Growth-promoting Factors Research Articles

Proteomic and Transcriptomic Analyses of Retinal Pigment Epithelial Cells Exposed to REF-1/TFPI-2

The authors previously reported a growth-promoting factor, REF-1/TFPI-2, that is specific to retinal pigment epithelial (RPE) cells. The purpose of this study was to determine the genes and proteins of human RPE cells that are altered by exposure to TFPI-2. Human primary RPE cells were cultured with or without TFPI-2. Cell extracts and isolated RNA were subjected to proteomic and transcriptomic analyses, respectively. Proteins were separated by two-dimensional gel electrophoresis followed by gel staining and ion spray tandem mass spectrometry analyses. Transcriptomic analysis was performed using a DNA microarray to detect 27,868 gene expressions. Proteomic analysis revealed c-Myc binding proteins and ribosomal proteins L11 preferentially induced by TFPI-2 in human RPE cells. Transcriptomic analysis detected 10,773 of 33,096 probes in the TFPI-2 treated samples, whereas only 2186 probes were detected in the nontreated samples. Among the genes up-regulated by TFPI-2 at the protein level were c-myc, Mdm2, transcription factor E2F3, retinoblastoma binding protein, and the p21 gene, which is associated with the c-myc binding protein and ribosomal protein L11. The mechanisms by which TFPI-2 promotes the proliferation of RPE cells may be associated with augmented c-myc synthesis and the activation of E2F in the retinoblastoma protein (Rb)/E2F pathway at the G1 phase of the RPE cells. Activation of ribosomal protein L11 and the Mdm2 complex of the p53 pathway may be counterbalanced by the hyperproliferative conditions.

Growth and mitogenic effects of arylphorin in vivo and in vitro

In insects, developmental responses are organ- and tissue-specific. In previous studies of insect midgut cells in primary tissue cultures, growth-promoting and differentiation factors were identified from the growth media, hemolymph, and fat body. Recently, it was determined that the mitogenic effect of a Manduca sexta fat body extract on midgut stem cells of Heliothis virescens was due to the presence of monomeric alpha-arylphorin. Here we report that in primary midgut cell cultures, this same arylphorin stimulates stem cell proliferation in the lepidopterans M. sexta and Spodoptera littoralis, and in the beetle Leptinotarsa decemlineata. Studies using S. littoralis cells confirm that the mitogenic effect is due to free alpha-arylphorin subunits. In addition, feeding artificial diets containing arylphorin increased the growth rates of several insect species. When tested against continuous cell lines, including some with midgut and fat body origins, arylphorin had no effect; however, a cell line derived from Lymantria dispar fat body grew more rapidly in medium containing a chymotryptic digest of arylphorin.

Endothelin signaling in osteoblasts: global genome view and implication of the calcineurin/NFAT pathway

Patients with prostate cancer develop osteoblastic metastases when tumor cells arrive in the bone and stimulate osteoblasts by secreting growth-promoting factors. Endothelin 1 (ET-1) is believed to be a key factor in promoting osteoblastic metastasis. Selective blockade of the ET(A) receptor is an established strategy in the development of cancer therapeutics. However, the molecular mechanisms whereby prostate cancer promotes abnormal bone growth are not fully understood. In this study, we have applied genomic approaches to elucidate the molecular mechanism of stimulation of osteoblasts by ET-1. To examine the ET-1 axis, we generated genomic signatures for osteoblasts treated with ET-1, in the presence and absence of a selective ET(A) antagonist (ABT-627). The ET-1 signature was comprised of several motifs, such as osteoblastic differentiation, invasion, and suppression of apoptosis. The signature also pointed at possible activation of the calcineurin/NFAT pathway. We showed that ET-1 activates calcineurin and causes nuclear translocation of NFATc1, implicating the pathway in the ET-1-mediated stimulation of osteoblasts. We also showed that ET-1 inhibits apoptosis in osteoblasts, implying that the suppression of apoptosis may be an important factor in the promotion of osteoblastic growth by ET-1.

