Biomedical subjects

J Lee

Publications and source records attributed to J Lee.

At least 631 records · Page 35Linked to original sources

PubMed1999

Fas ligand and Fas are expressed constitutively in human astrocytes and the expression increases with IL-1, IL-6, TNF-alpha, or IFN-gamma.

Fas ligand (FasL) and Fas are mediators of apoptosis, which are implicated in the peripheral deletion of autoimmune cells, activation-induced T cell death, and cytotoxicity mediated by CD8+ T cells. Fas is also believed to be involved in several central nervous system diseases, but until now, the effector cells expressing FasL in the brain have not been identified. We investigated the expression levels of Fas and FasL with the stimulation of cytokines and the possible effector cells targeting Fas-bearing cells. Our data demonstrated that: 1) FasL is expressed constitutively on astrocytes taken from a fetus or an adult and that its expression increases when these cells are treated with IL-1, IL-6, or TNF-alpha in which the pretreatment of IFN-gamma triggers astrocytes to express more FasL; 2) astrocytes induce apoptosis in MOLT-4 cells through FasL; 3) Fas is also expressed constitutively and is up-regulated by IL-1, IL-6, or TNF-alpha in which the pretreatment of IFN-gamma triggers astrocytes to express more Fas; 4) apoptosis occurs when fetal astrocytes are treated with agonistic anti-Fas IgM Ab after culture with IFN-gamma and TNF-alpha; and 5) TNF-related apoptosis inducing ligand is up-regulated in fetal astrocytes with stimuli of IL-1 or TNF-alpha. These findings suggest a possible role of astrocytes in the induction of apoptosis in central nervous system diseases.

Adult↗

PubMed1999

Wild-type Flp recombinase cleaves DNA in trans.

Site-specific recombinases of the Integrase family utilize a common chemical mechanism to break DNA strands during recombination. A conserved Arg-His-Arg triad activates the scissile phosphodiester bond, and an active-site tyrosine provides the nucleophile to effect DNA cleavage. Is the tyrosine residue for the cleavage event derived from the same recombinase monomer which provides the RHR triad (DNA cleavage in cis), or are the triad and tyrosine derived from two separate monomers (cleavage in trans)? Do all members of the family follow the same cleavage rule, cis or trans? Solution studies and available structural data have provided conflicting answers. Experimental results with the Flp recombinase which strongly support trans cleavage have been derived either by pairing two catalytic mutants of Flp or by pairing wild-type Flp and a catalytic mutant. The inclusion of the mutant has raised new concerns, especially because of the apparent contradictions in their cleavage modes posed by other Int family members. Here we test the cleavage mode of Flp using an experimental design which excludes the use of the mutant protein, and show that the outcome is still only trans DNA cleavage.

Amino Acid Sequence↗

PubMed1999

Different bioactivities of human thyrotropin receptors with different signal peptides.

For investigation of the mechanism and pathogenesis of Graves' disease, availability of a large amount of functional human thyrotropin receptor (TSHR) capable of recognition by Graves' autoantibodies is essential. Many attempts have been made to produce the extracellular domain of TSH receptor (TSHRE) in a baculovirus expression system. However, the receptor is expressed as an insoluble form and the refolded protein is often not recognized by the autoantibodies. In this study, we found that the TSHRE expressed with its own signal peptide (VL3-RE) in insect cells is retained inside of the cells and found in both soluble and insoluble fractions in equal proportion. The signal peptide is not removed. The receptor in the soluble fraction is not recognized by either TSH or Graves' autoantibodies. The TSHRE with an insect-specific mellitin signal peptide (Mel-RE) is also retained inside of the cell and found in both the soluble and insoluble fractions in equal proportion. However, the signal peptide is removed and the receptor is recognized by the Graves' autoantibodies but not by TSH. Also, the amount of Mel-RE expressed was 5-10-fold higher than VL3-RE. The two receptor preparations apparently have the same degree of glycosylation as evidenced by the same increased mass (approximately 15 kDa) due to glycosylation. However, the two receptors have different affinity for an anion-exchange resin and different pI. Deglycosylated receptors have the same pI. This suggests that the composition of sugars may be different. Taken together, the results suggest that the two receptors are modified and folded differently by different pathways due to the presence of different signal peptides. Use of an insect-specific signal peptide is recommended for expression of TSHR that is recognized by Graves' autoantibodies in a baculovirus system.

