We report the development of a new serum-free medium based on the use of factorial experiments. At first, a variety of hydrolysates were screened using a fractional factorial approach with High-Five cells. From this experiment yeastolate ultrafiltrate was found to have, by far, the most important effect on cell growth. Furthermore, Primatone RL^® was found to remarkably prolong the stationary phase of Sf-9 and High-Five cell cultures. The optimal concentrations for yeastolate and Primatone were determined to be 0.6 and 0.5%, respectively, on the basis of a complete factorial experiment. This new medium, called YPR, supported good growth of both Sf-9 and High-Five cells in batch cultures, with maximal densities of 5.4 and 6.1 × 10⁶ cells/ml, respectively. In addition, both cell lines achieved good growth in bioreactor batch culture and had a prolonged stationary phase of 3–4 d in YPR medium compared to Insect-XPRESS medium. The ability of the new medium to support recombinant protein expression was also tested by infecting Sf-9 or High-Five cells at high density (2 × 10⁶ cells/ml) with a baculovirus expressing secreted placental alkaline phosphatase (SEAP). The maximum total SEAP concentration after 7 d was about 43 IU/ml (58 mg/L) and 28 IU/ml (39 mg/L) for High-Five and Sf-9 cells, respectively.

Mechanisms for the transduction of photoperiodic and food signals to the corpus allatum (CA) are described. The focus of this paper is on the control of the CA by the brain in adult females of the firebug, Pyrrhocoris apterus. By using surgical interventions to the neuroendocrine complex of brain–subesophageal ganglion–corpora cardiaca–CA (BR–SG–CC–CA) in vivo and in vitro we were able to identify two regulatory pathways. (1) Slow regulation of the CA activity (stimulation or inhibition) needs a relatively long period of time to be accomplished (several d) in vivo and is associated with changes of the gland cell volume and ultrastructure. The stimulated or inhibited activity of the CA is maintained during short-term incubation of the isolated CA in vitro. (2) Fast inhibition of the CA activity is reversible during short-term incubation in vitro; the CA can be switched from lower to higher activity and vice versa, depending on the presence or absence of the BR–SG in the medium. Both slow and fast regulatory factors originate in the pars intercerebralis of the brain and in intact neuroendocrine complex they reach the CA via nerves. A slow inhibitor, induced by short d, causes reproductive diapause. A fast inhibitor prevents ovarian maturation in starved nondiapausing females. A slow stimulator, induced by feeding under long d, overcomes the fast inhibition of the CA, thereby stimulating vitellogenesis. Food signals are transmitted to the brain via humoral pathways.

Cultured cell lines that can be stably transformed with inducible gene constructs could prove extremely valuable for the continuous and economical production of recombinant proteins. Toward this goal, we have established 11 clones (designated NISES-BoMo-DK1 to 11) from a previously reported silkworm cell line, NISES-BoMo-DZ. Nine of these clonal lines showed a distinct morphological change, i.e., cell aggregation, in response to treatment with 1 μM 20-hydroxyecdysone (20E). DK10 cells transfected with various reporter assay plasmids under optimal conditions (i.e., 20–30% transfection efficiency) showed inducibility of gene expression by 20E. The 20E treatment of the prototypical DK10 cells resulted in a simultaneous, transient increase of the nuclear ecdysone (E) receptor levels. Further, this inducibility was also observed in a DK10 cell line stably transformed with the reporter plasmid that carries the hygromycin-resistance gene. This offers an opportunity to achieve efficient, continuous production of recombinant proteins. It could also allow high throughput screening for potential E agonists.

The objective of this study was to generate immortalized Sertoli cell lines from prepubertal lamb testes to facilitate investigations during the course of testicular differentiation. The Sertoli cells were enzymatically isolated and immortalized by transfection, with the sequences coding for the SV40 large T-antigen fused downstream of regulatory elements from the human vimentin gene. The different cell lines were positively stained with antibodies to vimentin and transferrin, in agreement with their Sertoli origin. Reverse transcriptase polymerase chain reaction was used to analyze the specific expression of molecular markers (clusterin/sulfated glycoprotein [SGP-2], follicle-stimulating hormone [rFSH], α-inhibin, anti-Müllerian hormone, Wilms' tumor gene [WT-1], steroidogenic factor 1 [SF-1], SRY-related HMG box gene g [SOX9], and sex-determining region of Y chromosome) normally expressed in this cellular type. All were shown to express messenger ribonucleic acids for SGP-2, α-inhibin, WT-1, SOX9, and SF-1 (except SF-1 for clone no. 1). Moreover, we performed alkaline phosphatase and receptor tyrosine kinase p145 (c-kit) detection to ensure the absence of contamination by peritubular, germ cells, and Leydig cells. Both tests were negative for all the seven cell lines. These ovine Sertoli cell lines are the first ones obtained from livestock that exhibit specific Sertoli cell characteristics resembling different stages of phenotypic development. They provide useful in vitro model systems for toxicological investigations, coculture, and transfection experiments, making it possible to study signal transduction pathways, cell–cell interactions, and gene expression in species other than rodents.

Mouse hepatocytes immortalized with a temperature-sensitive allele of the SV40 large T-antigen (CHST8 cells) were found to lack the high expression of the gap junction proteins Cx26 and Cx32 that characterizes normal mouse hepatocytes, expressing instead Cx43 and Cx45 at minimal levels. In order to examine the growth suppressive function of Cx32 on hepatocytes, we transfected these CHST8 cells with human Cx32 complementary deoxyribonucleic acid and measured the growth rates at 33, 37, and 39° C. Expression of human Cx32 and its messenger ribonucleic acid in the stable cell lines was confirmed by immunocytochemistry and by Western and Northern blots analyses. Dye transfer following lucifer yellow injection into the transfectants was extensive; Cx32 channels displayed unitary conductances of about 70 pS and were moderately voltage sensitive. When cultured at 33 and 39° C, growth rates of both parental cells and transfectants were of the same level. When examined at 37° C, growth rate of the transfectant, which highly expressed Cx32 at the membranes, was significantly decreased compared to the parental cells. However, no changes in the expression of Cx32 protein in the transfectants were observed between 33 and 37° C. These results suggest that Cx32 expression could inhibit hepatocyte growth in vitro using the conditional immortalized cells. Cx32 transfectants using a conditional immortalized mouse hepatocyte may be useful for examining the mechanisms of growth and differentiation in hepatocytes by gap junction expression.

