Quality validation of platelets obtained from the Haemonetics and Trima Accel automated blood-collection systems.

BACKGROUND: Platelet transfusion is required to treat haemo-oncology or trauma patients. Platelet apheresis (PA) performed with apheresis equipment has increased rapidly in recent years. Leucocyte-reduced platelet apheresis (LRPA) can reduce the risk of platelet refractoriness and febrile nonhemolytic transfusion reactions (FNHTRs) for transfusion. Accordingly, this study aimed to investigate and compare the platelet metabolic and functional responses between PA performed with Haemonetics and LRPA performed with Trima Accel cell separator. METHODS: The qualities of platelets collected through PA and LRPA were evaluated in terms of visual appearance, morphology, platelet-aggregation changes, metabolic activities, and bacterium-screening test during 5-day storage. Statistical analyses included two-sample t-test and generalised estimating equation(GEE) method. RESULTS: During 5-day storage in LRPA, residual leucocytes were all <1.0×106, and the parameters of platelet function were as follows: platelet aggregated to agonists such as adenosine 5'-diphosphate (ADP) and collagen, and the extent of shape change and pO2 showed no statistically significant difference between PA and LRPA. The hypotonic shock reaction (HSR) on days 0, 1, and 3 were significantly higher in LRPA than in PA (71.78±6.92 vs. 64.10±7.42; P=0.002; 71.53±8.98 vs. 62.96±9.84; P=0.007; 68.05±7.28 vs. 57.76±6.80; P<0.0001, respectively). Values of mean platelet volume (MPV) were statistically larger in PA than in LRPA on days 0, 1, and 3. On day 5, the swirling score was higher in LRPA than in PA. The mean lactate levels had no statistically significant difference between PA and LRPA. Moreover, no growth was observed through bacterium-screening test conducted on 40 samples. CONCLUSION: Comparison of LRPA and PA products collected from the Trima Accel and Haemonetics automated blood-collection systems, respectively, revealed that both products possessed good platelet qualities even though additional processes are needed to reduce leucocytes. Furthermore, investigating the outcomes of other apheresis instruments with focus on the safety of donors, products, and recipients is necessary.

The ERK-ZEB1 pathway mediates epithelial-mesenchymal transition in pemetrexed resistant lung cancer cells with suppression by vinca alkaloids.

High thymidylate synthase (TS) level in cancer tissue is considered to result in resistance to pemetrexed therapy for advanced stages of nonsquamous non-small cell lung cancers. To further investigate the mechanism of pemetrexed resistance and potential prognostic outcomes in lung cancer, we established pemetrexed-resistant lung adenocarcinoma cell sublines from CL1 harboring a mutated TP53 gene (R248W) and A549 harboring wild-type TP53. We found the TS expression is upregulated in both pemetrexed-resistant sublines and the reduced TS level achieved through shRNA inhibition resulted in higher pemetrexed sensitivity. We also demonstrated that the acquisitions of pemetrexed resistance enhances epithelial-mesenchymal transition (EMT) in vivo with a mice animal model and in vitro with CL1 and A549 sublines, which was associated with upregulation of ZEB1 which, in turn, downregulates E-cadherin and upregulates fibronectin. When ERK1/2 phosphorylation was reduced by an inhibitor (U0126) or siRNA inhibition, both pemetrexed-resistant sublines reduced their migration and invasion abilities. Therefore, the ERK-mediated pathways induce apoptosis with pemetrexed treatment, and may in turn mediate EMT when cancer cells are resistant to pemetrexed. We further demonstrated that the growth of pemetrexed-resistant tumors could be inhibited by vinblastine in vivo and vincristine in vitro. Our data indicate that pemetrexed resistance could be relieved by non-cross-resistant chemotherapeutic drugs such as vinca alkaloids and might be independent to TP53 status. Furthermore, the phosphorylation of ERK was reduced by vincristine. This finding provides a new insight for overcoming pemetrexed resistance and metastasis by application of vinca alkaloids.