Autoantibodies↗

PubMed1999

Identification of an intact sydnonimine ring depending on the reaction substituents of mesocarb by gas chromatography/mass spectrometry

Trifluoroacylated, trimethylsilylated and methylated mesocarb (3-(1-methyl-2-phenylethyl)-5-[[(phenylamino) carbonyl]amino]-1,2, 3-oxadiazolium) were analyzed by gas chromatography/mass spectrometry (GC/MS). In trifluoroacylation, N-trifluoroacylated sydnophen (SP-NTFA), where the exocyclic nitrogen atom of the sydnonimine ring attacks N-methylbis(trifluoroacetamide), was identified by GC/MS. However, in trimethylsilylation, C-trimethylsilylated sydnophen (SP-CTMS), where the C-4 atom of the sydnonimine ring attacks N-methyl-N-trimethylsilyltrifluoroacetamide, was detected. SP-NTFA and SP-CTMS are intermediates of mesocarb in pyrolysis, and retain an intact sydnonimine ring. In methylation, a ketene form of mesocarb reacts as a nucleophile with methyl iodide to produce methylated N-nitroso-N-cyanomethylamphetamine. Thus, depending on the reaction substituents, the intermediate of mesocarb in pyrolysis was identified to be either an intact sydnonimine ring skeleton or an open-ring compound using GC/MS. Copyright 1999 John Wiley & Sons, Ltd.

Journal Article↗

PubMed1999

Calculation of protein conformation by global optimization of a potential energy function.

A novel hierarchical approach to protein folding has been applied to compute the unknown structures of seven target proteins provided by CASP3. The approach is based exclusively on the global optimization of a potential energy function for a united-residue model by conformational space annealing, followed by energy refinement using an all-atom potential. Comparison of the submitted models for five globular proteins with the experimental structures shows that the conformations of large fragments (approximately 60 aa) were predicted with rmsds of 4.2-6.8 A for the C alpha atoms. Our lowest-energy models for targets T0056 and T0061 were particularly successful, producing the correct fold of approximately 52% and 80% of the structures, respectively. These results support the thermodynamic hypothesis that protein structure can be computed solely by global optimization of a potential energy function for a given amino acid sequence.

Algorithms↗

PubMed1999

Analysis of cyclic boronated and trimethylsilylated phenolalkylamines by gas chromatography and electron impact mass spectrometry

The side chain of phenolalkylamines containing a bifunctional group was derivatized as the cyclic boronate, and then the residual phenolic group was trimethylsilylated. The resulting derivatives were single reaction products in each case, with good gas chromatographic properties and informative mass spectra to afford prominent molecular ions by gas chromatography with electron impact mass spectrometry (GC/EI-MS). The cyclic boronated-trimethylsilylated derivatives yielded the isotope pattern for boron ((10)B:(11)B =-1:4.2) and characteristic ions [M](+), [M - H](+), [M - CH(3)](+), [M - RBO](+), [M - TMSO](+), and [M - TMSO - C(6)H(5)](+) ions in the mass spectra. In order to distinguish between m- and p-phenolalkylamines, the mass spectra of the cyclic phenylboronated-trimethylsilylated (PBA-TMS) derivatives were compared with those of the trimethylsilylated (TMS) derivatives. The TMS derivatives of octopamine (p-) and norfenefrine (m-) showed identical mass spectra, while the PBA-TMS derivatives had mass spectra sufficiently different from one other to distinguish between the isomers. The most prominent ion of the PBA-TMS derivative is the [M - H](+) ion (m/z 310) for octopamine and the [M](+) ion (m/z 311) for norfenefrine. Copyright 1999 John Wiley & Sons, Ltd.

Journal Article↗

PubMed1999

Significance of pretreatment hemoglobin level in patients with T1 glottic cancer.

