Primary Culture Medium Research Articles

Differences in nutrient uptake between the fat body and embryonic primary cultures of silkworm (Bombyx mori)

Abstract Nutrition utilization and by‐product formation in cultured insect cells has been investigated in several insect cells and has been of great interest to cell culturists and physiologists. In this research the biochemical changes in embryonic and fat body primary cultures of silkworm, Bombyx mori, have been compared. TC‐100 medium supplemented with 10% and 20% FBS was used in embryonic and fat body primary cultures, respectively. Medium was renewed every week and the amount of glucose, uric acid, urea, total protein and alkaline phosphatase were measured in the samples from medium of primary cultures using spectrophotometeric methods. All biochemical macromolecules except uric acid showed significant changes. Glucose decreased in embryonic tissues, while in fat body culture its amount increased. Urea accumulation in embryonic culture was higher than in the fat body cultures. Since urea is a by‐product, this accumulation could be due to higher utilization of amino acids. Total protein showed considerable changes and was consumed by embryonic culture more than the fat body's. Alkaline phosphatase showed stronger activity in embryonic cells.

44 NUCLEAR TRANSFER IN CATTLE USING SOMATIC CELLS FROM FROZEN TESTICLES WITHOUT CRYOPROTECTANTS

Obtaining somatic cells from preserved organs or tissues is useful for the conservation and regeneration of genetic resources by nuclear transfer (NT). Bovine cells for NT have been obtained from cooled carcasses stored at 0�C for several days (Arat et al. 2005 Reprod. Fert. Dev. 17, 164 abst) and from fetal skin tissue cryopreserved with DMSO (Fahrudin et al. 2001 J. Vet. Med. Sci. 63, 1151–1154). However, frozen storage of organs or tissues without cryoprotectants was considered to be quite inappropriate for obtaining viable cells. We report here that viable donor cells for NT were obtained from bovine testicles after frozen storage without cryoprotectants. In the first experiment, we investigated whether viable cells can be recovered from frozen testicles castrated from Japanese Black bulls. The testicles were frozen at -80�C in a freezer for several days; then some were stored in liquid nitrogen for 10 months without cryoprotectants. Before thawing, the testicles were divided into 3 pieces, caput epididymis, cauda epididymis, and testis. Each piece was then put in saline at 42�C for quick thawing. Thawed tissues were minced into 5-mm pieces and incubated at 39�C for 2 h in DMEM containing 0.1% collagenase and 0.2% dispase. After filtration through a 250-�m nylon mesh filter, the filtrates were centrifuged at 250 � 4g for 5 min. Then precipitates were resuspended with MF-start� primary culture medium (TM Cell Research Inc., Fukui, Japan) and incubated at 38.5�C under the atmosphere of 5% CO2 in air with high humidity. After 5 days of incubation, the medium was replaced and nonadherent debris was discarded. Viable cells were obtained from the caput epididymis. These cells actively proliferated and expanded. In the next experiment, to determine whether these cells can be used for NT, the cells were electrically fused with enucleated bovine oocytes. Bovine fibroblasts taken from unfrozen ear tissue were used as controls. The NT embryos were activated by Ca-ionophore treatment, followed by treatment with cycloheximide for 6 h, and then cultured in mSOF for 168 h. NT embryos reconstructed from testicle cells did not significantly differ from NT embryos made with control cells with regard to blastocyst rates (22.1% and 20.2%), cell number of blastocysts [130 � 43 and 121 � 43 (mean � SD)], and ICM ratio (21.1% and 22.6%), respectively (ANOVA). These results suggest that somatic cells derived from bovine frozen testicles can be used for nuclear transfer. Further studies are needed to examine whether viable cells can be obtained from other frozen organs or tissues. This study was partially supported by a grant from the Wakayama Prefecture Collaboration of Regional Entities for the Advancement of Technological Excellence, JST.

