Cell adhesion and migration of different human colon cell lines and primary tumors

Summary Several cell lines derived from colon tumors (HT-29, WiDr, PT4, PT5) and from human liver metastases (LM3) and primary human colon tumor cells (PTR) were compared with regard to their ability to migrate and to attach to different substrates (collagen G, laminin, fibronectin). Cells from the WiDr cell lines migrated actively, whereas the other cell lines, and LM3 and the PTs showed almost no migratory activity. The attachment efficiency was best in all cell lines assayed when tested on collagen followed by laminin and fibronectin as substrates. The HT-29 cells showed the strongest adhesion to all substrates, while the adhesiveness of PT4, PT5, and LM3 was reduced. The WiDr cells which migrated best showed the lowest adhesion to the substrates.     

Tumor cell adhesion of human colon carcinoma cells with different metastatic properties to extracellular matrix under dynamic conditions of laminar flow

Purpose: Shear forces have an important influence on cell adhesion and other cellular functions, and malignant cell lines appear to possess different adhesive properties under static and dynamic conditions. Thus, we analyzed human colon carcinoma cell adhesion under dynamic conditions and examined the interactions of HT-29 colon carcinoma cells of different metastatic properties with various immobilized ECM components. Methods: Wall shear adhesion threshold (WSAT), dynamic adhesion rate (DAR), and adhesion stabilization rate (ASR) were compared between the cell lines using dynamic conditions in a laminar flow chamber by decreasing the flow (wall shear stress) of cell suspensions. Patterns of cell adhesion under dynamic conditions were compared to adhesive interactions in static microtiterplate assays. Results: Poorly metastatic HT-29P cells adhered six times more than highly metastatic cells to type I collagen under laminar fluid flow, whereas only highly metastatic HT-29LMM showed adhesive interactions with fibronectin under static and dynamic conditions. High rates of cell adhesion to collagen IV were found under static, but not under dynamic, conditions. Conclusions: Although poorly and highly metastatic HT-29 cells express similar patterns of integrins, they differ in their adhesive properties to ECM components under static and dynamic conditions. Hydrodynamic shear forces appear to influence adhesive properties of HT-29 cells, and differences between dynamic and static cell adhesion were found. Received: 23 November 1999 / Accepted: 4 May 2000     

Antiproliferative activity of the non-myelotoxic antitumour agent of plant origin,Thaliblastine, on two human glioma cell lines

Summary The antiproliferative activity of the non-myelotoxic antitumour agent of plant origin, Thaliblastine, on two human glioma cell lines is described. Thaliblastine was added once one day following start of culture; proliferation was monitored over 7 days. The anti-proliferative activity of Thaliblastine was strongly dependent on concentration and time of incubation. The ID₅₀ of Thaliblastine in T406 and GW27 glioma lines was 5.1 g/ml and 8.2 g/ml (7.0 M and 11.2 M), respectively.Abbreviation TBL Thaliblastine     

Markers and characteristics of human SCLC cell lines

Summary Permanent human small cell lung cancer (SCLC) cell lines established in our laboratory were investigated for their expression of the enzymatic neuroendocrine markers l-DOPA decarboxylase (DDC), neuron-specific enolase (NSE), and creatine kinase (CK), including its BB isoenzyme (CK-BB), the classical tumor markers carcinoembryonic antigen (CEA), the and subunits of human chorionic gonadotropin (-HCG, -HCG), and -fetoprotein (-FP), and their chromosomal characteristics. DDC activities were detectable in 5/6 SCLC cell lines and absent in non-SCLC. NSE levels ranged from 160 to 1422 ng/mg soluble protein and were < 290 ng/mg soluble protein in non-SCLC. Activities of CK and levels of CK-BB clearly distinguished SCLC from non-SCLC with CK acticities > 1000 munits/mg soluble protein and CK-BB levels > 3000 ng/mg soluble protein in SCLC and < 300 munits/mg soluble protein and < 2000 ng/mg soluble protein in non-SCLC. CEA was detectable in 5/6 SCLC cell lines but absent in non-SCLC, and its level seemed to correlate with those of DDC, NSE, and CK. One cell line, SCLC-16H, lost some of its neuroendocrine properties and CEA after 1 year of in vitro cultivation. Generally, marker levels were low in fast growing cell lines and high in slow growing cell lines. HCG and subunit and -FP were not detectable in SCLC cell lines. All SCLC cell lines examined had near diploid DNA indices and modal chromosome numbers. Double minute chromosomes and homogeneously staining regions were found in 2/5 and 4/5 SCLC cell lines respectively. With respect to chromosomal aberrations, we found a deletion of the short arm of at least one chromosome 3 in all SCLC cell lines (5/5). These data show that (1) SCLC expresses neuroendocrine markers and CEA; (2) CK is the most sensitive marker, and DDC and CEA are the most specific markers for SCLC in vitro; (3) individual marker levels correlate with each other and the in vitro malignancy of SCLC; and (4) SCLC cell lines have relatively uniform chromosomal chracteristics. Our results suggest that patients whose tumors have high levels of DDC, NSE, CK-BB, and CEA have a better prognosis than those with low marker levels. This hypothesis could be proved by comparing pairs of patients that are matched for all known prognostic parameters, in particular tumor spread, for their serum and tumor marker levels with respect to the patients' outcome and prognosis.This work was supported by the SFB 215 of the German Research Society     

