A novel control scheme for inducing angiostatin-human IgG fusion protein production using recombinant CHO cells in a oscillating bioreactor

In this study, a novel control scheme for inducing protein production using a recombinant CHO cell line in a BelloCell bioreactor was developed. This control scheme was applied in a simple regular semi-batch process. Production of angiostatin-human IgG fusion protein in a suspension recombinant CHO cell culture and a protein-free medium was used for this study. The bottom holding time (BH) was the sole operating variable to control the exposure time of the cells immobilized on the carriers to the air and allow the nutrient remained on the liquid film of the carriers to be consumed to a threshold level so that the cells can be arrested and promoted for protein production. In the cell cultures with various BH (1.5-90 min), final cell densities of 1.6-4.0 x 10(9) have been obtained in 20 days while total angiostatin-human IgG production of 228-388 mg have been harvested. In general, low BH will minimize the nutrient limitation and favor the cell growth, while high BH will restrict the nutrient and promote the production in this type of non-growth associated production systems. It was found that specific production rate was generally inversely proportional to the specific growth rate. In this case of study, BH of 30 and 60 min were found to be about 72% better than BH of 1.5 min and 35% better than BH of 9 and 90 min in term of the total angiostatin-human IgG production. In comparison to a conventional spinner flask study, a 3.8-fold increase of the total angiostatin-human IgG production was realized in a 35-day culture. This study illustrated that a simple method of using BH in a semi-batch process can effectively control the apparent nutrient concentration to the cells, and thus regulate the cell growth and protein production in a novel oscillating bioreactor.     

Upregulation of neuronal nitric oxide synthase mRNA and protein in adrenal medulla of water-deprived rats.

Experiments were performed to investigate whether adrenal neuronal nitric oxide synthase (nNOS) mRNA and protein expression are responsive to alterations in body volume. Using an RT-PCR technique, the relative quantities of nNOS mRNA as well as the tyrosine hydroxylase and phenylethanolamine N-methyltransferase mRNA in the adrenals of water-deprived rats significantly increased from 12 hr to 4 days. In situ hybridization and immunohistochemical study showed that water deprivation activated nNOS mRNA and protein expression in the adrenal medulla. Four days after water deprivation, nNOS protein expression determined by Western blot significantly increased in the adrenal gland. Our results are the first to demonstrate that nNOS syntheses in the adrenal medulla are markedly increased in water-deprived rats. This study also indicates that the upregulation of nNOS synthesis of the adrenal medulla is associated with the activation of adrenal medullary function in the face of volume depletion.     

Structure and bioactivity of the polysaccharides in medicinal plant Dendrobium huoshanense

Detailed structures of the active polysaccharides extracted from the leaf and stem cell walls and mucilage of Dendrobium huoshanense are determined by using various techniques, including chromatographic, spectroscopic, chemical, and enzymatic methods. The mucilage polysaccharide exhibits specific functions in activating murine splenocytes to produce several cytokines including IFN-gamma, IL-10, IL-6, and IL-1alpha, as well as hematopoietic growth factors GM-CSF and G-CSF. However, the deacetylated mucilage obtained from an alkaline treatment fails to induce cytokine production. The structure and bioactivity of mucilage components are validated by further fractionation. This is the first study that provides clear evidence for the structure and activity relationship of the polysaccharide in D. huoshanense.     

Anion exchanger inhibitor DIDS induces human poorly-differentiated malignant hepatocellular carcinoma HA22T cell apoptosis

Anion exchangers (AEs) of the Cl^-/HCO3^- exchanger family contribute to the regulation of intracellular acid-base balance. Recently, we found that anion exchanger 2 (AE2) was significantly expressed in human hepatocellular carcinoma (HCC) and in poorly-differentiated human HCC HA22T/VGH cells. In the present study, we further explored the pharmacological function of AE in four human HCC cell lines (SK-Hep-1, HA22T/VGH, HepG2, and Hep3B) following the treatment of 4,4’-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), an AEs specific inhibitor. After administrations with 400–1000 μM of DIDS, cell proliferation was greatly inhibited in a dose-dependent manner from 47.5 to 65.0% in higher malignant HA22T/VGH cells, but not in other cell lines. The results of 4,6-diamidino-2-phenylindole (DAPI) staining, DNA fragmentation and flow cytometric analysis further revealed that cell apoptosis induced by DIDS was also observed in HA22T/VGH cells. Therefore, these findings suggested that AE may be involved, in part, in the proliferation and survival of HA22T cells and could be a new potential therapeutic target against specific human HCC. The authors Chih-Yang Huang and Jer-Yuh Lin contributed equally to this article.     

Endothelin and gelatinases in renal changes following blockade of nitric oxide synthase in hypertensive rats

We investigated the involvement of matrix metalloproteinases (MMPs), tissue inhibitor (TIMP) and endothelin-1 (ET-1) in the renal damage in spontaneously hypertensive rats (SHR) following nitric oxide (NO) deprivation. SHR received Nomega-nitro-L-arginine methyl ester (L-NAME) from 5 wk-old for a period of 30 days. An ETA antagonist, FR139317 was used. We gave SHR FR139317 alone and cotreatment with L-NAME. L-NAME caused systemic hypertension, decrease in plasma nitrate/nitrite, increases in blood urea nitrogen and creatinine, impairment of glomerular dynamics. NO deprivation reduced the renal tissue cGMP, but it increased the collagen volume fraction, number of sclerotic glomeruli, arteriolar injury score and glomerular injury score. In addition, L-NAME elevated the plasma ET-1 at day 5. Cotreatment with FR139317 alleviated the L-NAME-induced functional and structural changes of renal glomeruli. L-NAME administration for 5 to 10 days resulted in decreases in MMP2 and MMP9 with increasing TIMP2. After L-NAME for 15 days, opposite changes (increases in MMP2 and MMP9 with a decrease in TIMP2) were observed. FR139317 cotreatment ameliorated the L-NAME-induced changes in MMP2 and MMP9 throughout the 30-day observation period. The ETA antagonist cotreatment attenuated the L-NAME-induced increase in TIMP2 before day 15, but not after day 20. The results indicate that ET-1, MMPs and TIMP are involved at the early stage (before 10 days) of glomerular sclerosis and arteriosclerosis with functional impairment following NO deprivation. The changes in MMPs and TIMP at the late stage (after 20 days) may be a compensatory response to prevent further renal damage.     

