Replication of enterotropic and polytropic murine coronaviruses in cultured cell lines of mouse origin.

To understand the virus-cell interactions that occur during murine coronavirus infection, six murine cell lines (A3-1M, B16, CMT-93, DBT, IC-21 and J774A.1) were inoculated with eight murine coronaviruses, including prototype strains of both polytropic and enterotropic biotypes, and new isolates. All virus strains produced a cytopathic effect (CPE) with cell-to-cell fusion in B16, DBT, IC-21 and J774A.1 cells. The CPE was induced most rapidly in IC-21 cells and was visible microscopically in all cell lines tested. In contrast, the coronaviruses produced little CPE in A3-1M and CMT-93 cells. Although most virus-infected cells, except KQ3E-infected A3-1M, CMT-93 and J774A.1 cells, produced progeny viruses in the supernatants when assayed by plaque formation on DBT cells, the kinetics of viral replication were dependent on both the cell line and virus strain; replication of prototype strains was higher than that of new isolates. There was no significant difference in replication of enterotropic and polytropic strains. B16 cells supported the highest level of viral replication. To determine the sensitivity of the cell lines to murine coronaviruses, the 50% tissue culture infectious dose of the coronaviruses was determined with B16, DBT, IC-21 and J774A.1 cells, and compared to that with DBT cells. The results indicate that IC-21 cells were the most sensitive to murine coronaviruses. These data suggest that B16 and IC-21 cells are suitable for large-scale preparation and isolation of murine coronaviruses, respectively.     

Comparison of lentiviruses pseudotyped with S proteins from coronaviruses and cell tropisms of porcine coronaviruses

The surface glycoproteins of coronaviruses play an important role in receptor binding and cell entry. Different coronaviruses interact with their specific receptors to enter host cells. Lentiviruses pseudotyped with their spike proteins (S) were compared to analyze the entry efficiency of various coronaviruses. Our results indicated that S proteins from different coronaviruses displayed varied abilities to mediate pseudotyped virus infection. Furthermore, the cell tropisms of porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) have been characterized by live and pseudotyped viruses. Both live and pseudoviruses could infected Vero- CCL-81 (monkey kidney), Huh-7 (human liver), and PK-15 (pig kidney) cells efficiently. CCL94 (cat kidney) cells could be infected efficiently by TGEV but not PEDV. Overall, our study provides new insights into the mechanisms of viral entry and forms a basis for antiviral drug screening.

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Functional differences in the peplomer glycoproteins of feline coronavirus isolates.

The feline coronaviruses can be divided into two distinct antigenic groups on the basis of antigenic differences found on the peplomer (E2) glycoprotein of the virus. Because the E2 glycoprotein is responsible for many of the biological functions of coronaviruses, experiments were done to determine whether there were any E2 functional differences between these two antigenic groups. The avirulent feline infectious peritonitis virus (FIPV) isolate FIPV-UCD-2, which has one antigenic type of E2, was less rapidly internalized and could not spread from cell to cell in the presence of neutralizing antibody. Two virulent isolates, FIPV-DF2 and FIPV-79-1146, as well as the non-FIP-causing feline enteric coronavirus (FECV) isolate FECV-79-1683, all of which have the second antigenic type of E2, were very rapidly internalized and were able to spread from cell to cell despite the presence of neutralizing antibody. The avirulent FIPV-UCD-2 and FECV-79-1683 isolates were more labile at 37 degrees C at pHs of 6.5 and above than were the virulent FIPV-DF2 and FIPV-79-1146 isolates.     