Establishment,growth properties,and morphological characteristics of permanent human small cell lung cancer cell lines

Summary Cell lines from SCLC were established with a success rate of 43% from different metastatic sites of treated and untreated patients. All 6 SCLC cell lines grew as floating cell aggregates without substrate adherence. The degree of aggregation ranged from very tight spheroids to very loose sheets and chains. This gross morphological property showed a striking correlation to the PDT, with short PDTs in loose growing cell lines and long PDTs in tight growing cell lines. Cell size and nuclear features, i.e., chromatin pattern and nucleolar prominence, also seemed to correlate with the PDT and gross morphology. All SCLC cell lines had dense core granules by electron microscopical examination. Several different serum-free and serum-supplemented growth media were tested for their feasibility in estabilishing and permanently growing SCLC. Serum-free SIT medium and SIT 2.5 medium provided the best results in liquid culture. For semisolid SCLC cultivation, R10 medium was suprior to all other media tested. These cell lines are currently under intensive biochemical, molecular biological, and cytogenetical investigation in different laboratories and thus provide a tool for studying the biology of lung cancer.Abbreviations SCLC small cell lung cancer - FBS fetal bovine serum - R 10 Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS - D 10 Dulbecco's modified Eagle's medium supplemented with 10% FBS - SIT RPMI 1640 medium supplemented with selenium, insulin, transferrin - HITES RPMI 1640 medium supplemented with hydrocortisone, insulin, transferrin, estradiol, selenium - SIT 2.5 SIT medium supplemented with 2.5% FBS - PBS phosphate buffered saline - PDT population doubling time This work was supported by the SFB 215 of the German Research Society. The authors Thank Dr. Adi F. Gazdar for reviewing the morphology     

Effects of radiation fractionation on four squamous cell carcinoma lines with dissimilar inherent radiation sensitivity

Summary The effect of radiation fractionation was investigated using a new 96-well-plate clonogenic assay in four squamous cell carcinoma lines. Earlier experiments had shown that two of the cell lines (UT-SCC-1A and UM-SCC-14A) were inherently relatively sensitive to irradiation, and two (UM-SCC-1 and UM-SCV-1A) relatively resistant. All of the four carcinomas from which the cell lines were established had poor clinical outcome. The radiation doses were given as a single exposure, or split into two or three equal fractions with a 24-h interval. The two inherently sensitive cell lines showed enhanced survival after radiation fractionation as compared with a single dose, whereas the resistant cell lines did not. The result suggests that both the inherent resistance of cancer cells to irradiation and the repair of sublethal radiation damage may lead to treatment failure, and that shortening of the total irradiation time may overcome cancer cell recovery between fractions in some, but not in all carcinomas.Abbreviation SCC squamous cell carcinoma The study was financially supported by the Finnish Cancer Society     

Relationship between cell adhesion molecules expression and the biological behavior of gastric carcinoma

