PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “Insect cell lines”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 523 records · Page 29Linked to original sources
PubMed1995

Insect densoviruses may be widespread in mosquito cell lines.

A diagnostic PCR assay was designed based on conserved regions of previously sequenced densovirus genomic DNA isolated from mosquitoes. Application of this assay to different insect cell lines resulted in a number of cases of consistent positive amplification of the predicted size fragment. Positive PCR results were subsequently confirmed to correlate with densovirus infection by both electron microscopy and indirect fluorescent antibody test. In each case the nucleotide sequence of the amplified PCR fragments showed high identity to previously reported densoviruses isolated from mosquitoes. Phylogenetic analysis based on these sequences showed that two of these isolates were examples of new densoviruses. These viruses could infect and replicate in mosquitoes when administered orally or parenterally and these infections were largely avirulent. In one virus/mosquito combination vertical transmission to progeny was observed. The frequency with which these viruses were detected would suggest that they may be quite common in insect cell lines.

Animals↗
PubMed1991

The somatic envelopes around the germ-line cells of polytrophic insect follicles: structural and functional aspects.

The polytrophic ovarioles of three insect species, the fruit fly Drosophila melanogaster, the fungus gnat Bradysia tritici, and the honeybee Apis mellifera, were compared morphologically and with respect to the cytological organization of the peripheral somatic layers. Staining with rhodaminyl-phalloidin revealed differences in the organization of the muscle strands of the epithelial sheath and the microfilament pattern in the basal part of the follicular epithelium (mid-vitellogenic stages). Also, the size of the intercellular space between the follicle cells differed considerably in the three analyzed species. The basement membrane of Drosophila and Bradysia follicles was partially digested using purified collagenase. The observed morphological changes indicated that in both species the basement membrane of the follicular epithelium plays an important role in shaping the follicles. The possible functional significance of the species-specific structural differences is discussed.

Actin Cytoskeleton↗
PubMed2006

Onchocerca parasites and Wolbachia endosymbionts: evaluation of a spectrum of antibiotic types for activity against Onchocerca gutturosa in vitro.

BACKGROUND: The filarial parasites of major importance in humans contain the symbiotic bacterium Wolbachia and recent studies have shown that targeting of these bacteria with antibiotics results in a reduction in worm viability, development, embryogenesis, and survival. Doxycycline has been effective in human trials, but there is a need to develop drugs that can be given for shorter periods and to pregnant women and children. The World Health Organisation-approved assay to screen for anti-filarial activity in vitro uses male Onchocerca gutturosa, with effects being determined by worm motility and viability as measured by reduction of MTT to MTT formazan. Here we have used this system to screen antibiotics for anti-filarial activity. In addition we have determined the contribution of Wolbachia depletion to the MTT reduction assay. METHODS: Adult male O. gutturosa were cultured on a monkey kidney cell (LLCMK 2) feeder layer in 24-well plates with antibiotics and antibiotic combinations (6 to 10 worms per group). The macrofilaricide CGP 6140 (Amocarzine) was used as a positive control. Worm viability was assessed by two methods, (i) motility levels and (ii) MTT/formazan colorimetry. Worm motility was scored on a scale of 0 (immotile) to 10 (maximum) every 5 days up to 40 days. On day 40 worm viability was evaluated by MTT/formazan colorimetry, and results were expressed as a mean percentage reduction compared with untreated control values at day 40. To determine the contribution of Wolbachia to the MTT assay, the MTT formazan formation of an insect cell-line (C6/36) with or without insect Wolbachia infection and treated or untreated with tetracycline was compared. RESULTS: Antibiotics with known anti-Wolbachia activity were efficacious in this system. Rifampicin (5 x 10(-5) M) was the most effective anti-mycobacterial agent; clofazimine (1.25 x 10(-5) M and 3.13 x 10(-6) M) produced a gradual reduction in motility and by 40 days had reduced worm viability. The other anti-mycobacterial drugs tested had limited or no activity. Doxycycline (5 x 10(-5) M) was filaricidal, but minocycline was more effective and at a lower concentration (5 x 10(-5) M and 1.25 x 10(-5) M). Inactive compounds included erythromycin, oxytetracycline, trimethoprim and sulphamethoxazole. The MTT assay on the insect cell-line showed that Wolbachia made a significant contribution to the metabolic activity within the cells, which could be reduced when they were exposed to tetracycline. CONCLUSION: The O. gutturosa adult male screen for anti-filarial drug activity is also valid for the screening of antibiotics for anti-Wolbachia activity. In agreement with previous findings, rifampicin and doxycycline were effective; however, the most active antibiotic was minocycline. Wolbachia contributed to the formation of MTT formazan in the MTT assay of viability and is therefore not exclusively a measure of worm viability and indicates that Wolbachia contributes directly to the metabolic activity of the nematode.