SARS-Cov及其他冠状病毒基因组比较分析

摘要：对病毒种内和种间基因组的比较分析能获得很多关于病毒起源与演化的信息。对17株SARS-CoV的种内基因组变异分析发现共有137个变异位点,估算出SARS-CoV的突变率为8.04×10-3核苷酸替换/位点/年。变异位点在基因组上的分布不均匀,变异位点最多的是基因组中编码S1蛋白的区域,而在编码依赖于RNA的RNA聚合酶区域中几乎没有变异位点。核苷酸和氨基酸替换的偏性预示变异可能不仅仅是由随机漂变产生。对冠状病毒种间基因组结构比较分析发现,SARS-CoV的基因组结构与IBV很相似;而保守基因系统发育分析表明,SARS-CoV属于冠状病毒的一个新分支,并且与血清型第二组冠状病毒进化关系较近。对其他某些分子特征的分析发现,在不同的方面SARS-CoV和不同组冠状病毒有不同的相似点。进一步对基因组非保守开放阅读框(ORF)的基序(motif)和跨膜区分析发现,各组冠状病毒基因组中位于基因S-E间的非保守ORF可能是同源的,但不是绝对必要的;而IBV和SARS-CoV的基因组中位于基因M-N间ORF可能不是同源的。综合分析SARS-CoV与3组血清型冠状病毒进化关系、宿主分布,以及SARS-CoV和IBV的s2m的进化关系,可以推测SARS-CoV有可能来自禽类。 Abstract:The genome comparison of inter-species and intra-species can give us much information about the origin and evolution of viruses.There are 137 mutation sites in the 17 genomes of SARS-CoV,and the mutation rate is about 8.04×10-3 substitution/site/year.The distribution of the segregating sites is not steady,the most variable region appears in S1 protein,and the nucleotide sequence of RNA-dependent RNA polymerase has very few mutation sites.The substitution bias of nucleotide acids and amino acids indicates the non-random drift products.The comparison of genome structures of SARS-CoV and other coronaviruses shows that SARS-CoV and IBV share the same genome structure.Phylogenetic analyses of conserved genes of coronaviruses indicate that SARS-CoV is a new branch of coronaviruses and appears more close to the group II coronaviruses.Interestingly,SARS-CoV shares some different features with different groups of coronaviruses.Additional analyses show that the first ORFs between S and E genes of some coronaviruses are transmembrane proteins and share the common motif,indicating the possible common ancestor.From the host distribution of different groups of coronaviruses and the phylogeny of s2m,we can deduce that avian is the probable natural host of SARS-CoV.     

Different effect of the ''recombinant'' bradykinin on the contractile activity of cultured atrial and ventricular cardiomyocytes

The physiological activity of the "recombinant" bradykinin expressed by retrovirus recombinant pPS-3-neo (brd) was tested on cultural atrial (aCMC) and ventricular (vCMC) cardiomyocytes in newborn rats. The "recombinant" bradykinin was shown to have a chronotropic effect on aCMC and an inotropic effect on vCMC. The effects are in line with the action of the synthetic bradykinin preparation at a concentration of around 10(-15) M. A pretreatment of CMC by parmidine, i.e. a bradykinin antagonist, blocked the effect of bradykinin. The contractive CMC activity in the cultural cell medium, transferred by pPS-3-neo without the bradykinin gene, was not different from the control value.     

高致病性冠状病毒感染导致宿主免疫应答异常

近二十多年，全球范围内先后爆发了由严重急性呼吸综合征冠状病毒（severe acute respiratory syndrome coronavirus，SARS-CoV）、中东呼吸综合征冠状病毒（middle east respiratory syndrome coronavirus，MERS-CoV）和严重急性呼吸综合征冠状病毒2（severe acute respiratory syndrome coronavirus 2，SARS-CoV-2）3种高致病性冠状病毒导致的疫情。这3种高致病性冠状病毒感染通常伴随着免疫系统功能失调，临床表现有淋巴细胞减少症、细胞因子风暴、急性呼吸系统窘迫综合征，甚至多器官衰竭而导致死亡。揭示高致病性冠状病毒在免疫应答中的作用机制，对于预防与控制冠状病毒感染具有重要意义。本文总结了SARS-CoV、MRES-CoV和SARS-CoV-2的进入机制和受体特征、固有免疫应答和适应性免疫应答失调方面的研究进展，强调了高致病性冠状病毒与宿主免疫应答之间的复杂相互作用，以期为防治冠状病毒感染提供参考。     

Recombinant mouse hepatitis virus strain A59 from cloned, full-length cDNA replicates to high titers in vitro and is fully pathogenic in vivo

Mouse hepatitis virus (MHV) is the prototype of group II coronaviruses and one of the most extensively studied coronaviruses. Here, we describe a reverse genetic system for MHV (strain A59) based upon the cloning of a full-length genomic cDNA in vaccinia virus. We show that the recombinant virus generated from cloned cDNA replicates to the same titers as the parental virus in cell culture ( approximately 10(9) PFU/ml), has the same plaque morphology, and produces the same amounts and proportions of genomic and subgenomic mRNAs in virus-infected cells. In a mouse model of neurological infection, the recombinant and parental viruses are equally virulent, they replicate to the same titers in brain and liver, and they induce similar patterns of acute hepatitis, acute meningoencephalitis, and chronic demyelination. We also describe improvements in the use of the coronavirus reverse genetic system based on vaccinia virus cloning vectors. These modifications facilitate (i) the mutagenesis of cloned cDNA by using vaccinia virus-mediated homologous recombination and (ii) the rescue of recombinant coronaviruses by using a stable nucleocapsid protein-expressing cell line for the electroporation of infectious full-length genomes. Thus, our system represents a versatile and universal tool to study all aspects of MHV molecular biology and pathogenesis. We expect this system to provide valuable insights into the replication of group II coronaviruses that may lead to the development of novel strategies against coronavirus infections, including the related severe acute respiratory syndrome coronavirus.     

