanced proteolytic activities in cultured fibroblasts of Alzheimer patients are revealed by peculiar transketolase alterations; Paoletti F et al.; Characteristic alterations of transketolase (TK) in extracts from cultured Alzheimer fibroblasts have previously been reported (Paoletti et al . (1990) Biochem . Biophys . Res . Commun., 172: 396-401) . These abnormalities, encountered in 9 out of 13 Alzheimer patients, were revealed following isoelectric focusing and consisted of enzyme forms having unusually high alkaline pI values (alkaline bands) . The present work has shown that immunologically detected alkaline bands were progressively expressed when Alzheimer fibroblasts were incubated for three weeks without medium changes . Full expression of the altered enzyme pattern was not linked to relative cell density in the petri dish; rather, it appeared to be dependent directly on the time elapsed since cell confluence was reached . Alkaline bands could artificially be induced also in both crude and pure TK preparations from normal cells by a treatment with commercial proteases, particularly chymotrypsin . Moreover, specific inhibitors of endogenous cysteine-proteases were capable of abolishing TK alkaline bands in Alzheimer fibroblasts thus turning a pathological into a normal enzyme pattern . Results obtained suggest that Alzheimer fibroblasts contain enhanced Ca(2+)-independent cysteine-proteolytic activities as compared to normal and other pathological cells . These enzymes, exhibiting chymotrypsin-like activity, might exert their degradative effects at the time of cell extraction using TK and probably other cell components as potential substrates . However, peculiar TK abnormalities represent so far an useful biochemical marker detectable in fibroblasts of living Alzheimer patients and closely associated to this neurological disorder.

Eur Respir J, 1991 Oct, 4(9), 1066 - 75
Toxic effects of oxygen on cultured alveolar epithelial cells, lung fibroblasts and alveolar macrophages; Housset B et al.; Exposure to hyperoxia results in endothelial necrosis followed by type II cell proliferation . This suggests that type II cells are resistant to hyperoxia . Oxygen-induced lung injury may result from an overproduction of oxygen metabolites normally scavenged by antioxidants such as superoxide dismutase (SOD), glutathione peroxidase, catalase and reduced glutathione (GSH) . Therefore, resistance of type II cells to hyperoxia may be linked to high antioxidant activities . To test this hypothesis we compared in vitro the effects of a 24 h exposure period to 95% O2 on cultured type II cells, lung fibroblasts and alveolar macrophages isolated from rats . We show that type II cells, when compared with other cell types, are highly sensitive to hyperoxia as shown by increased lactate dehydrogenase (LDH) release, decreased deoxyribose nucleic acid (DNA) and protein content of Petri dishes and decreased thymidine incorporation into DNA . Synthesis of dipalmitoylphosphatidylcholine was also significantly reduced . Antioxidant enzyme activities as well as glutathione content were not higher in type II cells than in other cell types . However, hyperoxia results in a decreased SOD activity and glutathione content in type II cells which was not observed in fibroblasts . We conclude that adaptative changes in SOD and glutathione metabolism could be important defence mechanisms in cells exposed to hyperoxia.

Endocrinology, 1991 Aug, 129(2), 838 - 42
Rapid augmentation of prolactin cell number and secretory capacity by an estrogen-induced factor released from the neurointermediate lobe; Ellerkmann E et al.; 17 beta-Estradiol (E2) has been shown to exert an acute stimulatory effect on PRL secretion via an indirect action involving the neurointermediate lobe (NIL) . In the present study we used a reverse hemolytic plaque assay to determine whether this effect was manifested as an augmentation of the number of PRL secretors and/or an increase in the amount of hormone released per PRL cell . Cultures of anterior pituitary (AP) and NIL cells from ovariectomized rats were cultured overnight, exposed to the treatment (E2 or vehicle) for 3 h, and then subjected to a reverse hemolytic plaque assay that was carried out in the presence or absence of TRH . Concurrent exposure of AP cells to E2 and NIL cells evoked an 11-12% increase in the overall proportion of PRL-secreting cells . This was true when the AP and NIL cells were incubated as a mixed culture and when the two cell types were maintained in the same petri dish but on separate plastic supports, and TRH did not significantly influence this response . The effect of E2 on the number of PRL secretors was negated when NIL cells were not present throughout the experiment or when they were removed from the cultures just before commencement of E2 treatment . Simultaneous treatment with E2 and NIL cells also significantly augmented the sizes of PRL plaques produced under basal conditions by AP cells . Taken together, these results demonstrate that E2 stimulates NIL cells to release an activity that enhances PRL secretion in two ways: 1) by recruiting additional PRL cells into the secretory pool, and 2) by augmenting the secretory capacity of individual PRL cells.

Transfusion, 1991 Jul-Aug, 31(6), 483 - 90
Inactivation of viruses in platelet suspensions that retain their in vitro characteristics: comparison of psoralen-ultraviolet A and merocyanine 540-visible light methods; Dodd RY et al.; The ability of two fundamentally different photochemical procedures to inactivate model viruses in platelet suspensions was compared . Merocyanine 540 (MC 540) with visible light was used as an example of an oxygen-dependent chemical-directed at the viral membrane, and aminomethyl trimethyl psoralen (AMT) with ultraviolet A light (UVA) was used as an example of a nucleic acid-directed system . Antiviral conditions in petri dishes were identified and the effects of these procedures on platelet suspensions in plastic storage containers were studied . Concentrations of photochemicals in the 10 to 150 mumol range with 30 to 60 minutes of visible light (MC 540) or 1 to 2 minutes of UVA (AMT) readily inactivated 5 to 6 log10 of vesicular stomatitis virus (VSV) and other model viruses in platelet suspensions, provided the plasma concentration was reduced to about 15 percent by the use of a synthetic platelet storage medium . Extracellular pH, morphology scores, and aggregation response dropped markedly when platelets were treated with MC 540 and visible light . However, treatment with 136 mumol per L of AMT and 1 to 3 minutes of UVA could inactivate 5 log10 of VSV in platelet suspensions with retention of platelet characteristics for 4 days, particularly if oxygen levels were reduced during treatment . These studies demonstrate that AMT-UVA treatment meets the initial requirements for virus inactivation in platelet suspensions.

J Invest Dermatol, 1991 Jun, 96(6), 916 - 20
Pyrimidine dimer induction and repair in cultured human skin keratinocytes or melanocytes after irradiation with monochromatic ultraviolet radiation; Schothorst AA et al.; We compared the susceptibilities of cultured melanocytes and keratinocytes to dimer induction in DNA by monochromatic ultraviolet (UV) radiation . Keratinocytes as well as melanocytes were derived from human foreskin, grown as a monolayer in petri dishes, covered with phosphate-buffered saline containing 0.1% glucose, and irradiated . UV irradiation was carried out at 254, 297, and 302 nm as well as with a light source emitting predominantly 312 nm . The induction of pyrmidine dimers was assessed by determination of the number of T4 endonuclease V-sensitive sites (ESS) . We found a slightly higher response for dimer induction in melanocytes at 254, 297, and 302 nm; this difference was only significant at the 297-nm wavelength . Action spectra for pyrimidine dimer induction were derived from the exposure-response data obtained . The action spectra mimic to a large degree the action spectra for dimer induction in other cultured mammalian cells . The repair rate during a post-irradiation period lasting up to 24 h was substantially the same for the two cell types . The percentage of T4 endonuclease V-sensitive sites (ESS) remaining 9 and 24 h after irradiation was 45% and 30%, respectively.

J Invest Dermatol, 1991 Jun, 96(6), 888 - 97
Mediators, initiating the inflammatory response, released in organ culture by full-thickness human skin explants exposed to the irritant, sulfur mustard; Rikimaru T et al.; Mediators released from injured human skin that initiate the inflammatory response have not been adequately identified . Organ culture of full-thickness skin explants enables us to do so, because injury to the skin can be made in vitro, eliminating the rapid leakage of serum and infiltration of leukocytes that occur in vivo . In our studies, the military vesicant sulfur mustard (SM) (10 microliters of a 0.01 to 1.0% dilution) was topically applied to injure the epidermis of the explant . Then, the explants were cultured in small Petri dishes, usually for 18 h at 36 degrees C, and the organ-culture fluids were assayed for various inflammatory mediators . We found that the culture fluids from SM-exposed and control explants contained similar amounts of angiotensin-converting enzyme, trypsin-like and chymotrypsin-like proteases, acid phosphatase, beta-glucuronidase, beta-galactosidase, lysozyme, deoxyribonuclease, ribonuclease, interleukin 1, and lactic dehydrogenase . However, the culture fluids from SM-exposed explants contained increased amounts of histamine and plasminogen-activating activity, and often prostaglandin E2, when compared to culture fluids from control explants . After 3 to 4 d in culture, full-thickness human skin explants, when exposed to 0.2% SM (but not when exposed to 1.0% SM), sometimes showed separation of the epidermis and increased collagenase activity (i.e., hydroxyproline release) . Thus, histamine (from local mast cells), and prostaglandin E2 and plasminogen-activating activity (probably from both mast cells and epidermal cells) are apparently involved in early mediation of the inflammatory response.

Endocrinology, 1991 Jun, 128(6), 3299 - 309
Hormonal control of cell to cell communication: regulation by thyrotropin of the gap junction-mediated dye transfer between thyroid cells; Munari-Silem Y et al.; By microinjection of Lucifer yellow (LY) and analysis of the cell to cell transfer of the fluorescent probe, we have examined 1) the ability of thyroid cells in primary culture to reconstitute gap junctions and 2) the effects of extracellular signals on the functional activity of these junctions . Isolated thyrocytes cultured in tissue culture-treated petri dishes either formed monolayers or reorganized in follicular structures in the presence of the glycoprotein hormone TSH . In both culture conditions, LY-coupled cells were evident after 24-36 h . The communication between cells forming a reconstituted thyroid follicle was maintained for up to 9 days . In contrast, the dye coupling between cells in monolayer progressively decreased with time . The cell to cell communication, i.e., the number of dye-coupled cells in thyroid cell monolayer, was increased by TSH in a time- and concentration-dependent manner . The TSH action was not related to de novo protein synthesis . (Bu)2cAMP exhibited stimulatory effects similar, in terms of time course and amplitude of action, to those of TSH . The phorbol ester 12-O-tetradecanoyl phorbol 13-acetate rapidly inhibited both basal and TSH- or (Bu)2 cAMP-activated cell to cell communication . The dye coupling of cells in reconstituted follicles was also blocked by a short 12-O-tetradecanoyl phorbol 13-acetate treatment in both the presence and absence of TSH . Our data show that thyroid cells in culture, regardless of the full expression of the differentiated phenotype, rapidly reestablish intercellular gap junctions . The functional activity of gap junctions appears to be regulated 1) positively by a hormone, TSH, probably acting via the cAMP and protein kinase-A pathway, and 2) negatively by phorbol esters through the activation of protein kinase-C, the two regulatory pathways being interdependent.

Invest New Drugs, 1991 May, 9(2), 149 - 57
Comparative toxicity of fostriecin, hepsulfam and pyrazine diazohydroxide to human and murine hematopoietic progenitor cells in vitro; Du DL et al.; The in vitro myelotoxic potentials of three investigational antitumor agents, Fostriecin, Hepsulfam and pyrazine diazohydroxide (PZDH), were evaluated utilizing clonogenic assays . Human and murine marrow cells were exposed to each drug for 1 hr prior to culture in microcapillary (human) or Petri dish (murine) assays . Fostriecin (0.22-220 microM), Hepsulfam (0.34-340 microM) and PZDH (0.68-680 microM) inhibited myeloid (CFU-gm), erythroid (BFU-e, CFU-e) and megakaryocytic (CFU-meg) colony formation in a concentration-dependent manner . CFU-e from both species were more sensitive to Fostriecin than the other progenitors and murine cells more sensitive overall to Fostriecin than their human counterparts . Murine CFU-e were also more sensitive to Hepsulfam than human CFU-e, with CFU-gm and BFU-e being similarly affected in both species . Human BFU-e were greatly inhibited by PZDH, whereas murine BFU-e were relatively resistant to its toxic effects . Fostriecin was the most toxic of the three antitumor agents, with PZDH the least toxic.

J Clin Periodontol, 1991 May, 18(5), 305 - 11
Light and electron microscopic evaluation of biocompatibility, resorption and penetration characteristics of human collagen graft material; Quteish D et al.; This study was initiated to test the biocompatibility, resorption and penetration characteristics of human collagen graft material in vitro and in vivo using light (LM) and electron microscopy (EM) . To study this relationship, pieces of glutaraldehyde cross-linked collagen sponges (1 x 1 x 0.5 cm), were: (1) cultured in sterile Petri dishes with human gingival fibroblasts and human periodontal ligament fibroblasts for 2 weeks; (2) implanted in subcutaneous pockets made in both thighs (total 20 sites) of 10 Sprague-Dawley rats for 7-56 days . The behaviour of the growth of the fibroblasts was studied by inverted light microscopy (LM), then tissue culture specimens were studied from without and within using low-temperature scanning electron microscopy (LTSEM) . Blocks obtained from the graft sites of the rat were processed for LM and transmission EM . Long-term LM observations showed attachment and random orientation of cells on and around the collagen sponge in culture during the first 48 h . Between 7 and 14 days, the majority of the cells adjacent to the sponge were orientated at right angles to its margin with their long axes approximately parallel to each other . The LTSEM revealed that large numbers of HGF and HPLF grew onto the collagen sponges, but no cellular penetration to the middle of the sponge was seen . LM and TEM of the rat specimens showed a cellular reaction to the collagen graft, as well as slow resorption, and fibroblast invasion of the graft at 6-8 weeks . It was concluded that the human collagen graft was biocompatible with HGF and HPLF, with penetration first observed at 42 days post-implantation . In the in vivo study, the collagen underwent slow resorption over a period of 8 weeks.

Am J Respir Cell Mol Biol, 1991 May, 4(5), 440 - 8
Attachment characteristics of bovine bronchial epithelial cells to extracellular matrix components; Rickard KA et al.; Attachment of cells to extracellular matrix (ECM) plays an important role in the regulation of cell growth and differentiated function . We hypothesized that bronchial epithelial cells preferentially attach to ECM proteins and utilize specific receptors for ECM proteins . Bronchial epithelial cells were obtained from bovine lung by protease digestion . Both freshly isolated and cultured bronchial epithelial cells were plated onto plastic petri dishes coated with bovine serum albumin, type I collagen, type IV collagen, fibronectin, laminin, ECM synthesized by cultured bronchial epithelial cells, or uncoated . Freshly isolated cells demonstrated significant attachment to ECM but weak attachment to other matrix proteins . Cultured bronchial epithelial cells attached well to ECM; however, they had relatively increased attachment to type I collagen, type IV collagen, fibronectin, and laminin compared to freshly isolated cells . To determine whether the attachment of bronchial epithelial cells is arginine-glycine-aspartic acid (RGD)-mediated, an RGD-containing peptide known to block attachment mediated by many integrin receptors was added to the media (400 micrograms/ml) . There was no inhibition of attachment of freshly isolated cells; however, there was significant but not complete inhibition of the attachment of the cultured cells to type IV collagen, laminin, and fibronectin, but not to type I collagen or ECM . Thus, freshly isolated bronchial epithelial cells readily adhere to ECM, and the attachment does not appear to be mediated by RGD-dependent receptors . Cultured bronchial epithelial cells demonstrate increased attachment to component proteins of ECM, and this attachment is, in part, to RGD-dependent receptors.

Appl Microbiol Biotechnol, 1991 May, 35(2), 159 - 64
High density culture of anchorage-dependent animal cells by polyurethane foam packed-bed culture systems; Matsushita T et al.; Monkey kidney cells (Vero) and Chinese hamster ovary cells (CHO-K1) attached to the internal surface of polyurethane foam (PUF) and grew to a high cell density (1.1 x 10(8) cells/cm3 PUF and 4.2 x 10(7) cells/cm3 PUF, respectively) in a PUF-plates packed-bed culture system . This density of Vero cells was twice that obtained previously with a PUF-particles packed-bed culture system . A maximum cell density of 6.7 x 10(7) cells/cm3 culture vessel volume was obtained in a PUF-disc packed-bed culture of Vero cells . From the cell density of CHO-K1, growing in a monolayer on the surface of PUF and a petri dish, per bulk volume of PUF, we estimated that a surface area to volume ratio of PUF plates effective for cell growth was about 109 cm2/cm3.

Exp Parasitol, 1991 Apr, 72(3), 311 - 20
Schistosoma mansoni: possible involvement of protein kinase C in linoleic acid-induced proteolytic enzyme release from cercariae; Matsumura K et al.; The possible involvement of protein kinase C and Ca2+ metabolism in the proteolytic enzyme release from schistosome cercariae was studied . Cercariae were placed in dechlorinated tap water containing 0.37 mM calcium in the small glass petri dish and exposed to the stimuli (linoleic acid, phorbol esters, and Ca2+ ionophore) with or without inhibitors of protein kinase C or Ca2+ metabolism . The proteolytic activity of incubation medium of cercariae thus treated was measured by the azocoll assay . The penetration response of cercariae induced by linoleic acid, a physiological stimulus, was mimicked by phorbol esters . When exposed to phorbol esters, 0.02 to 2 microM of 12-O-tetradecanoylphorbol-13-acetate (TPA) and 0.2 to 2 microM of phorbol-12,13-dibutyrate (PDBu), cercariae ceased the swimming movement, began a rhythmic thrusting of the anterior tip of the parasite, and released the proteolytic enzyme, but they did not shed the tails . Lowering Ca2+ in water by addition of 5 mM ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA), phorbol ester-induced release of enzyme was completely inhibited . Phorbol ester-induced release of enzyme was partially inhibited by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), an inhibitor of protein kinase C, at a concentration of 100 microM . H-7 alone, at a concentration of 100 microM, did not affect the swimming movement of cercariae . The cercariae were stimulated to release the enzyme by high concentrations (10 and 100 microM) of the Ca2+ ionophore, A23187, but enzyme was not released by low concentrations (0.5 and 1 microM) of this drug . Cercariae exposed to A23187 behaved differently from those exposed to phorbol esters . They ceased swimming, showed strong muscle contraction, and shed their tail . A23187 stimulated cercariae to release the enzyme in the water containing 5 mM EGTA . A23187-induced enzyme release was not inhibited by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin antagonist, trifluoperazine (TFP), a better calmodulin antagonist on schistosome, or by verapamil, a Ca2+ channel blocker . Linoleic acid-induced release of enzyme was partially inhibited by 0.5 and 5 mM of EGTA and by 1 to 100 microM of H-7 . While it was not inhibited by N-{2-(methylamino)ethyl}-5-isoquinolinesulfonamide (H-8) and N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA-1004), inhibitors of cyclic nucleotide-dependent protein kinase which were used as negative controls of H-7, W-7, TFP, 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8), an intracellular Ca2+ antagonist, and verapamil.(ABSTRACT TRUNCATED AT 400 WORDS)

J Neurosci Methods, 1991 Apr, 37(2), 173 - 82
A simple method for organotypic cultures of nervous tissue; Stoppini L et al.; Hippocampal slices prepared from 2-23-day-old neonates were maintained in culture at the interface between air and a culture medium . They were placed on a sterile, transparent and porous membrane and kept in petri dishes in an incubator . No plasma clot or roller drum were used . This method yields thin slices which remain 1-4 cell layers thick and are characterized by a well preserved organotypic organization . Pyramidal neurons labelled by extra- and intracellular application of horse radish peroxidase resemble by the organization and complexity of their dendritic processes those observed in situ at a comparable developmental stage . Excitatory and inhibitory synaptic potentials can easily be analysed using extra- or intracellular recording techniques . After a few days in culture, long-term potentiation of synaptic responses can reproducibly be induced . Evidence for a sprouting response during the first days in culture or following sections is illustrated . This technique may represent an interesting alternative to roller tube cultures for studies of the developmental changes occurring during the first days or weeks in culture.

Clin Rheumatol, 1991 Mar, 10(1), 28 - 30
Mononuclear cells are not involved in BGP synthesis and secretion; Cantatore FP et al.; Osteocalcin or bone GLA protein (BGP) is found at high levels in only two tissues, the extracellular matrix of bone and dentine . Tissue culture experiments have demonstrated that BGP is synthesized by two osteoblastic osteosarcoma cell lines (ROS 2/3 and 17/2) and by normal osteoblastic cells in primary culture . BGP was not found in rat cartilage nor in liver, kidney, lung, spleen, brain, heart, thymus, skeletal muscle . In this study secretion of BGP was assayed by RIA in the supernatants of 48-hour cultures of peripheral blood lymphocytes or monocytes . Lymphocyte cultures were carried out using RPMI-1640 supplemented with L-glutamine and antibiotics at the concentration of 1 x 10(6) cells/ml and activated by PHA (10 ng/ml) . Peripheral blood monocytes were purified by adherence to plastic Petri dishes and treated with cold PBS supplemented with EDTA . Monocytes were cultured as previously described and stimulated with LPS (50 micrograms/ml) . Cell-free supernatants were obtained by centrifugation and stored at -20 degrees C, until the BGP assay was performed . The authors did not observe secretion of detectable amounts of BGP in the supernatants of short-term lymphocyte or monocyte cultures . These data indicate that circulating mononuclear cells are not involved in BGP synthesis and secretion.

Radiology, 1991 Mar, 178(3), 677 - 9
US-assisted aspiration thrombectomy: in vitro investigations; Schmitz-Rode T et al.; The authors describe the use of a new ultrasound (US)-aspiration thrombectomy technique . An oscillating US probe was inserted into a thin-walled, large-bore aspiration catheter . Experiments tested the ability of the new device and other catheter combinations to remove clot material from a Petri dish, as well as from small and large vessel models made of silicone and glass tubes, respectively . Results of the experiments demonstrated that an oscillating 1.0-mm US probe inserted into an aspiration catheter (7-9 F in diameter) promoted clot fragmentation and allowed continuous aspiration of thrombi of any size . When compared with simple large-bore catheter aspiration and with mechanical fragmentation by means of a US probe within a catheter that was flushed to cool the probe, US-assisted aspiration thrombectomy demonstrated significantly better results for percutaneous transcatheter removal of fresh thrombi.

AJNR Am J Neuroradiol, 1991 Mar-Apr, 12(2), 279 - 81
Metallic otologic implants: in vitro assessment of ferromagnetism at 1.5 T; Shellock FG et al.; MR imaging is contraindicated for patients with certain ferromagnetic implants because of potential risks related to movement or dislodgement . This is especially true for metallic implants located in sensitive areas of the body, such as those placed in and around the ear . Therefore, the ferromagnetic qualities of 35 different metallic otologic implants were assessed by placing them individually on a millimeter scale in a plastic petri dish that was slowly moved into the center of a 1.5-T MR imaging system . None of the metallic otologic implants moved during this procedure . The results demonstrate that each of these implants are made from nonferromagnetic materials and do not pose a risk to patients undergoing high-field-strength MR imaging . These data effectively expand the list of metallic implants that appear to be safe for MR imaging.

Prostaglandins, 1991 Mar, 41(3), 263 - 81
Metabolism of arachidonic acid by guinea pig Clara cells; Laporte J et al.; A method for the isolation of non-ciliated bronchiolar epithelial (Clara) cells from the guinea pig is described . Following digestion of the lung tissue with Type XXIV protease, the isolated lung cells showed a viability greater than 90% and contained 3% of Clara cells . Several cell populations were then separated on the basis of size using 2 centrifugal elutriations . The macrophages and endothelial cells were removed from the Clara cells enriched fractions by differential adherence on Petri dishes . The Clara cell-rich suspension was then further purified by centrifugation on Percoll non-continuous density gradients consisting of 48-52-55% Percoll solution . The lower interface and the pellet of the non-continuous gradient consisted of approximately 80% Clara cells . Identification of isolated Clara cells was confirmed by light microscopic observations after nitroblue tetrazolium staining and by ultrastructural characteristic features as observed by electron microscopy . The metabolism of arachidonic acid into prostaglandins and TxB2 by purified Clara cells was examined by enzyme immunoassay (EIA) and leukotriene formation was investigated by reverse phase high performance liquid chromatography (RP-HPLC) . Enriched guinea pig Clara cells incubated with arachidonic acid released TxB2, PGE2 and 6-keto PGF1 alpha, but did not produce leukotrienes . These cells could however transform exogenous leukotriene A4 into leukotriene B4 . These results suggest that guinea pig Clara cells possess the enzymes of the cyclooxygenase pathway required for TxB2, PGE2 and 6-keto-PGF1 alpha synthesis . Clara cells do not possess the 5-lipoxygenase enzyme but show some leukotriene A4 hydrolase activity since they can produce leukotriene B4 upon incubation with leukotriene A4.

Lancet, 1991 Feb 2, 337(8736), 269 - 70
Survival of scrapie virus after 3 years' interment; Brown P et al.; Supernatant fluid from a scrapie-infected hamster brain homogenate was mixed with soil, packed into perforated petri dishes that were then embedded within soil-containing pots, and buried in a garden for 3 years . Between 2 and 3 log units of the input infectivity of nearly 5 log units survived this exposure, with little leaching of virus into deeper soil layers . These results have implications for environmental contamination by scrapie and by similar agents, including those of bovine spongiform encephalopathy and Creutzfeldt-Jakob disease.

Anat Rec, 1991 Jan, 229(1), 125 - 8
A new technique for explantation and in vitro cultivation of chicken embryos; Dugan JD Jr et al.; A technique is described for explanting and cultivating chicken embryos in plastic drinking cups which have been modified with plastic wrap to reproduce the geometry and dimensions of the egg shell . Successful explantation rates of 97% are possible with a double-window technique, and survivability in cups exceeds that achievable in other in vitro systems (i.e., petri dishes) . Long-term survival to the 21st day of incubation is seen routinely . This system with cups is less expensive than that with petri dishes, and simpler than that with plastic wrap/tripods . Thus, this new method of in vitro cultivation of chicken embryos improves upon explantation rate, survivability and system design, and has a wide range of applications in developmental biology, angiogenesis, cancer, and pharmacology research.

J Clin Gastroenterol, 1991, 13 Suppl 1, S58 - 64
Gastric mucosal repair in vitro; Giebel J et al.; We tested whether cultured gastric mucosal cells would be suitable to study the two major steps of repair: restitution and proliferation . Preparations of freshly isolated epithelial cells of guinea pig gastric mucosa were used for the studies . The cells attached to Petri dishes within 10 h and formed monolayers after 48-72 h . Electron microscopy showed that each cell type was able to form lamellipodia (i.e., cell protrusions) during restitution in vivo . When monolayers were wounded with a razor blade, most cells at the edge of damage died within a few minutes, but some recovered from injury . Later, intact cells migrated from the edge into the denuded zone and restored the monolayer within 24-48 h . An increased number of cells near the edge started to synthesize DNA . In conclusion, this model allows one to study in vitro both aspects of mucosal repair.

Basic Life Sci, 1991, 58, 3 - 19; discussion 19-25
The molecular biology of radiation carcinogenesis; Hall EJ et al.; Major new insights into carcinogenesis have come from recent advances in cellular and molecular biology . The concept of oncogenes provides a simple explanation for how agents as diverse as radiation, chemicals or retroviruses can induce tumors that are indistinguishable one from another . Oncogenes may be activated by a point mutation, by a chromosome translocation, or by amplification . Ionizing radiations are efficient at the first two mechanisms . While oncogenes are frequently associated with leukemias and lymphomas, they are associated with only 10 to 15% of human solid cancers . The importance of the loss of suppressor genes was suggested first from studies with human-hamster hybrid cells, but has since been shown to be of importance in an increasing number of human solid tumors, from rare tumors such as retinoblastoma to more common tumors such as small cell lung cancer and colorectal cancer . The mechanism of somatic homozygosity clearly involves several steps, some of which, such as a deletion, could be readily produced by ionizing radiation . The multi-step nature of carcinogenesis can be demonstrated in the petri dish, where the transfection of multiple oncogenes is required to transform normal cells from short-term explants . It can be shown, too, in colorectal cancer in the human, where the activation of an oncogene and the loss of more than one suppressor gene may be involved in the progression from normal epithelium to a frank malignancy.

Nephrol Dial Transplant, 1991, 6 Suppl 3, 53 - 6
Suppression of beta 2-microglobulin release from lymphocytes by dialysis membranes; Schaefer RM et al.; As lymphocytes are one of the main sources of circulating beta 2-microglobulin (beta 2M), the direct effect of different dialysis membranes on beta 2M release from those cells was studied in vitro . Lymphocytes were isolated from 11 long-term haemodialysis patients and nine healthy controls . Cells were cultured on flat sheet membranes made from either Cuprophan, Hemophan, or polyacrylonitrile . Polystyrole petri dishes were used as controls . Beta 2M concentrations in the supernatant were measured after 3 and 7 days of culture by ELISA techniques . Beta 2M release from lymphocytes obtained from uraemic patients was almost identical to the release from healthy subjects . In the presence of all three membranes the release of beta 2M was less than that produced on polystyrole . This held true for lymphocytes isolated from both healthy and uraemic subjects . As for the three membranes the release of beta 2M into the supernatants was statistically the same when the adsorptive capacity of polyacrylonitrile was taken into account . However, there was a tendency for Cuprophan to exert the strongest inhibition, while Hemophan and polyacrylonitrile reduced beta 2M release to a lesser degree . Based on these data it seems that prolonged interaction between dialysis membranes and lymphocytes does lead to a reduction in beta 2M release.

Bioelectromagnetics, 1991, 12(5), 299 - 314
Morphological and electrophysiological changes produced by electrical stimulation in cultured neuroblastoma cells; Krauthamer V et al.; Electric fields, which were equivalent to those generated by medical devices, were applied to cultured neuroblastoma cells (mouse and human) to test for morphological damage and to establish damage thresholds . Each of two methods of applying fields permitted flow of electrical current and minimized exposure of cells to electrode-breakdown products . One method consisted of a pair of parallel wires in a Petri dish by which current was delivered within a fixed volume of flowing tissue-culture media . With the other method, the cells were held in a confined geometrical chamber and current was applied via agar bridges . Under a given set of stimulation parameters, damage was found to be variable from cell to cell . By changing the strength of the electric field (frequency and duration of stimulation held constant), thresholds of several V/cm were found above which cell damage could be reliably produced . Depending on the intensity of the field, damage took the form of cell lysis or damage to neurites . Intracellular recordings from the mouse neuroblastoma cells revealed a correlation between a decline in resting transmembrane potential and stimulus intensity . Human neuroblastoma cells were less susceptible to damage than were the mouse neuroblastoma cells, given the same strength of applied electric fields.

Lens Eye Toxic Res, 1991, 8(2-3), 281 - 309
Cell-substratum interactions and the cytoskeleton in cell shape-mediated growth regulation of lens epithelial cells; Iwig M et al.; Cell attachment to a suitable substratum is a precondition for the mitotic growth of nontransformed lens epithelial cells . Cultering of cells in suspension results in a strong decline of the DNA synthetic rate, whereas reattachment induces the reentrance into the cell cycle . Further studies revealed that not anchorage itself but cell flattening is prerequisite for the entrance of cells into the cycle . Flattened cells exert tension to the substratum via numerous filopodia . If the rigidity of the substratum is reduced by loosening of the collagen gel from the bottom of the petri dish, the gel becomes contracted by the traction forces of the cells and the cell shape becomes transformed from a flattened shape into a more spheroidal or longstretched one . This cell shape transition is connected with a decrease in RNA- and protein synthesis and a stop of DNA synthesis . During further experiments it was demonstrated that microfilaments are involved in gel contraction and cell shape alteration, respectively . Furthermore, intact microfilaments are needed for G0-G1-S-transition . Desintegration of microfilaments by cytochalasin is without influence on ongoing DNA synthesis but hinders strongly the entrance of cells into the S-phase . The survey gives some recent results on the molecular basis of cell substratum interactions as well as the structure and function of the cytoskeleton . The role of the cytoskeleton in cell shape-mediated growth regulation is discussed.

J Physiol (Paris), 1991, 85(3), 158 - 70
Behavioural effects of genetically engineered cells releasing dopa and dopamine after intracerebral grafting in a rat model of Parkinson's disease; Horellou P et al.; The relative importance of synaptic versus paracrine dopamine transmission for the occurrence of functional effects following intrastriatal grafting is not fully established . In the present study we grafted cell lines, expressing the form I of human tyrosine hydroxylase after infection with a recombinant retrovirus and selection in tyrosine-free-medium, to the denervated striatum in order to analyse the extent to which extracellular dopamine levels can be restored and the effect of a diffuse release of dopamine on motor impairement in a rat model of Parkinson's disease . In petri dish, the modified fibroblast cells (NIH.3T3) release DOPA constitutively whereas the modified endocrine cells (RIN) store and release dopamine in a regulated way . Interestingly, in denervated striatum, grafts of modified fibroblast cells produce DOPA which was efficiently converted into dopamine by the host striatal tissue . In the grafted striatum, both fibroblast and endocrine cells restore subnormal levels of diffuse release of dopamine which is notably unaffected and stimulated, respectively, by high concentration of potassium, in connection with the in vitro properties of the grafted cells . The intrastriatal grafts of modified cells partially reversed the apomorphine-induced but not the amphetamine-induced motor asymmetry . We discuss the implications of these results in the context of Parkinson disease.

Med Radiol (Mosk), 1991, 36(11), 22 - 3
{A comparative evaluation of 2 methods for cloning human lung tumors}; Sviridova IK et al.; Clonogenic capacity of tumor cells from 59 human lung cancer specimens was investigated in Petri dishes (24 specimens) and in capillaries (40 specimens), and parallel cloning was done in 5 cases . The results were as follows: (a) bacterial contamination in capillaries was noted less frequently than in Petri dishes (12.5 vs . 25%); (b) cells did not form colonies in capillaries in 42.5% and in Petri dishes in 25%; (c) colony-forming effectiveness in capillaries was 3.46-fold higher than that in Petri dishes; (d) the amount of tumor cells for analysis in capillaries is 10-fold less than that for analysis in Petri dishes . However, the absolute number of colonies, indispensable for assessment of tumor sensitivity to antitumor action (not less than 15 colonies per capillary or Petri dish) was obtained in 67% in Petri dishes and in 33% only in capillaries.

Differentiation, 1990 Dec, 45(3), 221 - 9
Filaggrin production by cultured human epidermal keratinocytes and its regulation by retinoic acid; Asselineau D et al.; Filaggrin is a basic protein normally present in the stratum corneum of epidermis . It derives from a high-molecular-weight precursor synthesized in the stratum granulosum of epidermis . This precursor, called profilaggrin, is thought to be associated with the keratohyaline granules of granular cells . It is known that profilaggrin, but not filaggrin, is present in conventional cultures of human keratinocytes grown on plastic petri dishes . In this study, we show that cultured human keratinocytes can convert profilaggrin into filaggrin, when they are grown on a collagen matrix and raised at the liquid-air interface in order to induce terminal differentiation . Moreover, the presence of terminally differentiating keratinocytes above the granular layer is necessary, but not sufficient, for the accumulation of filaggrin . Finally, we show that the accumulation of filaggrin in the outermost layers of submerged cultured human keratinocytes can be triggered by extensive removal (double delipidization) of retinoids from the serum supplement and inhibited when small concentrations (10(-11)-10(-10) M) of retinoic acid are readded to the culture medium . Altogether, the data reported suggest that not only the synthesis of profilaggrin, but also the conversion of profilaggrin into filaggrin are negatively controlled by retinoic acid . Further, it seems that retinoic acid acts directly on the conversion of profilaggrin into filaggrin rather than on the production of terminally differentiating cells capable of accumulating this protein.

Boll Soc Ital Biol Sper, 1990 Nov, 66(11), 1043 - 50
{Survival of bone tissue in organotypic culture under intermittent mechanical loading . Preliminary results}; Lozupone E et al.; With the aim to study the mechanism of transduction of mechanical stimuli in biological ones we have realized an experimental device for the application of intermittent mechanical forces on bone specimens in vitro . The scheme of the device is reported in Fig . 1 . It is constituted by a drive shaft which rotates on eccentric axis (1) supporting a longitudinal bar (2) with the load (3) . The latter rests on a piston (4) only during a limited period of every shaft revolution, so that the load becomes intermittent . The bone specimen (5) is placed under the piston and the two are placed in a tube containing the culture medium . This latter is BGJ mod . Fitton-Jackson (Gibco), enriched with fetal calf serum (10%) and ascorbic acid (70 microliters/ml) . Right metatarsi from 18-day-old rats were removed aseptically and placed under the piston for 2-6 days after resection of both ends . The homotypic ones, unloaded, were placed in 30 mm Petri dishes, and used as a control . The incubator environment was 5% CO2 in air (A group), or enriched with O2 (25-35%) (B group) . At the end of the experimental period the bone specimens were fixed in 4% formalin buffered and treated for conventional histologic methods . In the A group most of the osteocytic lacunae were empty . The osteoblasts disappeared already at the 2nd day; the periosteal fibroblast dedifferentiated and multiplied . The deposition or calcification of osteoid were completely lacking . The application of mechanical load promoted deposition of granular degenerative material around the bone, and the periosteal cells, well differentiated, were surrounded by metachromatic material, which resembles cartilage matrix.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Gen Genet, 1990 Nov, 224(2), 177 - 82
Localization by restriction fragment length polymorphism mapping in potato of a major dominant gene conferring resistance to the potato cyst nematode Globodera rostochiensis; Barone A et al.; A major dominant locus conferring resistance against several pathotypes of the root cyst nematode Globodera rostochiensis was mapped on the linkage map of potato using restriction fragment length polymorphism (RFLP) markers . The assessment of resistance versus susceptibility of the plants in the experimental population considered was based on an in vivo (pot) and an in vitro (petri dish) test . By linkage to nine RFLP markers the resistance locus Gro1 was assigned to the potato linkage group IX which is homologous to the tomato linkage group 7 . Deviations from the additivity of recombination frequencies between Gro1 and its neighbouring markers in the pot test led to the detection of a few phenotypic misclassifications of small plants with poor root systems that limited the observation of cysts on susceptible roots . Pooled data from both tests provided better estimates of recombination frequencies in the linkage interval defined by the markers flanking the resistance locus.