Selection of a standard culture medium for primary culture of Limulus polyphemus Amebocytes

This study provides information relevant to future research aimed at producing Limulus Amebocyte Lysate (LAL) in vitro, which would potentially reduce the need to harvest and bleed horseshoe crabs as in the current methods of LAL production. To address the need for primary culture of horseshoe crab amebocytes, this study tested the effects of a variety of standard insect cell culture media on amebocyte morphology and viability after 7 d of maintenance. Amebocyte morphology was least altered from in vivo form in Grace's Modified Insect Medium, with no observed degranulation of cells, as compared to the other media tested. There were significant differences in amebocyte viability among the six insect cell culture media tested. Grace's Modified Insect Medium sustained viability of 77.2 +/- 5.1% (mean +/- standard deviation) of amebocytes, followed distantly by Grace's Insect Medium with 35.1 +/- 8.7% amebocyte viability. Results indicate that Grace's Modified Insect Medium with horseshoe crab serum supplementation was the best candidate of the six media tested for future medium optimization for Limulus amebocyte requirements.

Single-chain Tissue-type Plasminogen Activator Is a Substrate of Mouse Glandular Kallikrein 24

Leydig cells of the adult mouse testis express at a detectable level three distinct glandular (tissue) kallikrein genes: mKlk21, mKlk24, and mKlk27. Recently, the proteins encoded by these genes were characterized using active recombinant proteases, but their roles in the mouse testis remained to be determined. The present study showed that among the proteases, mK24 markedly enhanced the activity of human recombinant single-chain tissue-type plasminogen activator when the two were incubated together. This activation was found to be due to proteolytic conversion of the single-chain enzyme to a two-chain form. The expression of tissue-type plasminogen activator in interstitial Leydig cells was demonstrated by RT-PCR and immunohistochemical analyses. The primary culture medium of adult male testicular Leydig cells contained immunoreactive substances recognized by anti-mK24 antibodies. In addition, the same medium was capable of converting the single-chain plasminogen activator to the two-chain protein. These results suggest that mK24 may play a role in the degradation of extracellular matrix proteins in the interstitial area surrounding the Leydig cells of the adult mouse testis, due not only to its own activity, but also to that of plasmin produced by the single-chain tissue-type plasminogen activator-converting activity of mK24.

An Approach to Studying Lung Cancer-related Proteins in Human Blood

Early stage lung cancer detection is the first step toward successful clinical therapy and increased patient survival. Clinicians monitor cancer progression by profiling tumor cell proteins in the blood plasma of afflicted patients. Blood plasma, however, is a difficult cancer protein assessment medium because it is rich in albumins and heterogeneous protein species. We report herein a method to detect the proteins released into the circulatory system by tumor cells. Initially we analyzed the protein components in the conditioned medium (CM) of lung cancer primary cell or organ cultures and in the adjacent normal bronchus using one-dimensional PAGE and nano-ESI-MS/MS. We identified 299 proteins involved in key cellular process such as cell growth, organogenesis, and signal transduction. We selected 13 interesting proteins from this list and analyzed them in 628 blood plasma samples using ELISA. We detected 11 of these 13 proteins in the plasma of lung cancer patients and non-patient controls. Our results showed that plasma matrix metalloproteinase 1 levels were elevated significantly in late stage lung cancer patients and that the plasma levels of 14-3-3 sigma, beta, and eta in the lung cancer patients were significantly lower than those in the control subjects. To our knowledge, this is the first time that fascin, ezrin, CD98, annexin A4, 14-3-3 sigma, 14-3-3 beta, and 14-3-3 eta proteins have been detected in human plasma by ELISA. The preliminary results showed that a combination of CD98, fascin, polymeric immunoglobulin receptor/secretory component and 14-3-3 eta had a higher sensitivity and specificity than any single marker. In conclusion, we report a method to detect proteins released into blood by lung cancer. This pilot approach may lead to the identification of novel protein markers in blood and provide a new method of identifying tumor biomarker profiles for guiding both early detection and therapy of human cancer.

Characteristics of the carbohydrate moiety and the intermolecular associations of the high-molecular-weight forms of alpha 2u-globulin.