Image analysis assessments of perfluorocarbon- and surfactant-enhanced protoplast division

Image analysis has been used to assess the growth of cell suspension-derived protoplasts of Petunia hybrida cv. Comanche at an interface between aqueous culture medium (KM8P), supplemented with 0.01% (w/v) Pluronic F-68, and oxygenated (10 mbar; 10 min) perfluorodecalin. Protoplasts synthesised a new cell wall and entered normal mitotic division which was sustainable to the cell colony/callus stage. This process was accentuated by the collective and additive effects of oxygen, perfluorodecalin and surfactant media supplements. The mean area (mm³) of protoplast-derived cell colonies after 68 days of growth was increased 35 fold over control (media alone) in the presence of these combined treatments. The new cultural regime, leading to improved cell throughput from protoplasts, is discussed primarily in relation to the role of perfluorodecalin as a gas carrier and possible effects of Pluronic F-68 in stimulating cellular uptake of nutrients and/or growth regulators. Image analysis provides a novel and accurate approach to quantifying cell growth responses.Abbreviations dpi dots per inch - FPE final plating efficiency - IPE initial plating efficiency - KM Kao & Michayluk (1975) - PFC Perfluorocarbon - UM Uchimiya & Murashige (1974)     

Regulation of cell death during infection by the severe acute respiratory syndrome coronavirus and other coronaviruses

Both apoptosis and necrosis have been observed in cells infected by various coronaviruses, suggesting that the regulation of cell death is important for viral replication and/or pathogenesis. Expeditious research on the severe acute respiratory syndrome (SARS) coronavirus, one of the latest discovered coronaviruses that infect humans, has provided valuable insights into the molecular aspects of cell-death regulation during infection. Apoptosis was observed in vitro, while both apoptosis and necrosis were observed in tissues obtained from SARS patients. Viral proteins that can regulate apoptosis have been identified, and many of these also have the abilities to interfere with cellular functions. Occurrence of cell death in host cells during infection by other coronaviruses, such as the mouse hepatitis virus and transmissible porcine gastroenteritis virus, has also being extensively studied. The diverse cellular responses to infection revealed the complex manner by which coronaviruses affect cellular homeostasis and modulate cell death. As a result of the complex interplay between virus and host, infection of different cell types by the same virus does not necessarily activate the same cell-death pathway. Continuing research will lead to a better understanding of the regulation of cell death during viral infection and the identification of novel antiviral targets.     

Influence of auxins and sucrose in monoterpenoid oxindole alkaloid production by Uncaria tomentosa cell suspension cultures

Growth and alkaloid production in Uncaria tomentosa cell suspension cultures were studied in Murashige and Skoog medium supplemented with 10 microM 2,4-dichlorophenoxyacetic acid, 10 microM kinetin, and 58 mM sucrose for maintenance and with 10 microM indole-3-acetic acid, 10 microM kinetin, and 58 mM sucrose for production. A U. tomentosa pale Uth-3 cell line, cultured in the production medium, showed a reduced lag phase and a specific growth rate (mu) of 0.27 day(-1), while cells growing in the maintenance medium showed mu = 0.20 day(-1). U. tomentosa cells growing in the production medium produced monoterpenoid oxindole alkaloids (MOA) in amounts of 10.2 +/- 1.6 microg g(-1) dry weight (DW). The chemical profile of MOA produced by in vitro cell cultures was similar to that found in the plant. After 10 subcultures, maximum MOA production decreased to 2.0 +/- 0.7 microg g(-1) DW, while tryptamine alkaloids (TA) were produced with a maximum of 6.2 +/- 0.4 microg g(-1) DW. The increase of initial sucrose concentration up to 145 mM in the production medium enhanced the cell biomass by 3.2-fold (from 10.2 +/- 0.1 to 32.8 +/- 1.1 g DW L(-1)), reduced mu from 0.27 to 0.23 day(-1), and provoked a substantial accumulation of TA (23.1 +/- 4.7 microg g(-1) DW). A high sucrose concentration stimulated MOA production in the maintenance medium (2.7 +/- 0.5 microg g(-1) DW), even in the presence of 2,4-dichlorophenoxyacetic acid.     