Neurosci Lett, 1990 Oct 16, 118(2), 172 - 6
Carbon filaments provide support and directionality to growing rat fetal spinal cord explants; Khan T et al.; Spinal cord explants obtained from 15 to 17-day-old fetal rats were cultured on bundles of 5-7 microns diameter carbon filaments attached to the bottom of Petri dishes . After a 3 week incubation period, the cultures were fixed and observed by light, scanning, and transmission electron microscopy . Neurites and glial processes were found to be growing both on and between the carbon filaments . The carbon filaments appeared to provide a biocompatible scaffold which promoted adhesion and gave directionality to the growing cell processes . These properties may make carbon filaments a suitable substrate for in vivo implantation into the damaged spinal cord.

Immunol Lett, 1990 Oct, 26(1), 11 - 5
MHC class II antigens on canine bronchoalveolar cells; Chang SC et al.; To evaluate the expression of MHC (major histocompatibility complex) antigens on canine bronchoalveolar cells (BAC), bronchoalveolar lavages (BAL) were performed in mongrel and German shepherd dogs . MHC class II antigens on canine BAC and peripheral blood mononuclear cells (PBMC) were detected by monoclonal antibodies (mAbs) B1F6, 7.5.10.1 and Q5/13 recognising canine MHC class II antigens, using cytofluorometry . These mAbs reacted with more than 20% of BAC and PBMC in both breeds of dog . The percentage of MHC class II positive cells in BAC were lower than those in PBMC . There was no significant difference in the percentages of MHC class II positive BAC and PBMC in mongrel and German shepherd dogs . To further identify the expression of MHC class II antigens on BAC, the cells were separated into adherent and nonadherent cells by petri dish adherence . The percentages of MHC class II positive cells in adherent and non-adherent cell populations were similar . Nearly half the lymphocytes in normal BAC were T cells detected by mAbs F3-20-7 and 1A1; B cells were scarce and represented less than 10% of nonadherent cells . Immunoprecipitation by anti-MHC class II mAbs, and SDS-polyacrylamide gel electrophoresis (SDS-PAGE) revealed MHC class II-like molecules on canine BAC and PBMC . After stimulation with phytohaemagglutinin (PHA), the percentages of class II positive cells in BAC and PBMC were significantly increased . Thus, these anti-MHC class II mAbs may prove to be of advantage in experiments designed to evaluate the changes in class II antigen expression on canine BAC during the course of immune response in the lung, as in pulmonary allograft rejection.

J Biol Chem, 1990 Sep 5, 265(25), 15261 - 6
Parathyroid hormone-induced alterations of protein content and phosphorylation in enriched apical membranes of opossum kidney cells; Reshkin SJ et al.; Parathyroid hormone (PTH) reduces Na/Pi co-transport activity in opossum kidney (OK) cells in a process mediated by protein kinases A and C . Further, inactivation of Na/Pi transport involves irreversible inhibition, possibly via internalization, of the transport system . This study analyzed alterations of concentration and phosphorylation of membrane proteins of an apically enriched preparation induced by short (10 min) and long (3 h) term incubation with 10(-10) M PTH of monolayer cultures of the OK-cell line . To this end, an apically enriched membrane fraction was isolated from cells grown on Petri dishes and analyzed by two-dimensional gel electrophoresis . Long term exposure of the cells to PTH induced changes in apical protein concentration . Four proteins were found to be decreased and one protein was found to be increased in its concentration . Addition of 10(-10) M PTH to the cells led to transient phosphorylation of five proteins . In contrast to transient phosphorylation, phosphorylation of one protein increased over the time period of 3 h . Combined analysis of silver staining and autoradiography led to the detection of an acidic 35-kDa protein in which specific phosphorylation increased over a time period of hours . The results document for the first time alterations in apical membrane protein content and phosphorylation state mediated by PTH when added to an intact cellular system . It is concluded that the identified proteins represent possible candidates for being involved directly or indirectly in PTH alterations of membrane transport.

J Invest Dermatol, 1990 Sep, 95(3), 325 - 32
Full-thickness human skin explants for testing the toxicity of topically applied chemicals; Nakamura M et al.; This report describes a model organ-culture system for testing the toxicity of chemical substances that are topically applied to human skin . In this system, the viable keratinocytes in the full-thickness skin explants are protected by the same keratinized layer as skin remaining on the donor, and toxicity can be assessed microscopically and/or biochemically . The human skin specimens were discards from a variety of surgical procedures . They were cut into full-thickness 1.0-cm2 explants, and briefly exposed to the military vesicant sulfur mustard (SM), which was used as a model toxicant . The explants were then organ cultured in small Petri dishes for 24 h at 36 degrees C . In the 0.03-1.0% dosage range, a straight-line dose-response relationship occurred between the concentration of SM applied and the number of paranuclear vacuoles seen histologically in the epidermis . Within the same SM dosage range, there was also a proportional decrease in 14C-leucine incorporation by the explants . Thus, the number of paranuclear vacuoles reflected decreases in protein synthesis by the injured epidermal cells . The epidermis of full-thickness untreated (control) human skin explants usually remained viable for 7 d when stored at 4 degrees C in culture medium . During storage, a relatively small number of paranuclear vacuoles developed within the epidermis, but the explants were still quite satisfactory for testing SM toxicity . Incubation (for 4 or 24 h at 36 degrees C) of such control skin explants reduced (often by 50%) the small number of paranuclear vacuoles produced during 4-7 d of storage . This reduction was probably caused by autolysis of many of the vacuolated cells . Two types of paranuclear vacuoles could be identified by both light and electron microscopy: a storage type and a toxicant type . The storage type seemed to be caused by autolysis of cell components . The toxicant type seemed to be caused by an invagination of the plasma membrane . Only toxicant-type vacuoles increased appreciably in number when skin explants were exposed to mustard, and to other toxicants.

J Am Mosq Control Assoc, 1990 Sep, 6(3), 406 - 10
Mosquito attraction to substances from the skin of different humans; Schreck CE et al.; Mosquito attraction responses to substances collected from human skin and placed on glass petri dishes were studied . Mosquito response varied according to the source of the substance . Substances removed from the head and hands elicited the greatest attraction response in laboratory-reared mosquitoes . Mosquito response lasted up to 6 h when the substance was aged and was increased by warming the samples from ca . 25 degrees C to 37 degrees C . Of the 12 mosquito species studied, attraction response was greatest in Aedes aegypti . It is cautioned that residues deposited by handling traps or other apparatus used in mosquito studies may influence test results.

Am J Respir Cell Mol Biol, 1990 Sep, 3(3), 217 - 26
Induction of surfactant protein (SP-A) biosynthesis and SP-A mRNA in activated type II cells during acute silicosis in rats; Miller BE et al.; The synthesis of the major surfactant protein, SP-A, was studied in activated alveolar type II cells isolated from the lungs of rats exposed to silica by intratracheal instillation . Exposure of rats to silica resulted in large increases in the levels of disaturated phosphatidylcholine and SP-A in the extracellular and intracellular surfactant compartments . Isolated type II cells were used to determine if the observed increases in SP-A were associated with increased SP-A synthesis . Type II cells were isolated by a combination of elastase digestion, centrifugal elutriation, and differential adherence on IgG-coated petri dishes . Type II cells from silica-treated lungs were separated into two populations, designated type IIA and type IIB . The type IIB, or activated population, consisted of type II cells that were larger than normal type II cells and, in addition, contained larger and more numerous lamellar bodies than normal type II cells . Type IIB cells contained 4.3-fold higher levels of SP-A compared to normal type II cells . SP-A synthesis was measured by incubating freshly isolated cells with {35S}Translabel (70% {35S}methionine, 15% {35S}cysteine) for up to 4 h in methionine-free medium, followed by immunoprecipitation of newly synthesized protein . The rate of SP-A synthesis was increased approximately 6.7-fold in the activated type II cells . Analysis of the newly synthesized protein by one-dimensional SDS-PAGE indicated three intracellular forms of SP-A with molecular weights of approximately 26,000, 30,000, and 34,000 . In type II cells from control rats, the 34-kD protein accounted for approximately 93% of the newly synthesized SP-A after 4 h of incubation; only a small amount of radioactivity was associated with the lower molecular weight species . The increased biosynthesis of SP-A in the activated type II cells was associated with a 7.3-fold increase in the level of SP-A mRNA . These results indicate that the content and synthesis of SP-A are both highly elevated in activated type II cells and that these increases may be due to increased levels of SP-A mRNA.

Can J Physiol Pharmacol, 1990 Sep, 68(9), 1226 - 30
Reexamination of dopamine as the prolactin-release inhibiting factor (PIF): supplementary agent may be required for dopamine to function as the physiological PIF; Shin SH et al.; A large number of studies have been performed concerning dopamine's inhibitory effect on prolactin release, but many of these studies have examined the effect of dopamine dissolved in a solution containing ascorbic acid . Ascorbic acid, routinely used to protect dopamine from oxidation, alone does not stimulate or inhibit prolactin release, but it can potentiate the inhibitory effect of dopamine in a static monolayer culture system by approximately 100 times . We have closely examined the inhibitory effect of dopamine on prolactin release in the absence of ascorbic acid using a perifusion system . Male rat adenohypophyses were dispersed with trypsin and cultured in a Petri dish to form cell clusters . Inhibition of prolactin release by dopamine (1 mumol/L) in the absence of ascorbic acid was sustained for only 63 min during the 2-h perifusion period . Following a 2-h period of incubation of dopamine in the same experimental solution, the dopamine concentration was reduced from 1 to 0.18 mumol/L, yet this "2-h-old dopamine" was still effective in inhibiting prolactin release (approximately 30 min) . This result suggests that the lactotrophs may be desensitized by chronic exposure to a high concentration of dopamine in the absence of ascorbic acid . In contrast, when a low concentration of dopamine (3 nmol/L) containing ascorbic acid (0.1 mmol/L) was perifused, inhibition of prolactin release was sustained for the entire 2-h perifusion period . Although there may be a large number of explanations for dopamine's transient inhibitory effect on prolactin release, the present results suggest that dopamine may require supplementary agent(s) to effectively inhibit prolactin release and thus function as the prolactin release inhibitory factor (PIF).(ABSTRACT TRUNCATED AT 250 WORDS)

Dtsch Zahnarztl Z, 1990 Aug, 45(8), 449 - 51
{In vitro growth of periodontal ligament fibroblasts between bone and root slices}; Sharaf MN et al.; In a first experiment the proliferation and growth kinetics of periodontal ligament fibroblast monolayers generated from human healthy (HPDL) and diseased (DPDL) periodontia were compared . In a second experimental series a periodontal ligament-like space was developed by laying a root slice inside a bony ring slice on the bottom of a tissue culture petri dish and leaving a space of about 0.5 mm between both structures . HPDL cells showed a higher proliferation rate than DPDL cells . Growing into the space between the slices, the PDL cells built up a matrix which was composed of cells and fibrils . HPDL cells built up the matrix more rapidly than DPDL cells.

Biochim Biophys Acta, 1990 Jul 12, 1053(2-3), 135 - 43
Proteoglycans synthesized in vitro by nude and normal mouse peritoneal macrophages; Petricevich VL et al.; Peritoneal macrophages from nude mice were found to be functionally similar to 'activated' macrophages from normal mice . The objective of the present study was to characterize the proteoglycans synthesized and secreted in vitro by peritoneal macrophages isolated from nude and normal Balb/c mice and to investigate the relationship between macrophage 'activation' and changes in the proteoglycan patterns . Macrophages obtained by peritoneal lavage were seeded in Petri dishes . After 2 h incubation at 37 degrees C, the adherent cells (macrophages) were exposed to {35S}sulphate for the biosynthetic labelling of proteoglycans . After incubation, the cell and medium fractions were collected and analysed for proteoglycans and glycosaminoglycans . The glycosaminoglycans were identified and characterized by a combination of agarose gel electrophoresis and enzymatic degradation with specific mucopolysaccharidases . It was shown that 3/4 of the total 35S-labelled glycosaminoglycans were in the extracellular compartment after 24-48 h . The macrophages synthesized dermatan sulphate (68%), chondroitin sulphate (7%) and heparan sulphate (25%) . Both cell and medium fractions of normal and nude mouse macrophages contained glycosaminoglycans with the same ratios, although the nude mouse macrophages synthesized 2-fold less glycosaminoglycans than the normal mouse macrophages . Lower levels of 35S-proteoglycans were also obtained from in vitro 'activated' macrophages, but the ratios of dermatan sulphate:chondroitin sulphate: heparan sulphate were altered in these cells as compared to the control . Furthermore, all the 35S-macromolecules found in the extracellular compartment of nude and normal control cells were of proteoglycan nature, in contrast to the medium fractions of 'activated' macrophages, which contain both intact proteoglycans and 'free' glycosaminoglycan chains . These results indicate that, at least as regards the proteoglycans and glycosaminoglycans, the nude mouse macrophages are not identical to the 'activated' macrophages from normal mice.

J Steroid Biochem, 1990 Jul 4, 36(4), 377 - 81
Testosterone micromilieu in staged rat seminiferous tubules; Parvinen M et al.; Endogenous testosterone concentrations in rat seminiferous tubules were measured in relation to different stages of the cycle of the seminiferous epithelium . For this purpose, the seminiferous tubules were mechanically separated from the interstitial tissue on a cooled (1 degree C) petri dish under a stereomicroscope without added medium . After recognition of the stages of the cycle by transillumination, the specimens were rapidly transferred by dry forceps into test tubes for testosterone radioimmunoassay . The results of the dry dissection method were compared with measurements on tubules that were kept after separation in phosphate buffered saline (PBS, pH 7.4), in order to reveal the possible leakage of testosterone from the tubules . The maximal concentration of testosterone per unit length of seminiferous tubule was found in stages VII and VIII of the cycle (288 +/- 60 fmol/cm, mean +/- SEM, n = 12), and the minimal in stages IX-XII (219 +/- 57 fmol/cm, P less than 0.01) . If the levels were correlated with unit volumes of the seminiferous tubules, identical concentrations of testosterone (521-542 fmol/mm3, approx . 500 nmol/l) were found in the different stages of the cycle . Despite the similarity of testosterone concentrations in the different parts of the seminiferous tubules the local concentrations of biologically active (i.e . free) testosterone may be modulated by extracellular and intracellular androgen binding components.

Peptides, 1990 Jul-Aug, 11(4), 625 - 32
The osteogenic stimulating effect of neuroactive calcitonin gene-related peptide; Bernard GW et al.; Silverman and Kruger (Somatosens . Res . 5(2):157-175; 1987) reported that sensory nerve fibers of the dental pulp secrete calcitonin gene-related peptide (CGRP) . These are localized exactly where secondary or tertiary dentin calcification occurs . Recently we found that CGRP has an osteogenic stimulating effect by increasing the number and size of bone colonies in vitro . The purpose of this study is to test whether there is a relationship between the effects of different doses of CGRP and bone colony numbers and/or size . Rat CGRP in different dosages (0.4, 4 and 40 micrograms/ml in BGJb medium) was added daily to 3 million light density (LD) bone marrow white cells which were harvested from adult Sprague-Dawley rats with the Ficoll-Paque density gradient separation method, then seeded onto a previously prepared feeder layer of fibroblasts in Petri dishes . Seven days after adding CGRP, in the controls without CGRP there were 2 bone colonies; with 0.4 microgram of CGRP there were 4 colonies; with 4 micrograms of CGRP there were 6 colonies; with 40 micrograms there were 9 colonies, indicating there was a significant increase in number of bone colonies with an increase in dose of CGRP between individual groups, respectively (p less than 0.0005 and p less than 0.0001) . In another experiment, intravenous injection of 10 micrograms of rCGRP/kg body weight was performed two hours before surgery . LD bone marrow white cells were collected and seeded onto a feeder layer in Petri dishes exactly as described above.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiovasc Res, 1990 Jul, 24(7), 555 - 9
Oxygen and extracellular fluid restriction in cultured heart cells: electron microscopy studies; Ne'eman Z et al.; STUDY OBJECTIVE--To evaluate the effects of "simulated ischaemia" on the structure of cultured heart cells . DESIGN--Cultured heart cells were subjected for 2 h either to anoxia or to anoxia with simultaneous extracellular volume restriction ("simulated ischaemia") . Cells maintained under normoxic conditions served as controls . The cells were then fixed in situ in Petri dishes with formaldehyde-glutaraldehyde . EXPERIMENTAL MATERIALS--Heart cells from one day old rats on day 5 in culture were used . MEASUREMENTS AND RESULTS--Electron microscope studies were carried out on control and injured cells . "Mildly ischaemic" cells featured raffled and invaginated cell surfaces, reduced matrix density, disorientated mitochondrial cristae due to swelling, and giant mitochondria . Dilatation of rough endoplasmic reticulum and electron dense membrane bound vesicles were observed in the cytoplasm . CONCLUSIONS--The model of simulated ischaemia is in keeping with the classical picture of irreversible cell damage caused by ischaemic injury.

J Biolumin Chemilumin, 1990 Jul-Sep, 5(3), 171 - 7
Different influences of cytochalasin B on the activation of human neurophils settled onto petri dishes displayed by simultaneously detected native and luminol-dependent luminescence; Roschger P et al.; Cytochalasin B (CB) is known to interfere reversibly with the cytoplasmic contractile filamental network of mammalian cells . The role of the microfilament system in the mechanism of the reactive oxygen intermediates release of polymorphonuclear leukocytes (PMNL) was studied for different kinds of stimuli . PMNL from fresh human blood were treated with CB and stimulated by adherence on plastic surfaces, by opsonized zymosan, by phorbol myristate acetate and by N-formylmethionyl-phenylalaline . The production of reactive oxygen species were monitored by simultaneous detection of native, luminol-independent, luminescence (NL) and luminol-dependent luminescence (LDL) using a method of spectral discrimination . Different influences of CB on NL with respect to LDL as well stimuli-dependent influences of CB on the luminescence response of PMNL were observed . Especially phagocytosis-associated activation of PMNL was strongly inhibited by CB, whereas LDL was reduced to a much greater extent in comparison with NL . A firm involvement of the microfilament system is indicated, but it depends on the kind of stimulus engaged.

Exp Neurol, 1990 Jul, 109(1), 111 - 30
Sulfated proteoglycans in astroglial barriers inhibit neurite outgrowth in vitro; Snow DM et al.; In vivo studies of the roof plate of the spinal cord and midline optic tectum in rodent and the developing subplate in the telencephalon of the chick showed that two glycosaminoglycans, keratin sulfate and chondroitin sulfate, possibly in the proteoglycan form (KS-PG, CS-PG, or KS/CS-PG), were present at times when axons approach closely but do not invade these territories . To address the question of whether KS/CS-PG actively inhibits growth cone elongation and to determine which component(s) of the proteoglycan may be critical to this phenomenon, we used a technique employing nitrocellulose-coated petri dishes onto which stripes of various purified macromolecules were attached . Isolated E9 chick dorsal root ganglia were grown on lanes of KS/CS-PG in alteration with lanes of the growth-promoting molecule laminin (LN) . Neurite outgrowth was abundant along stripes of LN . In contrast, upon encountering a stripe containing KS/CS-PG, neurites either stopped abruptly or turned and traveled along the KS/CS-PG stripe border . The effect was dependent upon the concentration of the proteoglycan with intermediate concentrations producing intermittent patterns of crossing . We mixed LN with the KS/CS-PG, where the LN was in concentrations which alone support outgrowth, and observed that the KS/CS-PG was still inhibitory when such a growth-promoting molecule was present . A 10-fold higher concentration of LN was able to overcome the inhibitory effect of the KS/CS-PG . These results suggest that the interaction of inhibitory and growth-promoting molecules can interact to produce a wide spectrum of neurite patterns ranging from complete inhibition to totally unimpeded outgrowth . Selective enzymatic removal of the KS or CS from the KS/CS-PG permitted various degrees of neurite outgrowth to occur across the previously inhibitory lanes, and digestion of both glycoaminoglycan moieties, leaving only the protein core of the molecule, resulted in a complete lack of inhibition . These assays demonstrated that KS/CS-PG is inhibitory to embryonic dorsal root ganglia neurites in vitro and that complete inhibition requires contributions from both KS and CS moieties.

Int J Radiat Biol, 1990 Jul, 58(1), 133 - 44
Respiration-induced oxygen gradients in cultured mammalian cells; Fengler JJ et al.; The effect of cell respiration on the availability of intracellular oxygen was investigated by comparing the radiosensitivities of respiring and non-respiring cells over a range of oxygen tensions . Monolayers of Chinese hamster V79 fibroblasts on glass Petri dishes were irradiated at respiration-inhibiting (4 degrees C) and normal (37 degrees C) cell-culturing temperatures . Desired extracellular oxygen concentrations were achieved by aspirating the culture medium above the cells prior to irradiation, leaving a residual thin film which prevented drying of the cells while allowing rapid equilibration with the overlying gas . Measurement of clonogenic survival revealed that, at equivalent extracellular oxygen concentrations, the cells irradiated at 37 degrees C were less radiosensitive than those irradiated at 4 degrees C . The difference in respiring and non-respiring cell radiosensitivity was dependent on cell shape, and decreased when the cells attached to the Petri dish surface were allowed to assume a flatter configuration . These results imply that at low extracellular oxygen tensions the oxygenation of critical cellular radiation target(s) is dependent on respiration and diffusion distance, as would be expected if oxygen gradients induced by respiration exist within and immediately around actively metabolizing cells.

J Leukoc Biol, 1990 Jul, 48(1), 74 - 80
Use of colloidal silica (Sepracell-MN) for enrichment of dendritic cells from human peripheral blood: comparison with other methods; Chehimi J et al.; Three methods are described for enrichment of dendritic cells from human peripheral blood . In method 1, mononuclear cells were incubated in plastic tissue culture flasks for two h . Nonadherent cells were removed . Adherent cells were washed to remove floating cells and incubated for 14 h at 37 degrees C in 5% CO2 . Carbonyl iron was added, and the flasks were incubated for another 2 h . Nonadherent cells were subjected to centrifugation over metrizamide gradient . Phagocytic cells containing ingested carbonyl iron, small lymphocytes, and free carbonyl iron particles passed through the metrizamide, while the interface cell population was enriched for dendritic cells . The purity and yield of enriched dendritic cells were 52.8% and 0.05%, respectively . In method 2, adherent mononuclear cells were cultured overnight, and the released cells (released adherent cells) were centrifuged over metrizamide to separate low-density cells . Monocytes from this low-density cell population were removed by panning over human gamma globulin-coated plastic Petri dishes . In this method the average purity and yield of DC were 63.8% and 0.1%, respectively . In method 3, released adherent cells were treated with anti-CD5 and anti-CD14 monoclonal antibodies plus baby rabbit complement for 15 min, washed, and centrifuged with colloidal silica (Sepracell-MN) . Centrifugation with Sepracell-MN was repeated three times . Low-density cells were panned twice over human gamma globulin-coated plastic Petri dishes . Nonadherent cells were highly enriched for DC . Contamination of T cells, B cells, and NK cells was undetectable by flow cytofluorometry . Contamination of monocytes was less than 2% . This method provided greater than 85.0% purity and 0.4% yield . This method (method 3) combines adherence, complement-dependent lysis, centrifugation with colloidal silica, and panning and provides the best yield and purity; it is therefore recommended for optimal purification of DC.

J Parasitol, 1990 Jun, 76(3), 425 - 8
Efficacy of agar-plate culture in detection of Strongyloides stercoralis infection; Arakaki T et al.; Agar-plate culture of feces using a modified petri dish proved to be highly efficient in the detection of Strongyloides stercoralis infection . Furrows left by S . stercoralis on the agar plate were distinguished readily in size from those left by Necator americanus.

Mol Cell Biol, 1990 Jun, 10(6), 2669 - 77
Biochemical characterization of the Drosophila dpp protein, a member of the transforming growth factor beta family of growth factors; Panganiban GE et al.; The decapentaplegic (dpp) gene of Drosophila melanogaster is required for pattern formation in the embryo and for viability of the epithelial cells in the imaginal disks . The dpp protein product predicted from the DNA sequence is similar to members of a family of growth factors that includes transforming growth factor beta (TGF-beta) . We have produced polyclonal antibodies to a recombinant dpp protein made in bacteria and used a metallothionein promoter to express a dpp cDNA in Drosophila S2 cells . Similar to other proteins in the TGF-beta family, the dpp protein produced by the Drosophila cells was proteolytically cleaved, and both portions of the protein were secreted from the cells . The amino-terminal 47-kilodalton (kDa) peptide was found in the medium and in the proteins adhering to the plastic petri dish . The carboxy-terminal peptide, the region with sequence similarity to the active ligand portion of TGF-beta, was found extracellularly as a 30-kDa homodimer . Most of the 30-kDa homodimer was in the S2 cell protein adsorbed onto the surface of the plastic dish . The dpp protein could be released into solution by increased salt concentration and nonionic detergent . Under these conditions, the amino-terminal and carboxy-terminal portions of dpp were not associated in a stable complex.

Boll Soc Ital Biol Sper, 1990 May, 66(5), 471 - 8
{A model of venous thrombosis induced by stasis and Feiba in rabbits}; Tettamanti R et al.; A model of venous thrombosis, induced by injection of Feiba(R) (Factor Eighth Inhibitor Bypassing Activity) plus stasis, was studied in the jugular veins of anaesthetized rabbits . Right and left jugular vein segments were isolated by surgical technique for a 3 cm length, which included the bifurcation of the vessel, and left "in situ" . Feiba(R) was injected through a marginal ear vein at the dose of 5 U/Kg/0.2 ml; 20 and 25 sec later the contralateral and the homolateral jugular vein segments were ligated respectively . Complete stasis lasted 10 or 20 min, then the vessels were removed, placed in a saline filled Petri dish and visually graded for red thrombus formation on a scale of 0 to 10 . A correlation was found between severity of thrombosis and duration of stasis . This model appears to be suitable for testing heparin or heparin-like substances, in fact a linear correlation was found between log dose of the drug (injected i.v . 5 min before Feiba(R} and its antithrombotic effect for each duration of stasis tested . In particular the DE50 value calculated for 10 min stasis was 20.5 mcg/Kg i.v . The reproducibility of the model was good even with a small number of animals (n = 6) for each treatment group.

Radiat Res, 1990 Apr, 122(1), 72 - 6
Induction of hypoxia in glass versus Permanox petri dishes; Zeman EM et al.; The survival of Chinese hamster ovary cells in culture following graded doses of X rays delivered under aerobic and hypoxic conditions, or treatment with the bioreductive drug SR 4233 under hypoxic conditions, was evaluated as a function of whether cells were plated onto glass or Permanox plastic petri dishes . In the case of treatment with SR 4233, the influence of varying the total volume of medium in the dishes was also studied . While the Permanox petri dishes were sufficient to yield "radiobiological" hypoxia, that is, oxygen enhancement ratios of approximately 3.0 were obtained for X irradiation, they were inferior to glass petri dishes with respect to the hypoxia-selective cytotoxicity of SR 4233 . For a 90-min hypoxic exposure to 40 microM SR 4233, the surviving fraction of cells plated on plastic dishes averaged about 50-fold higher than that of cells plated on glass dishes . Although varying the total medium volume did affect the extent of SR 4233-induced cytotoxicity for glass dishes--drug toxicity decreased slightly with increasing medium volume--this was not the case for the plastic dishes, in which the cell survival following a fixed SR 4233 exposure was essentially constant as a function of medium volume . These results suggest, at least for SR 4233, and under these experimental conditions, that Permanox petri dishes are not satisfactory for such studies.

Diabetes Res Clin Pract, 1990 Apr, 9(1), 65 - 73
Extreme insulin resistance due to anti-insulin receptor antibodies: a direct demonstration of autoantibody secretion by peripheral lymphocytes; Di Paolo S et al.; A 59-year-old woman with systemic lupus erythematosus was found to have marked hyperglycemia, extreme insulin resistance and abnormally high plasma immunoreactive insulin . Her circulating erythrocytes displayed a dramatic decrease of 125I-labeled insulin binding . Both the whole serum and purified IgG fraction strongly inhibited the binding of radiolabeled insulin to control erythrocytes . These results suggested, although indirectly, the existence of antibodies to insulin receptors in the serum of the patient . To directly investigate this issue, we used an enzyme-linked solid-phase immunoassay which allows the detection and enumeration of lymphocytes secreting antibodies towards insulin receptors . Peroxidase-conjugated anti-human immunoglobulin is used to reveal the binding of antibodies to insulin receptor-coated dishes . We demonstrated that the patient's mononuclear cells, when briefly incubated in Petri dishes with partially purified insulin receptor, were able to secrete immunoglobulins of G class specifically directed to the antigen . Moreover, only a fraction of the whole population of anti-insulin receptor antibodies was directed towards the insulin binding region of the receptor, seemingly corresponding to the auto-antibodies detected with conventional binding-inhibition assay.

Biomaterials, 1990 Mar, 11(2), 133 - 7
Screening of in vitro cytotoxicity by the adhesive film test; Srivastava S et al.; A novel procedure, the adhesive film test, is proposed as a screening method to predict potential cytotoxicity of biomaterials . This in vitro test utilizes a sterile strip of acrylate-based medical adhesive as an anchorage substrate in cytotoxicity studies . The adhesive film allows direct fixation of test samples to the base of the petri dish, ensuring close contact between sample and cells . The test is based on the principle that toxic components present in the test material will readily leach out into the culture medium and adversely affect the local cell population . The main advantage of the adhesive film test is that a viable cell population can be added directly to the test plate and after an incubation period of 24 h, the cellular response can be recorded as either cytotoxic or cytocompatible . Microscopic examination can be followed by quantifying the results using a micrometer to measure cellular attachment areas, migration distances and zones of inhibition . In addition, the adhesive film used to attach the test samples is shown to support extensive fibroblast growth and attachment to its surface and hence can also function as a negative nontoxic control in cytotoxicity studies.

Neurol Res, 1990 Mar, 12(1), 41 - 4
Cerebrospinal fluid factors following subarachnoid haemorrhage accelerate collagen lattice contraction by fibroblasts; Smith RR et al.; Proliferation in the intimal layer and medial necrosis are the most consistent findings in the cerebral artery following subarachnoid haemorrhage (SAH) in man . Recently, SEM studies from our laboratory have also shown marked endothelial injury as demonstrated by a profuse platelet carpet . Myofibroblasts proliferate in response to the platelet derived growth factor (PDGF), and abundant collagen is present in the vessel wall . We have employed experiments using fibroblast-populated collagen lattices to study cerebrospinal fluid (CSF) from patients with recent SAH . Isolated rat tail collagen and cultured human dermal fibroblasts are mixed together, placed in 35 mm Petri dishes, and allowed to gel . CSF samples are placed on the surface of the collagen lattice, using 0.2 ml saline for control . The collagen lattices are then incubated and daily measurements recorded . We found that CSF samples from patients with recently ruptured aneurysms significantly accelerate contraction of the collagen lattice . The factor in CSF is heat stable and has a molecular weight of less than 6000.

Cytokine, 1990 Mar, 2(2), 142 - 8
Leukocyte inhibitory factor activates human neutrophils and macrophages to release leukotriene B4 and thromboxanes; Conti P et al.; Recent evidence has proved that cytokines can stimulate the production of 5-lipoxygenase products . Leukotriene B4 (LTB4) is a major mediator of leukocyte activation in acute inflammatory reactions, which produce chemotaxis, lysosomal enzyme release, and cell aggregation . Leukocyte inhibitory factor (LIF) also causes biological responses related to inflammation, i.e., LIF directly induces specific granule secretion by polymorphonuclears (PMNs) and potentiates many formyl-methionyl-leucyl-phenylalanine (FMLPs) mediated responses . Since arachidonic acid products are important mediators of inflammation, we have studied the effects of LIF on the arachidonic acid cascade products LTB4 and thromboxane A2 (TxA2) . Resuspended at a final concentration of greater than 95% polymorphonuclear PMNs were isolated and tested with some cytokines on the release of LTB4 and TxA2 . Peripheral blood mononuclear cells were isolated and seeded in Petri dishes and incubated for 60 min . Adherent macrophages were used for the cytokine stimulation study . Both types of leukocytes were treated with LIF, interleukin 6 (IL 6), and granulocyte-monocyte colony stimulating factor (GM-CSF) at different concentrations, and test agents A23187 and FMLP . Radioimmunoassay for LTB4 and TxB2 was determined by the resulting supernatants . Treatment of PMNs and macrophages with LIF at different concentrations proved to generate significant increases in LTB4 and TxA2 production . This was compared with IL 6 and GM-CSF, which had no effects . In these experiments, TxA2 generations could not be attributed to platelet contamination of PMN suspensions . The quantity of platelet contamination was not sufficient to influence how much TxB2 was produced . The similarities of LIF to other arachidonate stimulating cytokines suggest a similar mode of action in producing hematologic changes typical of tissue injury.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood, 1990 Feb 15, 75(4), 874 - 80
Inhibition of receptor binding and neutralization of bioactivity by anti-erythropoietin monoclonal antibodies; D'Andrea AD et al.; We have generated four high affinity monoclonal antibodies (MoAbs) to recombinant human erythropoietin (EPO) . All four MoAbs immunoprecipitate radioiodinated native EPO, and the concentrations of MoAbs required for maximum binding range from 10 nmol/L to 100 nmol/L . Two MoAbs, designated Group I MoAbs, bind to an epitope within the N-terminal 20 amino acids of EPO and also immunoprecipitate sodium dodecyl sulfate (SDS)-denatured EPO . Two other MoAbs (Group II MoAbs) do not immunoprecipitate SDS-denatured EPO and do not bind to any of the eight endo C fragments of EPO . We first used murine erythroleukemia (MEL) cells to test the MoAbs for inhibition of EPO-receptor binding . MEL cells, although unresponsive to EPO, express 760 high affinity receptors for EPO per cell (Kd = 0.24 nmol/L) . To assay our MoAbs, MEL cells were grown as monolayers on fibronectin-coated Petri dishes and incubated at 4 degrees C with radioiodinated EPO . Group I MoAbs do not inhibit binding of radioiodinated EPO to the MEL EPO-receptor, but Group II MoAbs do inhibit binding in a dose-dependent manner . We next examined the neutralization of EPO bioactivity by our MoAbs, using EPO-dependent cell line . Only Group II MoAbs inhibit a newly developed EPO-dependent cell growth, demonstrating that inhibition of EPO-receptor binding correlates with neutralization of EPO bioactivity.

Wien Klin Wochenschr, 1990 Feb 2, 102(3), 77 - 80
Blood rheology during induced ischaemia of the lower limbs; Ciuffetti G et al.; The filterability rate of whole blood, the red and white blood cell sub-populations, and the erythrocyte and leucocyte count variations were studied during exercise in 20 male non-diabetic smokers, all with Stage II peripheral vascular disease (PVD), and 20 matched controls . A controlled ischaemia was induced using treadmill exercise . Blood samples were taken at rest, at the onset of calf pain and at haemodynamic recovery from peak exercise . Leucocytes were counted, separated using Ficoll-Hypaque into their sub-populations by centrifugation, and adherence to Petri dishes, re-suspended in buffer and filtered through 5 micron pore diameter filters . Whole blood filterability and the leucocyte count were significantly increased at the onset of calf pain . A significant increase was observed in the filterability of the monocyte sub-fraction and this persisted throughout the recovery period.

J Anim Sci, 1990 Feb, 68(2), 449 - 53
Incorporation and expression of a neo gene after transfer to caprine hematopoietic cells using a modified retrovirus; Casey DC et al.; The ability of the N2 retrovirus to introduce the selectable neo gene into (transform) caprine hematopoietic cells (CHC) was evaluated . Helper-free amphotropic retrovirus producing cells (RPC) were plated at approximately 1.0 x 10(5) cells per 25 cm2 tissue culture flask, cultured to 80% confluence and irradiated (1,500 rads) prior to CHC incubation . The CHC collected from three donor goats were washed and cultured for 24 h in either the RPC or control flasks . Cells were cultured in Dulbecco's Modified Eagles Medium containing 10% fetal calf serum (FCS) (DMEM) and 4 micrograms polybrene/ml . After 48 h of culture in fresh DMEM, cells were recovered and suspended in Iscove's DMEM supplemented with .3% gar, 12% FCS and 400 micrograms geneticin/ml (G418; neomycin) and transferred to 35-mm petri dishes (7.5 x 10(5) cells) for selection of G418 resistant cells . After 17 d of culture, plates were evaluated for total number of colony forming units (CFU, greater than 10 cells) . Total number of CFU was greater (P less than .01) in treatment samples (means = 175, SEM = 70) than in control cultures (mean = 0, SEM = 0) . The N2 retrovirus appears to be an effective vector for the transformation of CHC and may provide a means to introduce gene(s) into cells of domestic animals.

Dev Biol, 1990 Feb, 137(2), 219 - 32
Central nervous system antigen P84 can serve as a substrate for neurite outgrowth; Chuang W et al.; Neurite outgrowth promoting properties of neural cell surface proteins can be assessed by immobilizing isolated membrane proteins on nitrocellulose-coated petri dishes . Using this method, we have identified a unique cell surface antigen, designated P84, as a new neural cell adhesion molecule . Immunoaffinity purified P84 contains three polypeptides with molecular weights of 167, 85, and 66 kDa . When spotted onto nitrocellulose-coated plates, P84 supports adhesion of mouse cerebellar neurons and neurite outgrowth . Glial cell attachment was also observed . Intact monoclonal antibodies directed against P84 inhibit adhesion and outgrowth on a P84 substrate . This antigen is found on the surfaces of neurons in cultures of cerebellar cells . It is also found on a subclass of unidentified flat cells . P84 is not found on oligodendrocytes or GFAP-positive astrocytes . As early as E9, P84 could be detected in the floor plate region of the spinal cord . This pattern persists throughout embryonic development . Postnatally, widespread expression of P84 is observed in a variety of CNS tissues.