Role of ischaemic preconditioning in liver regeneration following major liver resection and transplantation

Liver ischaemic preconditioning (IPC) is known to protect the liver from the detrimental effects of ischaemic-reperfusion injury (IRI), which contributes significantly to the morbidity and mortality following major liver surgery. Recent studies have focused on the role of IPC in liver regeneration, the precise mechanism of which are not completely understood. This review discusses the current understanding of the mechanism of liver regeneration and the role of IPC in this setting. Relevant articles were reviewed from the published literature using the Medline database. The search was performed using the keywords "liver", "ischaemic reperfusion", "ischaemic preconditioning", "regeneration", "hepatectomy" and "transplantation". The underlying mechanism of liver regeneration is a complex process involving the interaction of cytokines, growth factors and the metabolic demand of the liver. IPC, through various mediators, promotes liver regeneration by up-regulating growth-promoting factors and suppresses growth-inhibiting factors as well as damaging stresses. The increased understanding of the cellular mechanisms involved in IPC will enable the development of alternative treatment modalities aimed at promoting liver regeneration following major liver resection and transplantation.

Catheter-based adenovirus-mediated anti-monocyte chemoattractant gene therapy attenuates in-stent neointima formation in cynomolgus monkeys

We have previously demonstrated great benefit from anti-monocyte chemoattractant protein-1 (MCP-1) gene therapy by “systemic” transfer of an N-terminal deletion mutant of human MCP-1 (called 7ND) gene into skeletal muscle for treatment of restenosis and atherosclerosis. However, recent evidence suggests that “local” gene transfer may be a clinically relevant approach. We therefore tested the hypothesis that catheter-based adenovirus-mediated anti-MCP-1 gene therapy attenuates stent-associated neointima formation. Bare metal stents were implanted in iliac arteries of cynomolgus monkeys fed a high cholesterol diet. Immediately after the stenting procedure, normal saline or recombinant adenoviral vector containing LacZ or the 7ND gene was administered locally into the stenting site through a Remedy channel-delivery catheter. Compared to saline infusion or LacZ gene transfer, 7ND gene transfer markedly reduced inflammatory changes at an early stage and attenuated neointima formation after 4 weeks. This strategy also reduced the increased production of pro-inflammatory and growth-promoting factors such platelet-derived growth factor. No systemic adverse effects of 7ND gene transfer were detected. There were no significant differences in serum cholesterol levels among the three groups. These data suggest that catheter-based adenovirus-mediated anti-MCP-1 gene therapy may be a clinically relevant and feasible strategy for treatment of in-stent restenosis.

Applied physiology: Understanding growth

The process of growth can be considered to occur in four phases: intra-uterine, infancy, early childhood and the pubertal growth spurt. Each of these components is regulated by different factors. During intra-uterine life, growth is regulated by genetic, maternal, nutritional, placental and growth-promoting factors. In infancy, growth is dependent upon nutrition, whereas in early childhood, growth hormone and thyroid hormones become important in regulating growth. Finally, the pubertal growth spurt is controlled by a combination of growth hormone and sex steroids. The process of growth can be considered to occur in four phases: intra-uterine, infancy, early childhood and the pubertal growth spurt. Each of these components is regulated by different factors. During intra-uterine life, growth is regulated by genetic, maternal, nutritional, placental and growth-promoting factors. In infancy, growth is dependent upon nutrition, whereas in early childhood, growth hormone and thyroid hormones become important in regulating growth. Finally, the pubertal growth spurt is controlled by a combination of growth hormone and sex steroids.