Recent reports have suggested that pretreatment hemoglobin level (Hgb) is significantly associated with local control (LC) and overall survival (OS) in patients with T1 and T2 squamous cell carcinoma of the glottic larynx. To further evaluate the association of pretreatment Hgb level and other factors with outcome, we performed a retrospective review limited to patients with T1 squamous cell carcinoma of the glottic larynx treated with external beam radiation therapy. One-hundred thirty-nine patients with T1 squamous cell carcinoma of the glottic larynx were analyzed. Median follow-up was 5 years (range 2-22). Median pretreatment Hgb was 14.4 gm/dl (range 8.2-17.2). The following parameters were analyzed for their impact on LC, OS, and disease specific survival (DSS): age; gender; pretreatment Hgb; tumor grade; anterior commissure involvement; field size; total dose; dose per fraction; and overall treatment time. Five-year actuarial LC was 84%. Pretreatment Hgb was not a significant predictor for LC when assessed as a continuous variable (P = 0.38), nor as a dichotomous variable with a cutoff at 13 gm/dl. Local control was 82% for patients with Hgb >13 vs. 92% for Hgb < or = 13 (P= 0.13). No other factor was significant for LC. Five-year actuarial OS was 74%. Univariate analysis revealed that, pretreatment Hgb, total dose, and patient age were significant factors for OS. Overall survival was 78% for patients with pretreatment Hgb > 13 gm/dl vs. 68% for patients with Hgb < or = 13 gm/dl (P = 0.004). Overall survival was 77% for patients treated with > 66 Gy vs. 67% for those treated with < or =66 Gy (P = 0.0013), and 80% for patients < or =61 years as opposed to 69% for patients older than 61 years (P = 0.017). Multivariate analysis revealed that only age (P = 0.014) and Hgb concentration (P = 0.001) retained significance. Five-year actuarial DSS was 92%. Pretreatment Hgb was not a prognostic factor for DSS, nor were any other analyzed factors. Pretreatment Hgb is not a significant prognostic factor for LC in patients with T1 squamous cell carcinoma of the glottic larynx, but it does predict for a poorer OS without affecting DSS. This suggests that patients with lower pretreatment Hgb may have confounding medical problems that detract from their overall survival.

Actuarial Analysis↗

PubMed1999

Chlamydia trachomatis nucleic acids can be found in the synovium of some asymptomatic subjects.

OBJECTIVE: The recent identification of antigens or nucleic acids of infectious agents in the joints of patients with reactive arthritis has raised questions about whether chlamydial or other infectious agent nucleic acids are also present in normal joints. We had the opportunity to study synovium from 30 asymptomatic volunteer subjects by use of polymerase chain reaction (PCR) for attempted identification of Chlamydia and other infectious agents. METHODS: All subjects had blind needle synovial biopsies with the Parker-Pearson needle. DNA was extracted and PCR performed using primers for Chlamydia trachomatis, Chlamydia pneumoniae, Borrelia burgdorferi, and pan bacterial 16S ribosomal RNA (rRNA). RESULTS: Two subjects were identified with nucleic acid for the 16S rRNA gene of C trachomatis. All other PCR reactions were negative except for the pan bacterial 16S rRNA in the C trachomatis-positive subjects. Both subjects, although symptom free, had some evidence of synovial reaction. CONCLUSION: C trachomatis appears to occasionally be disseminated to joints without producing overt disease.

Adult↗

PubMed1999

Developmental changes in brain serotonin synthesis capacity in autistic and nonautistic children.

Serotonin content, serotonin uptake sites, and serotonin receptor binding measured in animal studies are all higher in the developing brain, compared with adult values, and decline before puberty. Furthermore, a disruption of synaptic connectivity in sensory cortical regions can result from experimental increase or decrease of brain serotonin before puberty. The purpose of the present study was to determine whether brain serotonin synthesis capacity is higher in children than in adults and whether there are differences in serotonin synthesis capacity between autistic and nonautistic children. Serotonin synthesis capacity was measured in autistic and nonautistic children at different ages, using alpha[11C]methyl-L-tryptophan and positron emission tomography. Global brain values for serotonin synthesis capacity (K complex) were obtained for autistic children (n = 30), their nonautistic siblings (n = 8), and epileptic children without autism (n = 16). K-complex values were plotted according to age and fitted to linear and five-parameter functions, to determine developmental changes and differences in serotonin synthesis between groups. For nonautistic children, serotonin synthesis capacity was more than 200% of adult values until the age of 5 years and then declined toward adult values. Serotonin synthesis capacity values declined at an earlier age in girls than in boys. In autistic children, serotonin synthesis capacity increased gradually between the ages of 2 years and 15 years to values 1.5 times adult normal values and showed no sex difference. Significant differences were detected between the autistic and epileptic groups and between the autistic and sibling groups for the change with age in the serotonin synthesis capacity. These data suggest that humans undergo a period of high brain serotonin synthesis capacity during childhood, and that this developmental process is disrupted in autistic children.

Adolescent↗

PubMed1999

An evolving role of transesophageal echocardiography for the monitoring of interventional catheterization in children.