Production of monoclonal antibody using free-suspended and immobilized hybridoma cells: Effect of serum

The effect of serum on cell growth and monoclonal antibody (MAb) productivity was studied in a repeated fedbatch mode using both free-suspended and immobilized S3H5/gamma2bA2 hybridoma cells. In the suspension culture, serum influenced the cell growth rate but not the specific MAb productivity. The average specific growth rate of the suspension culture in medium containing 10% serum was approximately 0.99 +/- 0.12 day(-1) (+/-standard deviation), while that in medium containing 1% serum was approximately 0.73 +/- 0.12 day(-1). The specific MAb productivity was almost constant at 3.69 +/- 0.57 mug/10(6) cells/day irrespective of serum concentration reached a maximum at ca. 1.8 x 10(6) cells/mL of medium in 10% serum medium, and the cell concentration was gradually reduced to 1%. The specific MAb productivity of the immobilized cells was more than three times higher than that of the free-suspended cells. The amount of serum in the medium did not influence the specific MAb production rate of the immobilized cells. The maintenance of high cell concentration and the enhanced specific MAb productivity of the immobilized cell culture resulted in a higher volumetric MAb productivity. In addition, MAb yield in the immobilized cell culture with medium containing 1% serum was 2.2 mg/mL of serum, which was approximately three times higher than that in the suspension culture.     

A molecular arms race between host innate antiviral response and emerging human coronaviruses

Coronaviruses have been closely related with mankind for thousands of years. Communityacquired human coronaviruses have long been recognized to cause common cold. However, zoonotic coronaviruses are now becoming more a global concern with the discovery of highly pathogenic severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses causing severe respiratory diseases. Infections by these emerging human coronaviruses are characterized by less robust interferon production. Treatment of patients with recombinant interferon regimen promises beneficial outcomes, suggesting that compromised interferon expression might contribute at least partially to the severity of disease. The mechanisms by which coronaviruses evade host innate antiviral response are under intense investigations. This review focuses on the fierce arms race between host innate antiviral immunity and emerging human coronaviruses. Particularly, the host pathogen recognition receptors and the signal transduction pathways to mount an effective antiviral response against SARS and MERS coronavirus infection are discussed. On the other hand, the counter-measures evolved by SARS and MERS coronaviruses to circumvent host defense are also dissected. With a better understanding of the dynamic interaction between host and coronaviruses, it is hoped that insights on the pathogenesis of newly-identified highly pathogenic human coronaviruses and new strategies in antiviral development can be derived.

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Stimulation of release of Gal beta 1-4GlcNAc alpha 2-6 sialyltransferase from the FAZA hepatoma cell line by dexamethasone and phorbol ester

1. Gal beta 1-4GlcNAc alpha 2-6 sialyltransferase was assayed in FAZA hepatoma cells and the cell culture medium following growth of cells in presence of dexamethasone and phorbol ester. 2. There was about a seven-fold increase in sialyltransferase activities in cells and medium in presence of dexamethasone with the maximum effect occurring at 10(-6)-10(-7) M dexamethasone. 3. The presence of 10(-6) M phorbol ester in the culture medium increased sialyltransferase activities in cells and medium by ca 40% over the values found with dexamethasone alone. 4. The use of the FAZA hepatoma cell line for studies on sialyltransferase is compared with the primary hepatocyte system reported on earlier (Woloski et al., 1986).     

Levels of true thymidine kinase and nucleoside phosphotransferase in two strains of Tetrahymena pyriformis under different growth conditions.

Activities of typical thymidine kinase and nucleoside phosphotransferase are both present in logarithmically growing tetrahymena pyriformis, GL-1 and ST strains, contrary to previous reports. 2. Activities of thymidine kinase and nucleoside phosphotransferase are also found in both GL-1 and ST strains grown in the defined medium, PPL medium and Neff's medium. 3. The specific activities of both enzymes are very much influenced by the growth state. Both the specific activities of thymidine kinase and nucleoside phosphotransferase decrease steadily from the start of the experiments when the cell numbers were about 2-3 x 10(4) cells/ml in the PPL medium, while in the Neff's medium, the specific activities of thymidine kinase increase up to when the cell numbers reached 3-5 x 10(5) cells/ml and then decreased, but the specific activities of nucleoside phosphotransferase continuously decreased when the cell concentrations were 2-6 x 10(4) cells/ml. 4. In the PPL medium, the final cell numbers reached are about 6.5 x 10(5) cells/ml, while in the Neff's medium, the cell numbers increase further (to about 2 x 10(6) cells/ml). 5. No striking difference in activities of thymidine kinase and nucleoside phosphotransferase was observed when the cells were transferred from the defined medium to the Neff's medium, contrary to that reported by others for the activity of thymidylate synthetase.     