Isolation and Identification of F-Spondin in the Boar Testis and Its Production During Testis Growth

F-spondin/vascular smooth muscle cell growth-promoting factor (VSGP), purified from the follicular fluid of adult bovine ovaries, has been identified as a promoter of neuronal differentiation and vascular smooth muscle growth. The objectives of the present study were (1) to clarify whether F-spondin is also produced in the testis, which is ontogenically equivalent to the ovary, and (2) to examine whether production of this protein changes with testicular growth. To isolate F-spondin from the testis, testicular homogenates obtained from 8-week-old boars were sequentially subjected to heparin-Sepharose chromatography, diethylaminoethyl (DEAE)-Sepharose chromatography, and reverse-phase high-performance liquid chromatography (RP-HPLC). The isolated protein had a molecular mass of approximately 110 kDa and was cross-reactive with anti-F-spondin antibody by Western blotting. The purified protein was further characterized by amino acid sequence analysis of its internal peptide. The sequence obtained was GEQCNIVPDN VD, and a homology search indicated that the purified protein is a homologue of rat, human, and bovine F-spondin. By fractionation of the same amounts of testis tissue obtained from 1-, 8-, 16-, and 40-week-old boars, we analyzed age-related production of F-spondin in the testis. Western blotting of the fractions obtained from RP-HPLC revealed the presence of a band at approximately 110 kDa, corresponding to F-spondin, in the testes obtained from boars between 1 and 16 weeks old, but this band was not detected at 40 weeks. These results clearly indicate that (1) the porcine testis produces F-spondin and that (2) production of this protein is evident in the immature porcine testis, but not the adult testis.

Influence of amoebae and physical and chemical characteristics of water on presence and proliferation of Legionella species in hospital water systems

The reservoir for hospital-acquired Legionnaires' disease has been shown to be the potable water distribution system. The objectives of the present study were as follows: (1) to examine the possible relationship between physical-chemical characteristics of water such as temperature, pH, hardness, conductivity, and residual chlorine and the presence of amoebae as growth-promoting factors for Legionella species and (2) to determine eradication measures for water distribution systems to seek ways of reducing the risk of legionellosis. Ten hospitals in southwest France took part in this study. Water samples were collected from 106 hot water faucets, showers, hot water tanks, and cooling towers. Two analyses were performed to analyze the association between water characteristics and (1) the presence of Legionella species and (2) the proliferation of Legionella species. Of the 106 water samples examined, 67 (63.2%) were positive for Legionella species. Amoebae were detected in 73 of 106 (68.9%) samples and in 56 of 67 (86.6%) Legionella species-positive samples (P < 10(-6)). In these positive samples, conductivity was lower than 500 microOmega(-1).cm(-1) in 58.2% (P = .026), temperature was below 50 degrees C in 80.6% (P = .004), and hardness was significantly higher (P = 002) than in Legionella species-negative samples. Neither Legionella species nor amoebae were isolated from any sampling point in which the water temperature was above 58.8 degrees C. Multivariate analysis shows that high hardness and presence of amoebae were strongly correlated statistically with the presence of Legionella when showers, tanks, pH, and temperature promoted their proliferation. This study shows the importance of water quality evaluation in assessing environmental risk factors and in selecting the most appropriate prevention and control measures in hospital water systems.

Linear 3-Hydroxybutyrate Tetramer (HB4) Produced bySphingomonassp. Is Characterized as a Growth Promoting Factor for Some Rhizomicrofloral Composers

Sphingomonas spp. of alpha-proteobacteria often play a role in assisting the development of microfloral communities under adverse soil conditions. Using a Frateuria sp. as an indicator for bacterial growth assay, we investigated the bacterial growth-promoting factor in the culture fluids of Sphingomonas sp. EC-K085. This factor was successfully isolated and identified as linear (R,R,R,R)-3-hydroxybutyrate tetramer (HB4), having a hydroxy-end and a carboxy-end group. When 28 mug of HB4 was charged on a paper disc, impregnated Frateuria sp. cells in modified Winogradsky agar medium exhibited a promoted cell growth to form a clear colony emerging zone after a 2-day incubation.