BACKGROUND AND HYPOTHESIS: Transesophageal echocardiography (TEE) is increasingly utilized for the monitoring of interventional cardiac catheterization in children. The initial nondiscriminant use is giving way to a more selective approach. The current role and usefulness of this imaging modality are described. METHODS: Between 1991 and 1995, 44 patients (mean age 7.4 +/- 5 years, weight 22.8 +/- 14.2 kg) underwent interventional cardiac catheterizations performed under TEE monitoring. The interventional procedures included (1) those involving creation, enlargement, or closure of an interatrial communication; (2) balloon dilatation of stenotic lesions; and (3) occlusion of arterial ducts. Transesophageal echocardiographic monitoring was judged to be either of great value, contributory, or of little use if the interventional procedure and choice of instrument relied heavily upon, was guided, or not affected by the information obtained from the ultrasonic investigation, respectively. RESULTS: Transesophageal echocardiography was judged to be of great value in the monitoring of 18 (40%) procedures involving either the creation, enlargement, or closure of interatrial communications. This included 4 transseptal punctures, 4 blade septectomies for relieving restrictive atrial septal defects (n = 3) and an enlargement of the communication between the superior and inferior chambers of the divided left atrium in cortriatriatum, and 11 closures of a secundum atrial septal defect. Only the ultrasonic monitoring could clearly visualize the interatrial partition to provide real-time guidance on the positioning of the instrument to carry out the invasive procedures. Transesophageal echocardiography provided clear images of the hinge points of the aortic and mitral valves, the dimension of the narrowed pulmonary venous pathway in patients after venous switch operation. This facilitated the choice of the correct size of the balloon for dilatation in 11 patients (24%) and provided immediate postdilatation hemodynamic assessment. As for balloon dilation of subvalvar ventricular outflow tract obstruction (n = 3), aortic coarctation (n = 6), and occlusion of persistent arterial duct (n = 7), TEE offered no obvious advantage over fluoroscopy with angiography. The monitoring was judged to be of little value for the 16 (36%) procedures. CONCLUSION: Transesophageal echocardiographic monitoring is of great value in interventional procedures involving the atrial septum and enhances interventional procedures involving obstructive lesions of the left heart in children.

Cardiac Catheterization↗

PubMed1999

Neuropeptide Y expression and endogenous leptin concentrations in a dietary model of obesity.

OBJECTIVE: To determine how leptin concentrations and neuropeptide (NPY) are regulated in a model of dietary obesity in relation to relative growth (RG) and relative food consumption (RFC). RESEARCH METHODS AND PROCEDURES: Sprague-Dawley rats were fed a moderately high-fat diet for 14 weeks over which time animals diverged into obesity-prone (OP) and obesity-resistant (OR) populations. RG rates and RFC were calculated weekly. Following the study, an adiposity index was calculated and arcuate nucleus (ARC) NPY expression was determined by in situ hybridization (ISH) or ribonuclease protection (RPA) assays. RESULTS: Body weights were greater in OP rats after 2 weeks on the diet compared to OR rats and remained different throughout the study. RG and RFC were greater in OP rats compared to OR rats only during the first 2 weeks of the study. Leptin concentrations rose in both groups during the experiment, but the increase was greater in OP rats than in OR rats. Insulin changes paralleled those for leptin. ARC NPY mRNA expression was not different between OP and OR rats as measured by ISH and RPA. DISCUSSION: Although NPY expression has been reported to be different initially in OP and OR rats, this difference dissipates following divergence of body weight. RFC and RG data suggest the initial NPY elevation may contribute to increased weight gain of OP rats during the first 2 weeks of the diet. Higher relative leptin concentrations in OP rats may be necessary to normalize differences in adiposity and apparent leptin and insulin resistance of OP rats.

Animals↗

PubMed1999

Cloning of a proopiomelanocortin cDNA from the pituitary of the Australian lungfish, Neoceratodus forsteri: analyzing trends in the organization of this prohormone precursor.

