Fucoidan-dependent Increased Membrane Components in HepG2 Cells: Effect of Fucoidan Is Not Due to Gene Expression

Results and Discussion

We first constructed a correction table to estimate the amount of amino acids present in the protein sample from the provided PTH-amino acid (pmol) reading of the machine (Table I). Degradation of Ser and Thr during micro-sequencing was indeed large as expected by the use of TFA (Table I), however such a good yield line was obtainable following correction factors in Table I, using purified β-lactamase TEM fragment (from 24- of mature chain) of Escherichia coli O-157 (Figure 1). The binding efficiency of proteins by the EDC method was logarithmically-depended upon the MW of the proteins and peptides as shown in Figure 2. Therefore, we corrected the protein amount by using this log-log line of Figure 2 (correction factors are shown in the Materials and Methods section).

The component proteins of Hc cells (fetal liver cultured cells) were determined, and are presented in Table II (viral and bacterial proteins) and Tables III and IV (cellular proteins).

The component proteins of HepG2 (derived from an HCC liver of 15-year-old male from the USA) were also determined and are summarized in Tables V and VI (viral proteins with and without treatment of fucoidan), Tables VII, VIII, IX (cellular proteins; without treatment of fucoidan) and Tables X, XI, XII, XIII (cellular proteins; with treatment of fucoidan for 3 days).

Fetal cultured Hc liver cells contained 19% of the invaded microbes (virus, bacteria, and plant) (Table II), which was of similar level to human breast milk (16%; virus, bacteria, and yeast) and to cow’s milk (17%; virus, bacteria, and yeast) (data not shown). Interestingly, bacteria of Lactobacillus casei (Shirota) contained 12% of invaded bacteriophages (data not shown). Human biopsy tissues of hospitalized patients also showed similar results, but major virus and minor bacteria were detected; i.e., pseudo-cancer of liver (non-cancer) contained 19%, the LC portion of the leprosy patient 15%, HCC portion of primary biliary cirrhosis (PBC) patient 30%, respectively, of the invaded microbes (virus and bacteria) among total tissue proteins (data not shown). It is also interesting that the DNAJ protein (Hsp40; Heat shock protein 40 kDa of Mycobacterium leprae) appeared only in the LC portion of the leprosy patient, which suggested that the sensitivity of detection power of proteomics was highly and sufficiently performed by this microsequencing method. The liver of the leprosy patient also contained the unique soil-related plant-tumor making bacterium Agrobactrium tumefacience, and plant virus (Yellow mosaic virus) proteins, suggesting that co-infection of Mycobacterium leprae, Agrobactrium tumefacience, and plant virus (Yellow mosaic virus) in the patient might have occurred. Increased histone H3.1 in the LC portion of the liver of the leprosy patient has been also detected. High levels of total histone H3 transcripts (mRNA) in different tissues of plant Alfalfa has been reported showing a particularly high level in the somatic embryos (18). Thus, histone may be essential for immortality in plant cells and in human leprosy cells, but seemed not to be directly related to human cancer. Furthermore, the liver biopsy of HCC patient with HCV contained only viral proteins (LC tissue 12% and HCC tissue 16%, respectively, among the total tissue proteins), suggesting that antibiotics against the bacteria might have been used during hospitalisation of the patient. Fetal Hc liver cells contained 19% of invaded microbes; i.e., 4.7% viral and 14% bacterial proteins (Table II). Since fetal Hc cells were prepared from the mixed fetal-livers obtained from the pregnant women who died of traffic accidents in USA, immediate infection by the invading microbes (virus, bacteria, and plant) might have occurred. These invaded microbes might still persistently have been remained in cultured Hc cells (few times of the passages of culture) in spite of the presence of antibiotics against bacteria in the culture medium. On the other hand, established hepatocarcinoma HepG2 cells were cultured for numerous passages with antibiotics, and bacterial proteins fully disappeared (Table V). Thus, the content of viral proteins in Hc cells (4.7%; Table II) and in HepG2 cells (4.6%, Table VI, cultured with fucoidan) finally returned similar values. However, the viral content of HepG2 cells without fucoidan treatment was considerably higher (6.6%, Table V). The median viral content in the serum of non-cancer persons was 3.1% (n=9; range 0.4-4.5%) (19). Biopsies of livers of three HCC patients and a pseudo-cancer patient contained very high virus content, of 13.5% (n=5; range 8.1-22.2%), although the data of the virus content in the non-cancer and non-inflammatory healthy livers were not obtainable. Therefore, the metastasis of cancer might easily occur by infection or diffusion of virus to other organs (20). Viral protein content in washed cells and serum, higher than 5% among the total cellular proteins might be an indicator for “the state of cancer”. However, prompt determination of viral content may be difficult using the microsequencing method, which requires for analysis time of about one month using the presently used manual microsequencing algorithm.