Lymphocyte interactions with endothelial cells in microcirculation are an important regulatory step in the delivery of lymphocytes to peripheral sites of inflammation. In normal circumstances, the predicted wall shear stress in small venules range from 10 to 100 dyn/cm². Attempts to measure the adhesion of lymphocytes under physiologic conditions have produced variable results, suggesting the importance of studying biologically relevant migratory lymphocytes. To quantify the effect of shear stress on these migratory lymphocytes, we used lymphocytes obtained from sheep efferent lymph ducts, defined as migratory cells, to perfuse sheep endothelial monolayers under conditions of flow. Quantitative cytomorphometry was used to distinguish cells in contact with the endothelial monolayers from cells in the flow stream. As expected, migratory cells in contact with the normal endothelial monolayer demonstrated flow velocities less than the velocity of cells in the adjacent flow stream. The flow velocities of these efferent lymphocytes were independent of cell size. To model the inflammatory microcirculation, lymphocytes were perfused over sequential endothelial monolayers to directly compare the velocity of cells in contact with cytokine-activated and unactivated control monolayers. The tumor necrosis factor and interleukin-1–activated endothelial monolayers marginally decreased cell velocities at 1.2 dyn/cm² (3.6%), but significantly reduced cell velocities 0.3 dyn/cm² (27.4%; P < 0.05). Similarly, the fraction of statically adherent lymphocytes decreased as shear stress increased to 1.2 dyn/cm². These results suggest that typical wall shear stress in small venules, of the order of 20 dyn/cm², are too high to permit adhesion and transmigration of migratory lymphocytes. Additional mechanisms must be present in vivo to facilitate lymphocyte transmigration in the inflammatory microcirculation.

In order to characterize a fibroblast cell line representing normal human skin fibroblasts in three-dimensional cultures, we compared the fibroblast line MSU-1.1, derived from human foreskin and immortalized by v-myc, to primary human dermal fibroblasts (NDF). Our results demonstrate that in contrast to NDF, all MSU-1.1 fibroblasts die within 3–4 d when cultured within three-dimensional contractile collagen matrices. Also, in contrast to NDF, MSU-1.1 cells die markedly in anchored collagen gels as well. Death is due to apoptosis and is attenuated by addition of antibodies against collagen-recognizing receptors α1β1 and α2β1. Apoptosis of NDF in collagen lattices was repressed by an inhibitor of caspase-1, which was ineffective on apoptosis of MSU-1.1. Further, apoptosis by MSU-1.1 fibroblasts was also observed in anchored, i.e., restrained collagen lattices, an environment that supports proliferation of NDF.

Changes in oligosaccharide structures of glycoconjugates have been observed, and are postulated to have key roles in embryonic development and differentiation. N-Acetylglucosamine (GlcNAc) β-1,4-galactosyltransferase (β4GalT) AKI showed different expression patterns in time and space, and different enzymatic activity from the other known family members. The epidermis of mouse embryo included a high level of AKI activities, which transferred galactose (Gal) to endogenous glycoprotein (molecular weight 130 kDa) (GP130). The maximum activity was for 13.5-d postcoitum embryos. Specific antibody against AKI inhibited 81% of GlcNAc β4GalT activities, which indicates that AKI represents the major part of the embryonic epidermis enzymes. AKI shows 2.2 times higher galactosyltransferase activity toward Gal-acceptor glucose with α-lactalbumin (α-LA) than toward GlcNAc without α-LA. AKI is also expressed in mouse melanoma and leukemia cell lines and in human basal cell carcinoma specimens. The GP130 Gal acceptor once galactosylated by AKI may be directly involved in epidermal differentiation and oncogenesis.

Peroxidation products of polyunsaturated fatty acids may cause growth inhibition of cells in culture. This study was carried out to elucidate to what extent peroxidation products may be found in growth media, with and without cells and albumin, using thiobarbituric acid–reactive substances (TBARS) and protein carbonyl groups as measures of peroxidation. The growth of human microvascular endothelial cells was studied as influenced by docosahexaenoic (C22:6, n − 3), arachidonic acid (C20:4, n − 6), and serum albumin. Cell growth was strongly inhibited by the fatty acids, and the inhibition was related to the concentration of TBARS in the medium. Defatted albumin (0.5 g/100 ml) nullified the increase of TBARS in the medium and released the growth inhibition by the fatty acids. With polyunsaturated fatty acids (PUFA) there was a time- and concentration-dependent increase in media TBARS, observed both with and without cells, but the TBARS increase was somewhat greater in the presence of cells. Surprisingly, TBARS in cell-free media also increased somewhat upon increasing the albumin concentration from 0.5 to 5 g/100 ml, and the TBARS increase differed among various preparations of albumin. Unexpectedly, the albumin that had not been defatted gave the lowest TBARS values. The amount of protein carbonyl groups did not differ among various albumin preparations. It is concluded that PUFA may autooxidize in media used for cell cultures, and thereby cause an unspecific growth inhibition, which can be prevented by a low albumin concentration. However, even defatted albumin preparations may contain lipid peroxidation products, the causes and implications of which remain to be elucidated.