Beta2-microglobulin is a signaling and growth-promoting factor for human prostate cancer bone metastasis.

The protein factor beta2-microglobulin (beta2M), purified from the conditioned medium of human prostate cancer cell lines, stimulated growth and enhanced osteocalcin (OC) and bone sialoprotein (BSP) gene expression in human prostate cancer cells by activating a cyclic AMP (cAMP)-dependent protein kinase A signaling pathway. When beta2M was overexpressed in prostate cancer cells, it induced explosive tumor growth in mouse bone through increased phosphorylated cAMP-responsive element binding protein (CREB) and activated CREB target gene expression, including OC, BSP, cyclin A, cyclin D1, and vascular endothelial growth factor. Interrupting the beta2M downstream signaling pathway by injection of the beta2M small interfering RNA liposome complex produced an effective regression of previously established prostate tumors in mouse bone through increased apoptosis as shown by immunohistochemistry and activation of caspase-9, caspase-3, and cleavage of poly(ADP-ribose) polymerase. These results suggest that beta2M signaling is an attractive new therapeutic target for the treatment of lethal prostate cancer bone metastasis.

Leptin Signaling Promotes the Growth of Mammary Tumors and Increases the Expression of Vascular Endothelial Growth Factor (VEGF) and Its Receptor Type Two (VEGF-R2)

To gain insight into the mechanism(s) by which leptin contributes to mammary tumor (MT) development we investigated the effects of leptin, kinase inhibitors, and/or leptin receptor antagonists (LPrA2) on 4T1 mouse mammary cancer cells in vitro and LPrA2 on 4T1-MT development in vivo. Leptin increases the expression of vascular endothelial growth factor (VEGF), its receptor (VEGF-R2), and cyclin D1 through phosphoinositide 3-kinase, Janus kinase 2/signal transducer and activator of transcription 3, and/or extracellular signal-activated kinase 1/2 signaling pathways. In contrast to leptin-induced levels of cyclin D1 the changes in VEGF or VEGF-R2 were more dependent on specific signaling pathways. Incubation of 4T1 cells with anti-VEGF-R2 antibody increased leptin-mediated VEGF expression suggesting an autocrine/paracrine loop. Pretreatment of syngeneic mice with LPrA2 prior to inoculation with 4T1 cells delayed the development and slowed the growth of MT (up to 90%) compared with controls. Serum VEGF levels and VEGF/VEGF-R2 expression in MT were significantly lower in mice treated with LPrA2. Interestingly, LPrA2-induced effects were more pronounced in vivo than in vitro suggesting paracrine actions in stromal, endothelial, and/or inflammatory cells that may impact the growth of MT. Although all the mechanism(s) by which leptin contributes to tumor development are unknown, it appears leptin stimulates an increase in cell numbers, and the expression of VEGF/VEGF-R2. Together, these results provide further evidence suggesting leptin is a MT growth-promoting factor. The inhibition of leptin signaling could serve as a potential adjuvant therapy for treatment of breast cancer and/or provide a new target for the designing strategies to prevent MT development.

Identification of human hepatic stimulator substance gene promoter and demonstration of dual regulation of AP1/AP4 cis-acting element in different cell lines