HCV genome polyprotein is an anchored membrane glycoprotein (21, 22), and is detectable in the membrane fractions of cultured cells of Hc and HepG2, and of livers of diseases (n=4). Non-cancer fetal Hc cells contain 7.7 μg (0.77%; MW 250,000) HCV genome polyprotein (Table II), which may not be deanchored. Its level in hepatocarcinoma HepG2 cells is 7.2 μg/mg (0.72%; MW 250,000; Table V), but this may be deanchored. Fucoidan seems to reduce it to 2.0 μg/mg (0.20%; MW 83,000; Table VI), and the protein may not be of the deanchored state. The presence of HCV genome polyprotein in the liver tissues is also observed; i.e., pseudo-liver-cancer 0.44% (membrane MW 130,000), LC tissue of leprosy 2.2% (membrane; MW 230,000), HCC tissue of PBC 3.5% (soluble; MW 230,000), LC tissue of a HCC with HCV 0.79% (soluble; MW 40,000) and HCC tissue of a HCC with HCV 0.55% (membrane; MW 340,000), respectively (data not shown). MW of HCV genome polyprotein in the non-cancer state tissues and the fucoidan-treated HepG2 cells seems smaller compared to the cancer state. Therefore, the presence of a large molecular mass HCV genome polyprotein in liver cells may be a risk factor for HCC or liver cancer with immortality.

Envelope glycoprotein gp160 (HIV) was dramatically reduced by fucoidan in HepG2; i.e., from 8.2 μg/mg (0.82%; without fucoidan) to 0.0 μg/mg (0.0%; with fucoidan) (Tables V and VI). Although data in other hepatocarcinoma cells such as HuH-7 may also be necessary to derive a conclusion, fucoidan may be important for cancer cells to obtain the normal innate immune system by reducing HIV virus. Only a minor amount of the membrane protein Nef of HIV remained at the same levels (Table V; without fucoidan 0.6 μg/mg, and Table VI; with fucoidan 0.9 μg/mg) by fucoidan, however the HIV-1 Nef-interacting protein (T-complex protein 1 subunit eta; membrane protein made from the cellular DNA gene) is expressed in the fucoidan-treated HepG2 cells (Table X; 4.2 μg/mg). Therefore, the virulent viral Nef protein remaining in fucoidan-treated HepG2 cells may be neutralized by this cellular membrane protein (23). Fucoidan made from the edible Ishi-Mozuku at 0.102 mg/mL, dramatically reduces the proteins of plus-sense (+) single-stranded (ss) RNA virus from 5.3% to 0.29% (18.1-fold reduction) among the total cell-proteins (Table V and Table VI), while proteins in Hc cells are 3.6% (Table II). Hepatitis virus (HCV and HGV) plus retrovirus (HIV and Rous Sarcoma virus) are also reduced from 3.7% to 0.29% (12.6-fold reduction) among the total cell proteins (Tables V and VI), and those in the Hc cells is 1.1% (Table II). Reduction of retrovirus of HIV and Rous Sarcoma virus is also reduced from 2.3% to 0.2% (11.7-fold reduction) among the total cell proteins (Tables V and VI), and those in the Hc cells is 0.34% (Table II). HIV is a notorious virus responsible for human immunodeficiency (AIDS), and reduction of retroviral proteins seems to be essential for cure of HCC (24). Human papillomavirus (HPV) infection appears to be a necessary factor in the development of almost all cases of cervical cancer (25). Tumor formation in plants by the bacterium Agrobacterium tumefaciens has turned out to be caused by a symbiotic tumour-inducing (Ti) plasmid (or phage virus) (26). Helicobacter pylori infections have been linked to gastric adenocarcinoma (27), and the transformation of the gastric cancer cells may be speculated to have occurred by the plasmid or virus residing in this bacterium such as in the case of the tumour-inducing (Ti) plasmid residing in the plant bacterium Agrobacterium tumefaciens (26). Furthermore, virus or phage infection changes the surface glycochains of the dominated host cells (phage conversion), and prevents the invasion of other virus or phage to enter the conquered cell. Thus, glycochains of the cell surface may be changed by the invaded viral glycochain-converting enzymes (28). Therefore, the virus infection to the host cells may be an important issue for cancer and metastasis, and this glycochain conversion may be performed by the viral enzymes and viral genes but not by the cellular enzymes or cellular genes; i.e., cellular gene expression may not be directly related to cancer differentiation. Fucoidan may directly prevent the infection from virus, and the possibility of direct inhibition of HIV reverse trascriptase activity by fucoidan in vitro has already been reported (29). Other DNA viral proteins seem to increase within the three days of fucoidan treatment; 11.0 μg/mg (without fucoidan) to 38.4 μg/mg (with fucoidan) (Tables V and VI). However, this level of DNA virus might also be reduced during the longer treatment time by fucoidan due to the slow-preventing effect against DNA virus; i.e., oral administration of fucoidan for 3 weeks had been necessary in the mice cases (30). Following, fucoidan seems to dramatically reduce the viral proteins through direct inhibition of viral replication; i.e., immediate inhibitions on viral protein synthesis and viral genome replication.