Differential effects of grape juice on gastric emptying and renal function from cisplatin-induced acute adverse toxicity.

Grape skin and seeds contain large amounts of phytochemicals such as polyphenols, resveratrol, and proanthocyanidins, which possess antioxidant activities. Cisplatin is widely used in the treatment of cancer. High doses of cisplatin have also been known to produce acute adverse effects. The aim of this study was to investigate the protective effects of antioxidant properties of whole grape juice (with skin and seeds) on cisplatin-induced acute gastrointestinal tract disorders and nephrotoxicity in Wistar rats. Gastric emptying is significantly increased in whole grape juice-pretreated rats when compared to cisplatin treatment alone. The expression of ghrelin mRNA of stomach is increased in rats with whole grape juice. However, pretreatment with whole grape juice did not reduce renal function markers in acute renal toxicity. No significant changes were recorded in the oxidative stress/antioxidant status parameters of any study group. In contrast, pretreatment with whole grape juice slightly improved tubular cell vacuolization, tubular dilatation, and cast formation in renal tubules. These results show that consumption of whole grape juice induces somewhat beneficial effects in preventing cisplatin-mediated dyspepsia but does not offer protection against cisplatin-induced acute renal toxicity.

Low/negative expression of DDX3 might predict poor prognosis in non-smoker patients with oral cancer.

OBJECTIVE: DDX3 has diverse biological functions in translation control, cell growth regulation, and tumor progression. Oral squamous cell carcinoma (OSCC) is a common malignant tumor worldwide with a poor clinical prognosis. The impact of DDX3 expression in OSCC is seldom discussed. MATERIALS AND METHODS: Tumor tissues and adjacent normal tissues were obtained from 324 patients with OSCC. In this study, we used immunohistochemical staining methods to investigate the associations between DDX3 expression and the clinicopathological characteristics of OSCC. RESULTS: Low/negative DDX3 expression in tumor cells was significantly associated OSCC patient characteristics including male gender (P < 0.001), smoking (P < 0.001), alcohol consumption (P < 0.001), betel quid chewing (P = 0.002), poor relapse-free survival (P = 0.001), and poor overall survival (OS) (P = 0.001). Patients with low/negative DDX3 expression, and particularly non-smoker OSCC patients, had significantly worse OS as defined by the log-rank test (P = 0.020 for all cases; P = 0.008 for non-smoker patients). In non-smoker patients with OSCC, low/negative DDX3 expression in tumor cells was associated with poor prognosis (P = 0.024) and a 3.802-fold higher death risk, as determined by Cox regression. CONCLUSIONS: Low/negative DDX3 expression in tumor cells was significantly associated with aggressive clinical manifestations and might be an independent survival predictor, particularly in non-smoker patients with OSCC.

Expression and purification of a recombinant Fip-fve protein from Flammulina velutipes in baculovirus-infected insect cells.

AIMS: To develop an efficient and facile expression system supply of high purity and stable activity of rFip-fve for oral administration, medicinal study and applications. METHODS AND RESULTS: A recombinant virus that contained the chimera gene, encoding a bombyxin signal peptide sequence fused to a Fip-fve-6His sequence, was constructed. The rFip-fve was purified from the supernatant of the infected Sf21 cells using a nickel-chelated affinity column, and was verified by Western blot and MALDI-MS (matrix-assisted laser desorption ionization mass spectrometry) analyses. Results showed that a glycosylated mature rFip-fve was produced and secreted into the infected cell supernatant. The immunomodulatory activity of rFip-fve was evaluated by measuring the amount of interleukin-2 released from murine splenocytes. CONCLUSIONS: A reliable scheme to express and purify active rFip-fve in a baculovirus/insect cell system for medicinal applications and genetic study is a feasible means of solving potential problems related to the production and activity of rFip-fve protein. SIGNIFICANCE AND IMPACT OF THE STUDY: The rFip-fve expressed in insect cells was processed and modified in a manner more similar to that of its native counterpart than that in bacterial cells. Therefore, the potential applications of rFip-fve that is generated in Sf21 cells can be more effectively evaluated that produced in Escherichia coli.