We have investigated some of the biochemical characteristics of the glycosylated forms of α2u-globulin, the major protein of adult male rat urine. Labeled α2u-globulin that was secreted into the culture medium of primary monolayer cultures of hepatocytes was immunoprecipitated from the medium with antibody specific for urinary α2u-globulin and the complex used for further study. α2u-Globulin existed in the medium as a high-molecular-weight aggregate that could be dissociated with reducing agents and detergents into two glycosylated molecules of Mr 75000 and 50000 and one nonglycosylated molecule of Mr 20000. The glycosylated forms of α2u-globulin were shown by kinetic studies not to be precursors of the molecule of Mr 20000. When the glycoproteins were synthesized in the presence of radioactive sugar and amino acid precursors, the ratio of sugar to amino acid suggested that the highest-molecular-weight glycoprotein had 1.5–2 times the carbohydrate content of the faster-migrating glycoprotein. The oligosaccharide moiety, isolated from an exhaustive pronase digest of the combined glycoprotein molecules, was of Mr 5000 as determined by gel nitration methods. Sequential glycosidase digestion with neuraminidase, β-galactosidase, and β-N-acetylglucosaminidase produced a core oligosaccharide of Mr 1200. Anion-exchange chromatography demonstrated that the charge microheterogeneity of the α2u-globulin glycopeptides could be abolished by treatment with neuraminidase. Our results indicate that the oligosaccharide moiety of α2u-globulin is similar to those oligosaccharides found on other serum proteins, and suggest that formation of the species of α2u-globulin of Mr 75000 and 50000 requires either the attachment of many oligosaccharide chains to a single polypeptide of Mr 20000 or the covalent linkage of poly-peptides containing one or more oligosaccharide chains to form dimers and trimers.

Selection of a standard culture medium for primary culture of Limulus polyphemus amebocytes

Selection of a standard culture medium for primary culture of Limulus polyphemus amebocytes

UTILITY OF URICHROM II – A CHROMOGENIC MEDIUM FOR UROPATHOGENS

Purpose: Chromogenic media are increasingly being used as versatile tools in early differentiation and identification of bacterial isolates from clinical specimens. We have evaluated the Urichrome II, a newly introduced chromogenic medium, for its utility as a primary isolation and identification medium for urinary microbial isolates. Materials: This study included 5497 consecutively collected midstream and/or catheter catch urine samples obtained from patients attending several clinical services. They were inoculated on blood, MacConkey, cystine lactose electrolyte deficient, and Urichrom II agar plates for isolation of uropathogens. Results: The Urichrom II medium allowed the growth, and primary identification, of the predominant uropathogens like E.coli, Klebsiella spp. and Enterobacter spp. (KES group). The different coloured colonies produced by the breakdown of the chromogenic substrates by the specific enzymes of the bacteria were very useful in the presumptive identification of these organisms even from mixed cultures by the colour differences of the colonies. The medium also supported growth and differentiation of gram positive organisms like Staphylococcus aureus and enterococci. Conclusions: Urichrom II can be used as a primary culture medium for predominant uropathogens like E.coli, KES group and enterococci. It is an easy to use primary screening medium that considerably reduces the daily workload and thus minimizes or limits the use of identification tests.

63INTERFERON-TAU EXPRESSION FROM PRIMARY TROPHECTODERM OUTGROWTHS OF BOVINE BLASTOCYSTS: COMPARISON BETWEEN IVP, NT, AND PARTHENOGENIC EMBRYOS

Interferon-tau (IFN-tau) is expressed soon after bovine blastocyst formation and might be useful as a marker of appropriate biological function in embryos produced by nuclear transfer. To assess this possibility we have compared IFN-tau levels in the conditioned medium of primary trophectoderm cultures derived from IVP, nuclear transfer (NT), or parthenogenic bovine embryos. Embryos were produced from in vitro-matured cumulus-oocyte complexes processed from local slaughterhouse ovaries or obtained from Bomed, Inc. (Madison, WI, USA). In vitro fertilization, NT, and parthenogensis were as previously described (Talbot et al., 2000 Tissue and Cell, 32, 9–27) except that embryo culture was in G1/G2 medium in 5% oxygen (Lane et al., 2003 Theriogenology, 60, 407–419). Each 8–11-day embryo was cultured individually in a 4-well plate well (Nunc) with STO feeder cells using DMEM medium containing 10% fetal bovine serum as previously described (Talbot et al., 2000 Biol. Reprod. 62, 235–247). Any contaminating epiblast or endoderm was physically dissected and discarded so as to produce pure trophectoderm outgrowths. The success/failure ratio for colony formation was similar for IVP and NT embryos (IVP=155/29; NT=104/25), but was significantly different (P<0.05) for parthenogenic embryos (54/43). Trophectoderm colonies reached diameters of 1 to 1.5cm in 3–4wk, and, at this time, 72-h-conditioned cell culture medium was harvested, frozen, and measured for IFN-tau anti-viral activity as previously described (Talbot et al., 2000 Biol. Reprod. 62, 235–247). From 313 observations, IFN-tau production was analyzed as a two-factor mixed linear model. Differences in IFN-tau production by type of embryo were statistically significant (F=42.61; P<0.0001; df=2). Mean comparisons were done with Sidak adjusted P-values so that the experiment-wise error was 0.05. IFN-tau production means for IVP-, NT-, and parthenogenic-derived trophectoderm were 4311IUmL−1 (n=155), 626IUmL−1 (n=104), and 1595IUmL−1 (n=54), respectively. The results show that mean IFN-tau production from trophectoderm cultures derived from NT embryos is significantly reduced in comparison to IVP- and parthenogenote-derived cultures. Parthenogenote-derived cultures also produced significantly less IFN-tau than IVP embryos on average. IFN-tau production from trophectoderm outgrowths may be a useful measure of NT reprogramming success.