Gastroenterology, 1990 Feb, 98(2), 322 - 35
Basement membrane components are potent promoters of rat intestinal epithelial cell differentiation in vitro; Hahn U et al.; Basement membranes have been implicated in morphogenesis and cell differentiation . In this study, the effect of basement membrane components on intestinal epithelial cell maturation in a mesenchyme-free environment was investigated . Fetal rat small intestinal epithelial cells (from the 14th-17th day of gestation) were exposed to basement membrane-derived proteins (laminin, collagen type IV, and a complex basement membrane-enriched extract from the Engelbreth-Holm-Swarm sarcoma) and other extracellular matrix proteins (collagen type I and fibronectin) coated onto Petri dishes . The cells attached readily only to fibronectin and basement membrane proteins . For 5 days the developing epithelial colonies were monitored in vitro, assessing morphological and functional parameters of cell maturation . Colonies grown on laminin and the basement membrane extract were larger and of greater cell density . An increase in alkaline phosphatase and lactase activity was observed after 3-4 days in these colonies which could be enhanced to yield 90%-100% positive cells by the addition of dexamethasone to the medium while no sucrase-isomaltase activity was elicited . Electron microscopy confirmed a high degree of cellular polarization illustrated by tight junctions and apical microvilli in epithelial cells grown on a basement membrane-like support . In contrast, none of the other proteins stimulated the cells to mature in vitro . The authors conclude that certain basement membrane components actively promote fetal intestinal epithelial cell differentiation.

Arch Dermatol, 1990 Feb, 126(2), 175 - 80
Characterization of cellular elements in healed cultured keratinocyte autografts used to cover burn wounds; Petersen MJ et al.; Biopsy specimens from unburned skin were obtained from three severely burned patients and placed into tissue culture . After 2 to 3 weeks, the cultured keratinocytes were released from the Petri dishes and transplanted onto the patient's burn wound, which had been completely excised down to muscle fascia, thereby removing all cutaneous elements . Healing cultured autografts were found to become repopulated with Langerhans cells within 3 to 6 weeks . A neodermis rich in fibronectin rapidly formed between the autografts and muscle fascia . However, using monoclonal antibodies to cytokeratins as markers of differentiation, we found that the autograft keratinocytes expressed an abnormal pattern of differentiation that was similar to the differentiation seen in hyperproliferative states such as psoriasis . In contrast, healed split-thickness graft donor sites and reepithelialized interstices of mesh grafts maintained the basal keratinocyte staining pattern of normal skin with the AE-1 monoclonal antibody.

Biomater Artif Cells Artif Organs, 1990, 18(3), 437 - 46
The enhanced attachment and growth of endothelial cells on anhydrous ammonia gaseous plasma modified surfaces of polystyrene and poly(tetrafluoroethylene); Sipehia R; Anhydrous ammonia gaseous plasma technique was used for the surface modification of polystyrene petri dishes and poly(tetrafluoroethylene) (PTFE) membranes . Amino groups were added onto surfaces by exposing them to ammonia plasma . Plasma modified polymeric surfaces and control polymeric surfaces were seeded with bovine pulmonary artery endothelial cells (EC) . It was found that attachment of EC to control polystyrene surface was negligible . On the plasma modified polystyrene surface, there was improved attachment and growth of EC . At 96 hours, plasma modified surfaces yielded an order of 3 magnitudes more cells compared to those on control . Twenty four hours after seeding the cells, the percentage of EC attachment to control PTFE surfaces and modified surfaces were found to be about 36% and 92% respectively.

Histochemistry, 1990, 93(4), 359 - 62
Towards microfluorometric quantitation of polyamines in situ . Relationship between cellular polyamine concentration and fluorescence yield of the formaldehyde fluorescamine method; Hougaard DM et al.; Two different fluorescence cytochemical methods, the formaldehyde-fluorescamine (FF) method and the orthophthalaldehyde (OPT) method as well as an immunocytochemical method have been developed for the localization of spermidine and spermine . Of these three methods, the FF-method is the most easy to perform . We have studied the relationship between fluorescence intensity induced by the FF-method and cellular polyamine levels measured by HPLC in MCF-7 cells and HeLa cells . The experiments were designed to obtain different cell concentrations of polyamines . Cells grown on microscope slides in Petri-dishes were partly depleted of spermidine by two days inhibition of their ornithine decarboxylase activity using alpha-difluoromethylornithine . One hr before harvest the cells were exposed to different concentrations (0-30 microM) of spermidine . Microfluorometric results and chemical determinations of spermidine and spermine were obtained from each separate slide . The cellular total polyamine (spermidine + spermine) concentration on the slides varied between 4 and 15 nmol per mg protein (MCF-7 cells) and 5 and 26 nmol per mg protein (HeLa cells) and the corresponding microfluorometric results between 60 and 115 arbitrary units (MCF-7 cells) and 80 and 160 arbitrary units (HeLa cells) . Simple regression analysis showed a good linear relationship between cellular polyamine concentration and FF-fluorescence yield . The correlation coefficient for MCF-7 cells was 0.86 and for HeLa cells 0.82, significance of the correlations was p less than or equal to 0.0001 . Our results add further credence to the specificity of the FF-method and indicate that the method may be useful for microfluorometric quantitation of polyamines in situ.

In Vitro Cell Dev Biol, 1990 Jan, 26(1), 51 - 6
Cell proliferation on hydrogels; Nagaoka S et al.; The adhesion and proliferation of mammalian fibroblasts (Flow 7000) on the surface of hydrophilic (copolymer of N-vinyl-2-pyrrolidone and methyl methacrylate) and hydrophobic {polymethylmethacrylate (PMMA) stereocomplex} hydrogels with a wide range in water content were studied morphologically and quantitatively . It was demonstrated that cell proliferation on hydrogels by a static culture method decreased as the water content of the gels increased . However, it is remarkable that the cell proliferation on PMMA hydrogels with a high water content is equivalent to that on glass Petri dishes . The results obtained in the proliferation of cells on the surface of these hydrogels closely correspond to the state of cell adhesion . When fresh medium or air was perfused from the opposite side of the PMMA hydrogel membrane on which the cells were proliferating (perfusion method), the cells continued to grow into a higher density than with the conventional static culture method . In the case of fresh medium perfusion, the amount of proliferated cell was dependent on both the permeability of the membrane and the density of the membrane "scaffolding." Virus multiplication in the cultured cells increased in proportion to the cell density, whereas the cell function was similar in both culture methods.

Tsitologiia, 1990, 32(8), 852 - 7
{Benzopyrene hydroxylase induction and the functioning of the immunocompetent cells in human peripheral blood}; Lozovatskii AL et al.; Xenobiotics--inducers of benzo(a)pyrene hydroxylase (BPH)--exert different effects on mitogen-stimulated and mitogen-unstimulated human peripheral blood mononuclear cells (PBC) . In mitogen-stimulated culture xenobiotics highly increase BPH activity and suppress cell blast transformation . The incubation of the unstimulated PBC in the presence of xenobiotics increases insignificantly BPH activity, intensifies T-cell differentiation and concanavalin A-induced proliferation . The BPH activity is mainly associated with the PBC adhered to plastic Petri dishes . However, the control and induced levels of BPH activity depend on the interaction between adhered and nonadhered cells.

Growth Factors, 1990, 3(3), 181 - 90
Characterization of 5B12.1, a monoclonal antibody specific for IL-6; Sawamura M et al.; A monoclonal antibody (MAb) specific for interleukin-6 (IL-6) was generated by fusing SP2/0 cells with spleen cells from a mouse immunized with rat spleen cell derived plasmacytoma growth factor (rat PCT-GF) . This MAb inhibited the growth of an IL-6-sensitive murine plasmacytoma clone, MD90, in the presence of the immunogen, rat PCT-GF . More interesting, however, this MAb demonstrated species cross-reactivity by neutralizing murine (recombinant and P388D1 cell line-derived) and human (recombinant) IL-6 . IL-6 neutralization activity was also established in other IL-6 bioassays, such as the proliferation of spleen cells, plasmacytoma T1165, and a B-cell hybridoma 7TD1 . IL-6 neutralization was overcome partially by increasing the concentration of PCT-GF . The MAb had no effect on PCT-GF-independent plasmacytoma KI81 proliferation . Plastic petri dish-bound MAb removed rmIL-6 activity . These results suggest that this MAb specifically binds IL-6 and neutralizes bioactivity of various PCT-GF, rmIL-6, and rhIL-6.

Acta Biol Hung, 1990, 41(4), 387 - 97
Limitations of the use of plant material grown in Petri dishes for physiological experiments; Tari I et al.; Roots of plants growing "aeroponically" (AP) on moistened filter paper in Petri dishes for a few days are fairly often used for physiological experiments (e.g . measurement of root growth), for ion or herbicide uptake tests, before the establishment of hydroponic or aseptic cultures although their hormonal status is markedly different from that of the hydroponic (HP) control . On the 4th day of germination the ethylene production of cucumber (Cucumis sativus L . cv . Budai csemege) roots growing in AP under controlled conditions increased considerably and exhibited a maximum curve, HP roots evolved ethylene much more constantly . The morphological changes in AP roots (e.g . inhibited elongation and swelling of primary roots, and increased formation of root hairs), resembling those caused by exogenously applied ethylene, can be prevented with 10(-5) M Ag+, an inhibitor of ethylene action . In roots of one-week-old AP seedlings, the amount of an acidic inhibitor, which as judged from the Rf values is likely to be abscisic acid (ABA), is about twice as high as in HP seedlings . An elevated ethylene or ABA level of AP roots may result in a reduced elongation of the primary roots . Counteraction of this inhibition by Ag+ suggests that the effect of ethylene is the primary event in the reduction of root length . When using plant material grown in Petri dishes the possibility of similar changes in hormonal status of the roots must be taken into consideration.

J Lipid Mediat, 1990, 2 Suppl, S93 - 9
Modulation of the priming effects of platelet-activating factor on the release of interleukin-1 from lipopolysaccharide-stimulated rat spleen macrophages; Pignol B et al.; The pharmacologic modulation of the effect of platelet-activating factor (PAF) on the interleukin-1 (IL-1) activity present in the supernatants from lipopolysaccharide (LPS)-stimulated macrophages was investigated . Rat spleen macrophages were isolated by centrifugation on a Ficoll-Hypaque gradient followed by adherence on plastic petri dishes for 60 min at 37 degrees C under a 5% CO2/95% air atmosphere . The IL-1 content in the cell-free supernatants was assessed using the mouse thymocyte proliferation assay . Preincubation of macrophages for 10 min with 10 fM PAF prior to stimulation with 20 micrograms/ml LPS for 24 h markedly increased the IL-1 activity present in the supernatants from macrophages whereas no direct effect of PAF was noted . Although they had no direct effect, addition of L-651,392 (10 microM), a lipoxygenase inhibitor, or the oxygen-derived free radical scavenger mannitol (10 microM) during the 10-min preincubation period with PAF reversed by 105.0% and 79.9%, respectively, the action of the autacoid on IL-1 activity . Pertussis toxin (PT, 1 microgram/ml) decreased by 30% the LPS-induced IL-1 activity . Association of PT with PAF suppressed the enhancing effect of 10 fM PAF on the IL-1 activity present in the supernatants from LPS-stimulated macrophages . Thus, the enhancing effect of PAF on IL-1 release appears to be due to the production of lipoxygenase metabolites, leading to superoxide production and alterations of cAMP levels.

Medicina (B Aires), 1990, 50(4), 356 - 60
{Rabies transmission to rodents after ingestion of naturally infected tissues}; Delpietro H et al.; We describe seven trials in which Calomys musculinus and Mus Musculus were fed with naturally rabies-infected tissues extracted from vampire bats, dogs, and bovines . The tissues were not subjected to any kind of previous laboratory handling and were administered directly in Petri dishes; rodents ate them voluntarily . The only infectious tissues were bovine brains taken from outbreaks transmitted by vampire bats (Table 1) . It was possible to infect the two species of tested rodents, and there was no relationship between infection and amount of virus ingested . From the total number of 132 animals that ingested different kinds of rabies-infected tissues, 3 died of rabies infection . From 128 survivors of all the exposed mice, 22 presented seroconversion to rabies . In the infected Calomys musculinus there was evident nervous symptomatology consisting in excitability, aggressiveness, paralysis and isolation of rabies virus from their salivary glands . The possibility that rodents become rabies infected by the ingestion of naturally infected tissues would indicate that they may constitute a reservoir for rabies because cadaver ingestion is a natural feeding source for many species . Furthermore they may permit the passage of rabies virus in carnivorous, animals since they are an important prey for them . The present observations indicate two situations which may increase rabies risks to man through rodent bites.

Probl Tuberk, 1990, (9), 60 - 2
{Microscopic method of the determination of Mycobacterium tuberculosis sensitivity to chemotherapeutic agents}; Bil'ko IP; A microscopic method of testing M . tuberculosis sensitivity to chemotherapeutic drugs was developed and tried out . According to it, M . tuberculosis are inoculated and cultivated on agar-agar gel disks, put on a Petri dish with an egg medium and chemotherapeutic drugs . Following the incubation lasting for 5-7 days at 37 degrees C, agar-agar gel disks with inoculated material are removed from the medium and subjected to microscopic examination as to confirm or deny the growth of mycobacteria in the form of microcolonies . The developed method is simple and easy to use, and makes it possible to shorten the period of testing M . tuberculosis sensitivity to chemotherapeutic drugs up to 5-7 days since the examination is started.

Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi, 1990, 8(3), 199 - 202
{Successful axenic cultivation of a local human strain of Giardia lamblia in suckling gerbil}; Lu SQ et al.; G . lamblia cysts isolated from the fresh feces of a Giardia-infected boy in Beijing rural area were inoculated into suckling gerbils (Meriones unguiculatus) . Trophozoites of G . lamblia obtained from the intestines of infected gerbils were cultivated in modified TYI-S-33 medium enriched with dehydrated bovine bile . The parasites grew luxuriantly and formed an intensive monolayer on the surface of the culture tube on day 14 after initial cultivation . The culture has been maintained for more than 12 months and more than 120 subcultures have been made . The growth curve of the organism showed that the peak growth of the trophozoites was attained at the 120th hour after seeding . The generation time was 15 +/- 2.0 hours . Periodic examinations of Giardia cultures for bacteria contamination, with Petri dishes of blood agar and beef broth, proved negative . After being cryopreserved in liquid nitrogen for 1 week or longer, the average viable rate of the organism was 65.7% and the resuscitated parasites grew luxuriantly in subcultures.

Child Nephrol Urol, 1990, 10(4), 186 - 8
Significance of the monocyte crossmatch in living-related transplantation; Leichter HE et al.; It is claimed that antibodies to the vascular-endothelial (VEM) antigen system are responsible for early renal transplant rejection . To evaluate the significance of these anti-VEM antibodies, we performed a retrospective study in 16 patients aged 14.8 +/- 5 (SD) years, who received either a living-related HLA-identical (n = 5) or haplo-identical (n = 11) transplant between 4/79 and 3/84 . All patients had been transfused prior to transplantation . Lymphocyte and monocyte complement dependent cytotoxic crossmatches were performed using pre- and posttransplant recipients' sera and donor lymphocytes and monocytes . Lymphocytes were isolated with Ficoll-Hypaque; monocytes by adherence to plastic Petri dishes . Of the 16 patients studied, only 1 had a positive pre- and posttransplant monocyte crossmatch . Crossmatches using recipients' T and B lymphocytes were uniformly negative, indicating the presence of anti-VEM antibodies in the absence of anti-HLA antibodies in this 1 patient . This patient rejected the transplant in the immediate posttransplant period . Of the remaining 15 patients, 4 lost their kidneys within 16 days posttransplant, whereas 11 have good graft function . We conclude that anti-VEM antibody occurs rarely pretransplant and is an unusual cause of immediate rejection in the living-related transplant situation . However, when anti-VEM antibody is identified, transplantation should be avoided because of the likelihood of immediate early rejection.

Pathobiology, 1990, 58(6), 323 - 8
Cloning of primary human tumors in capillary tube versus Petri dish: a head-to-head comparison; Peng XG et al.; Plating efficiencies (PEs) of primary human tumors cloned in a capillary assay system were compared to those derived from the conventional two-layer agar Petri dish assay system . A total of 143 consecutively received primary human tumors of 24 different pathologies were simultaneously tested in both assay systems . The successful clonal growth rate in the capillary assay was 82.7%, while in the Petri dish it was 64.7% (p less than 0.001) . The median PE was 0.017% in the capillary assay demonstrating a 4.25-fold increase over the 0.004% PE of the Petri dish system . The data confirmed previous results showing that cancer cells of ovarian, breast, and lung origins clone with higher PE in capillary tubes . In contrast, we confirmed that stomach carcinoma cells were the only tumor type that showed a higher PE with the Petri dish method . In addition, this study shows for the first time that lymphomas and renal cell carcinoma, when they survive in vitro, clone equally well in both methods . However, the capillary cloning method resulted in a 66% success rate for lymphoma cell cloning, but Petri dish cloning resulted in only a 33% success rate . Thus, for some types of cancers (i.e., lymphoma), capillary cloning may be advantageous because it improves the probability of obtaining evaluable results . In other cases, the advantage of capillary cloning may be only the decreased amount of specimen and reagents needed for the assay.

J Egypt Soc Parasitol, 1989 Dec, 19(2 Suppl), 887 - 94
Dot-ELISA in diagnosis of schistosomiasis; Madwar MA et al.; One microliter of S . mansoni egg antigen was dotted directly on the nitrocellulose paper sheet acting as the adsorbent surface (9 dots/paper) . The sera of 25 Egyptian patients and 15 healthy persons (2 microliters of each) were dotted over the antigen dots, then 2 ml of each of the blocking, washing, HRP-conjugated IgG and DAB adding procedures, were added over the nitrocellulose paper in the petri-dish at room temperature . An intact brown circle (by naked-eye) indicates a positive in Dot-ELISA . There is an insignificant dot colour intensities in different clinical stages of S . mansoni infected Egyptians whereas, a direct relation was obtained between egg count and the colour intensity of the dots . The test had 100% sensitivity and 86% specificity thus it appears to be useful for both laboratory and field studies.

Cancer Res, 1989 Dec 1, 49(23), 6738 - 44
High invasiveness associated with augmentation of motility in a fos-transferred highly metastatic rat 3Y1 cell line; Taniguchi S et al.; We previously reported that v-fos transfer to a src-transformed rat 3Y1 cell line enhanced lung metastasis . To clarify the mechanism of this enhancement, we compared various biological factors related to metastatic potential between a fos-transferred highly metastatic cell line (fos-SR-3Y1-202) and the control cell line transferred with genetic marker (pSV2-neo) plus pBR322, neo-SR-3Y1-200 . Lung arrest, the effect of lung extract on cell growth, or sensitivity to natural killer cells could not explain the higher metastasis of fos-SR-3Y1-202, compared to findings with neo-SR-3Y1-200 . The invasiveness, assessed by penetration through a Matrigel-coated filter was about 5 times higher in fos-SR-3Y1-202 than in neo-SR-3Y1-200; high invasiveness in vitro was also observed in a fos-transferred mixed-population cell line (fos-SR-3Y1-200) and fos-transferred highly metastatic clones . Histopathological evidence of an in vivo tumor also showed the high invasiveness of fos-SR-3Y1-202 cells . To elucidate the causes of the increased invasiveness of fos-SR-3Y1-202, attachment of the cells to Matrigel and its components, such as laminin and type IV collagen, type IV collagenase activity, and motility were examined . Attachment of the cells to the substrate coated on Petri dishes or the activity of type IV collagenase did not differ significantly . On the other hand, cell motility, determined by a new method to directly quantitate alteration of cell shape continuously, using video image analysis and computer techniques, was higher in fos-SR-3Y1-202 than in neo-SR-3Y1-200 . Thus, the fos-transferred cell line, fos-SR-3Y1-202 has a high invasiveness, in association with augmentation of motility, hence the enhancement of metastatic potential.

Int J Radiat Biol, 1989 Dec, 56(6), 989 - 98
Cell density dependence of transformation frequencies in C3H10T1/2 cells exposed to X-rays; Bettega D et al.; The effects of cell density on transformation frequencies were studied in C3H10T1/2 cells exposed to 0.5 and 7 Gy of 200 kVp X-rays . Initial cell density strongly influenced transformation frequency; this decreased by a factor of between 4 and 10 when the initial seeding density was changed from 50 to 2500 cells/10 cm diameter Petri dish . The data were fitted with two equations: (a) an allometric function represented on a log-log scale by a straight line and (b) a sigmoidal function with plateaux between 50 and 250 cells/dish and above 600 . The two curves are compared and their probabilities discussed . Our data indicate that the region between 50 and 250 cells/dish would be the most suitable region for dose-effect measurements . A study of the growth curves at 0.5 and 8.5 Gy shows that cell growth rates are not influenced by initial cell density.

Neurochem Res, 1989 Dec, 14(12), 1203 - 7
Characterization of a co-culture system of neurons and hepatocytes; Westergaard N et al.; A co-culture system of cerebellar granule cells (glutamatergic neurons) and hepatocytes has been developed . Petri dishes divided in halves by a temporary septum were coated with poly-L-lysine and cerebellar granule cells plated in one of the compartments . Five days later hepatocytes were plated in the other compartment and after 2 days the septum was removed and the two cell types shared the same culture medium for a period of 5 days . During this period of time cultures of neurons and hepatocytes kept separately or in co-culture exhibited identical characteristics with regard to activities of pyruvate kinase and glucokinase (hepatocytes), aspartate aminotransferase (neurons) as well as evoked transmitter release (neurons) and content of cytochrome P-450 (hepatocytes) . The results show that it is possible to maintain neurons and hepatocytes in co-culture sharing the same culture medium for a prolonged period of time . Such a system may serve as a pharmacological model to study interactions between liver and brain cells with regard to neuroactive drugs.

Int J Cardiol, 1989 Nov, 25(2), 193 - 8
Leucocyte rheology in controlled coronary ischaemia; Ciuffetti G et al.; Since no studies have been carried out on the exact origin of the alterations in white blood cell rheology during the early stages of controlled ischaemia in coronary arterial disease, a model was set up using a cycle ergometer test (with a 25 watts increase every 2 minutes) . Blood samples were taken (before and after exercise and again 8 minutes later at recovery) from 18 patients with stable angina pectoris and a group of 22 matched controls . The filterability (through 5 micrometer diameter pore filters) of the polymorphonuclear leucocyte sub-population (separated by density gradient), the monocyte and lymphocyte sub-fractions (separated by adhesion to Petri dishes) as well as leucocyte activation (observed under a light microscope) were monitored . Our results showed that the total leucocyte count in patients and controls rose after exercise and was accompanied by a differential shift from the polymorphonuclear to the lymphocyte cells . The polymorphonuclear filterability rate increased significantly in patients when compared to their basal values at rest, and to the controls after exercise (+ 19.58%; P less than 0.002 vs basal values at rest; + 18.72%; P less than 0.002 vs controls) . This increase persisted throughout the recovery period (+ 19.86%; P less than 0.002 vs basal values; and + 23.52% P less than 0.001 vs controls), indicating that a reduced polymorphonuclear leucocyte filterability can be associated with the first signs of ischaemia.

Anticancer Res, 1989 Nov-Dec, 9(6), 1897 - 902
Homogeneous growth of tumor cell colonies in agar containing glass capillaries; Lathan B et al.; The capillary cloning system has been shown to have advantages over conventional cloning of human tumor cells in Petri dishes . In the present study a further optimization towards homogeneous colony distribution and high cloning efficiency is described . For reasons of reproducibility the study focused on cell lines, i.e . three human linew (MDA-231, HT-29, L363) and one rodent line (CHO-AB) . Major variables investigated were the gel length, the capillary tube diameter, the tube sealing and buffer system, and the cell number . Criteria for optimal tumor colony growth included homogeneous colony distribution along the gel, mean colony size and cloning efficiency . It was found that colony distribution as well as overall colony growth depended largely on the gel length, i.e . on the volume of tumor cell containing agar applied per capillary tube . The results showed that optimal tumor cell colony growth was achieved in 100 ul capillary tubes of 1.2 mm internal diameter filled with 30ul, yielding a gel length of 27 mm . Colony formation did not significantly differ between sealed and unsealed tubes, provided that HEPES buffer was added . It was concluded that, for practical reasons, sealing of tube ends and therefore utilization of HEPES buffer is not necessary . In a head to head comparison, cloning efficiency was equal or higher in capillary tubes than in Petri dishes . The capillary cloning system is an alternative for drug development as well as for predictive drug testing . Its major advantage is the utilization of fewer tumor cells.

J Neuroimmunol, 1989 Nov, 25(1), 57 - 61
Effector cells of autoimmune encephalomyelitis in the rat belong to the CD4-positive, OX22-adherent T cell subset; Hayosh NS et al.; We characterized the effector cells which mediate experimental autoimmune encephalomyelitis (EAE) on the basis of selective adherence properties . Nylon-nonadherent spleen cells (SpC) from Lewis rats challenged earlier with myelin basic protein (BP) in adjuvant were separated by 'panning' on Petri dishes coated with monoclonal antibody (MAb) OX22 . OX22 recognizes high molecular weight forms of the leukocyte-common antigen which is present on several cell types, including the CD4-positive T cells which mediate delayed hypersensitivity reactions . We found that the EAE effector cells were enriched in the OX22-adherent T cell population, which supports the hypothesis that delayed hypersensitivity is important in the pathogenesis of this autoimmune disease.

J Membr Biol, 1989 Oct, 111(1), 37 - 48
Fusion of cultured dog kidney (MDCK) cells: I . Technique, fate of plasma membranes and of cell nuclei; Kersting U et al.; The evaluation of the intracellular signal train and its regulatory function in controlling transepithelial transport with electrophysiological methods often requires intracellular measurements with microelectrodes . However, multiple impalements in epithelial cells are hampered by the small size of the cells . In an attempt to avoid these problems we fused cells of an established cell line . Madin Darby canine kidney cells, originally derived from dog kidney, to "giant" cells by applying a modified polyethylene glycol method . During trypsin-induced detachment from the ground of the petri dish, individual cells grown in a monolayer incorporate volume and mainly lose basolateral plasma membrane by extrusion . By isovolumetric cell-to-cell fusion, spherical "giant" cells are formed within 2 hr . During this process a major part of the individual cell plasma membranes is internalized . Over three weeks following cell plasma membrane fusion degradation of single cell nuclei and cell nuclear fusion occurs . We conclude that this experimental approach opens the possibility to investigate ion transport of epithelia in culture by somatic cell genetic techniques.

J Clin Pathol, 1989 Oct, 42(10), 1083 - 7
Leucocyte behaviour in controlled ischaemia of the calves; Ciuffetti G et al.; Whole blood filterability and leucocyte behaviour (number, activation, and subfraction filterability rates) were monitored at the earliest stage of peripheral ischaemia in 18 patients with stage II peripheral occlusive arterial disease (PAOD) and 20 matched controls . A model of controlled ischaemia, using exercise to stress leg circulation, was set up and blood samples were taken before exercise, at the onset of calf pain, and at recovery from peak exercise . Leucocytes were counted, separated into their subfractions on a Ficoll-Hypaque density gradient and by adhesion to Petri dishes, and filtered in buffer (like the whole blood suspensions) through 5 microns pore diameter Nucleopore filters . Unfractionated white cells, separated under gravity, with pseudopodia or cytoplasmic irregularities were regarded as activated . The whole blood filterability rate was significantly increased at the onset of calf pain and was associated with significant increases in the number of leucocytes and in the filterability rate of the monocyte subfraction, the latter persisting throughout the recovery period . No significant changes were observed in the other variables monitored, showing that impairments in white cell rheology may be associated with ischaemia.

J Cell Physiol, 1989 Sep, 140(3), 483 - 90
Cultures of fibroblasts in fibrin lattices: models for the study of metabolic activities of the cells in physiological conditions; Gillery P et al.; Two techniques for fibroblast culture in three-dimensional fibrin matrices (fibrin lattices) were used to study the behavior and metabolism of cells in this physiological support . When the fibrin lattices were prepared in silicone-coated glass petri dishes, fibroblasts induced retraction of lattices to an extent dependent on both cell density and serum concentration, the cells stopped dividing, and their protein and collagen syntheses proceeded at a lower rate, like that in collagen lattices . When fibrin lattices were prepared in plastic petri dishes, the matrix attached to the walls, there was no retraction, and the protein synthesis was very active even as regards collagen . The optimal concentration of ascorbic acid in nonretracting fibrin lattices was lower (10 micrograms/ml) than in monolayers (50 micrograms/ml) . The comparison of collagen and fibrin lattices showed that the collagenic nature of the lattice was not compulsory for supporting the phenomenon of retraction and that fibroblasts, when exposed to a stress in a fibrin matrix prevented from retracting, secreted far more collagen.

Fertil Steril, 1989 Sep, 52(3), 503 - 8
Coculture of human zygotes on fetal bovine uterine fibroblasts: embryonic morphology and implantation; Wiemer KE et al.; Zygotes from in vitro fertilization patients (n = 116) were randomly allocated to culture in either conventional plastic petri dishes or coculture on a monolayer of fetal bovine uterine fibroblasts . Embryos (n = 288) remained 26 to 32 hours in these culture systems . Video tape recording for later morphological analysis (11 parameters) was performed on 117 conventionally cultured and 104 cocultured embryos, shortly before replacement, by an independent observer, unaware of the culture conditions for each embryo . A significantly greater number of cocultured embryos (52%) had "good" morphology (zero or only one abnormal characteristic) as compared with conventionally cultured embryos (30%) . The most outstanding morphological characteristic of cocultured embryos was the expanded appearance of their blastomeres . The incidence of implantation per embryo increased from 13% to 19% when the coculture rather than conventional culture system was used, and the incidence of ongoing pregnancy per patient after coculture doubled to 35%.

Int J Cell Cloning, 1989 Sep, 7(5), 303 - 13
Microcapillary clonogenic assays for human marrow hematopoietic progenitor cells; Du DL et al.; The capillary clonogenic cell assay was developed and adapted to culture myeloid and erythroid colonies from human bone marrow cells . The plating efficiencies for femoral bone marrow granulocyte-macrophage progenitors (CFU-gm), erythroid colony-forming units (CFU-e) and erythroid burst-forming units (BFU-e) were 0.143%, 0.229% and 0.141%, respectively . Standard bone marrow progenitor Petri dish assays require a total culture volume of 1 ml per dish, and as such are not suitable for the small numbers of cells often obtained from human bone marrow samples . The microcapillary assay as developed and standardized in our laboratory has the unique advantage of being able to utilize small numbers of cells . This technique is suitable for evaluating the myelotoxicity of investigational new anti-cancer and anti-HIV agents and for further investigation of the mechanisms underlying chemotherapy-induced bone marrow toxicity.

J Protozool, 1989 Sep-Oct, 36(5), 451 - 4
Plasmodium falciparum gametocyte formation in vitro: its stimulation by phorbol diesters and by 8-bromo cyclic adenosine monophosphate; Trager W et al.; Phorbol 12-myristate 13-acetate increased the number of gametocytes by 50 to 100% in well or petri dish cultures of the HB-3 clone of Plasmodium falciparum . Phorbol dibutyrate had a similar effect . The optimal concentration for each of these agents was 20 ng/ml or approximately 30 nM . No effect of forskolin was found, other than a general inhibition of growth at concentrations over 10 microM . An inhibitor of phosphodiesterase, 8-bromo cyclic adenosine monophosphate (at concentrations of 0.1 and 1.0 microM) also significantly increased the number of gametocytes formed by this clone.

Stain Technol, 1989 Sep, 64(5), 225 - 7
An improved cellophane method for in vitro germination of recalcitrant pollen; Alexander MP et al.; The cellophane technique of La Cour and Faberge has been improved by the use of a booklet of filter paper . The booklet consists of seven squares of filter paper stapled together; the cellophane on which the pollen is germinated is placed between the two top leaves of the booklet and the whole soaked in a sucrose-based nutrient medium for 15 min . This arrangement keeps the cellophane flat as it absorbs medium . The top leaf of the booklet is then removed, the pollen dusted on it and the completed preparation closed in a plastic-wrapped Petri dish . The lower leaves of the booklet keep the cellophane moist for up to 24 hr . Proportions of pollen grains germinating are at least as high as in the hanging drop method; pollen of species that germinate poorly or not all in hanging drops do well in this technique . Because the pollen tubes adhere tightly to the cellophane, staining, observation, and studies of various sorts are facilitated.

Plant Foods Hum Nutr, 1989 Sep, 39(3), 267 - 78
Compositional and digestibility changes in sprouted barley and canola seeds; Chung TY et al.; Barley and canola seeds were sprouted over a 5 day period, in laboratory conditions under room temperature (22 degrees C) and room lighting . Following initial hydration, seeds were kept moist by wetting the germination trays at 9 a.m., 1 p.m . and 6 p.m . daily . A parallel germination experiment using 200 g quantities of seeds in petri dishes was conducted . Starting from the second day of germination, and every day, dishes of germinating seeds were removed, oven-dried, weighed and milled for proximate and chemical analysis . Seeds from the main germination experiment were fed in a digestibility trial to Wistar rats . Results indicated that sprouting was associated with depletion of many nutrients in both barley and canola, the major losses being in respect of dry matter, gross energy and triglycerides . In barley (but not in canola) sprouting was associated with significant increases in crude fiber and diglyceride content . In canola, there were significant losses in lipid content and increases in phytosterol and phospholipid content . Digestibility data showed an enhancement in digestibility of nutrients in barley but not in canola, implying that sprouting improved nutritional quality of barley but not canola.

Biochim Biophys Acta, 1989 Aug 7, 983(2), 264 - 8
Volatile anesthetics inhibit the ion flux through Ca2+-activated K+ channels of rat glioma C6 cells; Tas PW et al.; Ca2+-activated K+ channels in rat glioma C6 cells were investigated using monolayers of these cells in petri dishes . The ion flux through the channels was studied with 86Rb+ after addition of a Ca2+-ionophore to the incubation medium . Both the influx and efflux of 86Rb+ through these Ca2+-activated K+ channels were inhibited by the general anesthetic halothane (at clinical concentrations) . Other volatile anesthetics such as isoflurane, enflurane and methoxyflurane also inhibited the Ca2+-activated K+ channels at clinical concentrations . Inhibition of these channels by general anesthetics could have profound effects on signal transmission in the brain.

Int J Cancer, 1989 Jul 15, 44(1), 131 - 6
Clonogenic cell assay for anchorage-dependent squamous carcinoma cell lines using limiting dilution; Grenman R et al.; Clonogenic assays under either anchorage-dependent or -independent conditions are very useful for testing the sensitivity of tumor cells to cytotoxic drugs and radiation . These assays have not been widely used with squamous-cell carcinomas (SCC) because of poor tumor-cell viability and poor cloning efficiency, especially in semi-solid media . To find a clonogenic assay suitable for use with human squamous cancers we tested SCC lines, derived in our laboratory from patients with head and neck cancer, for the capacity to form colonies in soft agar and in 96-well plates . Of 13 UM-SCC lines tested for colony formation in agarose, only UM-SCC-11A was capable of growth in conventional semi-solid media . One other line, UM-SCC-14C, produced colonies in agarose only in the presence of epidermal growth factor . In contrast, all 17 of the SCC lines tested exhibited colony formation in adherent cell culture using limiting dilution in 96-well plates . The plating efficiencies of the SCC lines in the 96-well plate assay ranged from 0.02 to 0.52 colonies (wells)/cell whereas the PE values in soft agar were lower, ranging from 0.0055 to 0.0086 colonies/cell . The 96-well plate assay is not affected by cell migration, a problem encountered with some cell lines when clonogenic assays are performed in Petri dishes . UM-SCC-11A was tested for radiation sensitivity both in soft agar and in the 96-well plate assay . Comparable results were obtained . In summary, the majority of SCC cell lines did not form viable colonies in soft agar but the 96-well plate assay was applicable to a broad spectrum of anchorage-dependent human SCC cell lines and provides an efficient method for evaluating clonogenic cell survival.

Gene, 1989 Jul 15, 79(2), 333 - 44
Functional topology of human tissue-type plasminogen activator: characterization of two deletion derivatives and of a duplication derivative; Stern A et al.; Three types of permanent Chinese hamster ovary (CHO) cell lines with different amplified expression constructs that abundantly secrete derivatives of human tissue-type plasminogen activator (t-PA) were established . The first one expresses a deletion derivative in which the kringle 2 domain (K2) has been removed (FGK1L) . In the second derivative, the growth-factor-homologous domain (G) has also been deleted (FK1L); a third line expresses a duplication derivative of K2 (FK2K2L) lacking the (G) and kringle 1 (K1) . All deletion derivatives were constructed according to the exon-intron organization of the gene . We have analyzed the secreted proteins and the fibrinogen-stimulated plasminogenolytic activity as a function of different culturing conditions (fetal calf serum, aprotinin) of the cells . The specific activities of the two deletion derivatives (FGK1L and FK1L) were only 10-20% of the specific activity of t-PA . Surprisingly, the specific activity of the K2-duplication derivative, FK2K2L, was three times higher than that of t-PA . These data were correlated with the morphological properties of CHO cells constitutively secreting the described derivatives under different culturing conditions . CHO cells secreting the deletion derivatives (FGK1L and FK1L) remained attached to the surface of the petri dishes . Cell lines secreting the duplication derivative FK2K2L detached from the surface even in the presence of the protease inhibitor aprotinin.