Journal Article↗
PubMed1988

Posttranslational processing of endogenous and of baculovirus-expressed human gastrin-releasing peptide precursor.

The 27-amino-acid gastrin-releasing peptide (GRP1-27) is a neuropeptide and growth factor that is synthesized by various neural and neuroendocrine cells. The major pro-GRP hormone (isoform I) contains both GRP1-27 and a novel C-terminal extension peptide termed pro-GRP31-125. In order to define potentially active neuropeptides that could be generated from this novel protein domain, we analyzed the posttranslational processing of endogenous human pro-GRP1-125 in a small-cell lung cancer cell line. Because such studies are much easier in an overexpression system, we investigated at the same time the posttranslational processing of baculovirus-expressed human pro-GRP1-125 in an insect ovary cell line. In the small-cell lung cancer cell line, GRP1-27 was cleaved as expected from the endogenous prohormone at a pair of basic amino acids (29 and 30) and alpha-amidated at its C-terminal methionine; however, a number of novel peptides were generated by additional cleavages in the pro-GRP31-125 domain. In the insect ovary cell line, GRP1-27 was cleaved from the expressed prohormone by a different mechanism, as were a number of other peptides that appeared to be similar in size to those produced by the human neuroendocrine tumor cell line. These data show for the first time that an insect ovary cell line that is widely used to overexpress proteins can process a human neuropeptide precursor. They also reveal the existence of novel pro-GRP-derived peptides that are candidates for biologically active ligands.

Amino Acid Sequence↗
PubMedDate not supplied

On-line monitoring of physiological parameters of insect cell cultures during the growth and infection process.

On-line monitoring of insect cell cultures used for the production of recombinant proteins with the baculovirus expression vector system (BEVS) provides valuable tools for the optimization, operation, and control of the production process. The relative permittivity (epsilon') and CO(2) evolution rates (CER) were measured on-line using the biomass monitor and the infrared CO(2) analyzer, respectively. The growth and infection phases of two different cell lines, Spodoptera frugiperda (Sf-9) and Trichoplusia ni(High-5), were monitored using the above measurements. These in turn were correlated to the progress of the culture by using the off-line measurements of protein produced, virus titer, and biovolume, which is the product of viable cell density and mean cell volume. The epsilon', CER, and the biovolume profiles were closely matched during the growth phase of cells when grown in a batch or fed batch culture. The relationship became more complex when the cultures were either in stationary phase or in the postinfection phase. The epsilon' profile was found to be a good indicator of the process of synchronous baculoviral infection, showing a plateau between 18 and 24 h postinfection (hpi), the period during which budded virus is produced, and a peak at approximately 48 hpi correlated to the onset of accelerated cell lysis. The CER profile continues to increase after the growth period with a peak around the 24 hpi period, after which there is a decline in the profile corresponding to release of virus as seen from virus titer determinations. This was examined for Sf-9 cultures under conditions of cell densities from 3 to 50 x 10(6) cells/mL and MOI values ranging from 0.001 to 1000. The profiles were found to be similar also in the case of the High-5 cells. Thus both measurements give reliable information regarding the physiological status of the cells as seen from their correlation to virus and protein production. A further combination of these with the off-line measured parameters such as the biovolume and metabolite concentrations can give a more detailed understanding of the process and help in the better design and automation of these processes.

Animals↗
PubMed2000

PDGF and TGF-beta partially prevent 2-deoxy-D-ribose-induced apoptosis in the fat body cell line IPLB-LdFB from the insect Lymantria dispar.