Immunoreactivities of human nonmetastatic clone 23 and p53 products are disassociated and not good predictors of lymph node metastases in early-stage cervical cancer patients.

OBJECTIVE: To assess the relation between expressions of human nonmetastatic clone 23 (nm23-H1) and p53 in cervical cancer, their relationships with lymph node metastasis, and further to examine their predictive of lymph node metastases. MATERIALS AND METHODS: nm23-H1 and p53 expression profiles were visualized by immunohistochemistry in early-stage cervical cancer specimens. RESULTS: Immunoreactivities of nm23-H1 and p53 were disassociated. The independent variables related with lymph node metastases were grade of cancer cell differentiation (p < 0.029) and stromal invasion (p < 0.039). Sensitivity, specificity, positive and negative predictive values, and accuracy for lymph node metastasis were calculated to be 91.7%, 13.5%, 25.6%, 83.3%, and 32.7% for nm23-H1 and 66.7%, 51.4%, 30.8%, 82.6%, and 55.1% for p53. CONCLUSION: Nm23-H1 and p53 are disassociated and not good predictors of lymph node metastases in early-stage cervical cancer patients. However, stromal invasion and cell differentiation can predict lymph node metastasis.

Concurrent high expression of human telomerase reverse transcriptase and human nonmetastatic clone 23 in high-grade squamous intraepithelial neoplasia and squamous cell carcinoma of uterine cervix.

Human telomerase reverse transcriptase (hTERT) and human nonmetastatic clone 23 (nm23-H1) may be separately involved in tumor progression of uterine cervix. We therefore investigate the correlations of hTERT and nm23-H1 in cervical carcinogenesis and further check their application. One hundred and twenty-eight cervical tissues, including 48 squamous cell carcinoma (SCC), 36 high-grade cervical intraepithelial neoplasia (CIN) (CIN 2 and CIN 3), 20 low-grade CIN 1, and 24 normal cases, were collected for immunohistochemical expression of hTERT and nm23-H1. Spearman rank correlation analysis was applied to assess their correlation in these samples. The Fisher exact or Chi-square test was used to evaluate the expression of hTERT or nm23-H1 among each subgroup. The sensitivity, specificity, positive and negative predictive values (PPV and NPV), and accuracy of hTERT and/or nm23-H1 were calculated for the prediction of high-grade CIN and SCC. We found normal cervix and CIN 1 samples had concurrent low expression of hTERT and nm23-H1, whereas high-grade CIN and SCC samples had concurrent high immunoreactivities. The hTERT alone and hTERT or nm23-H1 in combination had better sensitivity, NPV, and accuracy. The nm23-H1 alone as well as hTERT and nm23-H1 in combination had better specificity and PPV. Our results reveal a significantly positive relationship between expression of hTERT and nm23-H1 in normal and neoplastic tissues of uterine cervix. We suggest high expression of hTERT alone and hTERT or nm23-H1 in combination can be offered additional molecular information correlated with high-grade CIN and SCC.

Implication of human telomerase reverse transcriptase in cervical carcinogenesis and cancer recurrence.