AIM： To evaluate the relationship between the expression of cell adhesion molecules （CAMs） and the biological behavior of gastric carcinoma. METHODS： Expression of syndecan-1, E-cadherin and integrin β3 were evaluated by immunohistochemical study in a total of 118 gastric carcinomas and 20 nontumor gastric mucosas. RESULTS： The expressions of syndecan-1 and E-cadherin were significantly lower in gastric carcinoma compared to non-tumor gastric mucosa, and the low expression rates were positively correlated to the tumor invasion depth, vessel invasion, lymph node metastasis and distant metastasis （P 〈 0.01 in all cases）. However, the expression of integrin β3 was significantly higher in gastric carcinoma compared to non-tumor gastric mucosa, and the high expression rates were positively correlated to the tumor invasion depth, vessel invasion, lymph node metastasis and distant metastasis （P 〈 0.01 in all cases）. In addition, the three protein expressions were correlated to the tumor growth pattern （P 〈 0.01, P 〈 0.01, and P 〈 0.05 respectively）, but not correlated to tumor differentiation （P 〉 0.05, P 〉 0.05 and P 〉 0.05 respectively）. Positive correlation was observed between the expressions of syndecan-1 and E-cadherin, but they which were negatively correlated to the expression of integrin β3 （P 〈 0.01 in all cases）. Univariate analysis demonstrated that the mean survival time and 5-year survival rate were lower in the cases with low expressions of syndecan-1 and E-cadherin and high expression of integrin β3 （P 〈 0.01, in all cases）. COX multivariate analysis showed that the expression level of syndecan-1 could be an independent prognostic index of gastric carcinoma （P 〈 0.01）, whereas E-cadherin and integrin β3 could not be independent indexes （P 〉 0.05, P 〉 0.05 respectively）.CONCLUSION： The low expression of syndecan-1 and E-cadherin and the high expression of integrin β3 are significantly correlated with the invasion and metastasis o     

永生化肝细胞系研究进展

原代肝细胞因体外生存期短、培养困难而限制了它的应用,近年来分子生物学技术的发展使正常细胞永生化成为可能.肝细胞的永生化对于药物毒理、生物人工肝脏支持以及肝脏组织工程的研究都具有非常深远的意义.该文综述近年国外学者构建的永生化肝细胞系,并讨论其应用前景和可能面临的问题.     

Establishment and characterization of four new squamous cell carcinoma cell lines derived from oral tumors

Summary Four cell lines were established from squamous cell carcinomas (SCC) of the oral cavity. Cell lines AW 13516 and AW 8507 were derived from poorly differentiated SCC and epidermoid carcinoma of the tongue respectively. Cell line AW 10498 was derived from moderately differentiated SCC of the lower alveolus, and AW 9803 grew from a well-differentiated SCC of a retromolar trigone. The cultures showed typical epithelial cell morphology, numerous mitotic figures, occasional multinucleated giant cells, individual cell diskeratosis and nuclear and nucleolar abnormalities. The cell lines AW 13516 and AW 8507 were fast growers with a doubling time of 35.5 h and 31.9 h, respectively, which was independent of the initial seeding density. Cell lines AW 10498 (doubling time 52.2 h) and AW 9803 (doubling time 66 h) showed slower growth and had shorter doubling times at higher seeding densities. The presence of cytokeratins was detected in all the four cell lines by using polyclonal antikeratin antisera in indirect immunofluorescence and in Western blotting. None of the cell lines expressed major histocompatibility complex (MHC) class II antigens. MHC class I antigens were expressed by three cell lines but not by AW 9803. Flow cytometric analysis of DNA content and chromosomal studies suggested the presence of polyploidy and aneuploidy in all the four cell lines. Ultrastructural studies revealed typical epithelial cell features, such as the presence of desmosomes, tonofilaments and keratin bundles.Abbreviations SCC squamous cell carcinoma - PBS phosphatebuffered saline - MHC major histocompatibility complex     

The role of tumor cell adhesion as an important factor in formation of distant colorectal metastasis