The IPLB-LdFB cell line from the fat body of the insect Lymantria dispar shows the presence of immunoreactive, platelet-derived growth factor (PDGF)-AB and transforming growth factor (TGF)-beta1 molecules, as well as the corresponding plasma membrane-like receptors, i.e. PDGFR-alpha, PDGFR-beta and TGFR-beta type II. Cytofluorimetric and morphological studies reveal that the reducing sugar 2-deoxy-D-ribose (dRib), an apoptotic agent for human cells, induces apoptosis in a concentration- and time-dependent manner even in IPLB-LdFB cells. PDGF-AB and TGF-beta1 partially counteract the effect of dRib, indicating a survival role of these factors in this apoptotic model of insect cells.

Journal Article↗
PubMed1998

Invertebrate cell culture considerations: insects, ticks, shellfish, and worms.

Establishment of cell lines from insect and arachnid invertebrates has become routine, whereas other invertebrate taxa have been frustratingly unproductive of cell lines. None is available for any marine invertebrate, despite a strong and well-recognized need for cell lines from species that are important in aquaculture, from parasite vectors and intermediate hosts of parasites, from parasites themselves, from certain biomedical models, and from other species that are pests. Drawing on experiences gained attempting to establish cell lines from molluscs and trematodes and on published and ongoing research with diverse invertebrates, this chapter attempts to anticipate the problems that are likely to be encountered in such endeavors and discusses possible solutions. Criteria to be considered in the selection of basic culture media, temperature, pH, and media additives; approaches that have been developed to yield sterile primary cultures; and factors to consider in decisions about feeding schedules, retention of tissue fragments and nonadherent cells, use of heterologous feeder layers, and other variables are described. Suggestions are made concerning means to objectively score the success of tested variables and means to induce cell replication. The chapter ends with notes on conventional means to characterize cell lines and an account of contemporary efforts to immortalize cells by means of genome manipulation. Enduring success with a single molluscan cell line, transient successes with crustacean and helminth cell lines, and promising developments in transgenesis with invertebrates all lead to the hopeful conclusion that the invisible barrier to cell propagation in historically refractory species will soon be a thing of the past.

Animals↗
PubMed1986

Fatty acid composition of whole bodies, specific tissues and cell lines of two lepidopteran insects.

We detail the fatty acid compositions of last larval instars of two lepidopterans, Spodoptera frugiperda and Trichoplusia ni, two tissues from T. ni, a cell line derived from each species and the respective larval and cell culture media. Larval whole-body and specific tissue fatty acid profiles exhibited the major features commonly found in previous lepidopteran analyses, whereas the cell-line fatty acid compositions were substantially different from the compositions of both their growth media and larvae of their respective species. It appears that these cell-line patterns result from increased monoene biosynthesis in response to low levels of exogenous polyunsaturated fatty acids, a commonly observed essential fatty acid deficiency symptom in whole animals.

Animals↗
PubMed2001

Ecdysone-inducible foreign gene expression in stably-transformed lepidopteran insect cells.

Cultured cell lines that can be stably transformed with inducible gene constructs could prove extremely valuable for the continuous and economical production of recombinant proteins. Toward this goal, we have established 11 clones (designated NISES-BoMo-DK1 to 11) from a previously reported silkworm cell line, NISES-BoMo-DZ. Nine of these clonal lines showed a distinct morphological change. i.e., cell aggregation, in response to treatment with 1 microM 20-hydroxyecdysone (20E). DK10 cells transfected with various reporter assay plasmids under optimal conditions (i.e., 20-30% transfection efficiency) showed inducibility of gene expression by 20E. The 20E treatment of the prototypical DK10 cells resulted in a simultaneous, transient increase of the nuclear ecdysone (E) receptor levels. Further, this inducibility was also observed in a DK10 cell line stably transformed with the reporter plasmid that carries the hygromycin-resistance gene. This offers an opportunity to achieve efficient, continuous production of recombinant proteins. It could also allow high throughput screening for potential E agonists.

Animals↗
PubMed1992

Domains regulating transcriptional activity of the inducible orphan receptor NGFI-B.