Colony morphology switching of Candida lusitaniae and acquisition of multidrug resistance during treatment of a renal infection in a newborn: case report and review of the literature

Candida lusitaniae is an emerging opportunistic pathogen which exhibits an unusual antifungal susceptibility pattern. We describe a case of fatal renal infection due to C. lusitaniae in a very low birth weight neonate who was treated with short courses of fluconazole given alternately with amphotericin B. A colony morphology switching was detected on the standard primary culture medium by changes in colony size. Switching was shown to affect deeply the susceptibility to amphotericin B. Afterwards, the switched phenotype developed a cross resistance to fluconazole and itraconazole. Several issues raised by this case are discussed in the light of an extensive review of the literature. Our observations point out the importance of both the detection of colony morphology switching and the close monitoring of antifungal susceptibility in the management of infections due to C. lusitaniae. A judicious therapeutic strategy should prevent the acquisition of multidrug resistance during antifungal therapy.

Role of tissue inhibitor of metalloproteases-1 in junction dynamics in the testis.

Using multiple high-performance liquid chromatography steps, we have identified and purified a polypeptide to apparent homogeneity from primary Sertoli cell conditioned culture medium that consisted of 2 molecular variants of 31 and 29 kDa when electrophoresed on a sodium dodecyl sulfate-polyacrylamide gel run under reducing conditions. Partial N-terminal amino acid sequence analysis of these 2 proteins revealed a sequence of NH(2)-IKMAKMLKGFDAVGNATG, which is homologous to tissue inhibitor of metalloproteases-1 (TIMP-1). Studies by semiquantitative reverse transcription-polymerase chain reaction using a primer pair specific to rat TIMP-1 demonstrated that both Sertoli and germ cells express TIMP-1. During maturation, the steady-state TIMP-1 mRNA level in the testis increased significantly from 40 to 60 days of age, which suggests its role in the restructuring of the epithelium during spermiation. This increase in testicular TIMP-1 expression was apparently not due to the increase in germ cell number, because TIMP-1 expression decreased approximately fivefold in germ cells isolated from testes of aging rats. Using Sertoli cells cultured at low (0.05 x 10(6) cells/cm(2)) and high (0.5 x 10(6) cells/cm(2)) densities, it was found that TIMP-1 expression increased transiently but significantly during junction assembly. A similar induction of TIMP-1 mRNA was also detected in Sertoli-germ cell cocultures during germ cell adhesion onto Sertoli cells. More important, the inclusion of either alpha(2)-macroglobulin (a protease inhibitor produced by Sertoli cells) or aprotinin (a serine protease inhibitor) into an in vitro germ cell adhesion assay facilitated the attachment of fluorescently labeled germ cells onto the Sertoli cell epithelium when compared to control, which suggests that the assembly of adherens junctions may involve protease inhibitors.