The polypeptide hormone precursor, proopiomelanocortin (POMC), was cloned and sequenced from the pituitary of the Australian lungfish, Neoceratodus forsteri, the only surviving species of the oldest extant lineage of lungfish. The Australian lungfish POMC cDNA had an open reading frame that coded for a 255-amino acid precursor. A comparison of POMC sequences from the Australian lungfish and the African lungfish indicated that the deduced amino acid sequences for ACTH, beta-MSH, and beta-endorphin were over 90% identical. Furthermore, within the open reading frames of the two lungfish POMCs, there was 84% identity at the nucleotide level. Although a gamma-MSH-like region was detected in the Australian lungfish POMC cDNA, this sequence contained mutations that have been detected in the gamma-MSH sequences of some ray-finned fish and are not found in the gamma-MSH sequence of the African lungfish or those of tetrapods. In addition, the sequence of beta-endorphin in the two species of lungfish has amino acid motifs that are found in the beta-endorphin sequences of cartilaginous fish and ray-finned fish but not in tetrapods. However, maximum parsimony analysis of the entire POMC open reading indicated that the lungfish POMC sequences form a clade with two amphibian POMC sequences rather than with POMC sequences from ray-finned fish. This result is consistent with the accepted view that the sarcopterygians (lungfishes and tetrapods) are a monophyletic assemblage. Analysis of rates of divergence for various POMC sequences indicate that point mutations are accumulating in the lungfish POMC sequences at a slower rate than in either amphibian or mammalian POMC sequences. The phylogenetic implications of these observations are discussed.

Adrenocorticotropic Hormone↗

PubMed1999

Mice lacking alpha-calcitonin gene-related peptide exhibit normal cardiovascular regulation and neuromuscular development.

alpha-Calcitonin gene-related peptide (alphaCGRP) is a pleiotropic peptide neuromodulator that is widely expressed throughout the Central and peripheral nervous systems. CGRP has been implicated in a variety of physiological processes including peripheral vasodilation, cardiac acceleration nicotinic acetylcholine receptor (AChR) synthesis and function, testicular descent, nociception, carbohydrate metabolism, gastrointestinal motility, neurogenic inflammation, and gastric acid secretion. To provide a better understanding of the physiological role(s) mediated by this peptide neurotransmitter, we have generated alphaCGRP-null mice by targeted modification in embryonic stem cells. Mice lacking alpha CGRP expression demonstrate no obvious phenotypic differences from their wild-type littermates. Detailed analysis of systemic cardiovascular function revealed no differences between control and mutant mice regarding heart rate and blood pressure under basal or exercise-induced conditions and subsequent to pharmacological manipulation. Characterization of neuromuscular junction in morphology including nicotinic receptor localization, terminal sprouting in response to denervation, developmental regulation of AChR subunit expression, and synapse elimination also revealed no differences in alphaCGRP-deficient animals. These results suggest that alphaCGRP is not required for the systemic regulation of cardiovascular hemodynamics or development of the neuromuscular junction.

Aging↗

PubMed1999

Lifestyle factors and chronic diseases: application of a composite risk index.

BACKGROUND: Assessing a combination of modifiable lifestyle practices may be a practical tool to modify patients' health behavior in counseling. Therefore, we developed a chronic disease risk index (CDRI) and investigated its relation with chronic disease in a multiethnic cohort. METHODS: A total of 15,693 men and 16,007 women in Hawaii who reported their diet and other lifestyle behaviors between 1975 and 1980 were followed until 1994. A semi-quantitative composite CDRI with scores ranging from 1 to 10 included the rankings for smoking, alcohol use, body mass index, fat intake, and fruit and vegetable consumption. Cox's proportional hazards regression was used to estimate the relative risk for chronic diseases. RESULTS: When comparing the highest to the lowest CDRI category, the respective relative risks (RRs) for total mortality were 2.9 (95% CI 2.3-3.8) and 3.8 (95% CI 2.9-5.0) for men and women. With higher CDRIs, the RRs for cancer incidence, mortality from cancer, coronary heart disease, and stroke increased significantly. Among the five components of the CDRI, smoking had the greatest influence on chronic disease risk, followed by body mass index. CONCLUSIONS: Positive health behavior reflected by the CDRI is associated with a lower risk of cancer and with greater longevity.

Adult↗

PubMed1999

Heterologous gene expression in avian cells: potential as a producer of recombinant proteins.