Human hepatic stimulator substance (hHSS) is a newly identified growth-promoting factor in the liver. HSS is capable of stimulating hepatic regeneration in partial hepatectomized rats, thus, promoting growth of hepatic tumor cells. To understand and elucidate the transcriptional regulation of h HSS gene, the 4890 bp of 5′-flanking region of the gene have been isolated and sequenced. The transcriptional start site, located at 248 nt upstream from the ATG starting codon, was identified by 5′-rapid amplification cDNA end (5′-RACE). The classical promoter sequences, such as TATA box or GAATT were not identified in the promoter region, instead a GC-rich segment was formed (>70%) by expanding to a longer than 400 bp, and immediately upstream from the ATG start codon. The transient transfection assays, using promoter deletion constructs, showed that h HSS promoter was efficiently capable in driving the reporter expression not only in HepG2 cells, but also in Cos7 cells. A region spanning nucleotides in the range of −447 to −358 bp revealed a negative regulation on promoter activity in HepG2 cells, but with positive regulation in Cos7 and Hela cells. The promoter activity was obviously influenced by AP1/AP4 (−375/−369 nt) mutation in these three cell lines. EMSAs showed that the site was recognized by AP1 in HepG2 cell, and only by an AP4 protein in Cos7 cells. The c-Jun bound to the promoter was further verified by supershift in HepG2 cells and human liver tissue. Chromatin immuno-precipitation (ChIP) demonstrated that there was a direct association of c-Jun with h HSS promoter in HepG2 cells. The c-Jun strongly suppressed h HSS promoter activity in transient expression analyses in HepG2 cells. Mutations in the AP1 binding sites rescued suppression caused by c-Jun, suggesting this was a direct regulation of the h HSS promoter. In contrast, there was no significant effect in c-Jun over-expressed Cos7 and Hela cells. The tissue-specific function of c-Jun in h HSS promoter activity may in part help explain the differences in biology function of hHSS between liver and non-liver cells.

Moclobemide upregulated Bcl‐2 expression and induced neural stem cell differentiation into serotoninergic neuron via extracellular‐regulated kinase pathway

1. Moclobemide (MB) is an antidepressant drug that selectively and reversibly inhibits monoamine oxidase-A. Recent studies have revealed that antidepressant drugs possess the characters of potent growth-promoting factors for the development of neurogenesis and improve the survival rate of serotonin (5-hydroxytrytamine; 5-HT) neurons. However, whether MB comprises neuroprotection effects or modulates the proliferation of neural stem cells (NSCs) needs to be elucidated. 2. In this study, firstly, we used the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay to demonstrate that 50 microM MB can increase the cell viability of NSCs. The result of real-time reverse transcription-polymerase chain reaction (RT-PCR) showed that the induction of MB can upregulate the gene expressions of Bcl-2 and Bcl-xL. By using caspases 8 and 3, ELISA and terminal dUTP nick-end labeling (TUNEL) assay, our data further confirmed that 50 microM MB-treated NSCs can prevent FasL-induced apoptosis. 3. The morphological findings also supported the evidence that MB can facilitate the dendritic development and increase the neurite expansion of NSCs. Moreover, we found that MB treatment increased the expression of Bcl-2 in NSCs through activating the extracellular-regulated kinase (ERK) phosphorylation. 4. By using the triple-staining immunofluorescent study, the percentages of serotonin- and MAP-2-positive cells in the day 7 culture of MB-treated NSCs were significantly increased (P<0.01). Furthermore, our data supported that MB treatment increased functional production of serotonin in NSCs via the modulation of ERK1/2. In sum, the study results support that MB can upregulate Bcl-2 expression and induce the differentiation of NSCs into serotoninergic neuron via ERK pathway.

Coronary Endothelium: A Key to Life Expectancy

Endothelial dysfunction occurs early in the development of atherosclerosis. Normal endothelium maintains vascular stucture and tone by balancing vasoconstriction with vasodilatation. With endothelial dysfunction, production of nitric oxide (NO) is impaired, leaving unopposed vasoconstrictive substances such as angiotensin II. The endothelium releases factors that balance growth promotion and growth inhibition. Physiologically, growth-inhibiting activity predominates, maintaining the blood vessel wall homeostasis. With endothelial dysfunction, growth-promoting factors dominate, resulting in vascular overgrowth and deformation, which lead to thickening of the media and reduction in lumen size. These vascular changes are common in hypertension, renal failure, myocardial infarction, stroke, and diabetes. QUESTIONS 1. Which of the 3 layers of an artery initiate atherogenic lesions? a. the media b. the intima c. the adventitia