NGFI-B is an early response gene which encodes a protein that has strong homology with nuclear receptors in the DNA binding domain and in carboxyl-terminal domains responsible for ligand binding and regulation of transcriptional activity. Previously, we have demonstrated that NGFI-B is transcriptionally active in cells grown in vitro in the absence of exogenously added ligand. However, the ligand for NGFI-B does not appear to be a component of cell culture medium, as NGFI-B remained active when expressed in cells grown in medium lacking phenol red, serum, essential vitamins, or essential amino acids. To define the transactivation domains, a mutational analysis was conducted which revealed that a serine/threonine-rich area of 18 amino acids within the amino terminus, termed TAB-1, is an important transcriptional activation domain. The mutation of two adjacent serine and threonine residues within TAB-1 significantly decreased transactivation by NGFI-B. An examination of the role of the carboxyl terminus in regulating NGFI-B transcriptional activity revealed that, in accordance with other nuclear receptors, mutants lacking portions of the carboxyl terminus had greatly decreased activity. The similarity with other receptors was further supported by studies with the mutant B delta 414-597 which encodes a fully active, truncated receptor analogous to a hormone-independent, constitutively active glucocorticoid receptor truncation mutant. This NGFI-B truncation mutant had activity similar to wild type NGFI-B in a number of mammalian cell lines; however, in contrast, it was 8-fold more active than the wild type receptor in the Drosophila S2 cell line, suggesting that insect cells either lack the NGFI-B ligand or obligatory accessory factors.

3T3 Cells↗
PubMed2006

Involvement of insect-derived growth factor (IDGF) in the cell growth of an embryonic cell line of flesh fly.

Insect-derived growth factor (IDGF) is the first adenosine deaminase-related growth factor (ADGF) purified from the conditioned medium of NIH-Sape-4, an embryonic cell line of Sarcophaga peregrina (flesh fly). Here we show the requirement of IDGF for the growth of NIH-Sape-4 cells. Growth factor activity was abolished by adsorption of IDGF from the conditioned medium of NIH-Sape-4 cells. In addition, knockdown of IDGF gene expression by RNA interference (RNAi) significantly reduced IDGF secretion from the cells following cell growth inhibition. The IDGF gene was strongly expressed in the hemocytes, and IDGF increased the viability of the larval hemocytes. These data provide evidence that IDGF is required for the growth of NIH-Sape-4 cells and possibly for hemocyte viability.

Animals↗
PubMed2005

Receptor-mediated endocytosis and intracellular trafficking of lipoproteins and transferrin in insect cells.

While the intracellular pathways of ligands after receptor-mediated endocytosis have been studied extensively in mammalian cells, in insect cells these pathways are largely unknown. We transfected Drosophila Schneider line 2 (S2) cells with the human low-density lipoprotein (LDL) receptor (LDLR) and transferrin (Tf) receptor (TfR), and used endocytosis of LDL and Tf as markers. After endocytosis in mammalian cells, LDL is degraded in lysosomes, whereas Tf is recycled. Fluorescence microscopy analysis revealed that LDL and Tf are internalized by S2 cells transfected with LDLR or TfR, respectively. In transfectants simultaneously expressing LDLR and TfR, both ligands colocalize in endosomes immediately after endocytic uptake, and their location remained unchanged after a chase. Similar results were obtained with Spodoptera frugiperda Sf9 cells that were transfected with TfR, suggesting that Tf is retained intracellularly by both cell lines. The insect lipoprotein, lipophorin, is recycled upon lipophorin receptor (LpR)-mediated endocytosis by mammalian cells, however, not after endocytosis by LpR-expressing S2 transfectants, suggesting that this recycling mechanism is cell-type specific. LpR is endogenously expressed by fat body tissue of Locusta migratoria for a limited period after an ecdysis. A chase following endocytosis of labeled lipophorin by isolated fat body tissue at this developmental stage resulted in a significant decrease of lipophorin-containing vesicles, indicative of recycling of the ligand.

Animals↗
PubMedDate not supplied

[A new cell line from Heliothis armigera (Hubn.) pupa ovaries].