We have explored the possibility of using avian cells for the expression of human proteins. We found that various avian cells including quail fibrosarcoma cells (QF), duck embryo cells (DE) and primary chicken embryo fibroblasts (CE) could efficiently be transfected with DNA by calcium phosphate coprecipitation, and that promoters which are transcriptionally active in mammalian cells also functioned well in these avian cells. Among the promoters we tested, the major immediate early promoter of human cytomegalovirus drove the highest level of chloramphenicol acetyl transferase (CAT) expression, outperforming the SV40 early promoter and the RSV LTR. Using the bacterial CAT gene as a reporter, we found that levels of CAT activity were higher in QF and DE cells than in mammalian cells such as CHO, HeLa, Vero and 293T cells. We further cloned a sequence encoding human erythropoietin (EPO) and compared its expression in QF and mammalian cells. Consistent with the CAT data, in transient transfection assays, QF cells produced higher levels of EPO than the mammalian cell lines tested. QF cells which can be passaged permanently were stably transfected with an EPO expression vector. The subcloned QF line was able to produce up to 1,700 U/ml EPO from 3 x 10(6) cells in 72 h. Purified QF-produced EPO showed a broad but discrete protein band, ranging from 33 to 41 kD and was as biologically active as CHO-produced EPO. Although a number of factors still remain to be optimized, our results demonstrate the potential of avian cells such as QF as producers of heterologous proteins.

Animals↗

PubMed1999

The effect of N-substituted alkyl groups on anticonvulsant activities of N-Cbz-alpha-amino-N-alkylglutarimides.

In order to examine the effects of N-substituted alkyl group on the anticonvulsant activities of N-Cbz-alpha-aminoglutarimides as novel anticonvulsants with broad spectrum, a series of (R) or (S) N-Cbz-alpha-amino-N-alkylglutarimides (1 and 2) were prepared from the corresponding (R) or (S) N-Cbz-glutamic acid and evaluated for the anticonvulsant activities in the maximal electroshock seizure (MES) test and pentylenetetrazol induced seizure (PTZ) test, including the neurotoxicity. The most potent compound in the MES test was (S) N-Cbz-alpha-amino-N-methylglutarimide (ED50=36.3 mg/kg, PI=1.7). This compound was also most potent in the PTZ test (ED50=12.5 mg/kg, PI=5.0). The order of anticonvulsant activities against the MES test as evaluated from ED50 values for (R) series was N-methyl > N-H > N-ethyl > N-allyl; for the (S) series N-methyl > N-H > N-ethyl > N-allyl > N-isobutyl compound. Against the PTZ tests, the order of anticonvulsant activities showed similar pattern; for the (R) series, N-methyl > N-H > N-ethyl > N-allyl; for the (S) series N-methyl > N-H > N-ethyl > N-allyl > N-isobutyl compound. From the above results, N-substituted alkyl groups were thought to play an important role for the anticonvulsant activities of N-Cbz-alpha-amino-N-alkylglutarimides.

Animals↗

PubMed1999

Galectin-1 and galectin-3 expression in human prostate tissue and prostate cancer.

Galectin-1 and galectin-3, two beta-galactoside-binding proteins, have been suggested to play a role in the development and progression of cancer. We have studied the expression of these molecules in normal human prostate tissue and prostate adenocarcinoma. Immunohistochemistry was used to examine formalin-fixed, paraffin-embedded sections of seven normal human prostates, eight cases of prostatic intraepithelial neoplasia (PIN), 20 primary adenocarcinomas of the prostate, and 12 prostate cancer metastases. Galectin-1 was expressed in most cases of all four histologic types. In contrast, galectin-3 expression was significantly decreased in primary carcinoma and metastatic disease compared with normal and premalignant tissue. Galectin-3 expression in primary tumors tended to be less than that of surrounding normal glands. We conclude that loss of galectin-3 expression may be associated with the progression of prostate cancer.

Adenocarcinoma↗

PubMed1999

Improved process for production of recombinant yeast-derived monomeric human G-CSF.

The human granulocyte colony-stimulating factor (hG-CSF) was efficiently secreted at high levels in fed-batch cultures of recombinant Saccharomyces cerevisiae. However, the secreted recombinant hG-CSF (rhG-CSF) was shown to exist as large multimers in the culture broth due to strong hydrophobic interaction. It was hardly monomerized even by urea at high concentration. This multimer has been reported to diminish specific receptor-binding activity of hG-CSF and causes undesirable problems in the downstream process. When the rhG-CSF was secreted to extracellular broth in the presence of a non-ionic surfactant (Tween 80) in the culture media, the multimerization of the secreted rhG-CSF was efficiently prevented in the fed-batch cultures. Also, the monomer fraction and secreted efficiency of rhG-CSF were significantly increased at the higher culture pH (6.5). Without using any denaturing agents, the secreted rhG-CSF monomer was easily purified with high recovery yield and purity via a simple purification process under acidic conditions, consisting of diafiltration, cation exchange, and gel filtration chromatography. A lyophilization process devoid of intermonomer aggregation was also designed using effective stabilizing agents.

Biopolymers↗