Arteriosclerosis, 1989 Jul-Aug, 9(4), 446 - 52
Effect of normolipemic and hyperlipemic serum on biosynthetic response to cyclic stretching of aortic smooth muscle cells; Grande JP et al.; Arterial smooth muscle cells synthesize matrix macromolecules in response to mechanical stimulation . Exposure to serum lipids also stimulates connective tissue fiber accumulation . To assess the effect of serum lipids on the biosynthetic response to tensile stress, we subjected rabbit aortic smooth muscle cells that were cultured on purified elastin membranes to cyclic stretching and relaxation 50 times per minute in the presence of serum-free medium (SFM), normolipemic serum (NLS), or hyperlipemic serum (HLS) . Incorporation of 14C-proline into proline and into hydroxyproline was taken as a measure of protein and collagen synthesis . When cells were grown in plastic Petri dishes, exposure to NLS or HLS increased both protein and collagen production to the same extent compared to synthesis in SFM (1.7 times for NLS and 1.6 times for HLS; p less than 0.001 compared to SFM) . For cells grown on stationary elastin membranes, NLS and HLS also increased protein and collagen synthesis compared to SFM . The effect of NLS was 1.35 times that of HLS for protein and 1.43 times greater for collagen (p less than 0.03) . Cyclic stretching in SFM doubled synthesis for both protein (p less than 0.002) and collagen (p less than 0.002) compared to stationary controls, but had no effect on synthesis in NLS . In HLS, however, cyclic stretching elevated synthesis to the same level as was found in NLS (p less than 0.003) . We conclude that the relative inhibition of synthesis on stationary membranes by HLS was not due to a toxic effect, since HLS increased synthesis both in Petri dishes and on elastin membranes, and the amplifying effect of cyclic stretching in HLS was similar to that seen in SFM.(ABSTRACT TRUNCATED AT 250 WORDS)

Appl Environ Microbiol, 1989 Jul, 55(7), 1808 - 10
Direct visual detection of aflatoxin synthesis by minicolonies of Aspergillus species; Lemke PA et al.; Single-spore colonies of Aspergillus flavus and Aspergillus parasiticus, grown for 4 to 5 days at 25 degrees C on a coconut extract agar containing sodium desoxycholate as a growth inhibitor, produced aflatoxin, readily detectable as blue fluorescent zones under long-wave (365 nm) UV light . Over 100 colonies per standard petri dish were scored for aflatoxin production by this procedure . Progeny from some strains remained consistently stable for toxin production after repeated subculture, whereas instability for toxin synthesis was revealed among progeny from other strains . Spore color markers were used to rule out cross-contamination in monitoring strains . A yellow-spored and nontoxigenic strain of A . flavus, reported previously to produce aflatoxin in response to cycloheximide treatment, proved to be toxin negative even after repeated exposure to cycloheximide . Extended series of progeny from another strain of A . flavus and from a strain of A . parasiticus were each compared by this plating procedure and by fluorometric analysis for aflatoxin when grown in a coconut extract broth . Both of these strains showed variation for toxin synthesis among their respective progeny, and specific progeny showed a good correlation for aflatoxin synthesis when examined by the two procedures.

Cell Biol Int Rep, 1989 Jun, 13(6), 547 - 53
Implication of activation of intraperitoneal macrophages with bio-degradable microspheres; Okawa T et al.; To estimate the possibility that biological degradable starch microspheres (DMS) activate abdominal or intraperitoneal macrophages (IMP), two sizes of DMS (Spherex, Pharmacia, Sweden) were injected into the peritoneum of the ICR mice of 4 to 8 weeks of age . Three days after the injection, peritoneal fluid was collected and incubated for one hour at 37 degrees C under 5% CO2 . The cells which adhered to the petri dish were IMP, to which DMS was added for 18 hrs . The cultured IMP were observed by scanning electron microscope (SEM) and the ratio of the active type to the total number of IMP was counted as an index of the effect of DMS to IMP . The activation effect of DMS on the incubated IMP was significant in the group which was cultured with 2 microns DMS after the 45 microns DMS injection . That indicated the possible DMS function as a potential IMP activating factor (MAF).

Radiat Res, 1989 Jun, 118(3), 420 - 36
The dose rate dependence of the relative biological effectiveness of 241Am versus 226Ra gamma rays; Schulz RJ et al.; The survival of Chinese hamster cells exposed to 59.5 keV 241Am gamma rays was compared with that obtained after exposure to 226Ra gamma rays . The Fricke dosimeter in conjunction with the calculational techniques of transition-zone dosimetry was employed to determine the dose rates to the cells at the petri dish/growth medium interface . The dose rates to the cells ranged from 11 to 133 cGy/h . In all cases, cell survival versus dose was best described by a simple exponential function of dose . For both radiations, graphs of D0 versus dose rate show complex but similar patterns of peaks and valleys . As the curve for 241Am is displaced toward lower dose rates compared with that for 226Ra, the relative biological effectiveness of 241Am vs 226Ra varies considerably with dose rate, ranging from 1.7 at 20 cGy/h to 1.1 at 40 cGy/h to 1.6 at 50 cGy/h . This phenomenon may be due to the LET-dependent accumulation of cells at the G2 + M interface in the cell cycle . The mean unrestricted track-average LET of 241Am (3.7 keV/microns) is 12 times higher than that for 226Ra (0.31 keV/microns) but only one-fifth that of carbon ions (18 keV/microns) for which G2 + M pile-up is observed . Application of the in vitro data derived from this study to the clinical situation, where the dose rate decreases rapidly with distance from the source, suggests that, dose for dose, 241Am will produce results little different from those obtained with 226Ra.

Taiwan Yi Xue Hui Za Zhi, 1989 Jun, 88(6), 545 - 50
Dual function of monocytes on the regulation of granulopoiesis; Wang SY et al.; The intrinsic balance of monocyte-derived mediators in the regulation of granulopoiesis was studied . Both the granulocyte macrophage colony-stimulating factors (GM-CSFs) and the colony inhibitory factor (prostaglandin E2; PGE2), were found to be elaborated by cultured monocytes, and most of these mediators were secreted during the first 4 days after monocyte cultivation . The concentration of monocytes in culture had a direct effect on the amount of mediator production . A maximum amount of mediators was noted to range from 2 x 10(5)-2 x 10(6) monocytes per 35 mm Petri dish . It was also found that the monocytes responded to the exogenous stimulus (lipopolysaccharide, LPS) in a biphasic manner . The optimal concentration of LPS in stimulating production of the tested mediators was 3.2 micrograms/ml . At higher concentrations (greater than 3.2 micrograms/ml), both the release of GM-CSFs and PGE2 were suppressed . The addition of indomethacin to the culture system resulted in a confirmatory increase in GM-CSFs production at LPS concentration higher than 3.2 micrograms/ml . The response of monocytes to endogenous regulators (GM-CSFs and PGE2) was also determined . The addition of GM-CSFs to the cultures promoted PGE2 production by monocytes in dose-dependent with an optimal concentration of around 450 units/ml . Alternatively, treatment of monocytes with PGE2 (10(-9) to 10(-10) M) also enhanced, to a certain extent, the production of GM-CSFs . The coincidence of the peaks of GM-CSFs and PGE2 release demonstrated in vitro suggested the existence of an intrinsic balance mechanism in the regulation of granulopoiesis by monocytes.

Taiwan Yi Xue Hui Za Zhi, 1989 May, 88(5), 523 - 5
An improved micro-incubation well for immunocytochemical staining procedures; Yu SM et al.; A shallow "micro-incubation well" was made with a piece of the square plastic slide and a glass slide . A circular hole (10-12 mm in diameter) was cut out of the center of the square plastic slide . This plastic slide was then attached to the glass slide with a drop of Epon-Araldite and cured in the 60 degrees C oven for 2 days . One to three small sections of tissue mounted within corresponding circles on an ordinary microscopic slide could be stained when placed upside-down on the well in a moisture chamber . The moisture chamber was modified by a disposable square Petri dish (100 x 15 mm) containing a small amount of water and a shallow slide stage which was made by cutting a half inch rim from the bottom of a round disposable Petri dish (100 x 15 mm) . This method provided the following advantages in immunocytochemical staining: (1) an even distribution of micro-amounts of antibody solution for long periods of time, and (2) greatly reduced the cost of the immunostaining technique by using micro-amounts of antibody solution.

Environ Res, 1989 Apr, 48(2), 287 - 95
An experimental study of the penetration of polycyclic aromatic hydrocarbons through a model of the bronchial lining layer; Gerde P et al.; The penetration of benzo{a}pyrene (BaP) through a nonbiological experimental model of the bronchial lining layer (BLL) was studied . The purpose was to investigate how the lipid-aqueous structure of the BLL might influence the rate of penetration of polycyclic aromatic hydrocarbons (PAHs) from the ambient air to the bronchial epithelium . The experimental model was built up in a petri dish by (A) a thin layer of paraffin at the bottom, simulating the lipophilic membranes of the epithelial cells; (B) an aqueous starch gel on top of the paraffin, simulating the viscous aqueous region of the BLL; and (C) a thin layer of phosphatidylcholine, simulating the surfactant lipid layer at the air interface . BaP was administered on top of the barrier either diffusely or from a point source, and the penetration was studied by measuring the concentration of BaP as a function of time both in the liquid phase and in the paraffin . Comparisons were made with a purely aqueous barrier without the thin phospholipid layer . The results show that the rate of penetration of BaP through the purely aqueous barrier is orders of magnitude higher than that of the lipid-aqueous barrier . A thin layer of phospholipids at the air interface thus has a tremendous influence on the rate of penetration of lipophilic substances and probably this, rather than the release rate of PAHs from their carrier particles, is the rate-determining step in the overall transport of PAHs from such particles to the bronchial epithelium.

Indian J Exp Biol, 1989 Apr, 27(4), 383 - 4
Cryopreservation of mouse embryos at -196 degrees C by vitrification; Agrawal KP et al.; Embryos (8-16 cell) were obtained from random bred albino mice (6-8 weeks old) that were induced to superovulate by injections of 5 I.U . PMSG and 5 I.U . hCG given 48 hr apart . Embryos were exposed to intracellular cryoprotecting medium (glycerol 10%, 1-2 propanediol 20% in PBS) for 10 min and then transferred to extracellular vitrification medium (25% glycerol, 25% 1-2 propanediol in PBS) . Vitrification medium containing embryos, and diluent (1 M sucrose) were loaded in a straw and immediately plunged into liquid N2 . After thawing at 20 degrees C, the contents of the straw were mixed by shaking (1 step dilution) and emptied in a petri dish . After 3 washings in culture medium the embryos were kept in CO2 incubator for further development . In 3-step dilution procedure the dilution of cryoprotectants was done in 0.5 and 0.25 M sucrose before culture . Embryos in 3-step dilution of cryoprotectants exhibited high survival as compared to 1-step dilution (20.23% vs 6.55%).

Histol Histopathol, 1989 Apr, 4(2), 217 - 22
Effects of glia-conditioned medium on primary cultures of central neurons; Conde Guerri B et al.; The effects of glia conditioned media on the survival and differentiation of embryonic neuron cultures of the Central Nervous System is described . We established glial cultures of peritumoral areas and the culture medium was changed serially and collected as glia conditioned medium (GCM) . Neuron enriched cultures plated on poly-L-lysine coated Petri dishes and after 4 days in vitro, the neuronal cultures were treated for the inhibition of glial cells . We established two groups of neuronal cultures that were respectively exposed to the GCM and to conventional culture medium . We evaluated the survival and differentiation of neuronal population in each group of cultures by contrast-phase microscopy and cellular uptake of Horse Radish Peroxidase . The cell cultures exposed to GCM showed a survival during the in vitro stages and an organization and differentiation pattern of mature neurons larger than the control cultures . This fact suggests that the glial cultures produce a diffusible neurotrophic factor that influences the neuronal response in vitro.

Chest, 1989 Mar, 95(3), 553 - 7
Chemiluminescence and antibody-dependent, cell-mediated cytotoxicity between human alveolar macrophages and peripheral blood monocytes in smokers, nonsmokers, and lung cancer patients; Lin CC et al.; Some evidence suggests a capability of peripheral blood monocytes to destroy tumor cells, while this ability by human alveolar macrophages, the main defense cells in the alveoli, is still debatable . We measured the chemiluminescence and antibody-dependent, cell-mediated cytotoxicity in the PBMs and HAMs of 12 lung cancer patients and 20 healthy subjects; the latter included ten smokers and ten nonsmokers . The PBMs were prepared by using a Ficoll-Hypaque density gradient, then separated by Petri-dish adherence . The HAMs were taken during the bronchoalveolar lavages . The chemiluminescence in the HAMs of smokers was significantly higher than nonsmokers, (p less than 0.05), which did not occur in the PBMs . Chemiluminescence in HAMs from the lung cancer patients was also significantly higher than the smoker control subjects (p less than 0.01) . However, the lung cancer patients had significantly lower ADCC activity than the smokers in the control group (4.52 +/- 2.96 vs 8.27 +/- 2.83 percent; p less than 0.05) . The chemiluminescence in the PBMs showed no statistical difference between the lung cancer patients and smoker control, but PBMs of the lung cancer patients had significantly lower ADCC activity than the smoker normal control group . The HAMs from lung cancer patients produced more superoxide anion, for an increased chemiluminescence reaction was noted, although ADCC activity was lower than in smokers, ie, HAMs were ineffective in killing tumors . Environmental factors such as cigarette smoking affect HAM's function by causing an increase of superoxide anion production . The chemiluminescence and ADCC activity in the PBMs does not always correspond to the HAMs findings . These results suggest that PBMs can not accurately reflect or predict the HAMs' function in lung diseases.

Clin Exp Metastasis, 1989 Mar-Apr, 7(2), 243 - 50
Plasminogen activator activity in clonogenic cell populations separated from a murine fibrosarcoma; Nagy B et al.; Tumor cells from a murine fibrosarcoma (FSa) produce plasminogen activator (PA), a protease that converts the zymogen plasminogen into the trypsin-like enzyme plasmin . Several studies indicate that tumor cell invasion is accompanied by proteolysis and that PA, generated by highly malignant cells, is by far the most ubiquitous protease associated with malignant transformation . Subpopulations of FSa cells were isolated by using density gradient centrifugation and the ability of these populations to form lung colonies was compared with their associated levels of PA production . Five populations of cells from a murine fibrosarcoma were separated in continuous gradients of Renografin in the density range 1.05-1.18 g/cm2 . The PA activities of an unseparated control cell lysate and cell lysates of the five separated populations were determined by using {125I}fibrin as a substrate in a reaction between cell lysate and plasminogen . The assay was based on the release of digested {125I}fibrin from the surface of Petri dishes into the supernatant solution, and the results were expressed as a percentage of the total radioactivity . The cell populations collected at densities of 1.05 and 1.09 (B1, B2) were the more clonogenic with relative clonogenic efficiencies of 2.6 and 3.3 times that of the unseparated tumor population, respectively . Analysis for PA demonstrated that enzyme formation was restricted mostly to these two populations . Cells from populations 4 and 5 did not secrete increased amounts of PA and had reduced clonogenic efficiencies compared with the unseparated FSa control population . These results are consistent with the hypothesis that PA activity is correlated with the clonogenicity of tumor subpopulations isolated from a heterogeneous and complex tumor system such as the FSa.

Microvasc Res, 1989 Mar, 37(2), 148 - 61
Isolation, characterization, and long-term cultivation of porcine and murine cerebral capillary endothelial cells; Tontsch U et al.; We present a simple method for isolation and long-term cultivation of porcine and murine cerebral capillary endothelial cells (cEC) . Two major points are made . First, that the "characteristic" morphology of the endothelial cells depends mainly on the presence of endothelial cell growth factors in the culture medium and second, that the identification of the cells as endothelial cells requires a special lectin instead of criteria used for large vessel endothelial cells, such as factor VIII staining or LDL uptake . Pure cerebral capillaries were isolated by means of a series of centrifugation steps; endothelial cells were released by collagenase treatment and cultivated on plastic petri dishes, which proved to be better for cell attachment than collagen or gelatin coating . The microvascular cells were cultivated in either the presence or absence of growth factors . Medium 199 + 10% FCS produced mainly spindle-shaped cells, growing in the "hills and valleys" pattern, which, if not passaged for weeks, showed three dimensional tubular structures . Cells of the "cobblestone" phenotype were promoted in medium 199 + 10% FCS, enriched with endothelial cell growth supplement (ECGS) and heparin (referred to as complete medium) . These cells retained their phenotype for months and could be passaged up to 35 times till now . If ECGS and heparin were omitted from these cultures, the cells became elongated and resembled smooth muscle cells . This effect was reversible when the cells were transferred to complete medium . With cEC, cloned by limiting dilution, we noticed this reversal phenomenon as well . We used several markers to characterize the microvascular cells and could show that the lectin of Bandeiraea simplicifolia is a highly reliable marker for endothelial cells and that the monoclonal antibody alpha-sm-1 (anti-smooth muscle cell actin) is excellent for determining smooth muscle cells.

Res Microbiol, 1989 Mar-Apr, 140(3), 235 - 41
Simplified diagnostic susceptibility testing of mycobacteria against thiacetazone, hydroxylamine, p-nitrobenzoic acid and picric acid; Burnens AP et al.; In order to simplify diagnostic susceptibility testing of mycobacteria against thiacetazone, hydroxylamine, p-nitrobenzoic acid and picric acid, we modified the standard procedure by using Middlebrook 7H11 agar in a single quadrant petri dish . This plate can be inoculated and read in parallel with routine susceptibility tests done by the proportion method . We present results of 449 consecutive routine strains tested with our method.

Comput Biomed Res, 1989 Feb, 22(1), 36 - 43
BUMP: a FORTRAN program for identifying dose-response curves subject to downturns; Simpson DG et al.; BUMP is a FORTRAN implementation of a modified Jonckheere-Terpstra test, proposed by Simpson and Margolin, to test nonparametrically for a dose-response curve when a downturn is possible at high doses . The Jonckheere-Terpstra statistic is commonly used to test for increasing or decreasing trends in dose-response relationships . In many experimental settings, however, a test agent has more than one effect, and a "bump"-shaped dose-response can occur . For instance, increasing the concentration of a certain nutrient on a petri dish may increase the growth rate at low doses yet decrease the growth rate at high doses because of toxicity . The modified test allows one to assess the significance of the initial increase in the dose-response curve and yet to minimize the effect on the conclusions of any downturn at higher doses . A complete system which operates directly on SYSTAT/MYSTAT files is available for the IBM-PC and compatibles; it includes a utility which converts ASCII data files to the SYSTAT/MYSTAT format . The FORTRAN 77 source code is available for those who would like to run BUMP on other machines.

Nippon Sanka Fujinka Gakkai Zasshi, 1989 Feb, 41(2), 167 - 72
{Role of sperm passage through cervical mucus: fertilizing capacity tested by in vitro fertilization with zona-free hamster eggs}; Ikuma K et al.; The effect of sperm passage through cervical mucus (CM) on the fertilizing capacity of human spermatozoa was examined in the in vitro fertilization system of zona-free hamster eggs . Each drop of ejaculated semen and BWW culture medium was connected by a small capillary tube filled with preovulatory CM, egg white or BWW medium under liquid paraffin oil in a plastic petri dish . After 2 hours, zona-free hamster eggs were added to the drop of BWW culture medium containing spermatozoa which had passed through the capillary tube and the mixture was incubated for various lengths of time at 37 degrees C under 5% CO2 in air . Human spermatozoa, which were washed and preincubated for 2 hours in BWW medium, were capable of fertilizing zona-free hamster eggs but needed a longer incubation time than spermatozoa which had passed through CM . Fertilization rates of spermatozoa which had passed through CM and egg white were very similar, but no fertilization occurred in the drop containing spermatozoa which had passed through BWW medium, presumably because of the contamination with seminal plasma . These results indicate that the most important role of CM may be to separate motile spermatozoa from seminal plasma components hostile to fertilization.

J Clin Invest, 1989 Feb, 83(2), 444 - 55
Interleukin 1 and tumor necrosis factor stimulate human vascular endothelial cells to promote transendothelial neutrophil passage; Moser R et al.; In an attempt to understand the regulatory mechanisms governing passage of neutrophils from the vascular bed to the interstitial tissue, we analyzed the effect of the pleiotropic monokines interleukin 1 (IL-1) and tumor necrosis factor (TNF) on transendothelial neutrophil traffic . Short-time preincubation of human umbilical vein endothelial cell (HUVE) monolayers with IL-1 and TNF led to an impressive time- and dose-dependent increase of endothelial cell-associated neutrophils when working in a full plasma system on petri dishes . Electron microscopic analysis revealed junctional penetration of monolayers by neutrophils . More quantitatively, when using a monolayer-on-filter-system, priming led to a severalfold increase in complete layer passage occurring in the absence of an external chemotactic gradient . Direct comparison with an upside-down modification of the system together with data demonstrating the vectorial behavior of such migration revealed that IL-1-stimulated transendothelial neutrophil traffic is polarized . The described enhancement of neutrophil transendothelial passage was found to be a unique feature of IL-1/TNF-activated HUVE since HUVE-dependent transmigration potentiation was not observed as a consequence of mere neutrophil attachment to endothelial cells (e.g., induced by Fc-mediated adherence of PMN to HUVE) . IL-1 acts selectively on endothelial cells as demonstrated by total inhibition of its effect by actinomycin D . Moreover, IL-1 does not induce HUVE monolayers to secrete a chemotaxin, and the neutrophil passage guiding principle is removable from the HUVE surface by short trypsin exposure . Congruent results were obtained with human adult arterial as well as saphenous vein endothelial cells . As shown by blockade of neutrophil migration with pertussis toxin, IL-1- and TNF-inducible transendothelial migration can be dissected into an initial anchoring step, which is succeeded by active neutrophil migration, possibly along a putative endothelial membrane-bound gradient.

Pharmacol Toxicol, 1989 Feb, 64(2), 193 - 5
A simple method for shell-less cultivation of chick embryos; Jakobson AM et al.; The chorio-allantoic membrane of explanted chick embryos can be used for the study of vascular development and of the vascular response to drugs and to tissue implants or tissue extracts . Three different types of vials for shell-less cultivation of chick embryos were compared in terms of short-term survival . Plastic cups with rounded bottoms were superior to Petri dishes and polyethylene film bags, in that they combined a high survival rate of the embryos with a possibility of using a large number of embryos in each experiment . With the use of plastic cups this method is simple, reliable and inexpensive.

Brain Res Dev Brain Res, 1989 Jan 1, 45(1), 49 - 57
Analysis of slow-onset neurite formation in NG108-15 cells: implications for a unified model of neurite elongation; Smalheiser NR; When undifferentiated NG108-15 cells are plated onto polylysine coated Petri dishes in serum-free medium, they form neurites within 1-4 h if plated in the presence of laminin or 5'-deoxy-5'-methylthioadenosine (rapid-onset neurites) . In the absence of such agents, serum-deprived NG108-15 cells extend axon-like neurites onto polylysine over several days; here we characterize the dynamic behavior of this slow-onset outgrowth pattern in detail . Individual cells plated on laminin expressed a gradual multipolar-to-unipolar transition due to rapid-onset neurites becoming remodelled into the appearance of slow-onset neurites . This phenomenon reflected the selective stabilization of certain rapid-onset neurites, along with the restriction of motility to their distal tips . Based upon the properties and interactions of both rapid- and slow-onset neurites in NG108-15 cells, a unified model for neurite formation is presented.

Radiat Res, 1989 Jan, 117(1), 79 - 92
Cell proliferation as a requirement for development of the contact effect in Chinese hamster V79 spheroids; Olive PL; Chinese hamster V79 cells grown for several hours in suspension culture form spheroids which are more resistant to killing by ionizing radiation than cells grown on petri dishes, a phenomenon known as the "contact effect." Previous results using the alkali-unwinding assay as a measure of DNA damage have implicated differences in DNA conformation as contributing to this effect; spheroid DNA denatures more slowly in dilute alkali than monolayer DNA, perhaps due to the presence of constraints to DNA unwinding . In this paper, the rate of development of radiation resistance is shown to be similar when either cell survival or DNA unwinding is used as an end point . At the midpoint for development of resistance, approximately 10 h, the unwinding kinetics indicate that either half of the cells contain constraints to DNA unwinding, or half of the DNA in all of the cells contains constraints . The latter explanation appears more likely since all cells seem to develop these constraints at the same rate, regardless of position in the cell cycle or the degree of contact with other cells . Results using the microelectrophoresis assay to measure damage to individual nuclei confirm the fact that 10-h cultures show a homogeneous radiation response intermediate between that of monolayers and spheroids . Incubation of cells at room temperature or in the presence of drugs which inhibit cell cycle progression prevents full development of the contact effect . Conversely, incubation of cells in medium containing inhibitors of polyamine synthesis, adenylcyclase, glutathione synthesis, poly(ADP-ribose)polymerase, topoisomerase II, or cell-cell communication does not inhibit development of the contact effect as measured by DNA-unwinding kinetics.

Surg Gynecol Obstet, 1989 Jan, 168(1), 63 - 4
A simple method for preventing blood-borne contamination to operating room personnel during vascular reconstructive operations; Patterson RB et al.; Although the risk of infection with hepatitis or HIV remains low, and the effectiveness of steps to reduce even this small risk is difficult to assess, the sequelae of the infection with either virus are often devastating . We described herein the use of a sterile Petri dish to inspect vascular anastomoses during arterial reconstructive operations . This simple, inexpensive precaution has been in use at the University of Cincinnati for two years and has proved so effective in preventing the contamination of operating room personnel with blood from a patient that it has recently become a routine part of all vascular reconstructive operations.

Tsitologiia, 1989 Jan, 31(1), 114 - 6
{Use of a gelatin substrate for culturing diploid human skin fibroblasts}; Nizheradze KA et al.; Gelatin coating of the growth surface of commercially available polystyrene Petri dishes is proposed for successful cultivation of human skin fibroblasts . A comparison of culture growth dynamics on gelatin, glass and polystyrene allowed to recommend gelatin as the substrate in order to receive an abundant similar material with a low population density viability . The absence of changes in carbohydrate metabolism changes during cell culturing on gelatin provides an opportunity to use these cells in studying regulation of carbohydrate metabolism.

Ann Rech Vet, 1989, 20(4), 473 - 83
{Rapid collection of swine lymphocyte subpopulations in large quantity}; Charreau B et al.; Lymphocyte subpopulations derived from different pig organs (blood, spleen, mesenteric lymph nodes) were separated by a simple, rapid and cheap panning technique, using either normal or ozone treated Petri dishes with bovine serum albumin . Slg+ lymphocytes could thus be obtained with a purity of up to 95% by adhesion onto Corning plastic dishes . The purified cells retained their proliferative activity with regard to lectins . The subpopulation including PT4 and PT8 was then separated by another panning on ozone-treated plastic dishes with a purity of 80-90%.

Acta Physiol Pharmacol Latinoam, 1989, 39(2), 127 - 32
Action of blood serum from rats with turpentine sterile inflammation on the development of CFU erythrocyte colonies . Possible role of erythropoietin; Gutnisky A et al.; Bone marrow CFUe mice cultures were prepared in Petri dishes and the number of full developed erythrocyte colonies were counted under various experimental conditions . In certain experiments, erythropoietin (EP = 0.4 U x ml-1 in the suspending medium) or sera from normal rats (100 microliters) or from animals with chronic turpentine sterile abscesses (60 or 100 microliters), were delivered to the CFUe colonies . The number of colonies in the group without EP was almost 5 times smaller than in the group with EP . Inasmuch as some few colonies are still able to develop, even in absence of added EP, the suggestion is advanced holding that the phenomenon may represent the effect of EP already bound to the group of cells initiating differentiation . Comparisons among all experimental groups indicate that the delivery of sera from turpentine rats to cultures containing added EP, reduced significantly the number of CFUe colonies seen in controls with EP but without inflammatory serum . It is suggested that the present findings could be explained assuming an inhibition of the influence of exogenous EP, subserved by the inflammatory serum, or alternatively that this kind of rat serum induces a partial blockade of EP at receptor sites whose activation is a mandatory step for the adequate and full development of cultured erythrocytes.

Oncology, 1989, 46(6), 391 - 9
Influence of fetal calf serum in combination with pharmacological doses of progesterone or estradiol on proliferation and cell cycle kinetics of cultured mammary cancer cells; Kiss R et al.; We describe an original method to monitor clonal cell density (hyperplasia) and the cell cycle kinetics of neoplastic cells simultaneously . We thus characterize the in vitro influence of two different types of fetal calf serum (FCS), defined as FCS-S and FCS-I, on the progesterone- or estradiol-induced effect on proliferation and cell cycle kinetic parameters of the MXT mouse and MCF-7 human mammary cancer cell lines . The two sera were treated with dextran-coated charcoal . The FCS-S serum showed a stimulatory influence on MXT and MCF-7 growth, whereas FCS-I was devoid of any clear-cut influence . The cells were cultured on glass coverslips, placed in Petri dishes containing either a control or a hormone-added medium, fixed for histology and submitted to the Feulgen reaction, which allows selective and quantitative (stoichiometric) staining of DNA . Proliferation and cell cycle kinetics were analyzed on the same sample of cells by means of the SAMBA 200 cell image processor . Our results show that steroid-mediated effects were dramatically modulated according to the type of serum used . Furthermore, they also show that pharmacological doses of progesterone or estradiol decrease MXT cell growth by at least two different mechanisms: the first is related to cell cycle kinetics, i.e . an inhibition of the cells into the S phase, while the second remains unknown but seems to be cell cycle independent . High dose estradiol, but not progesterone, induced the same inhibitory influence on the MCF-7 cells.

Immunopharmacol Immunotoxicol, 1989, 11(2-3), 309 - 20
Inhibition of natural killer activity by calcitonin gene-related peptide; Umeda Y et al.; The effect of calcitonin gene-related peptide (CGRP) on natural killer (NK) cell activity in spleen cells from Balb/c mice and nude mice was studied . CGRP dose-dependently (10(-9) to 10(-7) M) inhibited NK activity of spleen cells from both strains of mice . This inhibitory effect was observed at the effector to target ratios of 12.5:1 to 100:1 . Maximum inhibition by 10(-7) M CGRP was about 60% . The inhibition of NK activity by CGRP was also observed in anti-Thy 1.2 plus complement treated Balb/c spleen cells . Furthermore, when cells were treated with 10(-9) to 10(-7) M CGRP the concentration of intracellular cyclic AMP increased in spleen cells of nude mice . The characteristics of these cells were similar to those of NK cells, (1) being petri dish and nylon wool nonadherent, (2) expressing asialo GM1 antigen, and (3) lacking readily detectable Thy 1 antigen and immunoglobulin . In addition, the intravenous injection of asialo GM1 completely abolished NK activity in spleen cells from nude mice and the increase in intracellular cyclic AMP in spleen cells by CGRP was less in spleen cells from mice given an anti-asialo GM1 injection . Our present study suggests that CGRP inhibits NK cell activity by increasing the intracellular cyclic AMP concentration . CGRP may be implicated in the regulation of NK function.

Indian J Med Res, 1989 Jan, 89, 43 - 7
Establishment of a standard test method for determining susceptibility of Mesocyclops to different insecticides; Manonmani AM et al.; A method to determine the susceptibility of Mesocylops sp . to insecticides was devised . Matured adults were used for the bioassays . Mesocyclops sp . (25 in number) were placed in 24.9 ml of sterilized paddy field water in 5 cm diameter petri dish and 0.1 ml of appropriate stock solution of the insecticide was added . Mortality was scored 24 h after treatment . Among the insecticides tested, Permethrin, Dichlorvos, Temephos, DDT, Carbendazim, Fenitrothion, Zolone and Aldrin were effective against Mesocyclops sp.

J Biol Rhythms, 1989 Summer, 4(2), 201 - 16
A circadian rhythm of population behavior in Gonyaulax polyedra; Roenneberg T et al.; Like other flagellates, Gonyaulax polyedra exhibits diurnal vertical migration and pattern formation . Shape and size of the aggregations depend on container type, light intensity, and cell density . In Petri dishes, cells form oval "swarms"; within these, cells move downward in the highly dense center and rise up at the periphery . We have investigated the daily rhythm of this swarming activity in Petri dishes illuminated from the side, using time-lapse video recordings . At night, a "lawn" of cells forms at the bottom of the dish toward the light source (independent of light intensity) . Before dawn, cells rise toward the surface and aggregate in swarms . The daily vertical migration occurs independent of light direction and intensity . The diurnal swarms, however, form every day at the same location within the dish, at a distance from the light that depends on light intensity, indicating a self-selection of light intensity . In constant light and temperature and with negligible vertical nutrient differences, all aspects of the rhythm continue to oscillate for up to 3 weeks, when the rhythm of the population becomes desynchronized . Under cycles of bright white-dim red light (WR), cell entrain to WR 10:10 but free run in WR 8:8 and shorter cycles, showing relative coordination (von Holst, 1939) to the driving light cycle . They also entrain to the 24-hr multiple of WR 6:6 . Under nonentrained conditions, swarming activity is still influenced by light changes, and in spite of the apparent free run, the phasing of the averaged activity varies systematically with different T-cycle frequencies.

Folia Parasitol (Praha), 1989, 36(2), 107 - 12
Continuous culture of Plasmodium falciparum asexual stages in "normal" air atmosphere; Mirovsky P; The growth of six strains of Plasmodium falciparum in 5% CO2, 5% O2, 90% N2 and normal air atmosphere was determined daily by microscopical examination of blood films . All strains were able to grow in flasks without additional gas mixture but significantly lower parasitaemia was observed within the first five days of cultivation . Attempt at cultivating in petri dishes without candle jar technique failed but parasites survived in plasticine sealed dishes . The cultivation in air cannot be recommended for cultures initiated from cryopreserved material or low parasitaemia (0.1-0.3%) cultures.

Gegenbaurs Morphol Jahrb, 1989, 135(5), 779 - 93
Methylene blue-fast green staining of hemopoietic colonies in agar cultures; Zvetkova EB et al.; A simple technique is described for the routine in situ identification of the cellular composition of colonies and clusters in agar cultures of hemopoietic cells . The entire culture, dried and formalin vapor fixed within a Petri dish, is stained with a mixture of methylene blue and fast green . By this method cellular ribonucleoproteins (RNP), deoxyribonucleoproteins (DNP) and some cationic (arginine and lysine containing) proteins are detected . Different maturation stages of neutrophils, eosinophils, basophils, monocytes, and macrophages can be easily identified with colonies and clusters on the basis of the cytoplasmic and nuclear staining.

J Biomed Mater Res, 1988 Dec, 22(3 Suppl), 339 - 50
Tendon cell outgrowth rates and morphology associated with kevlar-49; Zimmerman M et al.; A rat tendon cell model was used to evaluate the in vitro biocompatibility of kevlar-49 . The cell response to kevlar was compared to carbon AS-4 and nylon sutures . Three trials were run and cell growth rates were statistically similar for all the materials tested . A separate experiment was conducted in which the same fiber materials were placed in the same Petri dish . Again, the rates were similar for each material . Finally, the cells were observed with a scanning electron microscope, and the three classic cell morphologies associated with this tendon cell model were observed . Also, cellular attachment to the fiber and cellular encapsulation of the fiber were identical for the three materials tested . Kevlar-49 proved to be comparable to carbon AS4 and nylon sutures in terms of cellular response and cell outgrowth rates.

Genetics, 1988 Dec, 120(4), 1111 - 23
Maize glutamine synthetase cDNAs: isolation by direct genetic selection in Escherichia coli; Snustad DP et al.; Maize glutamine synthetase cDNA clones were isolated by genetic selection for functional rescue of an Escherichia coli delta glnA mutant growing on medium lacking glutamine . The Black Mexican Sweet cDNA library used in this study was constructed in pUC13 such that cDNA sense strands were transcribed under the control of the lac promoter . E . coli delta glnA cells were transformed with cDNA library plasmid DNA, grown briefly in rich medium to allow phenotypic expression of the cDNAs and the pUC13 ampr gene, and challenged to grow on agar medium lacking glutamine . Large numbers of glutamine synthetase cDNA clones have been identified in individual 150-mm Petri dishes; all characterized cDNA clones carry complete coding sequences . Two cDNAs identical except for different 5' and 3' termini have been sequenced . The major open reading frame predicts a protein with an amino acid sequence that exhibits striking similarity to the amino acid sequences of the predicted products of previously sequenced eukaryotic glutamine synthetase cDNAs and genes . In addition, the maize glutamine synthetase cDNAs were shown to contain a 5' mini-ORF of 29 codons separated by 37 nucleotide pairs from the major ORF . This mini-ORF was shown not to be essential for the functional rescue of the E . coli delta glnA mutant . Expression of the cDNAs in E . coli is presumed to be due to the function of a polycistronic hybrid lac messenger RNA or translational fusions encoded by the pUC plasmids . Proteins of the expected sizes encoded by two different pUC clones were shown to react with antibodies to tobacco glutamine synthetase.

Pflugers Arch, 1988 Nov, 413(1), 90 - 2
The use of cytodex microcarrier beads in patch-clamp studies on cultured epithelial cells; Poronnik P et al.; This paper describes an economical attachment substratum for use in patch-clamp experiments on cultured epithelial cells . It consists of Cytodex microcarrier beads on which growing cells present a unique profile perspective that increases the ease with which patch-clamp seals can be obtained . Cells from the mouse mandibular gland cell line, ST885, were grown on the beads and studied using standard patch-clamp techniques . The results were indistinguishable from those obtained from cells grown in monolayers on Petri dishes . The method can be applied in many types of patch-clamp experiment and used with a wide variety of cell types . It is especially useful if one wishes to do experiments in the cell-attached or whole-cell configurations and needs to add drugs or hormones to the external surface of the cell.

Biol Reprod, 1988 Oct, 39(3), 546 - 52
The culture of bovine oocytes to obtain developmentally competent embryos; Sirard MA et al.; Bovine cumulus-oocyte complexes (COC) (n = 4230) were used in this study to assess the effects of culture method, hormonal supplementation, and cumulus cell concentration on maturation, fertilization and development of resulting embryos . Five treatments were evaluated . 1) 10 COC/50-microliter drops under oil in TCM 199 supplemented with 10% heat-treated fetal calf serum, follicle-stimulating hormone (0.5 microgram/ml), luteinizing hormone (5 micrograms/ml), and estradiol-17 beta (1 microgram/ml); 2) as in 1 without hormones; 3) as in 1 but in 3 ml TCM-199 in petri dishes without paraffin oil; 4) as in 2 but only 1 COC/50-microliter drop; and 5) as in 1 but with denuded oocytes . After 24 h maturation, the frequencies of oocytes reaching metaphase II were 98, 84, 92, 93, and 87%, respectively, for the five treatments . In the same order, percentages of normal fertilization were 73, 70, 62, 81, and 62%, and the frequencies of embryos containing two or more blastomeres at 65 h postinsemination were 69, 82, 66, 51, and 43% . The same five treatments were used in a second study in which 3,199 oocytes were fertilized, allowed to cleave in vitro to the 2- to 3-cell stage (42 h postinsemination), and transferred to oviducts of sheep (one treatment/oviduct) for 4 days . The frequencies of morulae or blastocytes obtained were 28, 18, 23, 24, and 11% for the five treatments, respectively . After nonsurgical transfer to bovine recipients (n = 8) using fresh or frozen-thawed embryos, three pregnancies past 50 days were obtained . Only one went to term with the birth of a live heifer calf.