Comparative biochemical studies among normal liver tissues and cultured cell lines (human Hc, HuH-6, HuH-7, HepG2, rat RLC-16, and retinoblastoma cells (Y79)) indicated that the following characteristics were the unique points of the immortal cell lines; i.e., [1] increase of +ssRNA virus, [2] occurrence of biotin production (adult humans could not synthesize biotin) (vitamin H) (6, 31, 32), [3] increase of biotinidase activity (Kip; repulsion power against produced biotin) (6), [4] appearance of D-aspartic acid (though the HepG2 showed a relatively low concentration) (5), [5] decreased ratio of membrane proteins (in the follows of this text), [6] increased production of histone H3.1 (3, 4). High concentration of biotin (both the total- and free-form) (32, 33) and high biotinidase activity (unpublished observation) was found in the chicken egg yolk. Interestingly, fucoidan seemed not to influence cellular gene expression in HepG2 at all; i.e., histone content of non-treated and treated with fucoidan HepG2 cells were unexpectedly similar.

Apoptotic mechanism by Wakame-fucoidan (containing 11% of uronic acid) from “Wakame” (Undaria pinnatifida) has been recently reported by using the human HCC cell line SMMC-7721 (the Cell Bank of the Chinese Academy of Sciences, Beijing, China) (34), however stopping of cell division at the S-phase (DNA-replication phase) by fucoidan is not significant. Furthermore, apoptosis-related proteins such as caspases and the gene products of Livin (a member of the inhibitors of apoptosis protein family) and XIAP could not be detected in HepG2 and Hc, and liver biopsies using the DMD method. The increased biotinidase Kip in the HCC tissue was inhibited only by the sulfated glycochains of fucoidan and heparin with the exception of chondroitin sulfate (6), indicating that both the sulfation and component sugar of glycochain was important to inhibit the glycoprotein enzyme biotinidase. Fucoidan and heparin were also known to inhibit blood coagulation via binding to the glycochains of glycoprotein antithrombin III (35). Furthermore, anti-tumor and anti-metastatic activities of lung adenocarcinoma in mice exerted by the fucoidan from Fucus evanescens have already been reported (36). In this case, intra-peritoneal injection method of 10 mg/kg/day of fucoidan were used (36). Therefore, fucoidan may inhibit the above mentioned characteristics from [1] to [5] in immortalized cancer cells possibly through glycol-chains; i.e. [3] decrease of Kip of glycoprotein enzyme of biotinidase, [1] decrease of viral glycoproteins, and [5] the increase of the membrane compartment rich in glycoproteins, although the putative human genes and information of glycochain for [4] aspartate racemase and for [2] biotin synthetase are not yet available in the protein database (Protein BLAST in NCBI).