The new continuous cells line derived from insect cells is perspective for the production of nuclear polyhedrosis viruses. The culture was prepared by fragmentation and trypsin treatment of the ovaries of Heliothis armigera (Hubn.) pupa with subsequent inoculation of viable cells in nutrient medium. By the present time the culture has undergone more than 100 passages in vitro and is characterized by all parameters required in a present-day passport.

Animals↗
PubMed2000

Perfusion chromatography for very rapid purification of class I and II MHC proteins.

Major histocompatibility complex (MHC) proteins are surface glycoproteins that are strongly associated with either self or foreign peptides. Their interaction with the T-cell receptor on the T-cells initiates an immune response and help in discriminating between self and non-self, respectively. We describe here a novel means of rapidly purifying human MHC molecules on either small scale or large scale from the cell lysate of lymphoblastoid B cell line and from insect cell culture supernatants by using affinity perfusion chromatography. As representative cases HLA-B2705, a class I MHC molecule, and HLA-DR1, a class II MHC molecule were purified from EBV-transformed human lymphoblastoid B cells, LG2. Soluble HLA-DR1 was also purified from the cell culture supernatant of insect cells. The peptides eluted from the purified HLA-B2705 were pool sequenced and found to have the same motif as has previously been published. This new method provides a very rapid means of purifying MHC protein molecules, applicable to both large scale and small scale purification, which in turn greatly enhances the accuracy of further analysis of the associated peptides through mass spectrometry.

Animals↗
PubMed1997

Recombinant fish neurotrophin-6 is a heparin-binding glycoprotein: implications for a role in axonal guidance.

Neurotrophin-6 (NT-6) was identified in the teleost fish Xiphophorus as a new member of the neurotrophin gene family. NT-6 binds specifically the glycosaminoglycan heparin. In this study NT-6 was expressed in a stably transfected mammalian cell line, and in insect cells via a recombinant baculovirus. It was purified to homogeneity and characterized by MS and N-terminal sequencing. NT-6 from both expression systems was proteolytically processed at one of two protease cleavage motifs and was found to be glycosylated. It supported the survival of embryonic chick sensory neurons; half-maximal survival was observed at 100 ng/ml. Furthermore, NT-6 elicited neurite outgrowth in explanted embryonic dorsal root ganglia. Addition of heparin into the medium did not potentiate the activity of NT-6 in survival assays. However, when a sensory ganglion explant was cultured in a collagen gel matrix assay adjacent to a heparin bead coated with NT-6, neurite outgrowth directed towards the bead was observed. This indicated that NT-6 was slowly released from the heparin bead generating a concentration gradient of NT-6 instrumental for axonal guidance in vitro. Thus the interaction of NT-6 with heparin might not be required for the activation of the cellular receptor for NT-6 on responsive cells but rather may serve to control, in vivo, the distribution of NT-6.

Amino Acid Sequence↗
PubMed2000

Characterization of a receptor for insect tachykinin-like peptide agonists by functional expression in a stable Drosophila Schneider 2 cell line.

STKR is an insect G protein-coupled receptor, cloned from the stable fly Stomoxys calcitrans. It displays sequence similarity to vertebrate tachykinin [or neurokinin (NK)] receptors. Functional expression of the cloned STKR cDNA was obtained in cultured Drosophila melanogaster Schneider 2 (S2) cells. Insect tachykinin-like peptides or "insectatachykinins," such as Locusta tachykinin (Lom-TK) III, produced dose-dependent calcium responses in stably transfected S2-STKR cells. Vertebrate tachykinins (or neurokinins) did not evoke any effect at concentrations up to 10(-5) M, but an antagonist of mammalian neurokinin receptors, spantide II, inhibited the Lom-TK III-induced calcium response. Further analysis showed that the agonist-induced intracellular release of calcium ions was not affected by pretreatment of the cells with pertussis toxin. The calcium rise was blocked by the phospholipase C inhibitor U73122. In addition, Lom-TK III was shown to have a stimulatory effect on the accumulation of both inositol 1,4,5-trisphosphate and cyclic AMP. These are the same second messengers that are induced in mammalian neurokinin-dependent signaling processes.

Amino Acid Sequence↗