The objective of this study was to evaluate the implication of human telomerase reverse transcriptase (hTERT) in cervical carcinogenesis and cancer recurrence. One hundred three cases of uterine cervix, including 20 normal, 13 low-grade squamous intraepithelial lesion (LSIL), 30 high-grade squamous intraepithelial lesion (HSIL), and 40 squamous cell carcinoma (SCC) tissues, were evaluated for hTERT immunoreactivity. The expressions of hTERT in normal, LSIL, HSIL, and SCC tissues were compared by Fisher exact or Chi-square test. The relationships between hTERT and clinicopathologic variables of SCC were also assessed. Furthermore, SCC patients were subdivided into negative and positive hTERT expression subgroups, and Kaplan-Meier curves were used to plot the cumulative recurrence hazard for 5 years. There was a significant difference for hTERT expression between LSIL and HSIL subgroups (P < 0.001) but no significant difference between normal and LSIL as well as HSIL and SCC subgroups. For SCC patients, hTERT expression was positive in lymph nodes, vagina, and parametrium metastastic cases. However, it did not reach a significant difference. The cumulative recurrence hazard for 5 years was about 29% in positive hTERT expression subgroup compared to 0% in negative hTERT subgroup (P = 0.2866). In conclusion, a point stage of HSIL exists in the progression of cervical carcinogenesis when the hTERT expression increases significantly. Moreover, SCC patients with positive hTERT expression may have higher cumulative recurrence hazard.

Nm23-H1 immunohistochemical expression in multisteps of cervical carcinogenesis.

To evaluate the expression of nm23-H1 protein in normal, precancerous, and cancerous tissues of the uterine cervix and its role in cervical carcinogenesis, metastasis and recurrence, 82 cervical specimens, including 30 squamous cell carcinomas (SCC), 19 high- and 13 low-grade squamous intraepithelial lesions (HSILs and LSILs) and 20 normal samples, were stained using immunohistochemical method with streptavidin-biotin peroxidase immunostaining. The Chi-square and Fisher's exact tests, as well as Chi-square test for trend, were used for comparing nm23-H1 expression among normal, LSIL, HSIL, and cancerous tissues. The correlation of nm23 expression with metastasis or recurrence was analyzed using Fisher's exact test. There were significant differences in levels of nm23-H1 expression between LSIL and HSIL (P = 0.016) and between LSIL and SCC (P = 0.004), but not between HSIL and SCC or normal and LSIL samples. Furthermore, a positive relationship was demonstrated for high nm23-H1 protein expression and degree of malignant transformation (P < 0.05). Among the 30 SCC cases, high nm23-H1 expression did not show significant association with either carcinomatous metastasis or recurrence (P = 0.123, 0.372, respectively). High nm23-H1 expression appears related to critical progression of LSIL to HSIL but not to metastasis or recurrence of SCC.

Cooking oil fume-induced cytokine expression and oxidative stress in human lung epithelial cells.

Epidemiological studies have shown an association between exposure to indoor air pollution from Chinese-style cooking and risk of lung cancer among Chinese females. Several toxic substances have been identified in cooking oil fumes (COF) collected from heated rapeseed oil. In this study, we examined the biological effects of COF on CL3 human lung epithelial cells. Exposure to 200 microg/ml COF significantly reduced cell growth within 4 days. In addition, we examined the effect of COF on TGFbeta1, TGFbeta2, IL-6, IL-8, and IFN-gamma gene expressions with the RT-PCR method. We found that TGFbeta1 mRNA levels increased after exposure to 200 microg/ml COF for 24 h. Similarly, exposure to 10 microM benzo[a]pyrene or 100 nM 12-O-tetradecanoylphorbol-13-acetate increased TGFbeta1 mRNA levels at 24 h. The mRNA levels of TGFbeta2, IL-6, IL-8, and IFN-gamma did not increase after treatment with COF, benzo[a]pyrene, or 12-O-tetradecanoylphorbol-13-acetate. COF-induced TGFbeta1 production was confirmed by quantification of TGFbeta1 in conditioned medium with enzyme-linked immunosorbent assay. Exposure to 200 microg/ml COF significantly increased TGFbeta1 secretion in a time-dependent and dose-dependent manner. It has been demonstrated that reactive oxygen intermediates induce TGFbeta1 gene expression. When CL3 cells were exposed to 200 microg/ml COF for 15 min, there was an increase in intracellular peroxide formation with the dichlorofluorescein method. Furthermore, treatment with 200 microg/ml COF for 12 h also significantly induced lipid peroxidation in CL3 cells. Our results show that exposure to COF inhibits cell growth, increases TGFbeta1 secretion, and induces oxidative stress in CL3 lung epithelial cells. This suggests that TGFbeta1 and oxidative stress play a role in the biological effects of COF on lung epithelial cells.