Correlation between airway responsiveness and proteoglycan production by bronchial fibroblasts from normal and asthmatic subjects

Asthma is characterized by an airway remodeling process involving altered extracellular matrix deposition such as collagen, fibronectin and proteoglycans. Proteoglycans determine tissue mechanical properties and are involved in many important biological aspects. Not surprisingly, it has been suggested that proteoglycan deposition may alter airway properties in asthma including airway hyperresponsiveness. In chronically inflamed airway tissues, fibroblasts likely represent an activated fibrotic phenotype that contributes to the excessive deposition of different extracellular matrix components. To investigate whether this was the case for proteoglycans, the production of hyaluronan, perlecan, versican, small heparan sulphate proteoglycans (HSPGs), decorin and biglycan was quantified in the culture medium of primary bronchial fibroblast cultures, established from four normal and six asthmatic subjects. Values were further correlated to the airway responsiveness (PC 20 methacholine) of donor subjects. Fibroblasts from subjects with the most hyperresponsive airways produced up to four times more total proteoglycans than cells from subjects with less hyperresponsive or normoresponsive airways. We observed a significant negative correlation between the PC 20 and perlecan, small HSPGs and biglycan, while such correlation was absent for decorin and close to significant for hyaluronan and versican. Altered proteoglycan metabolism by bronchial fibroblasts may contribute to the increased proteoglycan deposition in the bronchial mucosa and to airway hyperresponsiveness characterizing asthma.

Characterization of a cysteine proteinase inhibitor induced during neuronal cell differentiation.

A rat homolog of human cystatin E/M was identified by differential display of transcripts induced during neuronal cell differentiation. A member of the family 2 cystatins, rat cystatin E/M is secreted, glycosylated and developmentally regulated. Rat cystatin E/M is expressed in brain, and is induced during differentiation of a conditionally immortalized E17 rat hippocampal cell line (H19-7) by bFGF or activated Raf via MEK-dependent and -independent signaling pathways. Rat cystatin E/M protein is increased post-transcriptionally in PC12 cells, and the protein is secreted into the medium of primary embryonal hippocampal cultures. Analysis of the K (i) of recombinant His-tagged rat cystatin E/M toward cathepsins B and H revealed that rat cystatin E/M has an inhibitor profile distinct from that of other members of the cystatin family. Motif swapping between rat cystatin E/M and human cystatin C, a well-characterized cystatin, identified some residues that can contribute to the specificity of inhibition. Taken together, these results describe a member of the cystatin family that has a distinct inhibitor profile and may play a role in neuronal development.

An acidic fibroblast growth factor-like factor secreted into the brain cell culture medium upregulates apoE synthesis, HDL secretion and cholesterol metabolism in rat astrocytes

Production and release of apolipoprotein (apo) E and cholesterol were highly upregulated in the astrocytes prepared by 1-week secondary culture after 1-month primary culture of rat fetal brain cells (M/W cells) in comparison to the cells prepared by a conventional method of 1-week primary and 1-week secondary culture (W/W cells). Both cell preparations were mostly composed of astrocytes with small population of other glial cells, except that type-2 astrocyte-like cells accounted for 5–15% of M/W cells indicating more activated and/or matured status. The conditioned medium of the 1-month primary culture stimulated W/W cells to increase the release of apoE and cholesterol into the medium. The treatment of W/W cells by acidic fibroblast growth factor (aFGF) similarly upregulated biosyntheses and release of apoE and cholesterol. The effect of the conditioned medium was completely inhibited by pretreatment with an anti-aFGF antibody. The increase of the aFGF message was demonstrated in the brain cells after 1-month primary culture. The findings suggested that an aFGF-like trophic factor upregulates biosynthesis and secretion of apoE-high density lipoprotein (HDL) in astrocytes probably by autocrine stimulation in this culture system. Since this cytokine is highly expressed in the development or post-injury period of the brain, it putatively activates intercellular cholesterol transport to support construction or recovery of the brain.