Angew Parasitol, 1988 Sep, 29(3), 179 - 83
Environmental stress and parasitic infections: I . The effects of Gramoxone and Hexadrin on embryo and hatching of Fasciola gigantica miracidia; Igbinosa IB et al.; The effect of two chemical compounds commonly used in agriculture, Gramoxone (a herbicide) and Hexadrin (an insecticide) on embryonic development and hatching of Fasciola gigantica miracidia were experimentally assessed . These two pesticides were introduced in varying quantities into petri dishes containing unembryonated eggs of the trematode for a period of 30 days . LC50 (lethal concentration for 50% hatching) values were determined for them . For gramoxone it was 2 ppm and for Hexadrin it was 2.5 ppm . Results also show that at 4 ppm and 5.5 ppm the pesticides achieve 99.0% mortality of eggs . At sublethal concentrations they cause prolongation of embryonic processes and inhibition of hatching of miracidia.

J Leukoc Biol, 1988 Aug, 44(2), 111 - 21
Long-term cultivation of functional human macrophages in Teflon dishes with serum-free media; Helinski EH et al.; Reported herein are the results of studies demonstrating the utility of a chemically defined, serum-free medium designated as AIM-V (GIBCO) for the long-term (greater than 2 weeks) cultivation of functionally-defined human macrophages . The AIM-V medium is a mixture of HEPES-buffered Dulbecco's Modified Eagle Medium and Ham's Nutrient Mixture F12 that had been supplemented with purified human albumin, transferrin, insulin, and a proprietary mixture of purified factors . Nonadherent macrophages for serial studies were generated in petri dishes with a Teflon liner that had been seeded with a heterogeneous population of peripheral blood mononuclear cells of healthy human adults . For comparison, cultures were initiated with serum-free medium AIM-V and medium supplemented with AB/Rh+ serum or freshly collected autologous serum . Viability was defined by trypan blue dye, glass adherence, and phagocytosis of yeast . Macrophages were characterized by light microscopy, cytochemistry, and phenotypic analysis . Ultrastructural morphology was defined by scanning electron microscopy . These studies demonstrate that functionally defined human macrophages can be sustained in long-term culture with the use of serum-free medium that has not been augmented with mitogenic stimulants, growth-promoting lymphokines/monokines, or differentiation-inducing agents . Serum-free medium AIM-V, which has been approved for generating lymphokine, (i.e., interleukin-2; IL-2)-activated killer cells (LAK) for IL-2/LAK adoptive immunotherapy modalities, may also prove useful for studies defining and isolating regulatory proteins produced by activated monocytes and macrophages and for generating cytolytic macrophages for different antitumor regimens.

J Neurosci Methods, 1988 Aug, 25(1), 19 - 27
A chamber for electrophysiological recording from cultured neurones allowing perfusion and temperature control; Forsythe ID et al.; With the increasing use of cell culture in electrophysiology there has arisen a need for more rigorous control of the extracellular fluid composition and temperature . We describe here an environmental chamber designed to permit perfusion and temperature control of the extracellular medium during electrophysiological studies of cultured or acutely dissociated neurones in 35 mm Petri dishes . The chamber consists of a Peltier driven heat exchange, which allows temperature control above and below ambient . The Petri dish temperature is controlled in 3 ways: (1) through direct conduction from the controlled surface; (2) by flow of gas around the dish; and (3) by flow of extracellular medium to the Petri dish . We demonstrate the temperature control achieved with this equipment and the ability to exchange the extracellular medium in the whole dish . Using the whole-cell-patch recording technique to record from hippocampal neurones, Q10 parameters were estimated for the membrane time constant, input resistance and action potential halfwidth, over a range of 15-36 degrees C . The mean Q10 values (+/- S.E.M.) were 0.71 +/- 0.05, 0.58 +/- 0.03 and 0.35 +/- 0.1, respectively . Because of rapid diffusion and mixing of drugs when using puffer pipettes, quantitative pharmacological studies or manipulations of the extracellular recording medium are not possible . By perfusing the whole recording chamber the final concentration can be accurately known . Using this method we obtained an estimate of the relative potency of the non-specific excitatory amino acid antagonist, kynurenate, for synaptically activated N-methyl-d-aspartate (NMDA) receptors of 1:0.62.

J Virol Methods, 1988 Aug, 20(4), 285 - 93
Titration of avirulent Newcastle disease virus by the plaque assay method; Kournikakis B et al.; Parameters of a plaque assay for avirulent strains of Newcastle disease virus (NDV) were optimized for reproducibility and optimum titer in LLC-MK2 cells . Plaques were visible after 2 days and maximum virus titers were reached in 3 days . Virus titers were not affected by continued incubation through 6 days, although plaque size increased . An adsorption volume of 0.1 or 0.2 ml per 60 mm Petri dish was optimal, as was an adsorption time of 45 min . Trypsin (2.5 micrograms/ml) and magnesium sulfate (0.03 M) were essential requirements of the overlay medium and the presence of DEAE-dextran (0.02%) resulted in a 30% increase in titer . The use of cell monolayers, 1, 2, or 3 days old facilitated the performance of multiple assays per week and did not affect the virus titer.

In Vitro Cell Dev Biol, 1988 Aug, 24(8), 759 - 63
A minichamber device for maintaining a constant carbon dioxide in air atmosphere during prolonged culture of cells on the stage of an inverted microscope; Bavister BD; Construction details are described for a minichamber device that maintains a localized atmosphere of carbon dioxide in air over the stage of an inverted microscope . This device is easily constructed from Plexiglas and its specifications can be adjusted to fit virtually any inverted microscope . A flow of warm, humidified carbon dioxide in air gas mixture can be directed over a petri dish or unsealed culture flask to maintain the pH of bicarbonate-CO2 buffered media . By this means, prolonged culture of cells directly on the microscope stage is made possible without occurrence of detrimental pH changes . If the microscope is fitted with an environmental control chamber to maintain temperature, cells can be maintained on the microscope stage for days, permitting frequent observation of cell growth and activity . Alternatively, continuous cine or video recordings can be made . For example, using this device, hamster and rhesus monkey embryos have been cultured for 2 to 5 d on an inverted microscope while continuous time-lapse recordings were made of cell division and differentiation and activity of cellular organelles.

Am J Physiol, 1988 Aug, 255(2 Pt 1), C237 - 45
Effect of hormones on growth and function of cultured canine tracheal epithelial cells; Van Scott MR et al.; Insulin (INS), endothelial cell growth supplement (ECGS), transferrin (TF), cholera toxin (CT), hydrocortisone (HC), triiodothyronine (T3), and epidermal growth factor (EGF) were systematically examined for their effects on proliferation, ion transport activity, and morphological differentiation of canine tracheal epithelial (CTE) cells in culture . INS, ECGS, TF, and CT increase proliferation of CTE cells cultured on plastic Petri dishes but exhibit no acute effect on the bioelectric activity of freshly excised CTE . CT increases amiloride-insensitive transepithelial ion transport across both freshly excised canine trachea and CTE cultures . EGF has no effect on proliferation of CTE cells on plastic but induces hyperplasia of CTE cells on collagen matrices . EGF does not alter basal transepithelial ion transport across cultures but increases cellular responsiveness to beta-adrenergic stimulation . HC and T3 have no effect on proliferation or transepithelial bioelectric properties of CTE cells but improve morphological differentiation yielding cultures of complex epithelia containing cuboidal ciliated and nonciliated cells . These results demonstrate that growth and function of cultured CTE cells are affected by specific growth factors.

Anticancer Res, 1988 Jul-Aug, 8(4), 765 - 74
A new assay to evaluate cell growth and drug sensitivity in culture using a cell image processor; Kiss R et al.; The use of a cell image processor for the in vitro assessment of drug effects on cell growth, cell kinetics and chromatin organization is described . We have studied the influence of two well documented cytotoxic drugs, i.e . BCNU and vincristine (VIN), on the above mentioned parameters of the P-388 mouse leukemia, MCF-7 human mammary and HBL human melanoma cell lines . The cells were cultured for 1 to 4 days on glass coverslips put in Petri dishes containing or not (control) 10, 1 or 0.1 microgram/ml medium of the drug, after which they were fixed for histology, Feulgen-stained and analyzed through a cell image processor, i.e . the System for Analytical Microscopic Biomedical Applications (SAMBA 200) . Our results showed that both BCNU and VIN exerted a well-known antineoplastic effect that was assessed at three different but highly complementary levels on the same sample of cells in a very rapid and simple procedure.

J Periodontol, 1988 Jun, 59(6), 398 - 402
Hand versus ultrasonic instrumentation in the removal of endotoxins from root surfaces in vitro; Checchi L et al.; The goal of this study was to determine whether ultrasonic scalers are as effective as curettes in providing fibroblast attachment to the scaled root surfaces . Extracted, peridontally involved teeth were cut along the sagittal plane; then one half of the root was curetted, the other half ultrasonically scaled . In addition, monkey kidney fibroblasts were suspended in a petri dish containing root fragments of the tooth halves . At the same time, control dishes without fragments were mounted . All dishes were treated with radioisotopic techniques . There was no significant difference in fibroblast growth between peridontally involved root surfaces treated using curettes or ultrasonic scalers . Both treatments caused the roots to lose their toxicity . The limitations of ultrasonic scalers in terms of shape, size and awkward handling need to be considered when choosing the approach that best suits each case.

Cancer Res, 1988 Jun 1, 48(11), 3236 - 44
New chemotherapeutic drug sensitivity assay for colon carcinomas in monolayer culture; Schroy PC 3rd et al.; Ten previously untreated colon carcinomas were tested for chemotherapeutic drug sensitivity in primary monolayer culture . Colon carcinomas were partly digested to groups of epithelial cells which plated with a mean efficiency of 42 +/- 9% (SE) on a collagen I-bovine serum albumin substrate in serum-free medium, producing patches of tightly adherent epithelial cells . The cultured cells were judged epithelial by the presence of cytokeratins, an epithelial cell surface epitope, junctional complexes, and brush borders . Each carcinoma was plated in 40 to 60 Petri dishes (35 mm), yielding a mean of 28 +/- 8 (SE) colonies per dish (6832 +/- 1952 cells) . Drugs tested in duplicate plates were mitomycin C, cisplatin, streptozotocin, and 5-fluorouracil at 0.1, 1, 10, and 100 micrograms/ml, and at 0.1, 1, and 2x the peak tolerated drug concentration in serum . Twenty-four h after plating, any nonadherent cells were removed, and the adherent tumor cells were continuously exposed to the drugs for 3 days . Each drug induced colony lysis in a dose-dependent manner in responsive tumors . Drug-resistant, cycling cells were identified by {3H}thymidine incorporation in colonies which were not lysed by drug treatment . Each of the ten carcinomas exhibited inherent resistance to one or more chemotherapy drugs within the concentration ranges clinically achievable.

APMIS, 1988 Jun, 96(6), 531 - 6
Effect of PGE2 and LTB4 on vicia villosa binding lymphocytes; Gualde N et al.; Since there is a good deal of evidence that vicia villosa lectin (VVA) binds to contrasuppressor cells, mouse splenocytes and thymocytes were sorted by binding to VVA-coated Petri dishes . It was observed that vicia villosa adherent cells (VVA(+)) did not proliferate when mitogens were added to cultures, but they did enhance the thymidine uptake of PHA or ConA stimulated vicia villosa non-adherent (VVA(-)) splenocytes . PGE2 treatment of VVA(+) splenocytes or VVA(+) immature thymocytes did not very much affect the VVA(+) cell behavior . On the other hand, LTB4 increased the enhancing capability of VVA(+) splenocytes . Therefore, investigations dealing with the effects of LTB4 on lymphocyte subsets which may include VVA(+) cells should take into consideration the possible presence of contrasuppressor cells.

Blood, 1988 Jun, 71(6), 1581 - 9
Attachment of cultured human endothelial cells is promoted by specific association with S protein (vitronectin) as well as with the ternary S protein-thrombin-antithrombin III complex; Preissner KT et al.; The interaction of the multifunctional S protein (vitronectin) with cultured human endothelial cells of macrovascular and microvascular origin was investigated . Purified S protein, coated on polystyrene Petri dishes, induced dose-dependent and time-dependent attachment and spreading of human umbilical vein endothelial cells (HUVECs) as well as human omental tissue microvascular endothelial cells (HOTMECs) at 37 degrees C . Not only isolated S protein, but also the ternary S protein-thrombin-antithrombin III (STAT) complex promoted attachment of approximately 90% of the cells within 2 hours at an S protein concentration of 0.13 mumol/L . Inhibition of attachment in these experiments was achieved by the addition of the cell-attachment pentapeptide Gly-Arg-Gly-Asp-Ser and by monospecific antibodies against S protein, whereas nonrelated peptides or antibodies against fibronectin, fibrinogen, or von Willebrand factor (vWF) were ineffective . Direct binding of S protein to HUVECs and HOTMECs was studied with cells in suspension at a density of 1 x 10(6) cells/mL and was maximal after 120 minutes . S protein bound to both cell types in a dose-dependent fashion with an estimated dissociation constant Kd = 0.2 mumol/L . At a 200-fold to 500-fold molar excess of unlabeled S protein, greater than 80% of bound radiolabeled S protein was displaceable, whereas binding was reduced to 30% to 50% by addition of the pentapeptide, the STAT complex, or by physiologic concentrations of fibrinogen or vWF as well as Fab fragments of anti(human S protein)IgG, but not by Fab rabbit IgG . These findings present evidence for the specific association of S protein with endothelial cells ultimately leading to attachment and spreading of cells . Moreover, a novel function for the ternary STAT complex, which induced endothelial cell attachment and spreading virtually identical to free S protein, is described . These data further suggest a possible role for S protein during coagulation as major vessel wall-related adhesive protein at sites of vascular injury.

Radiobiologiia, 1988 May-Jun, 28(3), 317 - 22
{Postradiation changes in count and contact properties of neutrophilic granulocytes of the bone marrow in short-term culture}; Chukhlovin AB et al.; A study was made of adherence to plastic Petri dishes and viability (as non-pyknotic cell counts) of rat bone marrow cells cultured for 5 to 22 h in 199 medium containing 15 per cent fresh isologous serum . An overall decrease in the number of viable myelokaryocytes, including mature neutrophils, was observed in the cultures irradiated with doses of 3 to 12 Gy . In addition, gamma-irradiation increased substantially the adherence of neutrophils and, to a lesser extent, of other myelokaryocytes . A possibility of early radiation-induced disturbances in granulocytic maturation is discussed.

Biotechniques . 1988 May;6(5):408, 410, 412.
A rapid droplet method for Sanger dideoxy sequencing; Ner SS et al.; A method for performing the dideoxy sequence reaction on petri dishes is described . It allows rapid manipulation of clones and provides large amounts of sequence information quickly and without the need for elaborate laboratory equipment.

J Immunol, 1988 May 1, 140(9), 2880 - 5
Peritoneal B cells respond to phorbol esters in the absence of co-mitogen; Rothstein TL et al.; B cells obtained by irrigation of the peritoneal cavity differ from splenic B cells in signaling requirements for the initiation of DNA synthesis . Splenic B cells are stimulated to enter S phase by phorbol esters in conjunction with a second signal provided by calcium ionophore; however, splenic B cells are not stimulated by phorbol ester alone . In contrast, peritoneal B cells from NZB and BALB/c mice were stimulated to incorporate tritiated thymidine by each of the phorbol esters, PMA and phorbol dibutyrate, acting alone . Stimulation of peritoneal B cells was apparent when cells were cultured at lower than usual cell densities, and responses were unaffected by coculture with splenic B cells . Responding cells adhered to plastic petri dishes coated with anti-mouse IgM antibody, but were not completely removed by treatment with anti-Ly-1.2 antibody plus C . These results indicate that phorbol esters constitute a complete signal that stimulates some peritoneal B cells to enter S phase.

Gan To Kagaku Ryoho, 1988 Apr, 15(4 Pt 2-2), 1183 - 90
{Treatment of myelodysplastic syndrome and acute myelogenous leukemia with vitamin D3 {1 alpha(OH)D3}}; Irino S et al.; It is known that 1 alpha, 25(OH)2D3 is an inducer of nonlymphoid leukemic cell differentiation to monocyte-macrophage-like in vitro . The effects of oral administration of 4.5-15 micrograms/d 1 alpha (OH)D3, which is converted to 1 alpha, 25(OH)2D3 in vivo by liver cells, on leukemic cells were studied in two patients with AML and one with RAEB . In these three cases, 1 alpha (OH)D3 reduced the number of leukemic cells in the bone marrow and aggregated the dispersed chromatin of leukemic cells as heterochromatin . Furthermore, this drug induced atypical lymphocyte-like cells, which were considered to be differentiated from leukemic cells in case 1, and improvement of pancytopenia in case 3 . While hypercalcemia developed during 1 alpha (OH)D3 therapy in case 1, it disappeared within three days after discontinuation . We also studied the in vitro effects of 1 alpha, 25 (OH)2D3 on leukemic cells freshly isolated from the bone marrow in these three cases . After incubation with 10(-8) M 1 alpha, 25(OH)2D3 at 37 degrees C for 72 h, the number of adherent cells on the bottom of Petri dishes had increased . These cells were quite similar to monocyte-macrophages . These leukemic cells, after differentiation induced by 1 alpha, 25(OH)2D3, reacted strongly with monoclonal antibodies My-4 and My-7 . Both 100 microM D-cis and L-cis diltiazem (calcium channel blocker) enhanced the differentiation of HL-60 cells induced by 1 alpha, 25 (OH)2D3 . There was no significant difference between D-cis and L-cis diltiazem with regard to this enhancing effect . The cytoplasmic free Ca2+ concentration in HL-60 cells induced by 1 alpha, 25(OH)2 D3 and/or diltiazem, was increased significantly as compared with that in HL-60 cells incubated without inducers.

Eur J Cancer Clin Oncol, 1988 Mar, 24(3), 385 - 90
Estrogen receptor status and estradiol sensitivity of MCF-7 cells in exponential growth phase; Madeddu L et al.; Proliferative patterns of MCF-7 human breast cancer cells have been reported to influence their estrogen receptor (ER) contents . However, the experimental conditions under which these variations in ER contents were described differed from those commonly used for maintaining exponential growth . We, therefore, investigated whether or not MCF-7 receptor status also fluctuated under normal growth conditions . MCF-7 cells were cultured up to 4 days in 96-multiwell dishes . On each day, cell number was spectrophotometrically assessed after fixation and coloration of the cells with hematoxylin; corresponding ER content was measured by the Abbott enzyme immunoassay in KCl extracts . At the three plating densities tested (5, 10 and 20 x 10(3) cells/ml), an obvious parallel was found between the cell number and the ER content suggesting an unchanged receptor status throughout the culture period . Regression analysis confirmed this impression . Additional fractionation by SDS-PAGE of total MCF-7 proteins extracted at various times of the culture (up to 7 days in 35 mm Petri dishes) gave identical patterns suggesting that ER synthesis is regulated as the majority of proteins . Growth experiments indicated that this situation conferred a constant estrogenic sensitivity to the cells: 24 h exposure to 10(-8) M estradiol on either the 1st, 2nd, 3rd or 4th day after plating resulted in the same increase in cell number . All these data indicated that ER contents of MCF-7 cells were maintained at a constant level under exponential growth which resulted in a constant estrogenic sensitivity.

Virology, 1988 Mar, 163(1), 198 - 200
Effect of actinomycin D on tobacco mosaic virus (TMV) accumulation in isolated tobacco protoplasts under varying light conditions; Reunova GD et al.; The effect on Tobacco mosaic virus (TMV) accumulation of actinomycin D (AMD) introduced shortly after inoculation into isolated tobacco protoplasts under varying light conditions was examined . The emission spectrum of the light source contained lines in the visible range and in the ultraviolet band (300-400 nm) . AMD absorbed light in the visible (400-500 nm) and in the uv (200-400 nm) ranges . AMD substantially inhibited TMV multiplication in the light, and also when the protoplasts were incubated in petri dishes covered with a black filter that allowed only uv light in the range 260-390 nm to pass . In the dark, and also in petri dishes covered with blue or yellow filters that passed in the ranges 400-500 or 500-600 nm, respectively, AMD stimulated TMV reproduction . The suppression of TMV multiplication in isolated tobacco protoplasts was assumed to be associated with a photodynamic effect caused by absorption of uv light by AMD.

J Immunol, 1988 Feb 1, 140(3), 845 - 52
Pre-B cell generation potentiated by soluble factors from a bone marrow stromal cell line; Landreth KS et al.; Two bone marrow stromal cell lines isolated from the adherent layer of a Dexter-type long term bone marrow culture differ markedly in their hemopoietic support capacity . S17 supports myelopoiesis and the differentiation of early B cell precursors into B lymphocytes while S10 supports myeloid cell differentiation and not B lymphopoiesis . The identification of a stromal cell line with B cell support capacity prompted an investigation of whether the effects of S17 were mediated via soluble factors . Results presented herein indicate that medium conditioned by S17 but not S10 contains an activity that can induce the expression of the 220,000 m.w . 14.8 antigen and cytoplasmic mu H chain of Ig in B lymphocyte progenitors that have not yet expressed these markers . Bone marrow cells were depleted of 14.8+, cytoplasmic mu+ pre-B cells on antibody-coated petri dishes . After 24-h liquid culture newly generated pre-B cells were enumerated as cells that expressed cytoplasmic mu H chain of Ig but not Ig L chains by immunofluorescence . Expression of Ly5(220) was monitored by 14.8 antibody binding . This pre-B cell differentiation activity was abrogated by digestion with pronase, aminopeptidase, or carboxypeptidase . Isoelectric focusing data revealed the activity to have isoelectric point of 5.9 to 6.2 . S17-conditioned medium was fractionated using HPLC and each fraction tested for pre-B cell-generating activity . Fractions collected from a Superose 12 gel filtration column were found to have two peaks of activity associated with molecules of apparent m.w . of approximately 60,000 and 10,000 . Virtually identical peaks of activity were observed when medium conditioned by heterogeneous stromal cell cultures was fractionated . Separation of S10-conditioned medium revealed no cryptic activity . S17-conditioned medium was further characterized by anion exchange chromatography and the majority of the pre-B cell generating activity shown to be associated with the void volume that eluted from a MonoQ column . These fractions were rechromatographed on Superose and the activity again found to be associated with two fractions corresponding to apparent m.w . of 60,000 and 10,000 . The S17 pre-B cell differentiation activity appears to result from the presence of a novel molecule because other well characterized mediators had no activity in this short-term liquid culture system . No pre-B cell-generating activity was observed when IL-1 or conditioned medium containing IL-2, IL-3, or IL-4 (B cell stimulatory factor 1) were added to cultures.(ABSTRACT TRUNCATED AT 400 WORDS)

Infect Immun, 1988 Feb, 56(2), 457 - 61
Affinity-purified antibodies to ring-infected erythrocyte surface antigen do not correlate with merozoite invasion inhibition in Plasmodium falciparum; Coleman JP et al.; We affinity purified, from malaria-immune serum, antibody to the ring-infected erythrocyte surface antigen (RESA), using petri dishes containing a monolayer of Plasmodium falciparum ring-infected erythrocytes . Except for one out of eight samples, the purified antibody positive by RESA-immunofluorescent assay was not inhibitory to the in vitro invasion of merozoites into erythrocytes in three geographically distinct strains of P . falciparum . However, the initial high level of merozoite-inhibiting antibodies of the intact serum samples remained in the immunoglobulin G fraction from which the RESA antibodies had been removed by affinity chromatography . These results suggest that, although in some cases RESA-immunofluorescent assay-positive antibodies may be inhibitory to merozoite invasion, there are more important antibodies capable of merozoite invasion inhibition.

Cancer Res, 1988 Feb 1, 48(3), 715 - 24
Optimization and characterization of the capillary human tumor clonogenic cell assay; Ali-Osman F et al.; The capillary human tumor clonogenic cell assay (HTCA) has been shown to have important advantages over conventional HTCAs . In the present report, this promising novel HTCA was further optimized and characterized using 46 primary human tumor specimens, 6 human tumor cell lines (1 astrocytoma, 2 colon carcinomas, 1 melanoma), and 2 murine leukemias . Hydrocortisone, epidermal growth factor, heat-inactivated fetal calf serum, and horse serum were investigated for their ability to modulate tumor colony formation in the assay . Critical assay parameters that can affect tumor colony formation, namely, cell seeding density, agarose concentration, culture volume, capillary tube geometry, and capillary tube sealing, were also investigated . The results showed that serum (optimum concentration, 20%) was obligatory for tumor colony formation, and that both epidermal growth factor (50 ng/ml) and hydrocortisone (2.5 ng/ml), although supportive of colony growth, were not absolute requirements . Plating at 2.5-3 x 10(5) cells/ml in a culture volume of 50 microliters/capillary tube and an agarose concentration of 0.2% optimized colony formation (number, size, and distribution of colonies along the capillary tube) by primary human tumor cells . The cell lines generally formed colonies best at lower seeding densities and in lower culture volumes (30 microliters/tube) . Colony formation was significantly better in unsealed than in sealed capillary tubes and growth was just as good, and in some cases, better in round capillary tubes than in square ones . Using ovarian carcinoma cells, the Cellscan prototype system was demonstrated as feasible for automated counting and evaluation of tumor colony growth in capillary tubes . A comparison of the capillary HTCA and the agar double-layer assay in Petri dishes produced a median plating efficiency of 0.18 for the capillary HTCA and 0.036 for the Petri dish method . The overall success rate was 77% for the former and 53% for the latter assay.

J Electron Microsc Tech, 1988 Feb, 8(2), 217 - 22
A study of section wrinkling on single-hole coated grids using TEM and SEM; Abad A; To prevent section wrinkles usually encountered with the use of coated single-hole grids, a simple method was developed . Formvar film resting on a platform with holes (3.5 mm diameter) was heated with a slide warmer (60-65 degrees C) . The bottom of a glass petri dish was inverted over the platform to keep the ambient air at the desired temperature . Sections were picked up from the boat of the diamond knife with a single-hole grid and deposited at the orifice of the platform and allowed to dry . The grids were then carefully pushed through the orifice of the platform with the blunt head of a nail (3 mm diameter).

Zhonghua Min Guo Wei Sheng Wu Ji Mian Yi Xue Za Zhi, 1988 Feb, 21(1), 75 - 8
{Natural killer cell activity in nasopharyngeal carcinoma before and after radiotherapy}; Lynn TC et al.; From 1984 through 1986, a total of 20 patients with biopsy confirmed nasopharyngeal carcinoma (NPC) were randomly selected for studies on natural killer cell (NK) activity before and at the end of radiotherapy . Lymphocyte separation was done by centrifuge of heparinized peripheral blood with Ficoll-Hypaque and monocytes were removed by Petri dish adhesion . K562 cells were used as target cells of NK activity . The lymphocyte-target reaction of 4 hours was done and the ratio was 50:1, 25:1, and 12.5:1 . The results of 50:1 was taken as the NK activity . The mean value of NK activity in NPC patients after radiotherapy was 43.0 +/- 21.1%, this is not significantly different from 41.2 +/- 23.6% before radiotherapy . On the other hand, before radiotherapy, the patients with too high NK activity (greater than 40%) or too low NK activity (less than 20%) had a mild tendency toward median value (20%-40%) after radiotherapy.

Arch Androl, 1988, 20(1), 31 - 4
Sperm motility in fertile men and males in infertile units: in vitro test; Dahlberg B; An in vitro test evaluating spermatozooan motility was used to compare percentage of forward progressive spermatozooa at initial observation, within 1 h of obtaining samples, with forward progression after 8 h in Petri dishes (humid chambers) . A constant motility profile was found in the sperm of fertile men after 8 h, whereas a different profile was found in 94% of the infertile male groups.

Lasers Surg Med, 1988, 8(2), 125 - 9
Does low-energy helium-neon laser irradiation alter "in vitro" replication of human fibroblasts?
Hallman HO, Basford JR, O'Brien JF, Cummins LA.
Cultured human fibroblasts were treated in a controlled, randomized manner to assess the effect of low-energy (0.9 mW) helium-neon (HeNe) laser irradiation on cellular proliferation . Two trials were performed: one with fibroblasts in the third to fourth passage and the other with fibroblasts in the 13th to 14th passage . In each trial, separate plastic petri dishes were inoculated with the cells, maintained in a 5% CO2-95% air atmosphere, and nourished with HB 102 media . Treatment began 48 h after inoculation with daily 60-s irradiations of the "treated" cultures over a 5-d period . Control cultures underwent the same handling but were not irradiated . A significant stimulative, or inhibitive, effect on replication was not found in either trial.

Anat Anz, 1988, 165(1), 1 - 6
Human macrophages from skin: I . Isolation and culture; Michna H; Coarsely ground Lytta vesicatoria FABRICIUS (meloideae/coleoptera), with a cantharidine content of 0.5-1% was dissolved in ethanol and worked into a powdery solution . 1-2 cm2 large swabs were dissolved in the tincture, and with the help of a Karaya plate and an occlusive dressing was administered to the skin in the antebrachii anterior region . This led to a severe inflammation, which after a further 10 h was accompanied by pemphigus and vesiculation . The vesicular liquid was aspirated, centrifuged, the cells resuspended and coated on a small cover-glass in a petri-dish and incubated . The human macrophages were kept in a moisture-saturated atmosphere with 5% CO2 at a temperature of 37 degrees C . The blisters healed quickly and without scaring after a correct dressing of the wounds was administered . The safety of this method was thus tested on human subjects, at first on the author, and proven successful.

Tissue Cell, 1988, 20(3), 331 - 8
A microwell assay for anchorage independent cell growth; Macintyre J et al.; We describe modifications of the conventional assay for anchorage independent growth of fibroblasts that enable the assay to be carried out in microwell plates, as opposed to the conventional Petri dishes . The microwell assay is a good discriminator of final EGF concentrations in the range 10-100 picograms/ml, and can be used to detect absolute amounts of EGF below 2 pg . Addition of TGF beta to EGF enhances colony formation in the microassay in the usual manner . We describe the use of this microassay to identify and map local production of transforming growth factor activity by the component pieces of individual chick embryos . Transforming activity was identified in all the stages tested (Hamburger and Hamilton stages 13-23) . Highest levels were found near the mid-line of the embryo . No clear differences in the cranio-caudal axis have so far been identified . This technique will enable the spatial and temporal distribution of transforming activity throughout vertebrate embryos to be completely mapped . It seems likely that this mapping process will help elucidate the normal role of transforming growth factors in embryos.

Int J Dev Neurosci, 1988, 6(1), 89 - 102
Factors influencing neuronal growth in primary cultures derived from 3-day-old chick embryos; Mangoura D et al.; We compared neuronal growth patterns in primary cultures prepared by dissociating 3-day-old chick embryos, either whole embryo (E3WE) or head only (E3H) and plating the dispersed cells onto Petri dishes coated with either poly-L-lysine, collagen or laminin . The culture medium was Dulbecco's Modified Eagle's Medium (DMEM), supplemented with either 5 or 10% fetal bovine calf serum (FCS) . As we have previously described, in E3WE cultures on poly-L-lysine the neuronal primary growth patterns were aggregation with neuritic fasciculation, presence of growth cones with microspikes and very few flat cells . In contrast with cultures grown on poly-L-lysine, in cultures grown on collagen or laminin the distinct growth pattern was extensive networks of isolated and differentiated neurons lying on acquired monolayers of flat cells . When 5% FCS was used, as compared to 10% FCS, neuronal aggregates were fewer and smaller on poly-L-lysine; on collagen or laminin a tendency to aggregate was observed . Several differences were observed in the E3H cultures when compared to E3WE: (a) aggregates were less numerous with the prevailing pattern being a web-like, self-contained aggregate; (b) aggregates connected with other aggregates or flat cells were rare and the aggregate adhesivity was minimized; (c) neurons on collagen or laminin formed networks with the exception of a few, small aggregates displaying no fasciculation; (d) flat cells did not form a monolayer but islets which hosted the neuronal meshy networks . We attribute these differences in the growth patterns between the various types of cultures to be the combined result of a variety of environmental signals, derived from the provided substrata, the serum and the nonneuronal cell factors and cell surface, all primarily regulating neuronal adhesivity.

C R Acad Sci III, 1988, 307(20), 863 - 8
{Proliferation of oligodendrocytes is stimulated after their adhesion on a matrix made up of a nerve lectin, CSL}; Fressinaud C et al.; It is shown that oligodendrocytes (myelin producing cells in the central nervous system) can adhere to a substratum constituted by an endogenous cerebellar soluble lectin (CSL) adsorbed on plastic Petri dishes . This adhesion induces a rapid and important proliferation of cultured oligodendrocytes.

Adv Exp Med Biol, 1988, 237, 421 - 5
Suppression of immunoglobulin production by germinal centre HNK-1+ CD3+ cells; Banerjee D et al.; Germinal centre T cells co-expressing HNK-1 antigen have little lytic activity against NK targets (K562 cells) . In order to determine whether these cells regulate B cell function, they were purified from human tonsils by panning on anti-HNK-1 antibody coated Petri dishes and co-cultured with autologous and allogeneic tonsillar T and B cells in the presence of pokeweed mitogen . At the end of 7 days of culture, supernatants were assayed for immunoglobulin concentration by ELISA . A dose-dependent suppression of both IgG and IgM production was demonstrated at ratios from 1:125 to 1:16 of HNK-1+ cells to B cells, but enhancement was observed at very low ratios (less than 1:500) or ratios exceeding 1:16 in some tonsil preparations . Similar results were obtained with peripheral blood HNK-1+ cells but without enhancement in some cases at the extremes of HNK-1+ cells to B cell ratios . The suppression was not MHC-restricted . These preliminary experiments indicate that germinal centre HNK-1+ cells may be intrafollicular suppressor cells.

Int J Dev Neurosci, 1988, 6(2), 137 - 47
Synapse formation and development of neurotransmitter functions in neuronal cells from chick brain cultured in a serum-free, defined medium; Langui D et al.; Cells dissociated from cerebral hemispheres of 8-day-old chick embryos were seeded on poly-L-lysine coated Petri dishes in serum-containing medium . After 24 hr the culture medium was switched to a serum-free, chemically defined medium . These cultures contain mainly neuronal cells until day 14, characterized by the presence of acetylcholinesterase activity and neurofilament proteins . After 2 weeks glial cells progressively contaminated the neuronal culture . Cultures were maintained for a period of 4 weeks . From day 6 on numerous synapses with clear vesicles were observed . The activity of choline acetyltransferase remained low throughout the culture period, while GABA levels increased in parallel with synaptogenesis . Our observations indicate that chick cerebral hemisphere neuronal cultures grown in serum-free, chemically defined medium contain GABAergic neurons that undergo maturation.

Prikl Biokhim Mikrobiol, 1988 Jan-Feb, 24(1), 121 - 4
{A method for detecting restriction endonucleases in bacterial colonies}; Belavin PA et al.; A simple technique is proposed for detection of bacterial restriction endonucleases . Analysis is performed directly in the cells from colonies cultivated on Petri dishes . The cells collected with an inoculation loop are treated with lysozyme and Triton X-100 . After centrifugation the supernatant is tested for endonuclease activity . The technique enables up to 100 colonies to be tested for 3-4 h.

Arch Surg, 1987 Dec, 122(12), 1417 - 20
In vitro killing of melanoma by liposome-delivered intracellular irradiation; Pikul SS 2nd et al.; To better understand and optimize the mechanism of alpha particle killing of tumors, an in vitro model utilizing liposomes as carrier vehicles was developed to study the killing of melanoma via intracellular alpha-irradiation . The radionuclide 212Pb (lead), with its 10.6-hour half-life and alpha-emitting daughter 212Bi (bismuth), was encapsulated in liposomes to achieve the intracellular irradiation of melanoma cells in culture . In dose-response experiments, B16F10 mouse melanoma cells were incubated with liposomes 212Pb/212Bi bound to dextran 70 . Plating efficiency and growth of the melanoma cells cultured on gridded petri dishes after incubation were compared with controls at 24 and 48 hours . Greater than 85% cell killing occurred by 48 hours, with administered radioactivity levels of 1.6 dpm/mumol of lipid/cell, which corresponds to intracellular delivery of five to seven alpha particles per cell . These alpha doses can be exceeded in vivo with recirculation or in a perfusion circuit, and more efficient cytotoxic action may be possible.

Endocrinology, 1987 Dec, 121(6), 2161 - 70
A new model system for studying androgen-induced growth and morphogenesis in vitro: the bulbourethral gland; Cooke PS et al.; An organ culture system was devised for neonatal mouse bulbourethral glands (BUGs) in which androgen-dependent development parallels that in vivo . BUGs from 0-day-old (day of birth) mice were grown on Millipore filters placed on metal grids in petri dishes for 3 or 6 days in Dulbecco's Modified Eagle's Medium-Ham's F-12 medium (1:1) containing 10% fetal bovine serum . In medium supplemented with testosterone (T; 10(-7) or 10(-8) M), growth and epithelial morphogenesis of the cultured BUGs were comparable to those of the gland in situ . At lower doses of T (10(-9) -10(-12) M), BUGs showed dose-dependent decreases in the rate of growth and degree of epithelial morphogenesis . BUGs cultured without T contained only 38% as much DNA as those in T-supplemented medium, and epithelial morphogenesis did not occur . Thus, BUG development in vitro was dependent on androgens . The continued, albeit reduced, growth in cultures without T indicates that growth is also partially independent of androgens, but epithelial branching morphogenesis is totally dependent on this hormone . Growth and epithelial morphogenesis were reinitiated in glands that had developed in the absence of T, either in vivo or in vitro, by culturing the BUGs for 3 days in T-containing medium . The growth of BUGs in a serum-free medium with or without T paralleled that in comparable serum-containing cultures in vitro and in normal and castrated animals in situ . Coincubation of BUGs with T and 390 MSD (17 beta-N,N-diisopropylcarbamoyl-4-aza-5 alpha-androstan-3-one), an inhibitor of the enzyme 5 alpha-reductase, resulted in retarded development, indicating that T must be converted to 5 alpha-dihydrotestosterone (DHT) to promote normal BUG growth . Additionally, DHT (10(-8) M) alone could substitute for T in promoting BUG development . Thus, DHT must be the proximal androgen for BUG growth . The BUG is an excellent model system for examining androgen-dependent development and should be useful for studying epithelial morphogenesis, growth, and hormonal effects in vitro.