Attempts of growth-retardation tests directly by decreasing the intracellular concentrations of D-biotin and D-aspartic acid have not yet been successful. Thus, avidin addition in the culture medium was tested in order to reduce the intracellular biotin concentration, but the HeLa cells were not affected at all, since avidin-biotin complex (MW65,000) was actively taken-up by the cells (37). Since oral administration of the high-molecular-mass Okinawa-fucoidan attenuated DEN-induced liver fibrosis in Wistar rats (38), the liver cells seemed to be able to take-up the high-molecular-mass fucoidan (MW 200,000) via the gut. Furthermore, intraperitoneal injection of the high-molecular-mass fucoidan attenuated lung cancer in C57Bl/6 mice (36), and the lung cells also seemed to able to take-up fucoidan from different regions of the body. Then, a direct supply of amounts of biotinidase to promptly growing fetus and baby was suggested, since chicken egg yolk (unpublished observation) and human breast milk (39) contained relatively high biotinidase. Recently, the direct demonstration of dependence on biotin for the growth of the mutant bacterium Mycobacterium tuberculosis has been reported (40). Our testing of addition D-aspartic acid (10 mM) in the medium of RLC-16 (immortalised epitherial cell line established in Japan from the liver of Rattus norvegicus), which required L-glutamine (10 mM) in the medium, had also been unsuccessful (5); i.e., extremely high concentrations of D-aspartic acid (product) might have inhibited the aspartate racemase reaction. Then, reduction of intracellular biotin and D-aspartate might have been achievable by addition of mercury ion, which is a common inhibitor to biotin synthetase (41) and to aspartate racemase (42). Therefore, the growth of the HCC-derived cultured cells may be expected to be slowed-down by the edible fucoidan through the above suggested five signals.

Although data obtained from hepatocarcinoma, such as HuH-7 cells, might be necessary to draw the conclusion, fucoidan from edible Ishi-Mozuku also seemed to change the amounts of cellular proteins; i.e. (1) increase of ceruloplasmin (1.4-fold), transferrin (1.6-fold), glycol-chain synthesizing enzymes (2.2-fold) and membrane receptors (1.4-fold) (Tables VII, VIII, IX, X, XI, XII, XIII), and (2) decreases of non-liver proteins of albumin (3.3-fold; skeletal muscle), alpha-fetoprotein (AFP; 1.9-fold; fetal liver), myosin (1.4-fold; muscle), titin (1.2-fold; muscle), protocadherin (brain) 8.1-fold, hypoxia-inducible factor (brain) 3.5-fold, and lipoamidase (1.8-fold; brain) (Tables VII, VIII, IX, X, XI, XII, XIII). Increases in ceruloplasmin and transferrin turned-out to be non-general; i.e., a liver of pseudo-cancer showed similar ceruloplamin levels to HCC tissues, and LC tissue of a liver of an HCC patient showed a decreased transferrin amount compared to HCC tissue. Decrease of albumin and AFP might also not be general; i.e., although one HCC patient showed a decrease of albumin in HCC using chromato-graphic analysis (4), but increased albumin in HCC tissue was observed in an HCC patient, and increased AFP in the LC tissue of the leprocy patient was also observed using the microsequencing method. Instead, changes in the glycochain of AFP seemed more important; i.e., the highly-enhanced fucosylation of AFP in patients with germ cell tumor has already been reported (43). Since protein analysis of the liver tissues had been performed under the still unimproved micro-sequencing method, changes in the minor proteins seem to be meaningless. Thus, only the ubiquitously-expressed protein of lipoamidase, glycol-chain synthesizing enzymes, and membrane receptors might have certain meaning as described below. The above discussed results of changes in cellular protein amounts are in line with those of previous results using 2-dimensional electrophoresis analyses regarding sexual differentiation (8).

On the other hand, fucoidan seems not to change the amounts of biotinidase, histone, ankyrin, and membrane active-transport proteins at all (Tables VII, VIII, IX and X, XI, XII, XIII).

Membrane active-transport proteins are constitutively important proteins for cell viability through nutrition uptake (44) and against toxins (mercuric ion) (45). Histone is not detected in liver of pseudo-cancer as expected, but is highly expressed (1.2%) in the LC tissue of leprosy patients. Histone may not be important for the cancer, but may be essential for the immortality of cells. Ankyrin (an S-palmitoylated anchored membrane protein) is also essential for cell viability through maintaining the cell morphology (46), but does not appear in the liver tissues except LC and HCC tissues of one HCC patient with HCV. Therefore, biotinidase and membrane active-transport proteins seem to be essentially important for cell viability. Fucoidan does not influence gene expression of these essential proteins regarding viability of the cells; i.e., fucoidan may not act on the cell death mechanism of the cancer cells such as apoptosis. Then, this is the reason why fucoidan is a safe drug without side-effects on normal cells and organs.