Determination of tetracycline and streptomycin in mixed fungicide products by capillary zone electrophoresis.

A method of capillary zone electrophoresis (CZE) was used to determine tetracycline and streptomycin content in commercial agriculture products. The results indicated that this method was capable of analyzing the mixed fungicide in formulated products with instrument detection limit (IDL) of 0.50 microg/mL and a method detection limit (MDL) of 0.52 microg/mL for tetracycline, and IDL of 1.00 microg/mL and MDL of 1.22 microg/mL for streptomycin. Precision expressed by relative standard deviation (RSD) ranged from 1.44 to 4.37% of tetracycline and 1.00 to 4.20% of streptomycin. Recoveries were in the region of 98.2-102.5% for tetracycline and 95.3--103.0% for streptomycin. The low detection limit, the low RSD values, and the high percentage of recovery confirmed that the CZE technique is a sensitive and selective method. And the CZE method can analyze both tetracycline and streptomycin at the same time without complicated extraction and further derivative reaction.

Determination of destruxins, cyclic peptide toxins, produced by different strains of Metarhizium anisopliae and their mutants induced by ethyl methane sulfonate and ultraviolet using HPLC method.

Metarhizium anisopliae produces a family of cyclic peptide toxins, destruxins (DTXs), which exhibit various insecticidal activity. Four major DTXs have been separated by HPLC and identified by the liquid chromatography electrospray mass spectrometry (LC-ESI-MS) methods. Strain F061 of M. anisopliae produced large amounts of (DTXs), especially DTX-A (12.84+/-0.04 microg/ml), DTX-B (66.89+/-2.57 microg/ml) and DMDB (1.41+/-0.13 microg/ml). High levels of DTX-E (4.19+/-0.13 microg/ml) were produced by strain F007 of M. anisopliae. The results of our studies also showed that either ethyl methane sulfonate (EMS) or ultraviolet (UV) can significantly increase the production of DTXs. Mutant 61E-9 produced high levels of DTX-A (30.05+/-1.97 microg/ml), DTX-B (110.37+/-10.02 microg/ml) and DMDB (8.30+/-0.45 microg/ml). High levels of DTX-E (20.59+/-2.65 microg/ml) were produced by mutant 7E-3. Both mutant strains are suitable for industrial fermentation processes and possess a wide range of potential applications in the area of metabolic toxin production.

Single-stranded DNA-binding complex involved in transcriptional regulation of mouse mu-opioid receptor gene.

Previously, we reported the presence of dual (distal and proximal) promoters in mouse mu-opioid receptor (mor) gene, with mor transcription in mouse brain predominantly initiated by the proximal promoter. Sp factors, bound to double-stranded (ds) cis-regulatory elements, are critical for proximal promoter activity. Here, we further report that a single-stranded (ss) cis-regulatory element and trans-acting protein factor are also important for proximal promoter activity. A 26-bp mor polypyrimidine/polypurine region (PPy/u) can adopt ss DNA conformation, as demonstrated by S1 nuclease sensitivity. Using electrophoretic mobility shift analysis with nuclear extracts from mor-expressing SH-SY5Y cells, we demonstrate that the sense strand of PPy/u interacts with a major nuclear protein, termed mor polypyrimidine-binding protein (mPy), which is not related to Sp factors. Southwestern blot analysis indicated that mPy protein is approximately 25 kDa in size. Functional analysis suggests that mPy protein can trans-activate mor promoter as well as a heterologous promoter. Moreover, combinatorial activation of ss (mPy) and ds (Sps) DNA binding factors, interacting with an overlapping DNA (PPy/u) region, is necessary for proximal promoter activation. Thus our results suggest that transcription of mouse mor gene is regulated by an interplay of ss and ds DNA binding factors.