Atherosclerosis, 1987 Nov, 68(1-2), 87 - 93
Effect of an elastin growth substrate on cholesteryl ester synthesis and foam cell formation by cultured aortic smooth muscle cells; Grande J et al.; Exposure of smooth muscle cells cultured on plastic or glass to hyperlipidemic serum did not result in the formation of foam cells . Since elastin binds serum lipids, and vascular smooth muscle cells are normally closely associated with elastin, we investigated the effects of an elastin substrate on lipid metabolism and on the accumulation of lipid vacuoles by rabbit aortic smooth muscle cells in culture . When cells were grown in plastic petri dishes, cholesteryl ester synthesis, as measured by {14C}oleate incorporation into cholesteryl esters, was 3 times greater in rabbit hyperlipidemic serum (HLS) than in normolipemic serum (NLS) (P less than 0.001) . For cells of the same subculture grown on the elastin substrate, the synthetic rate was 6-fold greater in HLS compared to NLS (P less than 0.005) . The cells grown on the elastin membranes in the presence of HLS contained large numbers of Oil red O stainable lipid vacuoles and resembled foam cells, while those grown in petri dishes and exposed to HLS showed only an occasional cell containing a few vacuoles . Pre-incubation in lipoprotein-deficient serum markedly enhanced the stimulatory effect of HLS on cholesteryl ester synthesis for cells growing in plastic petric dishes but had much less stimulatory effect on the cells growing on elastin membranes . These studies indicate that close association with elastin modulates the response of smooth muscle cells to hyperlipidemia and suggest a role for elastin in the formation of foam cells of smooth muscle origin during atherogenesis.

Biochem Biophys Res Commun, 1987 Oct 14, 148(1), 392 - 6
Fibrinolysis by urokinase endowed with magnetic property; Inada Y et al.; The activated magnetic modifier was synthesized from magnetite, alpha, omega-dicarboxymethylpoly(oxyethylene) and N-hydroxysuccinimide (Biochem . Biophys . Res . Commun., 145, 908-914, 1987) . Urokinase was directly coupled with the activated magnetic modifier to obtain magnetic urokinase . The magnetic urokinase dispersed in saline and exerted high fibrinolytic activity (13.8 X 10(4) IU/mg protein), and was readily recovered from saline by magnetic force of 250 Oe . By applying magnetic force, the urokinase was attracted at our will and local fibrinolysis was achieved on fibrin gel in a petri dish.

Microvasc Res, 1987 Sep, 34(2), 239 - 49
Microvascular endothelial cells from human omental tissue: modified method for long-term cultivation and new aspects of characterization; Anders E et al.; A method for long-term cultivation of large amounts of human microvascular endothelial cells from the omental tissue (human omental tissue microvascular endothelial cells, HOTMECs) was devised . The method originally described by Kern, Knedler, and Eckel was modified: HOTMECs were isolated by enzymatic dissociation with collagenase . For primary cultivation and passages, HOTMECs were plated either onto fibronectin-coated petri dishes or onto a human fibroblast extracellular matrix (HFB-ECM) prepared from the same tissue . Omental tissue (10-15 g) yielded 4-8 X 10(5) HOTMECs; more than 90% of the cells adhered to precoated dishes and grew in Waymouth's culture medium supplemented with 20% heat-inactivated fetal calf serum . Confluence was reached 3-5 days after seeding with an average of 1-2 X 10(6) cells/dish . Confluent HOTMEC layers were subcultured at a split ratio of 1:3 up to 11 passages by plating the cells onto dishes coated with HFB-ECM and maintained in long-term culture for up to 3 months . The endothelial origin of these cells was demonstrated as follows . The cells in culture showed the typical "cobblestone" growth pattern and synthesized von Willebrand factor (vWF) as determined by metabolic labeling . Using an indirect immunostaining technique, the cytoplasm of the HOTMECs stained for vWF . A monoclonal antibody specific for human endothelial cells bound exclusively to the cultured cells . The expression of thrombomodulin on the surface of the cultured cells was demonstrated by the activation of protein C by thrombin . In control experiments, these features could be detected on neither fibroblasts nor mesothelial cells.

J Immunol, 1987 Aug 15, 139(4), 1046 - 53
The role of macrophages in anti-idiotypic antibody and T suppressor factor induction of timothy grass pollen antigen B-specific T suppressor cells; Malley A et al.; Cultures of normal spleen cells with anti-idiotypic antibody (anti-Id) or antigen B (AgB)-specific T suppressor factor (Tsf1) in mini-Marbrook chambers for 4 days at 37 degrees C lead to the in vitro induction of AgB-specific T suppressor (TS) cells . These TS cells significantly suppress a secondary AgB-specific IgE response, but they do not affect a secondary AgB-specific IgG response . Depletion of both B cells and macrophages from normal spleen cells by panning on anti-Ig-coated petri dishes provides an enriched T cell population . These enriched T cells when cultured with anti-Id or Tsf1 in mini-Marbrook chambers do not produce AgB-specific TS cells, and mice treated with cells harvested from the mini-Marbrook chambers have normal secondary AgB-specific IgG and IgE responses . The addition of as few as 1000 bone marrow-derived macrophages (BMDM) to cultures of the enriched T cells with anti-Id, or Tsf1 restores the ability of these cultures to produce significant levels of AgB-specific TS cells . Further studies reveal that the macrophage population must be histocompatible and express a cell surface I-J antigen . Attempts to pulse BMDM with anti-Id or Tsf1 at 4 degrees C and to culture in mini-Marbrook chambers 10(3) pulsed BMDM with enriched T cells were unsuccessful in producing AgB-specific TS cells . However, pulsing BMDM with anti-Id or Tsf1 at 37 degrees C, and adding 10(3) of these pulsed BMDM to enriched T cells in culture led to the formation of significant levels of AgB-specific TS cells.

Comput Methods Programs Biomed, 1987 Aug, 25(1), 31 - 8
A program which determines mutagenic concentrations of chemical carcinogens via a diffusion bioassay; Awerbuch TE et al.; A computer program implementing a mathematical model for determining mutagenic concentrations of chemical carcinogens was developed . The mathematical model describes the experiment in which a droplet of a suspected carcinogen is put at the center of a petri dish containing a bacterial lawn in an agar gel . After a period of incubation during which the chemical diffuses outward, one observes a concentric ring of mutants around the center . The largest radius at which mutation occurs, r mut, corresponds to the lowest (threshold) concentration of the chemical sufficient to produce bacterial mutation . Given a series of initial concentrations of a chemical and the resulting r mut's, the program computes and reports the threshold concentration and the decay time of the chemical . The program is also used as a method to determine the lowest mutagenic concentration for a particular time of exposure.

Scand J Immunol, 1987 Aug, 26(2), 161 - 73
In vitro and in vivo stimulation of murine lymphocytes by human respiratory syncytial virus strains; Alsheikhly AR et al.; Respiratory syncytial virus (RSV) strains of subtype A (A2, WV9894, and WV12138) and of subtype B (WV1293, WV4843, and WV6873) are mitogenic in vitro for unprimed BALB/c spleen cells . The virus also triggered splenocytes in vitro to secrete immunoglobulins . Plaque-purified and UV-irradiated materials of both RSV subtypes produced comparable levels of DNA synthesis . Infectious materials of both subtypes also induced pronounced responses . Lymphocyte activation with UV-inactivated RSV strain A2 was dose-dependent and maximal responses occurred after 4-5 days of incubation . The virus preparations were mitogenic for spleen cells depleted of T lymphocytes by treatment with anti-Thy 1.2 and complement and for lymphocytes of congenitally athymic mice (nu-nu) . They were also mitogenic for highly purified T lymphocytes separated by panning of spleen cells on anti-mouse Ig-coated Petri dishes, suggesting that both B and T lymphocytes respond to the mitogenic activity of RSV . Moreover, mice infected intranasally with RSV strain A2 generated local as well as peripheral cellular and humoral responses.

Allergol Immunopathol (Madr), 1987 Jul-Aug, 15(4), 221 - 4
Incidence of Alternaria Nees ex Fries in dwellings of Córdoba City (Spain); Infante F et al.; We have studied the occurrence of Alternaria, a genus of allergenic interest in the indoor atmosphere of 14 homes in Cordoba throughout 1984 . The sampling was carried out by sedimentation on a broad-spectrum mycological culture medium (2% Agar-Malt extract) in Petri dishes . Samples were collected every fortnight in 14 homes (three dishes per home were exposed in the following rooms, namely the kitchen, the toilet and the bedroom, for 20 minutes) located in different areas of the city and as much as possible representing the various physical conditions found . An overall of 727 Alternaria colonies belonging to six species (A . consortiale, A . crassa, A . dendritica, A . japonica, A . tenuis and A . tenuissima) were detected . Some of them were proven allergenics . They accounted for 5.2% of all fungal colonies found indoors . A . tenuis was by far the most frequently found species, followed by A . tenuissima (second in number) and A . consortiale (the most cosmopolitan) as they were the only species found in all 14 homes . On the other hand, A . crassa was the least frequent of all species collected . We have also determined the general variation of Alternaria and that of the different species found indoors throughout the year, finding a trend with the appearance of peaks towards spring and early summer, especially in June.

Anaesthesist, 1987 Jul, 36(7), 340 - 4
{Volatile anesthetics and n-alcohols inhibit the uptake of noradrenaline in pheochromocytoma cells}; Tas PW et al.; Studies with perfused animal organs did not show a significant inhibition of the norepinephrine uptake by volatile anesthetics However, the uptake of norepinephrine into isolated chromaffin granules proved to be halothane-sensitive , and there is indirect evidence for an inhibition of norepinephrine uptake by halothane in the dog saphenous vein . So far no literature is available on norepinephrine uptake into cultured cells in the presence of volatile anesthetics . The rat pheochromocytoma cell line PC 12, which shares many properties with adrenal medulla cells and also with sympathetic neurons, has been used as a model to study norepinephrine uptake and release . One of its properties in common with sympathetic neurons is the high-affinity uptake system for norepinephrine . Therefore, we have used PC 12 cells as a pharmacologically well-established model system to study the effect of general anesthetics on the uptake of norepinephrine . METHODS: The experiments were performed with monolayers of PC 12 cells on 3 cm petri dishes . The cells were allowed to take up tritiated norepinephrine (0.25 microCI/ml) for 20 min at 36 degrees C in the presence and absence of general anesthetics . RESULTS and DISCUSSION: The volatile anesthetics halothane, enflurane, isoflurane, and methoxyflurane in clinical relevant concentrations inhibited the high-affinity uptake of tritiated norepinephrine into the PC 12 cells (Fig . 1) . We could further show an inhibition of norepinephrine uptake by n-alkanols at concentrations that cause anesthesia in tadpoles . Fig . 2 illustrates that a good correlation (r = 0.997 for the n-alkanols, r = 0.934 for the volatile anesthetics) exists between anesthetic potency and the IC50 for norepinephrine uptake inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

Virus Res, 1987 Jul, 8(1), 43 - 59
Heat-regulated expression of the hepatitis B virus surface antigen in the human Wish cell line; Dreano M et al.; The DNA fragment coding for the hepatitis B virus surface antigen (HBsAg) was placed under the control of a human 70 kDa heat-shock protein (hsp70) promotor sequence . This plasmid construct has been used in transfection experiments to establish a stable amnion cell line of human origin (Wish), expressing an HBsAg in a heat-regulated fashion . Post-translational modifications, such as assembly, glycosylation, secretion and production of both major and middle S proteins appear to function normally . In addition, production of HBsAg under various protocols of heat induction is described . After inoculation into nude mice, development of tumours has been observed at the site of injection . Tumour cells, dispersed by means of collagenase or trypsin treatment from excised tumours, and subsequently seeded into Petri dishes, were able to secrete the same quantities of HBsAg after heat induction as were cells of the original cell line.

Brain Res, 1987 Jul, 431(1), 111 - 21
Kinetic analysis of 'rapid onset' neurite formation in NG108-15 cells reveals a dual role for substratum-bound laminin; Smalheiser NR et al.; Undifferentiated neural hybrid NG108-15 cells plated on laminin-coated, polylysine-treated plastic Petri dishes in minimal serum-free media formed long neurites within 1-4 h post-plating . Morphologic features and pharmacologic responses of these 'rapid onset' neurites were strikingly similar to those of neuronal growth cones . Cycloheximide (1-10 micrograms/ml) and forskolin (10(-7) to 10(-6) M) accelerated the initial formation of laminin-stimulated neurites, but did not cause rapid onset neurites to emerge upon Petri dishes coated with polylysine alone . Quantitative study of the substratum-dependent effect of cycloheximide showed that it was additive even with maximally effective amounts of laminin, independent of the magnitude of the laminin-stimulated baseline rate and not limited by an inherent ceiling in the rate at which neurites could form . The methylation inhibitor 5'-deoxy-5'-methyl thioadenosine (MTA) (3 X 10(-4) to 3 X 10(-3) M) did stimulate neurites to form on polylysine . MTA- and laminin-stimulated neurites were similar in their susceptibility to calmodulin antagonists and the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) . However, formation of MTA-stimulated neurites was not accelerated by cycloheximide . A simple two-compartment kinetic model of 'rapid onset' neurite formation is proposed: compartment A is common to both laminin- and MTA-stimulated neurites . Compartment B is affected by cycloheximide, and its access to the neurite formation machinery contained in compartment A is gated according to the nature of the substratum . In addition to its direct effects, laminin controls the relative dominance of the two kinetic compartments via modulating the effectiveness of other signals acting upon an endogenously active compartment B.

Dev Biol, 1987 Jun, 121(2), 510 - 25
Magnesium-dependent attachment and neurite outgrowth by PC12 cells on collagen and laminin substrata; Turner DC et al.; We report a study of the substratum and medium requirements for attachment and neurite outgrowth by cells of the pheochromocytoma-derived PC12 line . In attachment medium containing both Ca2+ and Mg2+, more than 50% of cells attached within 1 hr to petri dishes coated with native collagen Types I/III or II, native or denatured collagen Type IV, laminin, wheat germ agglutinin (WGA), or poly-L-lysine; attachment to dishes coated with nerve growth factor (NGF) was only about 20% and attachment to uncoated dishes or to dishes coated with fibronectin or gelatin was almost nil . Neither prior culturing in the presence of NGF nor addition of NGF to the attachment medium significantly affected the extent of attachment to collagen or laminin . With Ca2+ (1 mM) as the sole divalent cation, cells attached normally to WGA, polylysine, and NGF, but failed to attach to collagen or laminin . With Mg2+ (1 mM) as the only divalent cation, attachment to all substrata was about the same as in medium with both Ca2+ and Mg2+ . Like the ionic requirements, the kinetics of attachment, insensitivity to protease treatment of the cells, and inhibition by low temperature and sodium azide were similar for PC12 attachment to collagen and laminin, suggesting that a common molecular mechanism may underlie attachment to these substrata . The only significant difference observed was that addition of WGA (30 micrograms/ml) to the attachment medium inhibited attachment to collagen but promoted attachment to laminin . Finally, PC12 cells extended neurites on laminin, on native collagens I/III, II, and IV, and on denatured collagen IV; they did not extend neurites on denatured collagens I/III or II, NGF, or WGA . Neurite outgrowth on collagen and laminin occurred with Mg2+ as the sole divalent cation . These results suggest that the same Mg2+-dependent adhesion mechanism operates at the cell body and at the growth cone.

Dev Biol, 1987 Jun, 121(2), 376 - 88
Separation of the myogenic and chondrogenic progenitor cells of undifferentiated limb mesenchyme; Kosher RA et al.; Undifferentiated limb bud mesenchyme consists of at least two separate, possibly predetermined, populations of progenitor cells, one derived from somitic mesoderm that gives rise exclusively to skeletal muscle and one derived from somatopleural mesoderm that gives rise to the cartilage and connective tissue of the limb . In the present study, we demonstrate that the inherent migratory capacity of myogenic precursor cells can be used to physically separate the myogenic and chondrogenic progenitor cells of the undifferentiated limb mesenchyme at the earliest stages of limb development . When the undifferentiated mesenchyme of stage 18/19 chick embryo wing buds or from the distal subridge region of stage 22 wing buds is placed intact upon the surface of fibronectin (FN)-coated petri dishes, a large population of cells emigrates out of the explants onto the FN substrates and differentiates into an extensive interlacing network of bipolar spindle-shaped myoblasts and multinucleated myotubes that stain with monoclonal antibody against muscle-specific fast myosin light chain . In contrast, the cells of the explants that remain in place and do not migrate away undergo extensive cartilage differentiation . Significantly, there is no emigration of myogenic cells out of explants of stage 25 distal subridge mesenchyme, which lacks myogenic progenitor cells . Myogenic precursor cells stream out of mesenchyme explants in one or occasionally two discrete locations, suggesting they are spatially segregated in discrete regions of tissue at the time of its explantation . There are subtle overall differences in the morphologies of the myogenic cells that form in stage 18/19 and stage 22 distal subridge mesenchyme explants . Finally, groups of nonmyogenic nonfibroblastic cells which are fusiform-shaped and oriented in distinct parallel arrays characteristically are found along the periphery of stage 18/19 wing mesenchyme explants . Our observations provide support for the concept that undifferentiated limb mesenchyme consists of independent subpopulations of committed precursor cells and provides a system for studying the early determinative and regulatory events involved in myogenesis or chondrogenesis.

Blood, 1987 Jun, 69(6), 1587 - 94
Differential binding of erythroid and myeloid progenitors to fibroblasts and fibronectin; Tsai S et al.; Using a novel coverslip-transfer culture technique, we recently demonstrated that primitive erythroid burst-forming units (BFU-E) can migrate, proliferate, and differentiate in intimate association with stromal fibroblastoid cells in the presence of serum proteins and erythropoietin . No other exogenous hemopoietic growth factors are required . Most of the colonies that develop in this system are very large erythroid bursts, and very few granulocyte-macrophage (GM) colonies are observed . In this report, we present data indicating that the predominance of erythroid burst colonies in this culture system is due to preferential binding of primitive erythroid progenitors to the stromal fibroblastoid cells and not to differential stimulation of these erythroid progenitors by these cells . We next show that the binding of BFU-E to stromal cells is blocked by anti-fibronectin antibodies . Finally, we demonstrate the preferential binding of BFU-E to fibronectin by using glass coverslips or Petri dishes coated with purified human plasma fibronectin . The binding is blocked by a monoclonal antibody specific for the cell-binding domain of fibronectin . We conclude that: primitive erythroid progenitors bind strongly whereas G and/or M progenitors (CFU-G/M) bind only weakly to fibronectin; primitive erythroid progenitors bind to the cell-binding domain on the fibronectin molecule; and erythroid progenitors and precursors remain bound to fibronectin throughout differentiation.

Cancer Genet Cytogenet, 1987 Jun, 26(2), 261 - 70
Evidence for genetic predisposition for some nasopharyngeal cancers by in vitro hyperdiploidy in human dermal fibroblasts; Danes BS et al.; A numerical alteration in chromosome complement in human dermal fibroblast cultures, hyperdiploidy with a normal occurrence of tetraploidy (IVH), has been reported to be associated with some hereditary single tumors including squamous carcinoma of the nasopharynx (NPC) . Its incidence was compared in cultures derived from 39 NPC patients and 29 clinically normal subjects without a family cancer history by two different methods (percentage of numerically altered metaphases in chromosome preparations from nonconfluent monolayer Petri dish cultures in logarithmic growth, and by the distribution of DNA content of propidium iodide stained cells from plastic flask cultures in stationary growth phase as assayed by flow cytometry) to ascertain its usefulness in identification of such genetic predisposition . Concordance was observed between the two assays . There was a linear relationship between the percentage of hyperdiploid metaphases assayed in the chromosome preparations and the percentage of cells with a DNA index of greater than 1 as determined by flow cytometry . By metaphase assay, none of the 29 normals showed IVH . OF the 39 carcinoma of the nasopharynx patients studied, 19 had IVH and 20 did not . By flow cytometry there were significant differences in the flow cytometry DNA index (p less than 0.001) of IVH-negative (all normals and 20 carcinoma of the nasopharynx patients) and IVH-positive carcinoma of the nasopharynx patients . The percentage of cells with a DNA index of 2 and greater than 1 could be used to distinguish all IVH-negative from the IVH-positive subjects and, thus, were considered to be the parameters of choice in assaying IVH by flow cytometry . None of the subjects studied showed increased in vitro tetraploidy (IVT), which has been associated with some heritable colon cancer syndromes . Irrespective of family cancer history, approximately one-half of the NPC patients (19 of 39) had IVH, which has been reported to be associated with the in vivo expression of certain heritable tumors including carcinoma of the nasopharynx . The average age of carcinoma of the nasopharynx diagnosis was earlier (mean 52 yr) for the IVH-positive group than for the IVH-negative carcinoma of the nasopharynx group (mean 67 yr).

Biochim Biophys Acta, 1987 May 13, 919(1), 71 - 8
Cellular confluence determines injury-induced prostaglandin E2 synthesis by human keratinocyte cultures; Pentland AP et al.; When keratinocyte cultures become confluent, their prostaglandin E2 synthesis is suppressed . To determine whether the injury response is characterized by increased prostaglandin E2 synthesis, an in vitro injury model was developed . When confluent keratinocyte cultures were focally lethally irradiated using ultraviolet light B, a dose-dependent increase in prostaglandin E2 synthesis was induced by the injury . After irradiation, confluent cultures' prostaglandin E2 synthesis increased for 2 days to 8-fold more than controls, then decreased to control values by day 6 . Increased prostaglandin E2 synthesis was first detected 8 h after injury . Focal irradiation of non-confluent cultures (killing isolated colonies) caused no change in prostaglandin E2 synthesis, indicating that culture continuity must be disrupted before synthesis increases . In addition, partial irradiations of petri dishes demonstrated that enhanced metabolism was confined to cells adjacent to the injury site and was not mediated by a soluble factor . When confluent and injured cultures were incubated with {14C}arachidonic acid, and the products formed analyzed by thin layer chromatography, 10-fold more prostaglandin E2 microgram protein was seen in irradiated cultures relative to confluent controls . The products formed by each group were the same, and no consistent increases in metabolites other than prostaglandin E2 were observed . The increased synthesis of prostaglandin E2 by injured cultures was apparently due to an increase in cyclooxygenase activity as determined by kinetic experiments . These data indicate that the pattern of metabolism of arachidonic acid seen in non-confluent cultures is similar to that seen in injury, and that cell-cell contact modulates enhanced prostaglandin E2 synthesis.

Fundam Appl Toxicol, 1987 May, 8(4), 425 - 31
Application of microencapsulation for toxicology studies . I . Principles and stabilization of trichloroethylene in gelatin-sorbitol microcapsules; Melnick RL et al.; Microencapsulation is an innovative, alternative means of incorporating volatile, reactive, and/or unpalatable chemicals into animal feed for toxicologic studies . For such usage, the materials in the microcapsule shell must not adversely affect the laboratory animals, and the encapsulation process must not alter the chemical under study . Trichloroethylene (TCE), a volatile chemical identified in drinking water, was encapsulated in gelatin-sorbitol microcapsules . The concentration of TCE ranged from 40 to 43% (w/w) and most particles (approximately 85%) were between 300 and 420 micron in diameter . Under optimum storage conditions, loss of TCE from the microcapsules was less than 1% per month . Less than 2% of the TCE was lost from microcapsules held in uncovered petri dishes at ambient temperature and humidity for 14 days . Microencapsulated TCE was mixed with NIH-07 rodent feed at a level of 50 mg microcapsules/g feed (equivalent to 20.6 mg TCE/g feed) and stored at room temperature for 7 days in an open container or for 21 days in a sealed container . There was no detectable loss of TCE from the feed blends stored under these conditions . Thus, the stability of TCE in gelatin-sorbitol microcapsules is adequate for dosed-feed toxicity studies of this chemical.

Immunol Invest, 1987 May, 16(3), 227 - 40
Fluorescence-linked immunosorbent assay (FLISA) for quantification of antibodies to food antigens; Magnusson KE et al.; Serum antibodies of the IgG type from rabbits immunized with food antigens (beta-lactoglobulin, ovalbumin and gliadin) have been quantified using a fluorescence-linked immunosorbent assay (FLISA), with the antigens adsorbed as round spots (about 8 mm in diameter) on glass or plastic microscope slides . The indirect immunofluorescence intensities were determined using a microscope fluorometer, and compared to enzyme-linked immunosorbent assay (ELISA) in microtiter plates and diffusion-in-gel-ELISA (DIG-ELISA) in plastic petri dishes . It was found that FLISA in general became more sensitive when the antigens had been adsorbed onto a plastic (Nunclon) than onto a glass surface . When the antigens were adsorbed to the plastic slides, the relative sensitivity order (maximum serum dilution) of the assays was in general the following, ELISA greater than FLISA greater than DIG-ELISA . The fluorescence-linked method appeared to require equal or less antigen and conjugated antiserum per sample . Due to the visual inspection of the surface, inhomogeneities of the antigen-coating could be readily discovered and evaluated by several measurements within the field of antigen-antibody reaction . It is proposed that spot FLISA may be an alternative to ELISA especially when the amount of antigen or antiserum is limited.

J Chromatogr, 1987 Apr 17, 392, 333 - 47
High-performance liquid chromatographic determination of profiles of mycotoxins and other secondary metabolites; Frisvad JC; A reversed-phase high-performance liquid chromatographic determination of profiles of mycotoxins and other fungal secondary metabolites has been developed . Penicillium, Aspergillus and Fusarium polyketides, terpenes and alkaloids have been emphasized . In a gradient elution, using water-acetonitrile containing 0.05% trifluoroacetic acid, 134 secondary metabolites were eluted evenly with retention times from 1.08 to 34.48 min . Metabolites with the same retention time were usually not produced by the same species . As UV detection at 254 nm was used, some mycotoxins (type A trichothecenes, viridicatumtoxin, peptide-like compounds and xanthomegnin) could not be detected . The method appears to be valuable for chemotaxonomic studies of fungi . Unpurified concentrated chloroform-methanol extracts of petri dish cultures analysed by the proposed method presented gave species-specific characteristic profiles of known and unknown secondary metabolites and mycotoxins.

J Biochem (Tokyo), 1987 Apr, 101(4), 933 - 8
Transformation of macrophages into foam cells in vitro induced by cholesteryl oleate liquid crystals; Enomoto M et al.; Transformation of macrophages into foam cells after the uptake of cholesteryl oleate anisotropic liquid crystals was studied . A new technique to enhance the uptake of the liquid crystals by macrophages using an inverted petri dish was developed . Uptake of lipid droplets was found to increase in parallel with the amount of liquid crystals in the medium . A lysosomal enzyme was shown (by using lysosomotropic chloroquine) to be involved in the hydrolysis of the liquid crystals . About 47 and 72% of the {3H}cholesterol in liquid crystal-laden cells had disappeared after chase for 24 and 48 h, respectively . Thus the 50% clearance time of the liquid crystals by the macrophages was about 24 h, which was longer than that of denatured lipoprotein . A possible model of transformation of macrophages to foam cells is discussed.

Eur J Cell Biol, 1987 Apr, 43(2), 203 - 7
The biological clock of Chlamydomonas reinhardii in space; Mergenhagen D et al.; The overt circadian rhythm in a wildtype (wt+) and a short period (s-) strain of Chlamydomonas reinhardii has been studied in space using the photoaccumulation behavior as the recorded parameter . The period of the wt+ was 29.6 h, of the s- 21.4 h and did not deviate significantly from ground controls performed exactly at the same time . The phase was delayed in space by 4.2 h in the wt+, but was not altered in the s- . In both strains the amplitudes were significantly higher in space than in the ground controls . During the recording period of 6.5 days the cell density increased in both strains . The survival rate, i.e . the ability to form colonies on agar petri dishes, was higher in space than on ground . The period was in both strains by 1.1 h longer in Florida (Kennedy Space Center) in both the flight and the control samples than in Europe . The significance of these results is discussed with respect to the endogenous nature of the biological clock and the role of the microgravity environment.

J Immunol Methods, 1987 Mar 12, 97(2), 221 - 7
The effects of adherent cells on measurement of the hyper-responsiveness of rheumatoid B lymphocytes to Epstein-Barr virus; Winrow VR et al.; Rheumatoid peripheral blood mononuclear cells show an increased responsiveness to superinfection with Epstein-Barr virus (EBV) . We have investigated the role of adherent cells in this hyperresponsiveness using two different methods of adherent cell depletion . Depletion of adherent cells from both rheumatoid and normal mononuclear cells, using either dextran bead columns or plastic petri dishes, produced inconsistent changes in the response of autologous non-adherent cells to EBV . The addition of supernatants of cultured rheumatoid adherent cells also produced an inconsistent change in response although normal adherent cell supernatants increased the responsiveness of autologous non-adherent cells to EBV . The inconsistencies observed are discussed with respect to adherent cell subpopulations present in rheumatoid and normal peripheral blood mononuclear cell preparations when using recognized methods of adherent cell depletion.

Cancer Lett, 1987 Feb, 34(2), 157 - 63
Direct inhibition of murine B lymphocyte differentiation by 12-O-tetradecanoylphorbol-13-acetate; Schuman LD et al.; The tumor-promoting agent, 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibits the humoral immune response of lymphocytes to antigen . To test the hypothesis that this inhibition is due to a direct effect upon B lymphocytes, splenic lymphocytes or murine B lymphocytes, enriched by 'panning' splenic lymphocytes onto anti-IgM-coated petri dishes, were immunized in vitro with the thymus/accessory cell-independent antigen trinitrophenyl lipopolysaccharide (TNP-LPS) with or without TPA . The number of anti-TNP antibody-forming cells present in both lymphocyte populations after 5 days was almost completely inhibited to the same degree by TPA . These data unambiguously show that TPA can directly inhibit the differentiation of B lymphocytes to antibody-forming cells.

J Oral Pathol, 1987 Feb, 16(2), 69 - 74
Alterations in lipid fluidity induced by cholesterol and cholesterol hemisuccinate modulate the organization of microtubule skeletons in epithelial cells; Sauk JJ et al.; This study was designed to evaluate the role that cholesterol, and the more hydrophilic ester, cholesterol hemisuccinate (CHS) have on the ability of epithelial cells to attach, spread and reorganize microtubule skeletons on a defined substratum . A431 carcinoma cells were grown and incubated with cholesterol, or CHS in polyvinyl pyrrolidone . Subsequently the cells were plated either on plastic petri dishes or plasticware coated with collagen IV or laminin . The alteration in apparent membrane microviscosity was ascertained using fluorescence polarization measurements . Organization of microtubules was determined by immunofluorescence, and by transmission electron microscopy . Cholesterol and CHS inhibited attachment and spreading of epithelial cells . Cells previously attached and spread became spherical after treatment with cholesterol and CHS, but microtubules were unaffected . However, when the cells were pretreated in suspension with cholesterol or CHS the membrane microviscosities markedly increased, and upon subsequent plating those cells adhering neither spread nor organized microtubule skeletons . These results suggest that cholesterol-induced changes in lipid microviscosity modulate the membrane dynamics that control the ability of epithelial cells to attach, spread and organize microtubule skeletons.

Eur J Immunol, 1987 Feb, 17(2), 173 - 8
Functional activity in vivo of effector T cell populations . III . Protection against Moloney murine sarcoma virus (M-MSV)-induced tumors in T cell deficient mice by the adoptive transfer of a M-MSV-specific cytolytic T lymphocyte clone; Cerundolo V et al.; The functional activity of Moloney murine sarcoma virus (M-MSV)-specific T lymphocytes in vivo was assayed by the i.v . injection of virus-specific T lymphocytes into T cell-deficient "B mice" . Virus-specific T lymphocytes generated in mixed lymphocyte tumor cell cultures were transferred i.v . into syngeneic "B mice" injected simultaneously at a distant site with the virus . These experiments indicated that a low dose (1 X 10(6) cultured cells) of infused lymphocytes can afford protection . To define the T lymphocyte subpopulation which was active, Lyt-2+ lymphocytes were selected by "panning" on plastic petri dishes coated with anti-Lyt-2 monoclonal antibody, and Lyt-2- lymphocytes selected by treatment with anti-Lyt-2 monoclonal antibody and complement . The results indicated that a Lyt-2+ lymphocyte-enriched population was more efficient in conferring protection against M-MSV-induced tumors . To investigate if cytolytic T lymphocytes (CTL) alone had a protective effect, a M-MSV-specific CTL clone was transferred in the same model system . The results demonstrated that a M-MSV-specific CTL clone prevented M-MSV-induced tumor growth and also induced the destruction of syngeneic Moloney murine leukemia virus (M-MuLV)-induced MBL-2 leukemic cells in the peritoneal cavity . However, the cell dose required to obtain protection using a CTL clone was higher than that which was effective when mixed lymphocyte tumor cell culture cells were used . To assess the ability of the transferred cells to home and to repopulate the lymphoid organs of the "B mice", the frequency of virus-specific CTL precursors in the spleen was evaluated by limiting dilution analysis . The results indicated that lymphocytes from mixed lymphocyte tumor cell cultures can be recovered from the spleens of "B mice" injected i.v . 25 days earlier . On the contrary, following the transfer of an active CTL clone, a very low frequency (less than 1/200,000 cells) of virus-specific CTL precursors was present in the spleens of recipient animals . The same M-MSV-specific CTL clone did not yield protection against M-MSV-induced tumors or MBL-2 leukemic cells when injected i.v . into M-MuLV tolerant mice.

Nippon Sanka Fujinka Gakkai Zasshi, 1987 Feb, 39(2), 271 - 8
{Establishment of primary culture of cells derived from uterine leiomyoma}; Otsuka H et al.; To evaluate the effects of drugs on uterine leiomyoma and to clarify the histogenesis of uterine leiomyoma, we studied the establishment of primary culture of cells derived from uterine leiomyoma . Myoma tissues were cut into small pieces and suspended in trypsin . The primary cell culture, as a monolayer, was able to be passaged four times . To confirm that the cultured cells were derived from the myoma, the cells were stained with desmin by the enzyme-labelled antibody method . The cultured cells derived from uterine muscle cells were similarly treated with desmin staining . The results confirmed the morphological similarity between the two groups of cells . To confirm that the cultured cells were myoblast, fibroblasts derived from the myoma were cultured selectively . The cells that resembled myoblasts, morphologically appeared spindle-shaped . The cells that resembled fibroblast, appeared polygonal and extended over the bottom of the culture flask . The growth curve of cultured myoma cells (8.0 X 10(4) cells in 0.2 ml) in Petri dishes (60 X 15 mm) revealed logarithmic proliferation after about 5 days . The colony formation of cultured myoma cells (2 X 10(5) cells in 0.2 ml) in culture flasks (25 cm2 in the area of the base) morphologically appeared to have an irregular border and had many independent scattered cells around the colony when the medium was renewed twice a week . The myoma cells (8.8 X 10(4) in 0.2 ml) in the logarithmic phase were cultured, fixed after 12 days when the medium was not renewed, and stained with crystal-violet . Morphologically, the colony had a comparatively regular border, was round, and each cell in the colony was epithelioid . The plating efficiency was 0.07%.(ABSTRACT TRUNCATED AT 250 WORDS)

Biol Cybern, 1987, 57(3), 187 - 95
On the recognition of order and disorder; Markus M et al.; We compare several algorithms for the recognition of ordered and disordered images . As image sources we use waves of the Belousov-Zhabotinskii reaction coupled to convective motion in a petri dish . This device allows reversibly the generation of periodic (ordered) and aperiodic (disordered) patterns . The best match between the parametric description and the observations is obtained by an "autodifference function" . This function is computed by summing up intensity differences over all pairs of picture elements having a given distance on the picture plane . Then, the minimum of this function is determined upon variation of the distance . This algorithm is not only efficient for the recognition of order and disorder in "machine vision", but also plausible in biological visual perception.

Basic Res Cardiol, 1987 Jan-Feb, 82(1), 74 - 81
Effects of lidoflazine and mioflazine against potassium and veratrine induced shape changes in isolated rat cardiac myocytes; Ver Donck L et al.; The protective effects of lidoflazine and mioflazine against shape changes in isolated rat cardiac myocytes induced by depolarizing concentrations of potassium and veratrine have been examined . Myocardial cells, isolated from adult rat hearts and plated in Petri-dishes, yielded a population of nearly 100% rod-shaped calcium-tolerant myocytes . Addition of veratrine or potassium resulted in a calcium-dependent cell shortening and finally rounding up of nearly all cells . Ultrastructurally, the shape changes were accompanied by a complete loss of calcium associated with the sarcolemmal and T-tubular bilayer . Calcium deposits accumulated in the mitochondria, indicating intracellular calcium overload . Pretreatment of the myocytes with lidoflazine or mioflazine (10(-7)-10(-5) M) dose-dependently increased the number of remaining rod-shaped cells after potassium or veratrine addition . Such rod-shaped cells retained a normal pattern of calcium distribution along the sarcolemma and T-tubuli with no evidence of mitochondrial calcium overload . The protective effects of lidoflazine and mioflazine are explained in terms of preserving sarcolemmal integrity, whereby excessive calcium influx and subsequent cytosolic calcium overload could be prevented.