An increase of biotinidase activity (Kip; inhibition constant by the product biotin, and Cap) in HCC tissues has been recently found (6). However, the amount of biotinidase protein in HepG2 cells (from an HCC patient of 15 years) is not changed (from 16.4 to 14.9 μg/mg) by treatment with fucoidan (Tables VII, VIII, IX and X, XI, XII, XIII). Interestingly, the amount of serum biotinidase in the biotin-deficient patient is also not changed by administration of biotin (10 mg/day) (19). These findings suggest that biotinidase is the non-inducible (constitutively expressed) protein indispensable for growth and viability of adult humans.

The fetal liver biotinidase (Hc cells; 6.2 μg/mg) is firstly found at this time (Table IV), which suggests the important role of fetal biotinidase during fetal growth. Human serum biotinidase seems to be expressed in an age-depended differentiation manner; i.e., gradual increase of serum biotinidase to the adult level within 3 months after parturition, and the age-dependent gradual increase (highest activity; at 18.7 years of age), and decrease of the activity level to zero at the age of 174 years (47). Biotinidase is a known glycoprotein enzyme (48), and ubiquitous biotinidase activity in rat tissues are variable according to tissue differences; i.e., tissue difference of biotinidase activity occurs not by difference in biotinidase gene but the different glycochain of biotinidase (12, 47, 48). Then, decrease of biotinidase activity together with decrease of biotin by fucoidan may be caused by a possible binding of fucoidan to the glycochains of biotinidase.

Membrane-glycoprotein-enzyme lipoamidase is rich in the pig brain membrane (49) although not yet determined in the human brain, and lipoamidase was found to be decreased by fucoidan in HepG2 cells. Lipoamidase has been known to catalyze the de-anchoring reaction for N-myristoylated and glycosyl-phosphatidyl-inositol (GPI)-bonded membrane-bound enzymes (50). Lipoamidase is also thought to be the recycling enzyme of lipoic acid (49, 50). Therefore, lipoamidase activity (47) and lipoic acid concentration (16) may be correlated. Then, the decrease of de-anchoring amidase (tissue-type lipoamidase) protein also seems to occur through changes in glycol-chains of the enzyme biotinidase. Although lipoamidase could hydrolyze the S-palmitoyl-anchored membrane proteins or not is as yet not established (50), S-palmitoylated viral proteins of genome polyprotein (HCV) and envelope glycoprotein gp160 (HIV) in HepG2 cells seem to be de-anchored (Table V). The ratio of cellular membrane proteins to the total cellular proteins was found to be increased from 29.0% to 43.2% by fucoidan treatment in HepG2 cells after three days (membrane proteins are indicated in Italics in (Tables VII, VIII, IX and X, XI, XII, XIII). Thus, the immediate increase of the ratio of membrane by fucoidan may be related to the direct inhibition of glycoprotein enzyme of de-anchoring enzyme lipoamidase by fucoidan via its glycochain. A similar direct inhibition of glycoprotein enzyme of biotinidase by Okinawa-fucoidan was recently reported by our group (6). The ratio of membrane proteins to the total cellular proteins is usually approximately 50% in normal liver biopsy specimens; i.e., pseudo-liver cancer of 48.2%, LC tissue of leprosy 52.5%, LC tissue 36.0% and HCC tissue 26.5% of HCV infected patient, and HCC tissue of PBC patient 13.6%, respectively. Interestingly, Hc cells (immortal but non-cancer cell) from fetal tissue show that cellular membrane proteins are 23.9% of the total cellular proteins. This suggests that the ratio of membrane proteins seems directly linked to immortality (Tables II, III, IV). Since the ratio of membrane proteins to the total cellular proteins can be precisely, easily, and promptly determined within 3 h; i.e., tissue homogenate is ultra-centrifugated at 100,000 ×g for 1.5 h, and concentrations of proteins in the separated precipitated membrane- and soluble supernatant-fractions are determined by the fast SEC (3), thus, this ratio may become the most reliable diagnostic method for HCC and immortality. The incorporation of membrane proteins (or precursor membrane proteins) into the membrane structure requires for suitable metabolic conditions of glucose (51) and biotin concentration (52). Thus, the amount or ratio of membrane may be depended on the lipoamidase protein and/or lipoamidase activity regulated by glycolchain, and on the metabolic state of the cells. Therefore, immortalized cell lines and HCC tissues may be in the condition of “de-anchored” or “state of the decreased membrane-protein”.