Novel adaptation of brush cytology technique for short-term primary culture of squamous and Barrett's esophageal cells

Background: Esophageal disease research is impeded by a paucity of nonmalignant cell lines. Furthermore, culture of primary esophageal cells is difficult because of a lack of cell adhesion and contamination. The aim of this study was to develop a short-term culture method to facilitate cell physiologic studies by using primary esophageal cells. Methods: By using a cytology brush, squamous and Barrett's epithelial cells were obtained from the esophagus of patients undergoing upper endoscopy. Cells were brushed onto chamber slides and allowed to adhere to the surface. Primary culture media was then added and cells were maintained at 37°C for up to 72 hours. Cell yield and viability were calculated after trypan blue staining. For cell physiologic studies, cells were loaded with pH-sensitive dye BCECF-AM and intracellular pH measured on a dual excitation fluorescence microscope after an ammonium chloride prepulse. At the end of the physiologic experiments, cells were fixed in methanol and acetone and the cell type was verified with cytokeratin immunocytochemistry. Results: Viable human esophageal cells were maintained in culture for up to 72 hours. The cells extruded trypan blue, and BCECF-AM was cleaved to BCECF by an intact cell membrane and permitted intracellular pH measurements. The epithelial cell origin of the cells was confirmed by cytokeratin staining. There was no contamination over the culture period and no overgrowth by lymphocytes or fibroblasts. Conclusions: This novel adaptation of brush cytology technique enables short-term culture of esophageal cells for in vitro studies. This rapid, simple primary culture technique is suitable for endoscopy and does not require the immediate, time-consuming laboratory techniques. (Gastrointest Endosc 2001;54:186-9.)

Novel adaptation of brush cytology technique for short-term primary culture of squamous and Barrett's esophageal cells☆, ☆☆, ★

<h2>Abstract</h2> <b>Background:</b> Esophageal disease research is impeded by a paucity of nonmalignant cell lines. Furthermore, culture of primary esophageal cells is difficult because of a lack of cell adhesion and contamination. The aim of this study was to develop a short-term culture method to facilitate cell physiologic studies by using primary esophageal cells. <b>Methods:</b> By using a cytology brush, squamous and Barrett's epithelial cells were obtained from the esophagus of patients undergoing upper endoscopy. Cells were brushed onto chamber slides and allowed to adhere to the surface. Primary culture media was then added and cells were maintained at 37°C for up to 72 hours. Cell yield and viability were calculated after trypan blue staining. For cell physiologic studies, cells were loaded with pH-sensitive dye BCECF-AM and intracellular pH measured on a dual excitation fluorescence microscope after an ammonium chloride prepulse. At the end of the physiologic experiments, cells were fixed in methanol and acetone and the cell type was verified with cytokeratin immunocytochemistry. <b>Results:</b> Viable human esophageal cells were maintained in culture for up to 72 hours. The cells extruded trypan blue, and BCECF-AM was cleaved to BCECF by an intact cell membrane and permitted intracellular pH measurements. The epithelial cell origin of the cells was confirmed by cytokeratin staining. There was no contamination over the culture period and no overgrowth by lymphocytes or fibroblasts. <b>Conclusions:</b> This novel adaptation of brush cytology technique enables short-term culture of esophageal cells for in vitro studies. This rapid, simple primary culture technique is suitable for endoscopy and does not require the immediate, time-consuming laboratory techniques. (Gastrointest Endosc 2001;54:186-9.)

Determination of pyruvate and lactate in primary liver cell culture medium during hypoxia by on-line microdialysis–liquid chromatography

A microdialysis sampling device was constructed for the measurement of pyruvate and lactate in primary liver cell culture medium during hypoxia. It was composed of a Petri dish, a dialysis membrane and two transmission tubes within a hypoxia chamber. The dialysis membrane was located in the Petri dish such that it was immersed in the culture medium. Dialysates were collected and introduced by an on-line injector to a liquid chromatographic system for analysis of pyruvate and lactate. The detection limit of this assay was 0.2–2.0 μ M with acceptable intra- and inter-assay reproducibilities. In order to validate the assay, primary liver cells were incubated in the Petri dish within a hypoxia chamber in an incubator. The baseline concentrations of pyruvate and lactate in primary liver cell culture medium were 10.6±5.6 and 607±143 μ M, respectively. These levels drastically changed during hypoxia and reperfusion. In conclusion, the present assay provides a sensitive, direct measurement of pyruvate and lactate in culture medium while minimizing pretreatment procedures for sample preparation.