Toxicol Appl Pharmacol, 1987 Jan, 87(1), 32 - 42
Suppression of in vitro antibody production by dimethylnitrosamine in mixed cultures of mouse primary hepatocytes and mouse splenocytes; Kim DH et al.; The suppression of in vitro antibody responses by dimethylnitrosamine (DMN) was produced in a mouse hepatocyte and splenocyte co-culture system . Mouse hepatocytes were isolated from female B6C3F1 mice and cultured for 20-24 hr to allow the formation of a monolayer on collagen-coated plastic petri dishes . Spleen cells were isolated from the same hybrid and were co-cultured with the hepatocytes along with DMN . Cyclophosphamide (CP), an immunosuppressive agent requiring metabolic activation that was included as an initial positive control, produced a marked suppression of the in vitro antibody responses to LPS, DNP-Ficoll, and SRBCs in 4 hr in the co-culture system . Under comparable conditions DMN markedly suppressed the response to SRBCs, marginally suppressed the response to DNP-Ficoll, and did not suppress the polyclonal response to LPS . The suppression by DMN was related to the rocking speed during the 4-hr co-culture period and was optimally produced when the cultures were not rocked . Addition of serum into the medium (10% fetal calf serum) during the co-culture period did not change the effects of DMN on the antibody response . However, the addition of extracellular DNA (1 mg calf thymus DNA/ml) prevented the suppression of the antibody response by DMN . These results suggest that DNA represents the primary macromolecular target for the reactive intermediate of DMN, and indicate that a syngeneic co-culture system can be used to characterize the in vitro immunosuppression

Dis Markers, 1986 Dec, 4(4), 271 - 82
A standardized assay to identify colon cancer genotypes by in vitro tetraploidy in human dermal fibroblasts; Danes BS et al.; Increased tetraploidy in dermal fibroblast cultures (IVT), which is a putative biomarker for genetic predisposition to colon cancer, was assayed by two different methods: by per cent of tetraploid metaphases in chromosome preparations from non-confluent monolayer petri dish cultures in logarithmic growth (MA); by flow cytometry (FCM) distribution of DNA content of propidium iodide stained cells from plastic flask cultures in stationary growth phase . The assayed cultures were derived from split thickness skin biopsies from two clinical groups: 11 normal individuals without a family history of colon cancer and 17 clinically affected individuals with autosomal dominant polyposis coli cancer syndrome (6 with colonic adenomatosis, i.e . familial polyposis coli (FPC) and 11 also having extracolonic lesions, i.e . the Gardner syndrome, (GS) . Concordance was observed between the two assays . There was a linear relationship between the per cent of tetraploid metaphases assayed in the chromosome preparations and the per cent of cells with a DNA index (DI) of precisely 2, or of all cells with a DI greater than 1 as determined by FCM . In comparisons between the clinical groups, there was significant differences in the FCM DNA index (p values less than 0.001) of IVT- (normals and IVT- FPC) and IVT+ individuals (IVT+ GS and those FPC with IVT) . The per cent of cells with a DI of 2 and greater than 1 could be used to distinguish all IVT- individuals (normals and IVT- FPC) from IVT+ FPC individuals . However, only by per cent of cells with a DI greater than 1 could all the individual GS affecteds be distinguished from IVT- individuals . Thus, the per cent of cells with a DI greater than 1 is considered to be the parameter of choice to be used in assaying IVT by flow cytometry.

Biochem Pharmacol, 1986 Oct 15, 35(20), 3571 - 81
The rate of uptake of cardiac glycosides into human cultured cells and the effects of chloroquine on it; Algharably N et al.; HeLa cells grown on Petri dishes were either pulse labelled with various cardiac glycosides or grown in low concentrations of them for up to 2 days; either in the presence of chloroquine or not . The cells were then homogenised and the cell free homogenate layered on a continuous sucrose gradient; and the glycoside content and that of various markers measured . In another series of experiments HeLa cells were grown on plastic beads under the above conditions and then the content of glycosides and of some marker enzymes measured . The rate of internalisation of ouabain, digoxin and digitoxin from the plasma membrane preparation produced by the bead method is at 9% hr-1, similar to the rate of loss of digoxin and digitoxin from whole cells but much faster than that of ouabain . In the sucrose gradient experiments it was found that {3H}ouabain, digoxin and digitoxin all initially co-distribute with the plasma membrane marker, 5'-nucleotidase, and then leave this fraction of the homogenate at a fast rate when kept at 37 degrees, to co-distribute with the lysosomal marker, beta-hexosaminidase . At 2 degrees the ouabain remains co-distributed with the plasma membrane marker . The rate of transfer is estimated to be some 90% hr-1, much faster than previously thought . Chloroquine causes an increased retention of digoxin and digitoxin in the lysosomal fraction of the homogenate . These results are best explained by supposing that the sodium pump-glycoside complex rapidly enters a region of the peripheral cytoplasm, and that this region then controls the subsequent exit of digoxin and digitoxin from the cell . The main barrier for ouabain occurs at a stage later than this . The consequences of this model on other aspects of pump activity is discussed.

Brain Res, 1986 Oct 1, 384(1), 84 - 93
Cryopreservation of primary neurons for tissue culture; Kawamoto JC et al.; We have developed new cryopreservation methods which allow storage of fetal rat central nervous system tissues for more than 1 week at 3-8 degrees C or for several months at -70 or -90 degrees C prior to tissue culture . For refrigeration, small brain regions (less than 2 mm thick) were placed intact into 35 mm petri dishes of 'hibernation medium' inside a humidified chamber . Optimal preservation was obtained with hibernation media of pH 6.8-7.4, containing 30-70 mM K+, 10-30 mM Na+, 5-50 mM PO4(2-), 20 mM lactic acid, 5 mM glucose, and less than 0.1 mM Ca2+ . The media were made approximately isotonic by addition of sorbitol . For freezing, brain tissues were dissociated by gentle trituration (without enzymes) in the above medium supplemented with 5-10% dimethylsulfoxide . After refrigeration or freezing, neurons were very sensitive to damage from mechanical stress (e.g . centrifugation, harsh rinsing or trituration) . Rapid changes in osmotic pressure or excessive polylysine on the tissue culture substratum also reduced neuronal survival after cryopreservation . Pretreatment of tissue culture substrata with media from lung cell cultures, or plating of neurons at higher density (2000 cells/mm2) improved neuronal survival after cryopreservation.

J Clin Microbiol, 1986 Sep, 24(3), 460 - 1
Modified method for fungal slide culture; Harris JL; A modified slide culture method which combines advantages found in several slide culture methods is described . A block of inoculated nutrient agar sandwiched between two sterile cover glasses is placed in a plastic petri dish containing water agar . After adequate growth has occurred, the slide culture is disassembled and mounted in a conventional manner.

Appl Environ Microbiol, 1986 Sep, 52(3), 430 - 3
Practical direct plaque assay for coliphages in 100-ml samples of drinking water; Grabow WO et al.; A practical single-agar-layer plaque assay for the direct detection of coliphages in 100-ml samples of water was designed and evaluated . With this assay a 100-ml sample of water, an agar medium containing divalent cations, and the host Escherichia coli C (ATCC 13706) were mixed in a single container, and the mixture was plated on 10 14-cm-diameter petri dishes . It was more sensitive, reliable, and accurate than various other methods and proved rapid, simple, and economic.

J Am Mosq Control Assoc, 1986 Sep, 2(3), 350 - 4
Factors affecting storage of mycelial cultures of the mosquito fungal pathogen Lagenidium giganteum (Oomycetes: Lagenidiales); Su XQ et al.; Sunflower seed extract (SFE) agar cultures (in petri dishes) of Lagenidium giganteum (California isolate) were evaluated for zoospore production and ability to infect mosquito larvae, Culex quinquefasciatus, after periods of storage up to 93 days at 15 degrees C . Rates of decrease in zoospore production and infectivity were related to soluble protein concentration in the SFE-agar media but at all concentrations (0.7-6.0 mg/ml) about 50% of the initial levels were lost after 40-50 days of storage . Water loss from the SFE-agar did not affect zoospore production or infectivity except at extremely high levels (about 98% water loss).

Pharmazie, 1986 Aug, 41(8), 575 - 8
Film-coated zinc sulphate tablets and the effect of humidity on tablet properties; Gursoy A et al.; Preparation of film-coated zinc sulphate tablets and the effect of relative humidity and aging on the physical properties of these coated tablets were investigated in this study . Shellac; Eudragit E and L, hydroxypropyl methylcellulose (HPMC) and HPMC-Eudragit E mixture were used as film-forming materials . As zinc sulphate has efflorescent properties, the effect of humidity on the coated tablets was studied and physical stability tests were carried out . Coated tablets were kept in bottles and in open petri-dishes at different relative humidities (30, 55 and 85%) at room temperature over 12 weeks, and the weight loss, hardness, and disintegration times of these tablets were followed . Findings indicated that film-coated zinc sulphate tablets having good tablet quality can be prepared using the formulations (F-VI) HPMC-Eudragit E mixture and (F-III) Eudragit E as coating materials . Thus, the effect of relative humidity on tablet properties can be reduced.

Cancer Res, 1986 Aug, 46(8), 4012 - 7
Improved plating efficiencies for human tumors cloned in capillary tubes versus Petri dishes; Von Hoff DD et al.; As now constituted, the human tumor cloning assay performed in Petri dishes has several limitations including: (a) not all patients' tumors form colonies in the assay; (b) the plating efficiencies (number of colonies formed/number of cells plated) are low; and (c) a large number of tumor cells are required to perform drug sensitivity testing . In this study the use of capillary tubes, as vessels in which to clone human tumors, is compared to the use of 35-mm Petri dishes . In 100-microliters capillary tubes the optimal plating efficiencies are found with 50,000 cells/vessel (500,000 cells/ml), while in 35-mm Petri dishes the optimal plating efficiencies are found with 500,000 cells/vessel (250,000 cells/ml) . In head to head comparisons of plating efficiencies of 183 human tumors (18 different histological types), the median plating efficiency was 5-fold higher (range, 1.16-37.00) for the capillary tubes than for the Petri dishes . This improved plating efficiency was noted for nearly all of the histological tumor types examined . The improved plating efficiencies noted with the capillary system indicate that the Petri dish method may be too selective and not reflect the total number of clonogenic units in a human tumor . In addition, the higher plating efficiencies noted with the capillary system may be exploited to solve some of the problems noted with the conventional Petri dish method.

Zhonghua Min Guo Wei Sheng Wu Ji Mian Yi Xue Za Zhi, 1986 Aug, 19(3), 177 - 82
{Natural killer cell activity in patients with nasopharyngeal carcinoma}; Lynn TC et al.; In 1984 and 1985, a total of 30 patients with biopsy confirmed nasopharyngeal carcinoma (NPC) were randomly selected for studies on natural killer cell (NK) activity . Control study on 30 normal subjects were also done . Lymphocyte separation was done by centrifuge of heparinized peripheral blood with Ficoll-Hypaque and monocytes were removed by Petri dish adhesion . K562 cells were used as target cells of NK activity . The lymphocyte-target reaction of 4 hours was done and the ratio was 50:1, 25:1 and 12.5:1 . The results of 50:1 was taken as the NK activity . The mean value of NK activity in NPC patients was 34.8 +/- 22.7%, this is significantly different (t = 2.90, p less than 0.01) from 51.6 +/- 22.1% of the controls . If NK activity of 20% or more are taken for the normal standard, then the positive rate in NPC patients was 63.3% and that in the controls was 90.0% . The difference is significant, as chi 2 = 4.56, p less than 0.05 . The NK activity in NPC patients is not correlated with the sex and age of the patients, the disease extent and the EB virus associated antibody titers.

Zh Mikrobiol Epidemiol Immunobiol, 1986 Aug, (8), 3 - 5
{Cloning of the genes of hemolytic factors of Bordetella pertussis in Escherichia coli}; Rozinov MN et al.; The gene library of B . pertussis strain No . 475 (serovar 1 . 2 . 3.) was obtained on plasmid pUC19 in E . coli strain JM107 . The average size of the cloned fragments of chromosomal DNA was 4.9 Kb . Out of 1,700 tested recombinant clones, six caused visible hemolysis on petri dishes with agar containing 4% of sheep red blood cells after 16-hour incubation at 37 degrees C . Only two out of four isolated plasmids were similar in Pst I restriction . Consequently, the existence of several different genes responsible for the hemolytic factors of B . pertussis may be assumed . None of the cloned fragments had Hind III sites . Clones harboring hybrid plasmids possessed different modes of hemolysis, which may be indicative of gene expression in E . coli.

Int J Fertil, 1986 Jul-Aug, 31(3), 227 - 8
Hyaluronidase activity in split human semen; Savion M et al.; Hyaluronidase is an acrosomal enzyme which participates in the dissolution of the cumulus oophorus matrix, containing hyaluronic acid, and is essential for the fertilization process . In this study hyaluronidase activity was determined in two fractions of split normozoospermic and oligozoospermic human semen . The method consisted of measurements of areas of digestion of hyaluronic acid in agar (Petri dishes) as compared to activity obtained by commercial testicular hyaluronidase . It was found that the first splits of semen, which are generally characterized by higher sperm density and better quality of sperm, exhibit higher enzyme activity (136.0 +/- 11.6-207.0 +/- 15.1 micrograms/mL) as compared to the second splits (117.8 +/- 10-134.0 +/- 12.9 micrograms/mL).

Tumori, 1986 Jun 30, 72(3), 225 - 9
Motility of splenic cells from normal and fibrosarcoma-bearing mice in agarose assay; Aroskar VS et al.; When nonadherent splenic cells from normal and tumor-bearing (mouse fibrosarcoma, MFS) Swiss mice were added to wells made in agarose layers in plastic petri dishes, subpopulations of cells from tumor-bearing mice were seen to migrate out of the wells, whereas those from normal mice did not . The proportion of migratory cells among the lower density (less than 1.057 to less than 1.069 g/ml) cells was larger than that of higher density (1.069 to less than 1.087 g/ml) cells . When the plastic surface underneath the agarose layer was covered with a monolayer of MFS cells, the splenic cells from normal mice also migrated out of the wells . About 20% dead MFS cells were observed in the zone of migration when the migratory cells were from normal mice, and about 30% when the migratory cells were from tumor bearing mice . Apart from revealing the differences between the migratory behavior of splenic cells, the present work also suggests a novel application of agarose methodology in the study of interaction of cytotoxic cells with malignant cells.

Biochem Pharmacol, 1986 Jun 1, 35(11), 1801 - 4
Effect of doxycycline on oxygen-dependent killing mechanisms of human neutrophils; Sinico-Durieux I et al.; The effects of doxycycline on neutrophil adhesivity, ingestion rate, and oxidative burst by particle and soluble compounds have been analyzed . The rate of bacterial ingestion by neutrophils as well as its subsequently particle-induced oxidative burst comprising oxygen uptake, hydrogen peroxide and superoxide anion productions, and iodination were all inversely correlated to doxycycline concentration included in the assay medium . The neutrophil oxidative burst induced by phorbol myristate (a soluble stimulant) was also inversely correlated to doxycycline concentration . Drug effect was observed at lower concentrations when the neutrophil stimulant was a soluble compound than when it was particles . In contrast doxycycline did not affect neutrophil adhesivity to either nylon fibers or Petri dishes . Further studies are needed to assess whether the activity of the drug on the neutrophil is due only to its ability to chelate calcium and magnesium or to other properties.

Toxicol Appl Pharmacol, 1986 May, 83(3), 420 - 9
Immunosuppression induced by chemicals requiring metabolic activation in mixed cultures of rat hepatocytes and murine splenocytes; Yang KH et al.; Primary cultures of adult rat hepatocytes (Fischer 344) were used as an in vitro metabolic activation system in immunotoxicological assays . Rat hepatocytes were isolated by a collagenase perfusion technique and cultured for 20 to 24 hr to allow the formation of a monolayer on collagen-coated plastic petri dishes . Spleen cells isolated from (C57BL/6 X C3H)F1 mice were cocultured with the hepatocytes along with the chemicals . Cyclophosphamide (CP) and Aflatoxin B1 (AFB1) were effectively activated in this coculture system and produced a dose-related suppression of the in vitro antibody responses to LPS, DNP-Ficoll, and SRBC in 3 hr . Neither CP (1 mM) nor AFB1 (10(-4) M) cultured with spleen cells alone produced any effects . Both CP and AFB1 also produced a dose-related suppression of the proliferative responses to LPS, Con A, and PHA . In contrast, up to 100 mM of N-nitrosodimethylamine (DMN) did not suppress any of these assays after a 3-hr incubation in the coculture system . These results indicate that a coculture system can be used to characterize the activity of immunosuppressive chemicals requiring metabolic activation.

Endocrinology, 1986 May, 118(5), 2120 - 4
Subpopulations of lactotropes detected with the reverse hemolytic plaque assay show differential responsiveness to dopamine; Luque EH et al.; Cultured adenohypophysial cells secreting PRL were detected with a reverse hemolytic plaque assay . In this assay, PRL secretion from a pituitary cell results in hemolysis of cocultured protein A-coupled ovine erythrocytes in the presence of PRL antiserum and complement, so that a zone of hemolysis (a plaque) surrounds each lactotrope . The extent of hemolysis was related to the amount of PRL secreted by each lactotrope: batches of cohort cells incubated under similar conditions either in petri dishes for measurement of PRL secretion by RIA or in Cunningham chambers for measurement of plaque area revealed a significant relationship between secreted PRL and plaque area (r = 0.97; regression coefficient = 0.0007 pg/micron2) . Measurement of plaque area on lactotropes derived from proestrous rats revealed a bimodal frequency distribution that was composed of cells forming small plaques (35% of the total lactotrope population) and others forming large plaques (65%) . Treatment with 10(-7) M dopamine appeared to preferentially inhibit the large plaques; they decreased to 42% of the total with corresponding increases in the number of small plaques, but the total number of secretory lactotropes did not change . At 10(-5) M dopamine, large plaques virtually disappeared (only 9% remained), and small plaques appeared in increased numbers, but the number of secretory lactotropes decreased by about one third . These results suggest that the reverse hemolytic plaque assay can be used to quantify PRL secretion by individual lactotropes, that lactotropes from proestrous rats exist as two secretory subpopulations, and that dopamine may preferentially suppress the subpopulation secreting large amounts of PRL.

Clin Exp Immunol, 1986 Apr, 64(1), 214 - 22
Novel neutrophil chemotactic factor derived from human peripheral blood mononuclear leucocytes; Kownatzki E et al.; Human mononuclear leucocytes isolated from the peripheral blood by centrifugation on Ficoll-Hypaque cushions and adherent on plastic petri dishes, produced a chemotactic factor that attracted human neutrophilic granulocytes to the same extent as did optimal concentrations of the complement split product C5a and the leukotriene B4 . The active component eluted from a Sephadex G-50 gel filtration column as a single peak with an apparent molecular weight of 10,000 . The chemotactic activity was resistant to reductive cleavage of disulfide bonds and heating at 100 degrees C for 30 min but was lost when reduction and heating were combined . Digestion with a proteolytic enzyme eliminated the attractive potential . The data suggest that this is a novel chemotactic peptide . It is conceivable that it has been seen previously and was mistaken for a lymphokine or interleukin 1.

J Anat, 1986 Apr, 145, 1 - 12
Phase contrast and electron microscopical observations of adult mouse dorsal root ganglion cells maintained in primary culture; Smith RA et al.; Cultures of neurons and non-neuronal cells were prepared from adult mouse dorsal root ganglia and maintained for periods of up to six weeks upon uncoated petri dish substrates . Modifications were made to both existing isolation procedures and culture medium to ensure high cell survival . Cell structure was observed using phase contrast microscopy for living cells and standard light microscopy for Palmgren silver stained preparations . Scanning and transmission electron microscopy revealed the fine structure of the cells and showed the close relationship of satellite cells with the surviving dark neurons as culture establishment progressed . Satellite cells were associated with both the cell bodies and the regenerated neuritic processes . The use of the cultures for future cytotoxicity testing is assessed.

Acta Med Okayama, 1986 Apr, 40(2), 113 - 9
Suppression of natural killer cell activity by surgical stress in cancer patients and the underlying mechanisms; Yoshihara H et al.; The influence of surgical stress on the natural killer (NK) activity of peripheral blood lymphocytes in patients with carcinoma of the lung or gastrointestinal system was studied . The peripheral blood lymphocytes of the patients showed a marked decrease in NK activity against K-562 cells as target cells 1-2 days after surgery . The activity remained lowered for 2 weeks after thoractomy and for 1 week after laparotomy . No appreciable suppression of NK activity was observed with normal human peripheral blood lymphocytes preincubated with postoperative patient sera . Peripheral blood mononuclear cells obtained postoperatively from patients lost NK activity after ultraviolet irradiation, without any detectable loss of viability . Such irradiated mononuclear cells showed inhibition of NK activity after a 24-hour preincubation with peripheral blood lymphocytes from normal subjects . Similar suppressive activity was demonstrable in a fraction of mononuclear cells with adhesiveness to plastic petri dishes, while non-adherent cells had no such activity . When added immediately to the cytotoxicity assay system without the 24-hour preincubation, patient mononuclear cells caused no inhibition of NK activity, whereas adherent cells from normal subjects enhanced NK activity . The findings seems to indicate that, following surgical stress, plastic dish-adherent peripheral blood mononuclear cells become deprived of NK helper activity and exert suppression, thus causing postoperative depression of NK activity.

Allergol Immunopathol (Madr), 1986 Mar-Apr, 14(2), 101 - 6
{Fungi inside and outside the homes of subjects with immediate hypersensitivity in Zaragosa (Spain)}; Duce Gracia F et al.; In the study of patients with hypersensitivity to atmospheric fungi, the determination of species distribution in the personal environment of the patient is important . This distribution depends on climate variations, especially temperature and percent (%) humidity which favor the development of one or other genera . In the domiciliary surroundings, the technological advances in comfort (wetness, dryness, with refrigeration and heating systems) favor the establishment of a closed ecosystem which can sometimes contain fungi different from that found exteriorly; at times true colonization . Our objective is to compare the incidence of the different groups of intra and extra-domiciliary fungi in our environment (Zaragoza, Spain) and to verify their presence in the domiciles with their exterior surroundings, of 23 atopic patients, of which 8 had clinical asthma and/or rhinitis and demonstrable allergy to fungi, by PRICK and RAST . The other 15 patients were diagnosed as having asthma and/or rhinitis with a positive PRICK and RAST to house dust and Dermatophagoides or pollen allergens (non-allergic to fungi) . Five Petri dishes were given to each patient; one dish contained 5 mm . of Agar-Sabourad following Lumpkins' formula, and was used for extra-domiciliary exposure . The rest contained the same medium to which was added a 0.33% solution of Rose Bengal (an inhibitor) for internal exposure . This medium allows the development of a large number of genera of fungi . These 5 dishes were situated and remained in place for 60 minutes; the 4 interior ones were placed in different locations and always included the dormitory and living room.(ABSTRACT TRUNCATED AT 250 WORDS)

Exp Cell Res, 1986 Feb, 162(2), 401 - 10
Behavior of sea urchin primary mesenchyme cells in artificial extracellular matrices; Katow H; The primary mesenchyme cells (PMCs) were separated from the mesenchyme blastulae of Pseudocentrotus depressus using differential adhesiveness of these cells to plastic Petri dishes . These cells were incubated in various artificial extracellular matrices (ECMs) including horse serum plasma fibronectin, mouse EHS sarcoma laminin, mouse EHS sarcoma type IV collagen, and porcine skin dermatan sulfate . The cell behavior was monitored by a time-lapse videomicrograph and analysed with a microcomputer . The ultrastructure of the artificial ECM was examined by transmission electron microscopy (TEM), while the ultrastructure of the PMCs was examined by scanning electron microscopy (SEM) . The PMCs did not migrate in type IV collagen gel, laminin or dermatan sulfate matrix either with or without collagen gel, whereas PMCs in the matrix which was composed of fibronectin and collagen gel migrated considerably . However, the most active and extensive PMC migration was seen in the matrix which contained dermatan sulfate in addition to fibronectin and collagen gel . This PMC migration involved an increase not only of migration speed but also of proportion of migration-promoted cells . These results support the hypothesis that the mechanism of PMC migration involves fibronectin, collagen and sulfated proteoglycans which contain dermatan sulfate.

Liver, 1986 Feb, 6(1), 45 - 52
Characterization of effector cells in lymphocytotoxicity to autologous hepatocytes in HBsAg-positive and autoimmune chronic active hepatitis (CAH); Barnaba V et al.; Peripheral blood lymphocyte subsets involved in cytotoxicity to autologous hepatocytes have been characterized by isolation on antibody-coated Petri dishes in autoimmune and HBsAg-positive chronic active hepatitis (CAH) . In autoimmune CAH and in HBsAg-positive CAH without HBcAg in liver tissue, cytotoxicity is sustained by non-T lymphocytes and is confined to M1-positive cells bearing Fc receptors: M1 cytotoxicity inhibition by adding aggregated IgG suggests that these cells are responsible for an antibody-dependent cell-mediated mechanism (ADCC) . Moreover, when T-enriched fractions were separated in T4, T8 and 5/9 positive subsets, only the first one showed a significant cytotoxicity: T4 positive cells might act as cytotoxic T cells or might be involved in delayed type hypersensitivity (DTH) reactions . Cytotoxic T lymphocytes in HBsAg-positive CAH with HBcAg in liver tissue are confined in T8 positive subset, while helper/inducer T cells (T4 positive or 5/9 positive) seem to play an important role only in the induction of cell-mediated injury against hepatocytes . The inhibition of T cell-cytotoxicity by preincubating liver cells with monoclonal antibody (Mab) anti-HLA AB and not with Mab anti-HLA DR or aggregated IgG supports the involvement of the class I major histocompatibility complex (MHC) expressed on the hepatocyte surface.

Bioelectromagnetics, 1986, 7(4), 359 - 67
Microwave effects on isolated chick embryo hearts; Caddemi A et al.; This study was designed to examine the effects of microwaves on the electric activity of hearts as a means of elucidating interactive mechanisms of nonionizing radiation with cardiac tissue . Experiments were performed on isolated hearts of 9-12-day-old chick embryos placed in small petri dishes . Oxygenated isotonic Ringer's solution at 37 degrees C permitted heart survival . Samples were irradiated at 2.45 GHz with a power density of 3 mW/cm2 . The heart signal was detected with a glass micropipet inserted into the sinoatrial node and examined by means of a Berg-Fourier analyzer . Pulsed microwaves caused the locking of the heartbeat to the modulation frequency, whereas continuous wave irradiation might have induced slight bradycardia . Pulsed fields induced stimulation or regularization of the heartbeat in arrhythmia, fibrillation, or arrest of the heart.

Exp Brain Res, 1986, 63(2), 321 - 30
Influence of meningeal cells on the proliferation and maturation of rat neuroblasts in culture; Gensburger C et al.; Neuronal cells were obtained by dissociating cells from the cerebral hemispheres of rat embryos (10 to 17-day-old), either cleaned entirely or only partially of their meningeal membranes . These cells were seeded on poly-lysine-coated Petri dishes in serum-containing medium . The cultures most enriched in neuronal cells were obtained from brains of 13- to 15-day-old embryos and after 2 h, the culture medium was switched to Dulbecco's modified Eagle's medium, without serum, supplemented with the N1 supplements as described by Bottenstein et al . (1980) . The proliferation of neuroblasts from 13-day-old embryos in the presence or absence of meningeal cells was studied by using a combination of tritiated thymidine autoradiography and immuno-staining against neurofilament proteins . The neuroblasts seem to proliferate during the first 3 days . The proliferative activity was further enhanced in the presence of meningeal cells . The glioblasts multiply only after a period of one week in culture conditions as observed here . The subsequent development of the neuroblasts was followed over a period of 4 weeks and the ultrastructural appearance of these cells was investigated at 2 and 3 weeks . In the presence of meningeal cells, many neurons, intensely stained for neurofilament proteins, survived for 21 days, while in control cultures they underwent massive degeneration after 2 weeks . Synapses with numerous clear vesicles were abundant in cultures grown under the influence of meningeal cells; they were rare and possessed few vesicles in control cultures . The data indicate that meningeal cells affect the proliferation and maturation of rat neuroblasts in culture.

J Steroid Biochem, 1986 Jan, 24(1), 273 - 9
Steroid hormones induce cell proliferation and specific protein synthesis in primary chick oviduct cultures; Jung-Testas I et al.; A rapid method to obtain large amounts of tubular gland cells from chick oviduct was developed . Combined collagenase and trypsin treatment allowed within 1.5 h complete dissociation of the magnum portion of the oviduct . By differential attachment of cells, fibroblasts were separated from tubular gland- and ciliated cells . Tubular gland cells attached within 18 h to plastic Petri dishes, had large secretory granules and grew very actively . The responsiveness of cells to hormones and/or antihormone was tested by measurement of cell proliferation and specific protein synthesis . After 7 days of culture in the presence of estradiol (50 nM) or progesterone (100 nM), cell growth was increased by approximately 50 and 35% respectively . Tamoxifen (100 nM) inhibited the estradiol induced growth stimulation, but had also negative effects of its own . The anti-progesterone (in mammals) RU 486, inactive per se, did not antagonize progesterone induced growth . Ovalbumin- and conalbumin synthesis after 4-5 days of cultures under different hormonal conditions was assessed after immunoprecipitation of newly synthesized {35S}methionine labelled proteins . In the presence of estradiol (50 and 100 nM), progesterone (50 nM), and both estradiol and progesterone together (50 nM of each), ovalbumin and conalbumin synthesis was increased, when compared to control cultures without hormones, or to oviduct fibroblasts . Hormonal stimulation of ovalbumin synthesis was also shown in cell supernatant and culture medium after gel electrophoresis.

Invest New Drugs, 1986, 4(4), 367 - 71
Sodium azide is less suitable as a positive control of drug-induced lethality for in vitro clonogenic assays; Lelieveld P et al.; Sodium azide (6 mg/ml) was used as a positive control for drug-induced lethality in an in vitro clonogenic assay . Petri dishes containing control and sodium azide treated cultures of WiDr cells were placed together in a large Petri dish and incubated at 37 degrees C in an atmosphere of 10% CO2 in air . No growth was observed . Control cells formed colonies only when the dishes were separated from the sodium azide dishes . Using a microtiter plate the toxic effect was inversely related to the distance of the test cultures from the sodium azide treated cultures . These results suggested the formation of a toxic gas or vapour from sodium azide under cell culture conditions, probably an azide . Chemical analysis was based on characteristic reactions, such as the production of a precipitate with silver ions or formation of a red-coloured complex with ferric salts . On a microtiter plate, a gradient of the expected precipitate or red colour was observed, the highest amounts adjacent to the wells containing sodium azide . These results show that sodium azide acts as a positive control of drug-induced lethality for in vitro clonogenic assays . However, the formation of a highly toxic vapour, most likely hydrazaic acid, makes it a less suitable standard.

Ann N Y Acad Sci, 1986, 485, 349 - 68
Thrombin stimulates neutrophil adherence by an endothelial cell-dependent mechanism: characterization of the response and relationship to platelet-activating factor synthesis; Zimmerman GA et al.; Thrombin, a serine coagulation protease that is generated at sites of tissue injury and inflammation, stimulates the adherence of PMNs and neutrophils to EC . We found that thrombin enhanced the adhesion of neutrophils to primary monolayers of human umbilical vein EC when assayed by the binding of 111Indium-labeled PMNs to the EC, the recovery of unlabeled PMNs after incubation with thrombin-treated EC, and by phase contrast and scanning electron microscopy (SEM) . SEM demonstrated that thrombin caused PMNs to intimately adhere to the EC plasma membrane and under some conditions to become polarized . The thrombin-stimulated adherence was a rapid, time-dependent response with an onset within 1 minute of addition of thrombin, a peak at 5-10 minutes, and a decline thereafter . The response was concentration-dependent over the range 0.01-2 U/ml thrombin, and required active thrombin . Prothrombin, factor Xa, and fibrinogen were not effective . Thrombin-stimulated PMN adherence was dependent on the EC, because thrombin did not significantly stimulate neutrophils to adhere to albumin-coated petri dishes, subendothelial matrices, or primary cultures of smooth muscle cells . Human EC, when treated with thrombin, also produce platelet-activating factor (PAF; 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) . The concentration-response relationships and time courses for thrombin-stimulated PMN adherence and PAF production were tightly correlated . Furthermore, PAF itself stimulated the adherence of PMNs to EC, and pretreatment of PMNs with PAF selectively inhibited their adherence response to thrombin . These findings demonstrate two novel biologic activities of thrombin, the stimulation of EC-dependent adherence of PMNs and the production of PAF by EC, and suggest that they are functionally related . In addition, they suggest that thrombin may act as a plasma-derived humoral mediator of inflammation under some conditions.

Ann N Y Acad Sci, 1986, 486, 206 - 26
Growth-promoting factors in neurofibroma crude extracts; Riccardi VM; Crude extracts of neurofibromas from two unrelated neurofibromatosis (NF) patients were prepared by mincing, homogenizing, and ultracentrifugation in the absence of added solvents . Explant cultures of neurofibromas from other NF patients were grown at low density in culture medium with and without neurofibroma extract supplementation . Differences in growth were monitored by comparing monolayer densities, colony counts, or uptake of {3}H-thymidine . A consistent enhancement of growth rate was demonstrated, and titration curves showed an increasing effect with increasing dosage (ranging from 1.5 microliter/ml to 25 microliters/ml) . However, the extract could not substitute for fetal bovine serum . As determined by microscopic examination of Giemsa-stained petri dishes, small spindle-shaped cells, distinct in morphology from ordinary fibroblasts, were the overwhelmingly predominant cell type in most extract-treated cultures . While the specific identity of the growth factor(s) involved is unknown, the following may be stated: The presence of one or more growth factors that may act in an autocrine or paracrine manner in neurofibromas in vivo is demonstrated . There is a preferential effect of such a factor on spindle-shaped cells (presumably Schwann cells), allowing for the selective enrichment of these cells in vitro . There is an enhanced yield of clones derived from single cells, allowing further analysis of the cellular heterogeneity of neurofibromas at the biochemical and molecular levels . These considerations should help to distinguish between those models for neurofibroma growth that emphasize secondary somatic mutations (including allelic exclusion) on the one hand, and cellular interaction on the other hand.

Biol Cell, 1986, 58(3), 251 - 61
{In vitro development of blastema cells in axolotl limb regeneration: effect of insulin and nerve extracts on cellular proliferation}; Albert P et al.; For the purpose of investigating the nature of the nervous factor which controls cell proliferation in limb blastema of Newts, we have cultured primary mesenchymous cells from limb blastemas of Axolotl . The cultures were carried out in Petri dishes (Primaria, Falcon) with a basal medium with contained diluted MEM supplemented with hormones (insulin, somatotropin, hydrocortisone and thyroxine) . In this medium, the cells disperse from the explant from the 4th day of culture and begin to divide from the 7th day; 3 weeks later the culture begins to decline . During the course of culture, beginning at the 8th day, differentiation of myotubes and chondrogenesis occur . The mitotic index, measured on the 16th day after 48 hr of colchicine treatment, is about 1.6% . Addition of foetal calf serum to the basal medium favours cell migration and survival and stimulates proliferation (mitotic: index 6%); beef embryo extract has no effect on cell migration and a small effect on proliferation (mitotic index: 2.3%) . Addition to the basal medium of insulin or nerve extracts (brain and spinal cord of adult newts, brain of 12 days chick embryos) 6 days before we measure the mitotic index stimulates proliferation in proportion to the dose, up to 6 times the mitotic index in basal medium . These results are discussed with respect to the problem of cell proliferation control during limb regeneration.

Scand J Immunol, 1986 Jan, 23(1), 25 - 33
Concanavalin A induction of suppressor activity in the T-helper subset defined by monoclonal antibodies; Davidsen B et al.; Human monocyte-depleted peripheral blood mononuclear cells were separated in T4+ and T8+ populations by a panning technique . Petri dishes coated with goat anti-mouse antibodies were plated by peripheral blood mononuclear cells coated by monoclonal antibodies, either T4 or T8 . The cell populations were separated into adherent and non-adherent populations based on binding to the goat anti-mouse-coated plastic dishes . The purity of the adherent populations was 96% . T4+ and T8+ populations were used as effector cells in the concanavalin A-induced suppressor test . The T4+ population revealed a pronounced suppressor activity similar to that exhibited by the T8+ population . This finding was independent of two different sources of monoclonal antibodies, T4/T8 and OKT4/OKT8 . The registered suppressor activity in the monoclonal antibody-defined helper population could not be explained either by a switch of the membrane phenotype from T4+ to T8+ cells or by an increased interleukin 2 consumption of the concanavalin A-treated cells.

Int Arch Allergy Appl Immunol, 1986, 80(4), 395 - 400
Characterization of an idiotype-binding helper cell required for the formation of timothy grass pollen antigen-B-specific IgE; Malley A; Experiments were conducted using an excess of biosynthetically labeled timothy grass pollen antigen-B-specific (AgB-specific) T helper factor (THF) with enriched T cells, enriched B cells, or normal spleen cells to determine the site of the THF action . These studies indicated that the labeled THF bound preferentially to T cells, and analysis of the cell surface characteristics suggested that these T cells were Lyt 123+ cells . A panning technique using partially purified AgB-specific THF-coated Petri dishes depleted a population of idiotype-binding T cells that appears to be required for the formation of AgB-specific IgE antibody.

Exp Cell Res, 1985 Dec, 161(2), 473 - 83
Fibronectin-independent attachment of human gingival fibroblasts to interstitial and basement membrane collagens; Farsi JM et al.; We have studied the ability of human gingival fibroblasts (HGF) to attach to different interstitial (types I, II and III) and basement membrane (types IV and V) collagens . HGF cells were plated onto collagen-coated Petri dishes under various conditions and the percentage of cells attaching to the collagen was determined . HGF were found to attach to all the different types of native collagens, but attached poorly to the corresponding denatured collagens . When plated in the presence of 15% fetal bovine serum (FBS) or fibronectin-depleted FBS, similar percentages (approximately 85%) of cells attached to both interstitial and basement membrane collagens, demonstrating an attachment mechanism that is independent of plasma fibronectin . That the attachment in the presence of serum was also independent of cellular fibronectin was shown by the inability of fibronectin antibodies to block attachment to any of the collagen types . HGF were also capable of attaching to all of the collagen types in the complete absence of serum . In previous studies, investigators using cell lines have suggested that cell attachment in the absence of serum is non-physiological . However, the serum-free attachment of HGF to collagen was found to be dependent on cellular protein synthesis indicating that this attachment mechanism has biological significance.

Arch Otolaryngol, 1985 Dec, 111(12), 820 - 1
Effects of magnetic resonance imaging fields on stapedectomy prostheses; Applebaum EL et al.; Seven different types of widely used metallic stapedectomy prostheses were individually placed on a millimeter scale in a plastic Petri dish . The Petri dish was moved within the core and around the magnet of a 1.5-tesla magnetic resonance imaging unit (General Electric Sigma) . No movement of any of the prostheses was seen . We conclude that there is no apparent danger of these prostheses becoming displaced in stapedectomy patients subjected to the electromagnetic fields of magnetic resonance imaging units.

J Exp Med, 1985 Dec 1, 162(6), 1852 - 61
Physiology of IgD . VI . Transfer of the immunoaugmenting effect of IgD with T delta-containing helper cell populations; Coico RF et al.; We show that the IgD-induced augmentation of the immune response to trinitrophenylated keyhole limpet hemocyanin can be transferred to syngeneic mice with spleen cells from IgD-injected donors . The augmenting activity is present in the Lyt-1+2-, L3T4+ T cell population and is absent from B cells . The ability of transferred T cells to augment the immune response correlates with the presence of a high frequency of Lyt-1+2- T cells that form rosettes with IgD-coated sheep erythrocytes (T delta cells) . Such rosette-forming cells can also be induced by incubation of spleen cells from normal donors in IgD-coated petri dishes . Injection of normal spleen cells exposed to IgD-coated petri dishes together with antigen also augments the immune response of recipients . The existence of a regulatory circuit based upon interactions between T delta cells, antigen, B cell surface IgD, and serum IgD, is proposed.