Fucoidan increases glycochain-synthesizing enzymes (from 2.2 to 4.9 μg/mg; 2.2-fold; Tables VII, VIII, IX and X, XI, XII, XIII in bold brown), although not yet conclusive since the database of genes of glycochain-synthesizing enzymes were uploaded only recently. However, this increase may be important for acquiring the normalized immunity through normalized glycolchains of the glycoproteins. Therefore, it is also interesting that fucoidan per se has the ability to perform innate immunity in the invertebrate shrimp (53). Since the reduction of +ssRNA virus, HCV and HIV are over 10-fold, the effect of fucoidan on the cellular protein synthesis linked to the differentiation (or gene expression) seems to be small or slow. Therefore, fucoidan may have a direct effect on glycol-chain synthesis and/or metabolism of the glycoproteins. Indirect therapies using glycol-chain compounds or immunological polysaccharides as antigens have already been performed on cancer patients in Japan; i.e., glycochain antigen prepared from human-type tubercle bacilli SSM (Special Substance of Maruyama; Maruyama vaccine) (54) and glycol-lipid antigens prepared from the own or another patient’s urines (Hasumi vaccine) (55). Because fucoidan seems to have a direct effect on glycobiology and immunology together with the reduction of HIV and HCV on HepG2, the experiment for the therapy of chemically induced HCC by edible fucoidan from Silky-Mozuku in the rat has already been performed by us (56). It has been found that the successful therapy is observed only in the SD rat (outbred; constitutively possessing the normal immunity), but the tests with the LEW rat (inbred; without constitutively possessing the immunity genetically; generally used in the transplantation experiment) are all in vain (56).

In addition to the effects on proteins and glycoproteins, fucoidan from Japanese edible Ishi-Mozuku (Sphaerotrichia divaricate) at 0.102 mg/mL surely retarded the growth of HepG2 cells at 3 day’s treatment; collected and washed cell volume was decreased to 1/2 of the cell volume of untreated control, and two-fold lower volumes of homogenate were obtained, which was safely employed to comparative studies (Tables V, VI, VII, VIII, IX, X, XI, XII, XIII). However, this fucoidan at 0.20 mg/mL did not retard the growth of normal Hc cells. On the other hand, it is noteworthy that fucoidan from Ireland’s non-edible Mozuku (Fucus vesiculosus) at 0.20 mg/mL killed and detached the Hc cells immediately within 3 days, and Hc cells could not be harvested using the cell scraper. With respect to apoptosis (or programmed cell death), apoptosis-related proteins (deoxyribonuclease, caspases, products of the genes p53, Livin, and XIAP) were not detected in HepG2 cells (both with and without fucoidan), in Hc cells, and liver tissues (both HCC and LC tissues) at all. However, by MTT assay measuring the mitochondrial-reducing ability of living cells with deteriorated cell membrane is widely employed for detecting apoptosis in biotechnological studies. The trypan blue method, by using another dye, is also used to measure dead cells with disrupted cell membranes. Therefore, the cellular-toxicity test (or apoptosis test) using lower concentrations of Ireland’s non-edible Mozuku (Fucus vesiculosus) on Hc cells by the MTT assay may still be remained to be performed together with the direct volume-based growth test.

Analysis of gene expression is important for biological and clinical researches; i.e., differentiation, development, ageing, cancer, and diseases researches. Analysis of messenger RNA (mRNA) instead of proteins is an indirect method, and it uses a hybridization technique with expensive DNA probes. Two-dimensional (2D) electrophoresis seems to be a direct protein analytical method, however electrophoresis of hydrophobic membrane proteins and the very-large MW proteins, such as titin, still remains a difficult issue (57). Therefore, sexual differentiation in response to mating hormone (S-farnesyl-dodecapeptide) has been previously studied by using only the soluble hydrophilic cellular proteins by our group, which indicated that unexpectedly small differences in the gene expression have occurred (8). On the other hand, this micro-sequencing method analyzes directly and generally both the membrane and soluble proteins expressed in the cells including viral and bacterial invaded-microbe proteins.

Thus, this micro-sequencing method is proven to be powerful tool for the identification and determination of the component proteins of biological specimens, which are useful in biosciences, such as clinical chemistry, diagnosis and therapy in medicine, biology, biochemistry, and biotechnology.