Control of alkaline phosphatase activity in C3H10T1/2 cells: role of retinoic acid and cell density.

The enzyme alkaline phosphatase (AP) has been shown to be lost or inappropriately expressed during carcinogenesis in some tissues. Because retinoic acid (RA) appears to play a role in the normal regulation of the enzyme (RA up-regulates AP in a variety of cell types) we have suggested that altered AP expression in some cancers may be caused by a defect in the ability of the cells to respond normally to retinoid. We have begun to use the chemically transformable mouse embryo fibroblast cell, C3H10T1/2, to investigate this possibility. In this initial study we characterized AP regulation in normal C3H10T1/2 cells and show that: (1) 10(-7) M RA increases AP activity within 3-4 h in serum-free medium; (2) serum inhibits short-term induction (0-8 h) in a concentration-dependent manner (10% serum causes complete inhibition); (3) during long-term RA exposure (24 h and 48 h), induction can be detected in serum-containing medium; (4) AP induction is dose related at RA concentrations from 10(-10) M to 10(-6) M in serum-free medium; (5) 10(-5) M RA is ineffective at inducing AP in serum-free medium during 8 h but is the most effective concentration in serum-containing medium during 24 h and 48 h exposures; (6) AP inducibility by RA requires near-confluent cell densities; and (7) when cultures become confluent, cells become constitutive for AP and no longer require RA for enzyme expression. The effects of serum and cell density on AP inducibility by RA and implications of the RA up-regulation of AP for teratogenesis are discussed.     

Interdigitated microelectrode (IME) impedance sensor for the detection of viable Salmonella typhimurium

Interdigitated microelectrodes (IMEs) were used as impedance sensors for rapid detection of viable Salmonella typhimurium in a selective medium and milk samples. The impedance growth curves, impedance against bacterial growth time, were recorded at four frequencies (10Hz, 100Hz, 1kHz, and 10kHz) during the growth of S. typhimurium. The impedance did not change until the cell number reached 10(5)-10(6) CFUml(-1). The greatest change in impedance was observed at 10Hz. To better understand the mechanism of the IME impedance sensor, an equivalent electrical circuit, consisting of double layer capacitors, a dielectric capacitor, and a medium resistor, was introduced and used for interpreting the change in impedance during bacterial growth. Bacterial attachment to the electrode surface was observed with scanning electron microscopy, and it had effect on the impedance measurement. The detection time, t(D), defined as the time for the impedance to start change, was obtained from the impedance growth curve at 10Hz and had a linear relationship with the logarithmic value of the initial cell number of S. typhimurium in the medium and milk samples. The regression equations for the cell numbers between 4.8 and 5.4 x 10(5) CFUml(-1) were t(D) = -1.38 log N + 10.18 with R(2) = 0.99 in the pure medium and t(D) = -1.54 log N + 11.33 with R(2) = 0.98 in milk samples, respectively. The detection times for 4.8 and 5.4 x 10(5) CFUml(-1) initial cell numbers were 9.3 and 2.2 h, respectively, and the detection limit could be as low as 1 cell in a sample.     

Complete genomic sequence of human coronavirus OC43: molecular clock analysis suggests a relatively recent zoonotic coronavirus transmission event