Cancer Res, 1985 Nov, 45(11 Pt 1), 5436 - 41
Development of a miniaturized, improved nucleic acid precursor incorporation assay for chemosensitivity testing of human solid tumors; Kern DH et al.; Two technological problems limit the usefulness of chemosensitivity assays: low success rates (generally 30-60%); and the requirement for large numbers of tumor cells (5 X 10(5)/dish) . To solve these problems, we developed a miniaturized, improved, nucleic acid precursor incorporation assay (MINI-assay) . In this new assay, 0.3-1.5 X 10(5) tumor cells were plated in double-layer agarose in 16-mm wells of a Costar (No . 3524) 24-well cluster dish . After 72 h of incubation, 5 microCi {3H}thymidine were added to each well . After an additional 24 h of incubation, the trichloroacetic acid-precipitable material was collected and counted by liquid scintillation . We found that 280 of 351 (80%) human solid tumors gave evaluable chemosensitivity results . Labeling efficiency was optimum when the plating density was between 1.5 and 3 X 10(4) cells/well . Radioisotope uptake was less efficient in 35-mm Petri dishes and in the 7-mm wells . The MINI-assay was particularly suitable for small specimens (less than 1 g) and for tumor types that usually yield small numbers of viable tumor cells (19 of 30 breast cancers and 56 of 71 sarcomas were evaluable) . The artifacts of colony counting (cell clumps, debris, clots) were also eliminated with this assay . With high evaluability rates, the requirement of fewer cells, a short duration (5 days), and ease of quantitation, the MINI-assay is widely applicable to chemosensitivity testing in human tumors.

Cancer Res, 1985 Nov, 45(11 Pt 2), 5648 - 55
Surface expression of tumor-associated antigens in primary cultured human colonic epithelial cells from carcinomas, benign tumors, and normal tissues; Friedman E et al.; A new method for the analysis of the binding of monoclonal antibodies to cell surface tumor-associated antigens utilizes 1- to 2-day primary cultures of human colonic carcinomas, adenomas, and normal epithelial tissue . The antibodies are added to the live cells which form monolayer epithelial patches of several hundred cells on the surface of the Petri dish by migration in a continuous sheet from a small explant . These epithelial patches are then fixed with methanol and processed in situ using the indirect immunoperoxidase assay . Three monoclonal antibodies (MAbs) prepared against membrane-enriched fractions of human metastatic breast cancer were assayed . MAb B1.1 bound to each of 11 benign and each of 18 malignant colonic tumors tested . MAb B6.2 displayed similar reactivity, binding to each of 7 adenomas and each of 15 carcinomas assayed . Both MAbs also bound to normal colonic epithelial cells in both the live cell studies presented here and in earlier studies (D . Stramignoni et al., Int . J . Cancer, 31: 543-552, 1983) . MAb B72.3 bound only to tumor cells and not to normal epithelial cells in the live cell assay . This epitope was rapidly lost in culture . B72.3 reactivity on each of two carcinomas was decreased 9- to 36-fold when primary culture continued for 5-6 days . B72.3 bound to each of 20 tumors (15 carcinomas, 5 adenomas) when the cells were cultured for 1 or 2 days but on only 2 of 8 tumors when the cells were cultured for 3 to 8 days . The B72.3 epitope was more strongly expressed on the live cells in the explant and on those monolayer cells directly adjacent to the explant than on the cells more towards the edges of the patch colony . This implied that the cell flattening which occurred when cells migrated from the explant may have played some role in antigen loss . A very similar fraction of primary cultured carcinoma and adenoma cells bound each MAb, indicating that these MAbs in live cell assay do not distinguish between benign, noninvasive colonic tumors and invasive carcinomas . The live cell assay was compared to the standard assay utilizing sectioned, fixed tumors . In parallel assays of eight tumors the fraction of cells reactive in the indirect immunoperoxidase assay was consistently higher on live cells for each of these MAbs than on fixed tissue . Due to this greater sensitivity the live cell assay was able to detect reactive cells in two cases which were scored as negative (less than 1% positive cells) in the fixed tissue assay.

S Afr Med J, 1985 Oct 26, 68(9), 651 - 2
Osmolarity studies with different containers and volumes in a human in vitro fertilization programme; Kruger TF et al.; In performing in vitro fertilization, a stable osmolarity in the medium surrounding the egg or embryo is of the utmost importance if a good fertilization and pregnancy rate is to be achieved . This study evaluated osmolarity changes in different volumes of fluid in the Falcon 3001 and 3037 Petri dishes and the Falcon 2058 tissue culture tube over a 24-hour period . It was found that the osmolarity was more stable in the Falcon 3037 Petri dish and in the tissue culture tube . The 3037 Petri dish was chosen for culturing human embryos.

Cell Biol Int Rep, 1985 Oct, 9(10), 893 - 9
Automated counting of human tumour colonies in the Courtenay-Mills assay system; Verheijen RH et al.; A procedure for using the Omnicon automated image analysis system for counting colonies grown from a human tumour cell line (COLO 205) in the Courtenay-Mills assay is described . This involves the transfer of the agar medium from culture tubes into petri dishes . Comparisons of observer and instrument counts were done on a blinded basis . Run-to-run correlation coefficient was 0.996 for automated counting and the inter-observer correlation coefficient was 0.984 . Both assessments showed a linear relationship between the number of cells plated and the number of colonies grown . Automated colony counting is fast, reliable and provides additional information on colony size distribution, not obtainable with manual counting . This automated procedure will greatly facilitate in vitro drug sensitivity evaluation.

Cancer Res, 1985 Oct, 45(10), 5027 - 34
Isolation of mouse T-cell lymphoma lines from different long-term interleukin 2-dependent cultures; Giglia JS et al.; A number of different biological properties have been ascribed to the hormone-like protein interleukin 2 (IL-2) . However, the most salient feature of this lymphokine is its ability to sustain the long-term proliferation of T-cells from humans and mice . Reported herein are the results of studies demonstrating the isolation of growth factor-independent cell lines from the long-term IL-2-dependent murine T-cell line CTLL-2 that is used frequently as the source of target cells in IL-2 bioassays . Sustained log-phase growth of these T-cells in vitro has been achieved using Petri dishes of polymethylpentene; growth could not be sustained in similar dishes of glass, untreated polystyrene, polystyrene that had been treated for cell culture, or polycarbonate . The IL-2-independent line grew as a T-cell lymphoma when injected i.p . into pristane-treated, but not untreated, syngeneic C57BL/6 mice . In contrast, cells from the IL-2 parental line CTLL-2 did not grow in vivo . Characterization of the IL-2-independent lines propagated in vitro (denoted as line CEC) or in vivo (denoted as line CEP) demonstrated that they retained their dependency for 2-mercaptoethanol and expressed phenotypic profiles of their parental line CTLL-2 (Thy 1.2+, Lyt-1-; Lyt-2-) . Isolation of an IL-2-independent T-cell lymphoma from a CTLL-2 line obtained from another investigator using a protocol that has proven reproducible under carefully controlled laboratory conditions and defined phenotypic traits of the syngeneic T-cell isolates provided evidence that the tumors were not a cross-culture contaminant arising as a result of a laboratory accident . Moreover, karyotypic analysis using a quinacrine:Hoechst banding technique revealed similar marker chromosomes in the IL-2-dependent and -independent lines . IL-2-independent lines have also been established from the IL-2-dependent murine T-cell line CT-6 . Accordingly, the results of these studies suggest that, during prolonged cultivation that has included exposure to crude IL-2 preparations known to contain phorbol ester, possibly viruses, and other contaminants, the IL-2-dependent lines have developed subpopulations that are thought to have undergone malignant transformation of unknown etiology to generate IL-2-independent murine T-cell lymphomas that can be passaged repetitively either in vitro or in vivo.

In Vitro Cell Dev Biol, 1985 Oct, 21(10), 569 - 74
Mechanical damage to murine neuronal-enriched cultures during harvesting: effects on free fatty acids, diglycerides, Na+,K+-ATPase, and lipid peroxidation; Demediuk P et al.; The most commonly used procedure to harvest cultured cells from petri dishes is to scrape the cells off the plates with a rubber or Teflon policeman . However, the results reported herein demonstrate that this technique, with its associated mechanical trauma, significantly perturbed cell membranes in neuronal-enriched cultures derived from the ventral half of fetal murine spinal cords . This is evidenced by liberation of free fatty acids and diglycerides, partial inhibition of Na+,K+-ATPase activity, and increased malondialdehyde production . Harvesting the cells by freezing, either on liquid nitrogen or dry ice, significantly attenuated these effects . This important observation indicates that mechanical manipulation of cultured cells during harvesting significantly affects subsequent biochemical analyses, particularly those associated with the cell membrane (e.g., membrane lipid metabolism and assay of intrinsic membrane enzymes).

J Immunol Methods, 1985 Sep 3, 82(1), 3 - 15
Study of cell deformability by a simple method; Mege JL et al.; Cell deformability plays an important role in many immunological processes, such as phagocyte chemotaxis and endocytosis . The most widely used method of assay consists in aspirating cells into glass micropipettes and measuring the length of the protrusion induced by a given pressure, or the minimum pressure required to drive cells into the micropipette . This procedure requires specialized equipment and delicate manipulation . The present report describes a simpler procedure: cells are centrifuged in petri dishes floating on a water cushion, then fixed and coated with 0.8 micron diameter latex beads, which allows rapid and accurate determination of their height . This method is compared with the micropipette technique by studying lymphocyte and macrophage-like cell lines in physiological medium and in the presence of a divalent cation chelator or a microfilament inhibitor . In addition to simplicity, the main advantages of this technique are that (i) many cells may be examined within a reasonable period of time, which allows testing of heterogeneous cell populations, and (ii) unexpectedly, centrifugation was quite harmless under our experimental conditions, since it did not impair cell proliferative ability nor phagocytic ability . It is concluded that the method may be used in clinical laboratories to explore phagocyte dysfunctions, as well as in experimental studies.

J Immunol Methods, 1985 Sep 3, 82(1), 161 - 7
A simple method for the solubilisation of reduced NBT, and its use as a colorimetric assay for activation of human macrophages by gamma-interferon; Rook GA et al.; We describe a simple method by which the insoluble blue formazan dye produced by the reduction of nitro blue tetrazolium can be dissolved without heating using potassium hydroxide and dimethyl sulphoxide . This modification enhances the sensitivity and increases the applications of tests performed using the microELISA method and removes variations caused by uneven cell monolayers . It also allows quantification of NBT reduced by cells adherent to coverslips or in larger wells or Petri dishes, and can be used as a sensitive assay for macrophage activation by gamma-interferon.

J Hosp Infect, 1985 Sep, 6(3), 326 - 32
Effective design for a laboratory discard sterilizer; Line SJ; A modification of the direct air displacement system of processing laboratory discard material in an autoclave is described . The method overcomes many of the drawbacks encountered previously with existing laboratory sterilizers and yet provides an effective means of replacing air with steam in difficult laboratory discard loads . Plastic Petri dishes were used as the standard load and plastic boxes as the container . Thermocouple results indicate that the described method gives good steam penetration of the load and allows safe handling by the operator.

J Immunol Methods, 1985 Jul 16, 81(1), 7 - 13
Detection and enumeration of monocytes in human blood with peanut agglutinin; Rosenberg M et al.; Binding of peanut agglutinin (PNA) to normal human peripheral blood mononuclear cells was analyzed on a cell sorter, and compared to the binding of the monocyte specific monoclonal antibodies Mac-1 and Leu-M3 . Each of the reagents labeled 9-11% of the mononuclear cells and similar binding patterns were observed . Of the PNA+ cells, 67% adhered to plastic petri dishes, whereas 76% of Mac-1+ cells were adherent . No competition for binding was observed between PNA and Mac-1 on the one hand, or PNA and Leu-M3 on the other . In double staining experiments, about 10% of the cells, comprising 80% of the monocytes, were PNA+ Leu-M3+ . Our results show that PNA can serve for the identification and enumeration of monocytes in human peripheral blood.

Cancer Res, 1985 Jul, 45(7), 3236 - 42
Altered actin cytoskeletal patterns in two premalignant stages in human colon carcinoma development; Friedman E et al.; Primary culture of human colonic biopsies converts the single cell thick epithelial layer from a highly indented sheet in vivo into a flat patch on the surface of a Petri dish . Migration of cells from biopsies in a continuous sheet to form the patch cultures allows the cultured cells in large part to retain the junctional complexes and membrane interdigitations which connect adjacent cells in vivo and therefore to maintain their spatial relationships to neighboring cells . Migration of the cells onto a flat surface also allows visualization of their actin cables (E . Friedman, M . Verderame, S . Winawer, and R . Pollack, Cancer Res., 44: 3040-3050, 1984) . Actin organization patterns have been studied in primary patch cultures of colonic epithelial cells from four stages in the development of colon cancer: normal tissue, normal-appearing but preneoplastic cells characteristic of familial polyposis patients, benign tumors or adenomas from familial polyposis patients, and benign and malignant tumors from patients in the general population . Carcinomas exhibited the least number of actin cables, while adenomas contained the greatest concentration . Similar actin patterns were seen in both familial polyposis and nonpolyposis adenomas . The preneoplastic prebenign tumor stage characteristic of familial polyposis patients had less actin cables than either normal cells or benign tumor cells . Thus actin organization loss characterized the transition from the normal colonic epithelial cell to the preneoplastic nontumor cell . The ability to form actin cables was then regained with the transition from the preneoplastic pretumor cell to the benign tumor cell and lost again with the benign tumor to malignant tumor transition . The complexity of these changes in actin organization during the step-wise transformation of colonic epithelial cells was not predicted from the simple model of actin cable loss accompanying fibroblast transformation.

Jpn J Cancer Res, 1985 Jul, 76(7), 596 - 602
Recombinant gamma-interferon and lipopolysaccharide enhance 1,25-dihydroxyvitamin D3-induced cell differentiation in human promyelocytic leukemia (HL-60) cells; Murao SI et al.; The induction of cell differentiation by a combination of 1,25-dihydroxyvitamin D3 {1,25-(OH)2D3}, recombinant gamma-interferon (rec gamma-IFN), and a lipopolysaccharide from E . coli (LPS) was studied in a clonal population (clone-9) of human promyelocytic HL-60 leukemia cells in vitro . Treatment of clone-9 cells with 10(-9) to 10(-7)M 1,25-(OH)2D3 yielded a macrophage cell differentiation . The addition of 10 or 100 U/ml of gamma-IFN and 2 or 10 micrograms/ml LPS caused a further increase in expression of the different differentiation markers . The most pronounced effects involved increases in cell attachment to the surface of tissue-culture Petri dishes and in lysozyme, nonspecific esterase, and cytolytic activities . The combined treatment with 1,25-(OH)2D3 and rec gamma-IFN and LPS also caused an increase in the percent of multinucleated giant cells . These results indicate the effectiveness of combining different agents in inducing cell differentiation in HL-60 cells . A similar approach may be useful in controlling myeloid leukemias in vivo.

Br J Dermatol, 1985 Jul, 113 Suppl 28, 118 - 23
Model for detection of membrane-associated antigens on epithelial cells (HLA-A, -B, -C alloantigens and pemphigus antigens); Vermeer BJ et al.; Incubation of skin explant culture with HLA-A, -B and -C alloantibodies results in detachment of cells at the periphery of the outgrowth . This detachment can be observed within 1 1/2 h, using the reflection contrast microscope . During the detachment of the epithelial cell, characteristic changes occur, such as disorganization of the filopodia and lifting up of the cell borders from the surface of the Petri dish . The same changes were seen using antibodies against pemphigus antigens.

J Gen Virol, 1985 Jul, 66 ( Pt 7), 1469 - 77
Ia antigens and Fc receptors of mouse peritoneal macrophages as determinants of susceptibility to lactic dehydrogenase virus; Inada T et al.; The relationship between susceptibility of mouse peritoneal macrophages to lactic dehydrogenase-elevating virus (LDV) infection and expression of I region-coded antigens (Ia) on these cells was investigated . The proportion of Ia-positive cells in resident peritoneal macrophages from adult and suckling mice were 4 to 10% and 50 to 70% respectively . Approximately the same percentage of the cells were susceptible to LDV, as detected by fluorescent antibody staining . In adult mice, double-labelling experiments showed that most of the Ia-positive cells were LDV-infected . When the cells were cultured for more than 24 h in vitro, Ia-positive cells rapidly disappeared and the culture became resistant to LDV . Removal of Ia-positive cells by treatment with anti-Ia plus complement or enrichment using an anti-Ia-coated Petri dish simultaneously removed or enriched for LDV-susceptible cells . Treatment of cells with trypsin (1 mg/ml) removed their I-A and I-E antigens and simultaneously abolished susceptibility for LDV . When LDV was preincubated with subneutralizing amounts of antibody, infectivity for macrophages was enhanced and the proportion of LDV-infected cells was higher than that of Ia-positive cells . This suggests that Fc receptors on macrophages can act as receptors for LDV coated with antiviral IgG.

Biochem Biophys Res Commun, 1985 Jun 28, 129(3), 611 - 8
Electric field mediated transformation: isolation and characterization of a TK+ subclone; Zerbib D et al.; Transformation of mammalian TK- cells by a plasmid carrying the TK gene from Herpes virus simplex 1 (pAGO) was mediated by electroporation . The cells were treated either in suspension or growing in monolayers directly in the petri dish . The yield of transformation was between 8.10(-5) and 2.10(-4) per microgram DNA depending on the experimental conditions . The structure of the integrated DNA was investigated proving the occurrence of a duplication process that affected preferentially the pBR322 part of the pAGO DNA (60 copies per cell) . The TK gene that gave the TK+ phenotype to the selected clone was present in less than 6 copies.

J Helminthol, 1985 Jun, 59(2), 119 - 25
Laboratory experiments to evaluate the ability of Arthrobotrys oligospora to destroy infective larvae of Cooperia species, and to investigate the effect of physical factors on the growth of the fungus; Gronvold J et al.; Laboratory investigations were designed to study the influence of temperature, pH and oxygen tension on the growth of Arthrobotrys oligospora, a nematode-trapping microfungus . Experiments were performed to evaluate the potential role of A . oligospora in destroying third-stage larvae of Cooperia spp . on agar plates and in cattle faeces . The fungus had a growth rate optimum at 23 degrees C and pH 6 . Anaerobic cultivation for 23 hours at 23 degrees C and 39 degrees C inhibited fungal growth, but it did not destroy the fungus, which regained growth upon a subsequent shift to aerobic conditions at 23 degrees C . Under experimental conditions in petri-dishes containing agar, the nematode-trapping efficiency of the fungus was striking in that 100% of a population of third-stage larvae of Cooperia spp . was captured within three days of the experiment . The trapping efficiency in faeces was shown to depend upon the inoculation level . At a concentration of approximately 2500 conidia per g faeces, 99% of the larvae were destroyed . The possibilities of using nematode-trapping fungi in controlling animal-parasitic nematodes are discussed.

Biomaterials, 1985 May, 6(3), 193 - 7
Poly(2-hydroxyethyl methacrylate)--collagen composites which promote muscle cell differentiation in vitro; Stol M et al.; A new simple method has been developed which allows the mixing of poly(2-hydroxyethyl methacrylate)--polyHEMA--and fibrillar collagen in any desired ratio . PolyHEMA alone was shown to be an unsuitable cultivation substrate for primary cultures of chicken embryonic skeletal muscle cells . Composites containing polyHEMA and 50% (w/w) or more collagen supported myogenesis . Such layers, firmly adhered to the bottom of plastic Petri dishes, were mechanically stable and biologically active, thus favourably combining properties of both the original materials . It is suggested that polyHEMA-collagen composite layers may be used for cultivation of differentiating cells in vitro.

Acta Endocrinol (Copenh), 1985 May, 109(1), 25 - 31
Functional integrity of anterior pituitary cells separated by a density gradient; Scheikl-Lenz B et al.; Using a continuous Percoll density gradient, endocrine cells of the anterior pituitary were separated . The cells were obtained from adult female Sprague-Dawley rats which had been ovariectomized for 7 days . The gradient revealed two equally sized populations of cells with densities of about 1.02 and 1.09 g/ml . Ninety-two per cent of the cellular GH content, 64% of LH, and 60% of TSH were found in the high density peak . Sixty-one per cent of the cellular Prl appeared in the low density peak . Immunocytochemical staining of the LH containing cells showed that 74% of the gonadotrophs were in the high density peak . After separation, the cells retained their responsiveness to LRH, TRH and GRF . Culture conditions influenced stimulated hormone release . Before stimulation, the cells were cultured either in tissue culture flasks (attached cells) or in Petri dishes (cells in suspension) for 3 days . After TRH-stimulation, suspended thyrotrophs released more TSH than attached thyrotrophs . Comparing the cells of both peaks, attached thyrotrophs of the high density peak showed higher stimulated TSH-release than those of the low density peak . The response of the gonadotrophs and somatotrophs to stimulation did not differ when culture conditions were changed . The present results demonstrate that the secretory activity of endocrine cells is influenced by culture conditions and should be evaluated fore each cell type.

Am J Physiol, 1985 May, 248(5 Pt 1), C410 - 8
Epithelial properties of human colonic carcinoma cell line Caco-2: effect of secretagogues; Grasset E et al.; Human colonic carcinoma Caco-2 cells grown in vitro form epithelial layers of highly polarized cells . Unlike colonic adsorptive cells they possess a mucosal membrane with very limited ionic conductance, even after exposure to aldosterone . When grown on filters, Caco-2 cells were sensitive to various secretagogues; these included 10(-5) M dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP) and 10(-10) M vasoactive intestinal peptide, both of which, added serosally, enhanced the short-circuit current . The same applied to mucosal forskolin . Caco-2 cell sensitivity to serosal epinephrine was lower . Ion substitutions and 22Na-36Cl flux measurements indicated the possibility of secretagogue-dependent chloride secretion . Measurements on cells grown on Petri dishes and exposed to 1 mM DBcAMP for 1 h enabled detection of more profound modifications . Sustained 20-mV cell depolarization and a large reduction in the relative electrical resistance of the mucosal membrane were concomitant with a sizable decrease in 36Cl accumulation . These results suggest that Caco-2 cells, which to some extent resemble colonic crypt cells, possess the cAMP-dependent mucosal chloride conductance characteristic of secretory cells.

Biochim Biophys Acta, 1985 Apr 25, 834(2), 238 - 48
GM2-ganglioside metabolism in cultured human skin fibroblasts: unambiguous diagnosis of GM2-gangliosidosis; Raghavan S et al.; The metabolism of GM2-ganglioside was studied in situ using cultured skin fibroblasts from normal individuals and patients with different forms of GM2-gangliosidosis . {3H}Sphingosine-labeled GM2 was provided in the culture medium to confluent cells in 6-cm petri dishes . After 10 days, the cells were washed free of radioactivity and harvested by trypsinization . The cellular lipids were extracted and analyzed for radioactivity in GM2 and its metabolic products . In fibroblasts from healthy subjects, 50-60% of the total cellular radioactivity was found in the neutral glycosphingolipids, ceramide, sphingomyelin and fatty acids . Degradation of the labeled GM2 progressed rapidly via GM3, ceramide dihexoside and ceramide monohexoside with a build-up of radioactivity mainly in the ceramide pool of the cell . The labeled ceramide is also reutilized for the synthesis of ceramide trihexoside, globoside and sphingomyelin or is converted to fatty acid and incorporated in ester linkages . In contrast, cells from patients with GM2-gangliosidosis representing Tay-Sachs, Sandhoff and AB variant forms of the disease did not metabolize the ingested labeled GM2-like controls . Nearly all of the radioactivity was present in the ganglioside fraction in the lipid extracts from these cells and consisted of unhydrolyzed GM2 . High-performance liquid chromatographic analysis of monosialogangliosides from cells grown without added labeled GM2 in the medium indicated accumulation of endogenously synthesized GM2 in cell lines from all patients with GM2 gangliosidosis compared to healthy controls . This approach provides a reliable tool for pre- and post-natal diagnosis of all forms of GM2-gangliosidosis without ambiguity.

Poult Sci, 1985 Apr, 64(4), 629 - 33
Evaluation of four disinfectants under poultry grow-out conditions using contact agar sampling technique; Fate MA et al.; Four commercial disinfectants used in poultry house sanitation procedures were evaluated for efficacy using a specialized petri dish (Rodac plate) for contact sampling . The samples were taken from a variety of surface materials commonly found in poultry rearing facilities . Two media were used, one for bacteria and one for mold . Predisinfection samples were taken following removal of chickens and litter . Postdisinfection samples were taken 4 hr after room treatment with a high pressure spray apparatus . The most effective disinfectant for reducing bacteria colony counts was a product that contained glutaraldehyde . The other products, ranking in order of efficacy for bacteria, contained cresylic acid, iodophors, and a combination of quaternary ammonium compound and formaldehyde . The most effective disinfectant for reducing mold colony counts was the product containing cresylic acid . The other products, ranking in order of efficacy for mold, contained iodophors, the combination of quaternary ammonium compound and formaldehyde, and glutaraldehyde . The most effective overall disinfectant was the cresylic acid product.

J Invest Dermatol, 1985 Apr, 84(4), 257 - 61
A murine monoclonal antibody (VM-1) against human basal cells inhibits the growth of human keratinocytes in culture; Oseroff AR et al.; Using epidermal cells from psoriatic plaques as the immunogen, an IgG1 murine monoclonal antibody, VM-1, has been produced which stains basal keratinocytes on frozen sections of skin obtained from normal individuals and from psoriatic plaques . In some areas of both normal and psoriatic epidermis, the cell layer immediately above the basal cells is also stained . Cells in the external root sheath of the hair follicles also bind VM-1 . The antibody binding site is trypsin-resistant, and is not blocked by bullous pemphigoid serum . If dispersed epidermal cells are preincubated with VM-1 for 1 h or more before plating, the majority of the cells do not attach and spread out on a collagen-coated Petri dish surface or on a fibroblast feeder layer . When added to attached, preconfluent cultures of keratinocytes, VM-1 inhibits growth and alters cell morphology . The growth inhibition is specific for keratinocytes, and viability studies show that it is not due to an immediate toxic effect of the antibody . The VM-1-induced inhibition of keratinocyte growth is not reversed by soy bean or lima bean trypsin inhibitors added at the time of cell plating or at the time of addition of antibody.

J Cell Sci, 1985 Apr, 75, 35 - 42
A new vacuum-operated stress-providing instrument that applies static or variable duration cyclic tension or compression to cells in vitro; Banes AJ et al.; An instrument providing cyclic stress to cells cultured in vitro has been developed . The unit uses a vacuum to deform a plastic Petri dish yielding 0.13% compression to cells on the inner surface, measured by strain gauge recordings . A regimen of 25 s stress and 5 min relaxation induced no significant change in synthesis of a 45 X 10(3) Mr protein that comigrates with actin, whereas a 52 X 10(3) Mr protein that comigrated with tubulin decreased from 12.7 +/- 0.451% of the total protein synthesized in control, static cells to 8.53 +/- 0.182% in stressed cells . The unit may have a broad application in monitoring biochemical changes in response to stress in cells such as muscle, lung, tendon, ligament and bone that are normally subjected to tension or compression.

Hepatology, 1985 Mar-Apr, 5(2), 215 - 9
Long-term maintenance of taurocholate uptake by adult rat hepatocytes co-cultured with a liver epithelial cell line; Foliot A et al.; Taurocholate (TC) uptake by adult rat hepatocytes co-cultured with other rat liver epithelial cells (RLEC) was studied comparatively to hepatocytes in primary culture . Cells were cultured on Petri dishes for desired times prior to measuring their ability to transport TC . TC uptake was linear for 150 sec in both culture conditions . In hepatocytes cultured alone, the initial rate of TC uptake at an extracellular concentration of 100 microM was 0.19 +/- 0.02 nmole per min per 10(6) cells after 48 hr of culture and decreased by 75% after 4 to 6 days . In hepatocytes co-cultured with RLEC, the rate of uptake at 48 hr (0.31 +/- 0.01 nmole per min per 10(6) cells) was significantly higher than in hepatocytes cultured alone (p less than 0.01); in addition, TC uptake remained stable at an average rate of 0.17 +/- 0.01 nmole per min per 10(6) cells for up to 56 days . No detectable uptake was found in RLEC cultured alone . TC uptake exhibited both saturable (Vmax = 0.30 +/- 0.03 nmole per min per 10(6) cells and Km = 42.6 +/- 4.4 microM) and nonsaturable components . These kinetic parameters were similar to those previously reported in isolated hepatocytes and in short-term cultured hepatocytes . TC uptake exhibited sodium dependence and was significantly reduced when extracellular sodium was replaced by lithium and sucrose, or in the presence of 1 mM ouabain . After 18 days of co-culture, TC uptake had qualitatively the same characteristics as at 48 hr, with a saturable and a nonsaturable component.(ABSTRACT TRUNCATED AT 250 WORDS)

J Immunol Methods, 1985 Feb 28, 77(1), 37 - 43
A method of purifying sheep sIg+ lymphocytes as a tool for class II MHC antigen analysis; Chasset P et al.; A method is described for the purification of sheep lymphocytes carrying class II MHC antigens . After incubation of purified blood lymphocytes on anti-IgM-coated petri dishes, the adherent fraction contained 95% sIg-positive cells determined by immunofluorescence . When tested with cross-reacting anti-class II (bovine and human) monoclonal antibodies, more than 95% of these cells were positive either by immunofluorescence or cytotoxicity . This technique will permit studies of the polymorphism of sheep class II antigens.

Anal Biochem, 1985 Feb 1, 144(2), 329 - 35
Biosynthetic labeling with 32P: radiation damage to mammalian cells; Cooper PC et al.; Theoretical calculations showed that biosynthetic radiolabeling of cells using typical concentrations of 32P (1 mCi/ml) resulted in high radiation doses (200-500 rad/h) being absorbed by the cells . Subsequent investigations with a mouse myelomonocytic leukemia cell line (WEHI-3B(D+)) showed significant loss of replicative ability during brief (less than 1 h) exposures to 1 mCi/ml of 32P . Complete loss of cell replicative ability was found with isotopic doses less than 100 rad (i.e., 100 muCi/ml for 5 h) . Experiments employing a less radiosensitive pre-B-cell line (18.81) revealed that significant loss of viability occurred during incubation with 32P under identical conditions to those employed for the WEHI-3B(D+) cell line . Control experiments utilizing decayed batches of 32P and physical separation of the isotope solution from the cells confirmed that the cytotoxicity was caused by radiation emission rather than the presence of toxic components in the isotopic solution . The radiation doses absorbed by cells biosynthetically labeled with 59Fe, 33P, 35S, and 14C were calculated . Although significant levels of radiation can be absorbed 32P was considerably more radiotoxic than the other isotopes . The results of calculations indicated that the judicious choice of container geometry could reduce the absorbed radiation dose from 32P solutions . In particular the biosynthetic radiolabeling of cells in capillary tubes (diameter less than 1 mm) can reduce the absorbed rate to less than one-tenth of the dose received by cells suspended in Petri dishes or centrifuge tubes.

J Parasitol, 1985 Feb, 71(1), 10 - 6
Life cycle and postembryonic development of Oochoristica anolis (Cyclophyllidea: Linstowiidae); Conn DB; Gravid proglottids of Oochoristica anolis from naturally infected anole lizards, Anolis carolinensis, were placed in covered Petri dishes with laboratory-reared beetles, Tribolium confusum and Tenebrio molitor . After maintenance at 25 C, metacestodes developed in 29 of 61 T . confusum (48%), but in none of 5 T . molitor . Beetles contained from 1 to 22 metacestodes (means = 3.3), which were fully developed by day 40 postexposure . A primary lacuna was never observed, but the possibility of its presence could not be ruled out without histological study . No cercomer was formed and metacestodes retained larval hooks throughout development . Scolices were invaginated at removal from the hemocoel, but usually evaginated quickly in Ringer's . On day 60 postexposure, metacestodes were fed by stomach tube to 5 anoles, 2 lacertid lizards (Podarcis muralis) and 2 mice . Worms developed only in anoles, 3 of which were infected upon examination . Oncospheral hooks were present in worms after 7 days development in the lizard; a median excretory pore was present at the posterior tip of all stages examined, including the terminal mature proglottid of a worm after 105 days in a lizard . Scolex growth rate was linear throughout metacestode and adult development, but growth rate in body length was diphasic, punctuated by change of hosts, associated with strobilization . Attempts to establish parenteral infections in anoles were unsuccessful . Present data constitute the most complete life history study thus far for any species of Oochoristica.

Eur J Immunol, 1985 Feb, 15(2), 189 - 92
Evaluation of accessory cell heterogeneity . II . Failure of dendritic cells to activate antigen-specific T helper cells to soluble antigens; Ramila G et al.; The activation of antigen-specific T cells requires Ia+, antigen-presenting accessory cells (AC) . Dendritic cells (DC) and macrophages (M phi) isolated from spleen an peritoneal exudate were tested as AC for the activation of the activation of T helper cells and the induction of T cell proliferation . The cell separations to obtain DC and splenic M phi were performed by discontinuous bovine serum albumin gradients, adherence on petri dishes and rosetting with opsonized sheep erythrocytes . DC as well as the M phi were able to induce antigen-specific T cell proliferation, but only the M phi and not the DC activated antigen-specific T helper cells which help B cells for antibody production to soluble antigens . Keyhole limpet hemocyanin-specific T cells repeatedly stimulated with DC and antigen also did not express helper activity . The failure of DC to induce T helper cells was not due to the activation of a suppressor pathway . Thus, dendritic cells, although very efficient as AC in the induction of various T cell functions, are not able to activate T helper cells required for carrier-specific T-B cooperation and therefore cannot be the sole accessory cells . Based on these results and on previous data using Ia+ tumor cell lines as AC, we confirm the existence of functional AC heterogeneity.

Prog Clin Biol Res, 1985, 178, 229 - 31
Rearing Culicoides obsoletus (Diptera, Ceratopogonidae) on agar cultures of nematodes; Boorman J; Culicoides obsoletus midges were reared on a culture of nematodes in small Petri dishes on an agar-based medium . At 25 C pupae were formed from 12 to 71 days after the eggs hatched . The efficiency of the method was low but, if improved, could be useful for investigating relationships between members of the Avaritia subgenus . An unexplained feature was the almost total absence of females among these laboratory-reared midges.

Lab Invest, 1985 Jan, 52(1), 77 - 84
Direct toxic effects of paraquat and oxygen on cultured endothelial cells; Ody C et al.; Paraquat (PQ) is a herbicide known to generate O2 radicals and to injure lung epithelial cells, leading eventually to pulmonary fibrosis . To test for the possible existence of a direct cytotoxic action of PQ on endothelial cells, we have studied, for up to 5 days, the action of 10(-6) to 10(-4) M PQ on primary cultures of pig aortic endothelial cells and compared these effects to those obtained with exposure to 95% O2-5% CO2 . The decrease in DNA and protein content of Petri dishes and the increase in lactate dehydrogenase release were found to depend on PQ concentration and the duration of exposure to PQ . The toxic effects of hyperoxia were intermediate, ranging between those obtained with 10(-5) and 10(-4) M PQ . Hyperoxia and 10(-4) M PQ produced a similar marked inhibition of DNA synthesis after a 1-day period of exposure . Combined exposure to both PQ and hyperoxia resulted in changes comparable to those obtained with hyperoxia alone (decrease in protein and DNA content) or PQ alone (lactate dehydrogenase release) . Additive effects were seen only for the inhibition of DNA synthesis . The selenomethionine-related increase in glutathione peroxidase activity had a protective effect against hyperoxia-induced lactate dehydrogenase release but not against PQ induced cytolysis . Finally, shorter exposures to O2 and PQ revealed the existence of a trend toward recovery only for cells exposed to hyperoxia . The prolonged toxic action of PQ could not be related to PQ accumulation and storage by endothelial cells . These studies indicate that PQ can exert a direct, dose-dependent, and prolonged cytotoxic effect on cultured endothelial cells.

J Cell Biol, 1985 Jan, 100(1), 56 - 63
Isolation of a cell-surface receptor for chick neural retina adherons; Schubert D et al.; Embryonic chick neural retina cells release glycoprotein complexes, termed adherons, into their culture medium . When absorbed onto the surface of petri dishes, neural retina adherons increase the initial rate of neural retina cell adhesion . In solution they increase the rate of cell-cell aggregation . Cell-cell and adheron-cell adhesions of cultured retina cells are selectively inhibited by heparan-sulfate glycosaminoglycan, but not by chondroitin sulfate or hyaluronic acid, suggesting that a heparan-sulfate proteoglycan may be involved in the adhesion process . We isolated a heparan-sulfate proteoglycan from the growth-conditioned medium of neural retina cells, and prepared an antiserum against it . Monovalent Fab' fragments of these antibodies completely inhibited cell-adheron adhesion, and partially blocked spontaneous cell-cell aggregation . An antigenically and structurally similar heparan-sulfate proteoglycan was isolated from the cell surface . This proteoglycan bound directly to adherons, and when absorbed to plastic, stimulated cell-substratum adhesion . These data suggest that a heparan-sulfate proteoglycan on the surface of chick neural retina cells acted as a receptor for adhesion-mediating glycoprotein complexes (adherons).

Immunol Lett, 1985, 10(2), 81 - 6
Spleen cell populations in normal and tumor-bearing hamsters; Merdrignac G et al.; Spleen cell subpopulations from normal and tumor-bearing hamsters (TBH) were quantified using Petri dishes coated with specific antibodies, and by flow cytometric immunofluorescence analysis . The relative numbers of T cells (Thy + cells) decreased, by both methods, as a function of tumor growth, while the number of B cells (bearing surface Ig) increased . Cells without T or B markers (null cells) were more numerous in the spleen of TBH and a large number of them expressed the receptor for the Fc fragment of IgG . Splenic cells were also sorted according to their light-scattering properties, and electron microscopic analysis was performed on the sorted fractions . It showed the presence of secreting plasmocytes and activated macrophages in the spleen of TBH.

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