PURPOSE: The interactions of blood-borne colorectal carcinoma cells with vascular endothelium are important during hematogenous formation of distant metastases. To adhere to the vessel wall, circulating carcinoma cells that come into contact with the microvasculature must resist the tractive forces of the flow of plasma and other circulating cells that tend to detach them from the wall. METHODS: Hydrodynamic adhesion assays have been introduced to mimic the microcirculation and investigate cell adhesion under flow conditions. Different aspects of colorectal cancer cell adhesion during hematogenous formation of distant metastases are summarized and discussed in this review. RESULTS: Adhesion of colorectal carcinoma cells to endothelial cells and extracellular matrix is influenced by the presence of fluid flow. Shear forces alone are able to induce signal transduction events in these cells that result in cell activation and modification of adhesive behavior. CONCLUSIONS: Consideration of fluid dynamics of circulating colorectal cancer cell movement in the microcirculation leads to new knowledge ofin vivo processes that are involved in tumor cell adhesion to the vessel wall in host organs. Shear forces have been found to influence adhesive properties of colorectal carcinoma cells to endothelial cells and underlying subendothelial extracellular matrix. Understanding the complex processes involved in tumor cell adhesion may result in the development of novel therapeutic strategies.Presented in part at the meetings of the American Society of Colon and Rectal Surgeons, Washington, D.C., May 1 to 6, 1999, and Boston, June 24 to 29, 2000. Winner of the New Jersey Society for Colorectal Surgeons Award 1999.     

Combined chemo- and radiosensitivity testing with ifosfamide and ACNU in human lung cancer cell lines

The combination of radiotherapy and cytostatic drugs is of interest in the treatment of several solid tumors. In these preclinical investigations we tested whether ifosfamide and ACNU are able to enhance radiation effects. The experiments were performed by using the MTT assay. Two small cell and 2 non small cell lung cancer cell lines were involved. Ifosfamide, ACNU or both drugs together were tested in 6 different concentrations adjusted to the peak blood level. During the 1 hour drug incubation time, the cell lines were either irradiated with a single dose of 4 Gy or not. The main results were that ACNU possessed only little cytostatic activity in the cell lines under examination. In contrast, ifosfamide caused a dose related cytostatic activity in all cell lines. Concentrations of 26 g/ml (NCC-SCLC H 82) or 10–12 g/ml (3 other cell lines) were able to reduce the surviving cell fraction to less than 50% (IC₅₀). While ACNU showed no clear outlined radiosensitizing properties, ifosfamide reinforced the radiation effects in 3 out of 4 cell lines indicating radiosensitizing properties of this drug. Synergistic effects of ifosfamide and ACNU have not been noticed. These preclinical investigations may constitute the basis for combined ifosfamide and irradiation therapy in future clinical trials.Presented at the Satellite Symposium Ifosfamide in Tumor Therapy: Questions for the Nineties; 15th International Cancer Congress, Hamburg, August 16–22, 1990     

Growth requirements,growth factor responsiveness,and growth factor secretion of three human embryonal carcinoma cell lines

Summary The in vitro growth requirements of three human embryonal carcinoma cell lines (H 12.7, 2102 EP, 1428 A) were investigated. The basal medium DME/F12 supplemented with insulin, transferrin, and low-density and high-density lipoproteins was sufficient to support substantial multiplication of all three lines. The most efficient attachment factor was either fibronectin (for 2102 EP and 1428 A) or collagen type I (H 12.7). In a serum-free system the influence of epidermal growth factor (EGF), insulin-like growth factor I, multiplication stimulating activity (MSA), a platelet extract, and the glucocorticoids dexamethasone and hydrocortisone, as determined by the DNA synthesis rate of the cells, was generally minimal. However, the DNA synthesis rate of cell lines H 12.7 and 2102 EP was increased by MSA, and the line with the highest potential to differentiate (H 12.7) was stimulated by EGF. All three cell lines secreted growth factors in a heterologous stimulation assay. Insulin-like growth factors I and II were not part of the growth promoting activity. The inhibitory effect of a monoclonal anti-EGF antibody on the ³H-thymidine incorporation of cell line 2102 EP might indicate autocrine secretion of EGF or an EGF-like factor by this cell line.     