MDM2 mRNA expression is a favorable prognostic factor in non-small-cell lung cancer.

MDM2 is one of the downstream target genes for transcriptional activation by the product of the p53 tumor-suppressor gene. Transactivation of MDM2 gene expression is represented by the presence of a functional p53 protein. We hypothesized that MDM2 mRNA expression may be a more suitable prognostic factor than p53 or MDM2 protein expression and p53 gene mutations. In this study, expression of MDM2 mRNA, p53 protein, and MDM2 protein and mutations of the p53 gene were assessed in 81 lung tumor tissue specimens using RT-PCR, immunohistochemistry, and direct sequencing among exons 5-8, respectively. By immunohistochemistry, 33 and 42 of 81 patients with p53 (40.7%) and MDM2 (51.5%) protein expression were found in lung tumor specimens, respectively. The p53 direct sequencing data indicated that 13 of 81 patients (16.0%) had p53 mutations. However, Kaplan-Meier analysis showed that p53 protein and MDM2 protein expression and p53 mutation were not useful as prognostic factors. Interestingly, the survival of patients with MDM2 mRNA expression was longer than that of patients without MDM2 mRNA expression, though MDM2 mRNA expression was not associated with clinicopathological parameters, including tumor grade, tumor stage, tumor type, and TNM values. Moreover, Cox regression analysis showed that MDM2 mRNA expression was a significantly independent favorable prognostic factor in non-small-cell lung cancer (NSCLC) patients. Thus, measuring MDM2 mRNA expression using RT-PCR may be a simple, useful approach for predicting the survival of NSCLC patients.

Transcriptional regulation of mouse delta-opioid receptor gene.

Three major types of opioid receptors, mu (MOR), delta (DOR), and kappa (KOR), have been cloned and characterized. Each opioid receptor exhibits a distinct pharmacological profile as well as a distinct pattern of temporal and spatial expression in the brain, suggesting the critical role of transcription regulatory elements and their associated factors. Here, we report the identification of a minimum core promoter, in the 5'-flanking region of the mouse DOR gene, containing an E box and a GC box that are crucial for DOR promoter activity in NS20Y cells, a DOR-expressing mouse neuronal cell line. In vitro protein-DNA binding assays and in vivo transient transfection assays indicated that members of both the upstream stimulatory factor and Sp families of transcription factors bound to and trans-activated the DOR promoter via the E box and GC box, respectively. Furthermore, functional and physical interactions between these factors were critical for the basal as well as maximum promoter activity of the DOR gene. Thus, the distinct developmental emergence and brain regional distribution of the delta opioid receptor appear to be controlled, at least in part, by these two regulatory elements and their associated factors.

Visualization of time-dependent redistribution of delta-opioid receptors in neuronal cells during prolonged agonist exposure.

To date, the visualization of delta-opioid receptor (DOR) internalization has been largely focused on the events of short-term agonist treatment in transfected non-neuronal cells. In this study, we followed DOR trafficking upon prolonged agonist exposure in the neuronally derived neuro2a cells, stably transfected with the fusion DOR (HA-DOR) cDNA. Internalization of surface DOR was clearly visualized in 5 min of exposure to agonist (100 nM DADLE), and the cell surface DOR remained low throughout the entire 24 h agonist exposure. Significant intracellular accumulation was visible at 20 min exposure, and increased to a maximum at 4 h, after which intracellular DOR staining gradually diminished. DOR intracellular staining was enhanced in the presence of agonist and chloroquine, a lysosomotropic agent, suggesting that internalized receptors were targeted to lysosomes and degraded upon prolonged treatment. Time-dependent colocalization of DOR with transferrin and LAMP-2 following short-term and prolonged agonist exposure further confirmed that receptor was distributed to early endosomes (sequestration) and subjected to lysosomes for degradation (down-regulation), respectively.