Collagen Matrix in Spinal Cord Injury

The fibrous scar that develops after central nervous system (CNS) injury is considered a major impediment for axonal regeneration. It consists of a dense collagen IV meshwork, which serves as a binding matrix for numerous other extracellular matrix components and inhibitory molecules like proteoglycans and semaphorins, but also growth-promoting factors. Inhibition of collagen matrix formation in brain and spinal cord lesions leads to axonal regeneration and functional recovery, although collagen IV per se is not inhibitory for axonal outgrowth. This review focuses on the molecular properties of the collagen IV matrix and its interactions with various molecules that are expressed after CNS lesion. Moreover, studies on collagen expression and matrix formation after injury of regenerating versus non-regenerating nervous systems are reviewed. Major differences in collagen deposition in the CNS and the peripheral nervous system (PNS) and differences in specific cell responses to extracellular matrix deposition in the lesion area are discussed. Therapeutic treatments aiming at suppression of fibrous scarring have been shown to promote axon regeneration in various lesion paradigms of the mammalian CNS.

Growth-promoting effect on iron-sulfur proteins on axenic cultures ofEntamoeba dispar

A growth-promoting factor (GPF) that promotes the growth of Entamoeba dispar under axenic culture conditions was found in fractions of mitochondria (Mt), hydrogenosomes (Hg) and chloroplasts (Cp) obtained from cells of six different protozoan, mammalian and plant species. We were able to extract the GPF from the Cp-rich leaf cells of a plant (spiderwort: Commelina communis L.) in an acetone-soluble fraction as a complex of chlorophyll with low molecular weight proteins (molecular weight [MW] approximately 4,600). We also found that on treatment with 0.6% complexes of 2-mercapthoethanol (2ME), complexes of chlorophyll-a with iron-sulphur (Fe-S) proteins (e.g., ferredoxins [Fd] from spinach and Clostridium pasteurianum) and noncomplex rubredoxin (Rd) from C. posteurianum have a growth-promoting effect on E. dispar. These findings suggest that E. dispar may lack a sufficient quantity of some essential components of Fe-S proteins, such as Fe-S center.

Cardiac and Vascular Hypertrophy in Fabry Disease

Fabry disease is an X-linked disorder resulting from alpha-galactosidase A deficiency. The cardiovascular findings include left ventricular hypertrophy (LVH) and increased intima-media thickness of the common carotid artery (CCA IMT). The current study examined the possible correlation between these parameters. To corroborate these clinical findings in vitro, plasma from Fabry patients was tested for possible proliferative effect on rat vascular smooth muscle cells (vascular smooth muscle cell [VSMC]) and mouse neonatal cardiomyocytes. Thirty male and 38 female patients were enrolled. LVH was found in 60% of men and 39% of women. Increased CCA IMT was equally present in males and females. There was a strong positive correlation between LV mass and CCA IMT (r2=0.27; P<0.0001). VSMC and neonatal cardiomyocyte proliferative response in vitro correlated with CCA IMT (r2=0.39; P<0.0004) and LV mass index (r2=0.19; P=0.028), respectively. LVH and CCA IMT occur concomitantly in Fabry suggesting common pathogenesis. The underlying cause may be a circulating growth-promoting factor whose presence has been confirmed in vitro.

Angiotensin II type 1 receptor expression in ovarian cancer and its correlation with tumour angiogenesis and patient survival