Regulation of growth hormone receptors in human prostate cancer cell lines

We previously demonstrated the gene expression of two growth hormone (GH) receptor (GHR) isoforms in prostate cancer (PCa) patient tissues and human PCa cell lines. In that initial study, we characterized LNCaP cell GH binding characteristics to GHR and its activation of relevant signal transduction pathways. We now show that GH binding to GHR and GHR mRNA expression in the cell lines studied are hormonally regulated. In the androgen-dependent LNCaP cells, the potent, specific and stable androgen analogue, mibolerone, caused a time- and biphasic dose-dependent, stimulation of ¹²⁵I-hGH specific binding to cells cultured in serum-free medium (SFM); however, when LNCaP cells were grown in chemically defined Gc full medium, long-term mibolerone-induced inhibition was observed. This effect of Gc on the androgen response was mimicked by the triiodothyronine (T₃) contained in GC. In contrast, oestradiol (E₂), cortisol, and insulin-like growth factor (IGF)-I and -II all caused stimulation of GH binding. Furthermore, we also observed homologous and heterologous, isoform- and cell-type-specific regulation of GHR mRNA expression in all three cell lines. In LNCaP cells, GH caused stimulation of both GHR mRNA and of its exon 9-truncated isoform, GHR_tr; however, mibolerone, E₂ and T₃ all stimulated GHR_tr mRNA more potently than they did GHR. In androgen-independent PC3 cells, GH stimulated GHR_tr expression, but almost not GHR, while in contrast, in androgen-independent DU145 cells, GH caused a clear reduction in GHR and less so in GHR_tr. The differential regulation of GHR isoform gene expression in human PCa cell lines and of GHR functional capacity (GH binding), by hormones and growth factors relevant to disease progression, suggests that GHR may prove to be an additional therapeutic target to slow down/prevent progression of human prostate cancer.     

Patterns of antibody reactivity against selected human leukemia cell lines

Summary Absorption procedures which allow the production of a selectively cytotoxic anti-human lymphocyte serum are described. Although the production of a reagent whose reactivity is restricted exclusively to lymphocytes may be achieved by exhaustive absorption steps using fresh human erythrocytes, CML cells, and fetal liver cells, a more realistic alternative is the use of appropriately selected cultured human leukemia cell lines. Data are presented which show how these cell lines may be employed to selectively manipulate the crossreactivity spectrum of ALS.Pre-treatment of donor bone marrow cells prior to transplantation with a selectively lymphocytotoxic ALS has been shown to allow transplantation of bone marrow across major histocompatibility barriers in rodents without the occurrence of GvH reactions, and it is the purpose of the present investigations to show that an analogous anti-human ALS can be prepared which possesses the required degree of selectivity to allow its application for human bone marrow transplantation.Abbreviations ALS anti-lymphocyte serum - CFU-c colony-forming unit, in vitro - CFU-dc colonyforming unit, diffusion chamber - CML chronic myelogenous leukemia - FCS fetal calf serum - GvHR graft-versus-host reaction - MLC mixed lymphocyte reaction - SAL selective anti-lymphocyte serum     

Hsp70 response to 5-fluorouracil treatment in human colon cancer cell lines

Background and aims Colorectal cancer is a common disease with high rate of mortality. Although there is evidence of some benefits of 5-fluorouracil (5-FU), the most commonly used drug in colon cancer therapy, it still remains unsatisfactory because of intrinsic or acquired drug resistance. Heat shock proteins (Hsps) synthesis can be increased by cellular insults, such as chemotherapy-induced damage. Inducible Hsp70 has been suggested to be involved in cytoprotection against apoptosis. In the present study, we investigated whether the content of Hsp70 is associated to 5-FU resistance. Methods HT-29 and SNU-C4 human colon cancer cell lines were treated with 5-FU and their relative chemoresistance, and Hsp70 were determined. Results Comparison of IC₅₀ values showed that the HT-29 cells were relatively resistant to 5-FU, whereas the SNU-C4 cells presented greater sensitivity to this drug. Further, 5-FU treatment leads to a hypodiploid population in HT-29 cells significantly lower compared to SNU-C4 cells. In the HT-29 cell line, 5-FU treatment promoted an increase of 5.5 times in Hsp70 concentration after 12 h. Then, within 24 h, the increase in Hsp70 levels was still about two times. In contrast, in the SNU-C4 cell line, 5-FU induced an increase of about two times in the Hsp70 content after 12 h and, after 24 h, did not significantly affect Hsp70 content. Conclusions These data suggest that 5-FU induced Hsp70 synthesis in the HT-29 resistant cell line and that this Hsp70 accumulation could protect against 5-FU-induced apoptosis. Thus, Hsp70 protection against 5-FU-induced apoptosis might underlie colon cancer chemoresistance.     