Identification of Macrophage Migration Inhibitory Factor as a Potent Endothelial Cell Growth-Promoting Agent Released by Ectopic Human Endometrial Cells1

The growth of endometrial cells in ectopic locations (endometriosis) is dependent on the establishment of an adequate blood supply. Neovascularization (angiogenesis) is therefore a vital step toward the progression of this disease. We first revealed the presence of a potent mitogenic activity for human endothelial cells in the culture medium of immortalized human endometriotic cells. The activity, measured by the level of [(3)H]-thymidine incorporation into the DNA of human coronary artery endothelial cells, was then purified by anion exchange high-performance liquid chromatography. Electrophoretic analysis of one of the bioactive fractions that markedly enhanced endothelial cell proliferation showed three distinct bands with apparent molecular masses of 15.8, 12.6, and 6.5 kDa. N-terminal microsequencing of an internal peptide from the 12. 6-kDa protein showed 100% homology with human macrophage migration inhibitory factor (MIF). The protein was positively identified as MIF by Western blot analysis using a specific anti-MIF antibody. Anti-MIF antibody inhibited the bioactivity found in the evaluated fraction and the conditioned medium of primary endometriotic cell cultures, and commercial recombinant human MIF displayed a high mitogenic activity for endothelial cells. Our findings reveal that MIF is released by endometriotic cells and acts as a potent mitogenic factor for human endothelial cells in vitro. This may have a considerable interest, in view of the crucial role of angiogenesis in ectopic endometrial cell growth and activity and in numerous tissues undergoing dynamic physiological changes, such as human endometrium.

Changes in beta-2 microglobulin expression in prostate cancer

Increased shedding of beta-2 microglobulin (B2m, 11.8 kDa), a component of the major histocompatibility complex class I (MHC I), by tumor cells has significance with regard to escape from immune-surveillance, cellular proliferation, and tumor development and progression. Aberrant expression of MHC I is known to occur in prostate cancer (PCa) but not in benign prostatic hyperplasia. We have determined B2m released by PCa cells in culture and in the urine of 101 patients with advanced PCa by Western blotting and radioimmunoassay. B2m levels in the conditioned medium of human PCa cell lines and primary cultures derived from distant metastasis as well as in the urine of patients with bone and visceral metastasis were higher than normal and also higher than those from patients with local/regional extensions of the disease. In the group of patients with bone metastasis (66 patients), high urine B2m was associated with significantly shortened survival. In addition to the 11.8 kDa B2m, a high molecular weight B2m immunoreactivity of approximately 38 kDa was found in highly tumorigenic PC-3 cell line, but not in the relatively indolent DU-145 and LNCaP human PCa cell lines. The approximately 38 kDa B2m was found in the urine of several PCa patients but not of healthy controls examined by Western analysis. The conditioned medium of a prostatic small cell carcinoma cell line, NCI-H660, had high levels of chromogranin A and B2m, but prostate specific antigen was absent. In conclusion, increased B2m shedding was associated with distant metastasis of PCa and an abnormal B2m immunoreactivity was found in PCa.

Insulin-like growth factor (IGF) system in the bovine mammary gland and milk.

Primary bovine mammary cells express the two IGF receptors (IGF-IR, IGF-IIR), insulin receptor, and four IGFBPs (IGFBP-2, -3, -4, and -5). Examination of the IGF-IR during the mammary gland lactation cycle shows that IGF-IR number declines at parturition, a change that coincides with decreases in the blood level of its ligand, IGF-I. IGF-II and IGF-IIR are largely unchanged. IGFBP-3 is the predominant mammary IGFBP and its concentration also declines in blood and milk during lactation compared to prepartum and involution periods. Time of lactation and pregnancy were the main determinants of milk but not blood IGFBP-3 levels. IGFBP-3 binds to membrane proteins of bovine mammary tissue; an IGFBP-3 binding protein has been identified as bovine lactoferrin. Lactoferrin has the capacity to compete with IGF binding to IGFBP-3. Appearance of both IGFBP-3 and lactoferrin in conditioned media of primary cultures of bovine mammary cells was stimulated by all trans retinoic acid (atRA). Furthermore, atRA was necessary for the entry of exogenously added lactoferrin into the mammary cell nucleus, while IGFBP-3 entry into the nuclei of atRA treated cells required the presence of lactoferrin. These findings reveal a novel role for lactoferrin, suggesting that lactoferrin is critically involved in the regulation of the IGF system during the involution period.