Angiotensin II, a main effector peptide in the renin–angiotensin system, acts as a growth-promoting and angiogenic factor via type 1 angiotensin II receptors (AT1R). We have recently demonstrated that angiotensin II enhanced tumour cell invasion and vascular endothelial growth factor (VEGF) secretion via AT1R in ovarian cancer cell lines in vitro. The aim of the present study was to determine whether AT1R expression in ovarian cancer is correlated with clinicopathological parameters, angiogenic factors and patient survival. Immunohistochemical staining for AT1R, VEGF, CD34 and proliferating cell nuclear antigen (PCNA) were analysed in ovarian cancer tissues (n=67). Intratumour microvessel density (MVD) was analysed by counting the CD34-positive endothelial cells. Type 1 angiotensin II receptors were expressed in 85% of the cases examined, of which 55% were strongly positive. Type 1 angiotensin II receptors expression was positively correlated with VEGF expression intensity and MVD, but not with histological subtype, grade, FIGO stage or PCNA labelling index. In patients who had positive staining for AT1R, the overall survival and progression-free survival were significantly poor (P=0.041 and 0.017, respectively) as compared to those in patients who had negative staining for AT1R, although VEGF, but not AT1R, was an independent prognostic factor on multivariate analysis. These results demonstrated that AT1R correlated with tumour angiogenesis and poor patient outcome in ovarian cancer, suggesting its clinical potential for a novel molecular target in strategies for ovarian cancer treatment.

CLIP-170 Homologue and NUDE Play Overlapping Roles in NUDF Localization inAspergillus nidulans

Proteins in the cytoplasmic dynein pathway accumulate at the microtubule plus end, giving the appearance of comets when observed in live cells. The targeting mechanism for NUDF (LIS1/Pac1) of Aspergillus nidulans, a key component of the dynein pathway, has not been clear. Previous studies have demonstrated physical interactions of NUDF/LIS1/Pac1 with both NUDE/NUDEL/Ndl1 and CLIP-170/Bik1. Here, we have identified the A. nidulans CLIP-170 homologue, CLIPA. The clipA deletion did not cause an obvious nuclear distribution phenotype but affected cytoplasmic microtubules in an unexpected manner. Although more microtubules failed to undergo long-range growth toward the hyphal tip at 32 degrees C, those that reached the hyphal tip were less likely to undergo catastrophe. Thus, in addition to acting as a growth-promoting factor, CLIPA also promotes microtubule dynamics. In the absence of CLIPA, green fluorescent protein-labeled cytoplasmic dynein heavy chain, p150(Glued) dynactin, and NUDF were all seen as plus-end comets at 32 degrees C. However, under the same conditions, deletion of both clipA and nudE almost completely abolished NUDF comets, although nudE deletion itself did not cause a dramatic change in NUDF localization. Based on these results, we suggest that CLIPA and NUDE both recruit NUDF to the microtubule plus end. The plus-end localization of CLIPA itself seems to be regulated by different mechanisms under different physiological conditions. Although the KipA kinesin (Kip2/Tea2 homologue) did not affect plus-end localization of CLIPA at 32 degrees C, it was required for enhancing plus-end accumulation of CLIPA at an elevated temperature (42 degrees C).

A METHOD FOR PROLONGED SURVIVAL OF PRIMARY CELL LINES

We have established a means for prolonged survival of primary cell cultures and establishment of continuous cell lines without genetic manipulations. Primary cultures of granulosa cells degenerate rapidly in vitro by a spontaneous onset of apoptotic cell death. Earlier attempts to circumvent this limitation have included transformation with oncogenes, spontaneous immortalization of primary cultures, and chemical carcinogenesis. We have found that addition of a complex of growth-promoting compounds, carrier proteins, and factors isolated from porcine follicular fluid to standard culture medium allows, reproducibly, the establishment of continuous porcine primary granulosa cell lines with genetic stability. This same supplement allows the prolonged survival of primary cell cultures derived from adult rat ovaries. The rat ovary primary cultures consisted of mixed phenotypes, including epithelial, neuron-like, and mesenchymal cell types. Numerous cells stain positive for alkaline phosphatase in these cultures. Other primary cell lines were established from embryonic rat liver and from adult rat lungs, using the same supplement. The survival effect is reversible because cells degenerate when the supplement is removed. Therefore, the cell lines have neither acquired properties of a tumor cell line nor have they been immortalized by a virus infection. We expect that our approach will open the door to prolonged survival of other primary cell types.