Coronaviruses are enveloped, positive-stranded RNA viruses with a genome of approximately 30 kb. Based on genetic similarities, coronaviruses are classified into three groups. Two group 2 coronaviruses, human coronavirus OC43 (HCoV-OC43) and bovine coronavirus (BCoV), show remarkable antigenic and genetic similarities. In this study, we report the first complete genome sequence (30,738 nucleotides) of the prototype HCoV-OC43 strain (ATCC VR759). Complete genome and open reading frame (ORF) analyses were performed in comparison to the BCoV genome. In the region between the spike and membrane protein genes, a 290-nucleotide deletion is present, corresponding to the absence of BCoV ORFs ns4.9 and ns4.8. Nucleotide and amino acid similarity percentages were determined for the major HCoV-OC43 ORFs and for those of other group 2 coronaviruses. The highest degree of similarity is demonstrated between HCoV-OC43 and BCoV in all ORFs with the exception of the E gene. Molecular clock analysis of the spike gene sequences of BCoV and HCoV-OC43 suggests a relatively recent zoonotic transmission event and dates their most recent common ancestor to around 1890. An evolutionary rate in the order of 4 x 10(-4) nucleotide changes per site per year was estimated. This is the first animal-human zoonotic pair of coronaviruses that can be analyzed in order to gain insights into the processes of adaptation of a nonhuman coronavirus to a human host, which is important for understanding the interspecies transmission events that led to the origin of the severe acute respiratory syndrome outbreak.     

Carbonic anhydrase of blue-green alga Spirulina platensis]

Carboanhydrase (carbonate-hydroliase EC 4.2.1.1.) is found in the extract of Spirulina platensis cells. A linear dependency of the enzyme activity on the protein concentration; pH optimum is found to be 8.0. Specific activity of carboanhydrase is 3 muM/min-mg of protein under the concentration of CO2 of 4-10(-3) M, appearing Michelis constant being 4.9-10(-3) M. The enzyme was stabilized with 10 mM of cisteine, its activity was inhibited by 50% with sulphanylamide (1-10(-5) M), acetazolamide (8--10(-7) M) and Cl- ions (5-10(-2) M). The activity of carboanhydrase, as well as the rate of NaH14CO3 fixation, depended on the pH value of cultural medium.     

Bat origin of human coronaviruses

Bats have been recognized as the natural reservoirs of a large variety of viruses. Special attention has been paid to bat coronaviruses as the two emerging coronaviruses which have caused unexpected human disease outbreaks in the 21st century, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV), are suggested to be originated from bats. Various species of horseshoe bats in China have been found to harbor genetically diverse SARS-like coronaviruses. Some strains are highly similar to SARS-CoV even in the spike protein and are able to use the same receptor as SARS-CoV for cell entry. On the other hand, diverse coronaviruses phylogenetically related to MERS-CoV have been discovered worldwide in a wide range of bat species, some of which can be classified to the same coronavirus species as MERS-CoV. Coronaviruses genetically related to human coronavirus 229E and NL63 have been detected in bats as well. Moreover, intermediate hosts are believed to play an important role in the transmission and emergence of these coronaviruses from bats to humans. Understanding the bat origin of human coronaviruses is helpful for the prediction and prevention of another pandemic emergence in the future.     

CCK stimulates growth of six human pancreatic cancer cell lines in serum-free medium

The growth responses of six human pancreatic cancer cell lines (SW-1990, PANC-1, MIA PaCa-2, BxPC-3, RWP-2 and CAPAN-2) to cholecystokinin (CCK) were evaluated in serum-free medium (SFM). In each experiment cells were initially plated in media containing fetal calf serum (FCS) grown for 48-72 h, and then washed with saline. Cells were incubated for an additional 72 to 96 h in medium devoid of FCS in the absence (control) or presence of synthetic CCK analogue (Thr4,Nle7)CCK9 (10(-13) to 10(-9) M), or CCK8 (10(-12) to 10(-9) M), or CCK39 (10(-12) to 10(-9) M). Viable cell counts were performed with a hemocytometer. Growth of each cell line was stimulated in the presence of CCK in serum-free medium, although the magnitude of responses differed. The concentrations of (Thr4,Nle7)CCK9 which stimulated the greatest increase in cell counts as compared to controls for each cell line were: SW-1990, 39% (10(-12) M, P less than 0.05); PANC-1, 45% (10(-9) M, P less than 0.005); MIA PaCa-2, 42% (10(-12) M, P less than 0.005); BxPC-3, 32% (10(-13) M, P less than 0.05); RWP-2, 37% (10(-11) M, P less than 0.005). Maximal response to CCK8 occurred at the 10(-9) M dose for each cell line: MIA PaCa-2, 40% (P less than 0.025); PANC-1, 85% (P less than 0.001); RWP-2, 68% (P less than 0.001) and CAPAN-2, 52% (P less than 0.001). The maximal increase in cell count with CCK39 ranged from 44-74% and occurred with either 10(-11) or 10(-10) M. CCK8 in SFM also stimulated cell growth as well as or better than FCS alone in three out of four pancreatic cell lines.(ABSTRACT TRUNCATED AT 250 WORDS)