活动性幼年特发性关节炎患者sICAM-1 sVCAM-1的表达及意义

目的测定各型活动性幼年特发性关节炎(JIA)患儿血清中可溶性细胞间黏附分子-1(sICAM-1)、可溶性血管间黏附分子-1(sVCAM-1)、白细胞介素(IL)-1、IL-4、肿瘤坏死因子(TNF)-α水平,探讨sICAM-1、sVCAM-1与疾病活动、疾病分型以及疾病严重程度的关系。方法用酶联免疫吸附试验(ELISA)检测30例活动性JIA患儿与8名健康对照儿童sICAM-1、sVCAM-1水平;用放射免疫法(RIA)检测IL-1、IL-4、TNF-α水平。结果30例JIA患者血清中sICAM-1、sVCAM-1、IL-1、IL-4、TNF-α水平明显高于健康对照组(P<0.01);在各型JIA中sVCAM-1与血沉(ESR)、C反应蛋白(CRP)呈正相关(P<0.05或0.01);而sICAM-1仅在全身型及多关节型JIA患儿中与关节肿胀指数、夜间痛呈正相关(P<0.01),与炎性指标ESR、CRP等无关。结论JIA患者血清中sICAM-1、sVCAM-1、IL-1、IL-4、TNF-α水平显著升高,可能参与了JIA的发病过程,sVCAM-1、sICAM-1可与ESR、CRP一起作为判断病情严重性的指标,且可能与JIA分型有关。     

四株人结肠腺癌细胞CEA表达水平的检测及其意义

目的:检测四株结肠癌细胞株中癌胚抗原(carcinoembryonic antigen,CEA)的表达,并探讨其意义．方法:四株人结肠腺癌细胞(LS174T,LoVo, SW480及HCT-8)分别应用ELISA法测定细胞培养上清中CEA含量;免疫组化检测细胞CEA蛋白表达;半定量RT-PCR检测细胞CEA mRNA表达丰度．结果:LS174T和SW480细胞在蛋白和mRNA水平均可检测到CEA的表达且前者表达量高于后者(细胞培养上清中CEA含量:1050±25．0 ng／107 cells vs 66±5．6 ng／107 cells,P<0．0001;半定量检测CEA mRNA表达丰度:1．137±0．155 vs 0．399±0．135,P=0．003);而LoVo细胞在蛋白和和mRNA水平均未检测到CEA的表达:HCT-8细胞免疫组化有弱阳性染色．结论:四株结肠癌细胞株的实际CEA表达情况与既往文献报道不尽相同．这种细胞株CEA表达特性的改变有可能影响其体外生物学行为从而导致实验结果的不确定性．     

Carbohydrate-dependent binding of human myeloid leukemia cell lines to neoglycoenzymes,matrix-immobilized neoglycoproteins,and bone marrow stromal cell layers

Summary The presence of sugar receptors on human myeloid leukemia cells was comparatively assessed by a highly sensitive binding assay, employing a panel of 14 types of neoglycoenzymes (chemically glycosylatedEscherichia coli -galactosidase). The selected carbohydrate ligands mainly encompass common components of natural glycoconjugates as mono- or disaccharides. The monocytoid cells of the THP-1 line, the very young myeloblasts and the myeloblasts of the lines KG-1a and KG-1, the promyelocytes of the HL-60 line, and the early myeloblasts/erythroblasts of the K-562 line displayed a nonuniform pattern of specific binding with quantitative differences at a fixed, nonsaturating concentration of the probes. Scatchard analysis in four cases corroborated the indication of cell-type-related differences between the various cell lines. To test whether the detectable cellular sugar-binding sites can mediate adhesion to glycoligands, a rather simple model matrix of nitrocellulose-immobilized neoglycoproteins was first used. In comparison to the carbohydrate-free carrier protein significant cell adhesion was observed primarily with neoglycoproteins that exposed galactose,N-acetylgalactosamine,N-acetylglucosamine, mannose, and fucose moieties among the 11 tested types of carbohydrate residue. Subsequently, human bone marrow stromal cell layers were tested as a model matrix with increased levels of physiological relevance and complexity. Mixtures of carbohydrate and neoglycoprotein were employed as inhibitors of an interaction via lectins between the stromal and the tumor cells. The carbohydrate-dependent alterations of this parameter revealed cell-type-associated properties. Tumor cell binding was significantly decreased for not more than two lines with the effective sugars, namelyN-acetylgalactosamine, mannose, fucose, and sialic acid.