Mouse mu opioid receptor gene expression. A 34-base pair cis-acting element inhibits transcription of the mu opioid receptor gene from the distal promoter.

The 5'-flanking region of the mouse mu opioid receptor (MOR) gene has two promoters, referred to as distal and proximal, and the activities of each in the brain are quite different from each other. The 5'-distal promoter regulatory sequences (5'-DPRS), positioned between these two promoters, have strong inhibitory effects on the reporter gene expression driven by the MOR distal promoter. In our studies, detailed 3' deletion mapping of the 5'-DPRS narrowed down the negative cis-acting element to a 34-base pair (bp) segment (position -721 to -687). This 34-bp cis-acting element functions in both neuronal (NMB) and non-neuronal (CHO and RAW264.7) cultured cells. S1 nuclease protection assays indicated that this 34-bp cis-acting element suppresses distal promoter activity at the transcriptional level. Linker scanning mutagenesis demonstrated that nucleotides around position -721 and -689 in the 34-bp cis-acting element are essential for the regulation of distal promoter activity. Operational characterization of the 34-bp cis-acting element in the homologous MOR distal promoter and the heterologous SV40 promoter showed that its effects are position- and promoter-dependent while being orientation-independent in both promoters. Collectively, these data suggested that this 34-bp segment is a conditional transcriptional cis-acting element that blocks mouse MOR gene expression from the distal promoter.

Transcriptional regulation of mouse mu-opioid receptor gene.

Previously, the existence of dual promoters was reported in mouse mu-opioid receptor (mor) gene, with mor transcription in the mouse brain predominantly initiated by the proximal promoter. In this study, we further analyzed the proximal promoter region, base pairs -450 to -249, to identify cis-DNA regulatory elements and trans-acting protein factors that are important for mor promoter activity. The results revealed that a mor inverted GA (iGA) motif and a canonical Sp1 binding site are required for the promoter activity. Using electrophoretic mobility shift analysis, we identified nuclear proteins that specifically bind to the mor iGA motif and that are immunologically related to Sp1 and Sp3. Mutation of the mor iGA motif, resulting in a loss of Sp binding, led to a 50% decrease in activity. Mutation of the canonical Sp1 binding site yielded a lesser (approximately 25%) loss of activity. Mutation of both motifs together resulted in an approximately 70% decrease in activity. In cotransfection assays using Drosophila SL2 cells, Sp1 trans-activated the promoter in a manner dependent on the presence of mor iGA and canonical Sp1 binding motifs. Sp3 can also trans-activate the promoter, and furthermore, Sp1 and Sp3 can trans-activate the mor promoter additively. Our results suggest that combined or cooperative interaction of Sp transcription factors within the proximal promoter is necessary for activation of mor gene transcription.

Increased growth promoting but not mast cell degranulation potential of a covalent dimer of c-Kit ligand.

The native form of soluble c-kit ligand (KL) is a noncovalent dimer. We have isolated a soluble, disulfide-linked dimer of murine KL (KL-CD) by expressing KL in Escherichia coli and refolding the denatured protein under conditions that promote the formation of both noncovalent dimers (KL-NC) and KL-CD. KL-CD exhibits a 10- to 15-fold increase in the ability to stimulate the growth of both the human megakaryocytic cell line MO7e and murine bone marrow-derived mast cells relative to KL-NC. Colony-forming assays of murine bone marrow progenitor cells also reflected this increased potency. However, KL-CD and KL-NC are equally able to prime mast cells for enhanced IgE-dependent degranulation in vitro and activate mast cells in vivo. Improving the growth-promoting activity of KL without changing its mast cell activation potential suggests that KL-CD or a related molecule could be administered in the clinic at doses that stimulate hematopoietic recovery while avoiding significant mast cell activation.