Cloning and functional characterization of the HRASLS2 gene

The HRAS-like suppressor 2 (HRASLS2) gene belongs to the H-REV107 gene family involved in the regulation of cell growth and differentiation. HRASLS2 is expressed at high levels in normal tissues of the small intestine, kidney, and trachea. We cloned HRASLS2 cDNA from human SW480 colon cancer cells. Most wild-type, and some N- and C-terminal truncated HRASLS2 (HRASLS2DeltaNDeltaC) were expressed as a granular pattern located at perinuclear region in HtTA cervical cancer cells, while truncation at the C-terminus only (HRASLS2DeltaC) resulted in a diffuse pattern. Wild-type HRASLS2 significantly suppressed colony formation of HeLa and HCT116 cells. HRASLS2DeltaNDeltaC significantly inhibited colony formation of HCT116 cells, but HRASLS2DeltaC did not affect cell growth. HRASLS2 suppressed the RAS-GTP levels and total RAS protein by 44% and 25%, respectively in HtTA cells; however, the suppression was not observed in truncated HRASLS2 variants. In conclusion, the HRASLS2 protein suppressed growth and RAS activities of cancer cells, and the C-terminal hydrophobic domain appeared to be indispensable for both activities.     

Highly efficient classification and identification of human pathogenic bacteria by MALDI-TOF MS

Accurate and rapid identification of pathogenic microorganisms is of critical importance in disease treatment and public health. Conventional work flows are time-consuming, and procedures are multifaceted. MS can be an alternative but is limited by low efficiency for amino acid sequencing as well as low reproducibility for spectrum fingerprinting. We systematically analyzed the feasibility of applying MS for rapid and accurate bacterial identification. Directly applying bacterial colonies without further protein extraction to MALDI-TOF MS analysis revealed rich peak contents and high reproducibility. The MS spectra derived from 57 isolates comprising six human pathogenic bacterial species were analyzed using both unsupervised hierarchical clustering and supervised model construction via the Genetic Algorithm. Hierarchical clustering analysis categorized the spectra into six groups precisely corresponding to the six bacterial species. Precise classification was also maintained in an independently prepared set of bacteria even when the numbers of m/z values were reduced to six. In parallel, classification models were constructed via Genetic Algorithm analysis. A model containing 18 m/z values accurately classified independently prepared bacteria and identified those species originally not used for model construction. Moreover bacteria fewer than 10(4) cells and different species in bacterial mixtures were identified using the classification model approach. In conclusion, the application of MALDI-TOF MS in combination with a suitable model construction provides a highly accurate method for bacterial classification and identification. The approach can identify bacteria with low abundance even in mixed flora, suggesting that a rapid and accurate bacterial identification using MS techniques even before culture can be attained in the near future.     

Identification of collapsin response mediator protein-2 as a potential marker of colorectal carcinoma by comparative analysis of cancer cell secretomes

The cancer cell secretome may contain many potentially useful biomarkers. We therefore sought to identify proteins in the conditioned media of colorectal carcinoma (CRC) cell lines but not in those from other cancer cell lines. The secretomes of 21 cancer cell lines derived from 12 cancer types were analyzed by SDS-PAGE combined with MALDI-TOF MS. Among the 325 proteins identified, collapsin response mediator protein-2 (CRMP-2) was chosen for evaluation as a potential CRC biomarker, since it was selectively detected in the CRC cell line secretome and has never been reported as a cancer biomarker. Immunohistochemical analysis of 169 CRC specimens showed that CRMP-2 was positively detected in 58.6% of the tumors, but weakly or not detected in >90% of the adjacent nontumor epithelial cells. Moreover, the CRMP-2-positive rate was significantly increased in earlier stage tumors and lymph node metastasis. Plasma CRMP-2 levels were significantly higher in CRC patients (N = 201) versus healthy controls (N = 201) (61.3 +/- 34.6 vs. 40.2 +/- 24.3 ng/mL, p = 0.001). Our results indicate that comparative analysis of cancer cell secretome is a feasible strategy for identifying potential cancer biomarkers, and that CRMP-2 may be a novel CRC biomarker.     

Feasibility study of exponential feeding strategy in fed-batch cultures for phenol degradation using Cupriavidus taiwanensis

An indigenous phenol-degrading bacterial isolate Cupriavidus taiwanensis R186 was used to degrade phenol from an aqueous solution under fed-batch operation. An exponential feeding strategy combined with dissolved oxygen control was applied based on kinetic characteristics of cell growth and phenol degradation to meet sufficient metabolic needs for cellular growth and achieve the best phenol removal efficiency. Without the stress of phenol inhibition, the optimal set point of specific growth rate of exponential feeding for fed-batch phenol degradation was found to be 0.50–0.55μ_max (μ_max denotes the maximum specific growth rate from Monod model). Meanwhile, the sufficient set point of dissolved oxygen for maximal phenol degradation efficiency was approximately at 10–55% air saturation. With the optimal operation conditions, the best phenol degradation rate was 0.213 g phenol h⁻¹, while a shortest treatment time of 15 h was achieved for complete degradation of 11.35 mM (ca. 3.20 g) of phenol.