Polymer Thin Film-Induced Tumor Spheroids Acquire Cancer Stem Cell-like Properties.

: Although cancer stem cells (CSC) are thought to be responsible for tumor recurrence and resistance to chemotherapy, CSC-related research and drug development have been hampered by the limited supply of diverse, patient-derived CSC. Here, we present a functional polymer thin film (PTF) platform that promotes conversion of cancer cells to highly tumorigenic three-dimensional (3D) spheroids without the use of biochemical or genetic manipulations. Culturing various human cancer cells on the specific PTF, poly(2,4,6,8-tetravinyl-2,4,6,8-tetramethyl cyclotetrasiloxane) (pV4D4), gave rise to numerous multicellular tumor spheroids within 24 hours with high efficiency and reproducibility. Cancer cells in the resulting spheroids showed a significant increase in the expression of CSC-associated genes and acquired increased drug resistance compared with two-dimensional monolayer-cultured controls. These spheroids also exhibited enhanced xenograft tumor-forming ability and metastatic capacity in nude mice. By enabling the generation of tumorigenic spheroids from diverse cancer cells, the surface platform described here harbors the potential to contribute to CSC-related basic research and drug development. SIGNIFICANCE: A new cell culture technology enables highly tumorigenic 3D spheroids to be easily generated from various cancer cell sources in the common laboratory.

Polyphosphoinositides are derived from ether-linked inositol glycerophospholipids in rat brain.

Membrane inositol glycerophospholipid (IGP) is metabolized to phosphatidylinositol-4-phosphate (PIP), phosphatidylinositol-4, 5-bisphosphate (PIP2), and inositol triphosphate (IP3) in signaling transduction. This study was carried out to determine the subclasses of IGP involved in signaling pathway. The acyl chain moieties of the phospholipids are easily modulated by dietary fatty acids. We analyzed acyl chain composition of IGP 3-subclasses, PIP and PIP2 from rat brain after feeding sunflower seed oil enriched with linoleic acid or fish oil high in eicosapentaenoic acid and docosahexaenoic acid. Long chain polyunsaturated fatty acids (LCPUFA) as eicosapentaenoic acid and docosahexaenoic acid were not incorporated into ether-linked IGP (alkenylacylglycerophosphoinositol and alkylacyl-glycerophosphoinositol), PIP and PIP2, while diacyl-glycerophosphoinositol (GPI) contained high LCPUFA. These results suggest that PIP might be phosphorylated from only the ether-linked IGP (alkenylacyl- and alkylacyl species) but not from diacyl subclass for signals to intracellular responses in the plasma membrane of rat brain.

Cloning and expression of male-specific protein (MSP) from the hemolymph of greater wax moth, Galleria mellonella L.

Male-specific protein (MSP) is a soluble protein that accumulates in high amounts in the hemolymph and other organs of adult male wax moth. The MSP was purified from adult male wax moth by gel filtration and reversed phase column chromatography, and its amino acid sequence was determined. Because of blocked N-terminus, several internal amino acid sequences of MSP were obtained by the in-gel digestion method using trypsin. RT-PCR was conducted using degenerate primers designed from the internal amino acid sequences. 5'-RACE PCR was used to obtain the complete coding region and 5'-UTR sequence. The full length MSP cDNA sequence encodes a 239 amino acid polypeptide with an 18 amino acid signal peptide. The putative mature MSP has a molecular mass of 24,317 Da and an isoelectric point (pI) of 6.00, but shows a molecular mass of 27 kDa on SDS-PAGE. Sequence alignment showed a significant similarity between MSP and juvenile hormone binding proteins (JHBPs) of several lepidopteran species, including G. mellonella.

Wax moth, Galleria mellonella fat body receptor for high-density lipophorin (HDLp).

To identify and characterize the HDLp (high-density lipophorin) receptor from Galleria mellonella (LpRGm), we used techniques of ligand blotting. This method was, to our knowledge, first used to characterize the lipophorin receptor (LpR) in insects. LpRGm had an approximate molecular weight of 97 kDa under non-reducing conditions and bound the HDLp specifically. The time-course of lipophorin binding to their receptor protein was rapid. The binding of lipophorins to their receptors was saturable with a Kd of 34.33+/-4.67 microg/ml. Although Ca2+ was essentially required in the binding of HDLp to their receptors, interestingly increasing concentration of Ca2+ has shown to have a slight inhibitory effect. EDTA was used here as Ca2+ chelating reagent, because Mg2+ in the binding buffer did not affect the binding of HDLp to their receptors, and inhibited the binding of HDLp and LpRGm absolutely. Suramin (polysulfated polycyclic hydrocarbon), known to inhibit the binding of lipoproteins to their receptors, effectively abolished the binding of HDLp to their receptors. LpRGm showed the stage specific binding activity especially in day 1-3 last instar larval, prepupal, and day 1-3 adult stages.

Purification and cDNA cloning of inducible antibacterial peptides from Protaetia brevitarsis (Coleoptera).

Three antibacterial peptides, named protaetins 1, 2, and 3, were purified and characterized from immunized larval hemolymph of Protaetia brevitarsis, a fruit tree pest in Korea. Also, protaetin 1 was cloned. Acid extraction, gel filtration, preparative acid-urea PAGE, and reversed-phase FPLC were used for purification of peptides. Protaetins 1 and 3 had molecular masses of 7.5 and 12 kDa on Tricine SDS-PAGE, respectively, and the molecular mass of protaetin 2 was 9,283.95 Da as determined by MALDI-TOF mass spectrometry. In an antibacterial assay, protaetins showed antibacterial activities against a panel of Gram-positive and -negative bacteria. For the RT-PCR (reverse transcription polymerase chain reaction) to obtain the complete primary sequence, the primer was designed according to the N-terminal amino acid sequence of protaetin 1. Amino acid sequence homology of protaetin 1 with holotricin 2, an antibacterial peptide from Holotrichia diomphalia, showed 99% identity. Northern blot analysis showed that the protaetin 1 gene was strongly expressed in the fat body after Escherichia coli injection, but not in normal fat body. Also, it was expressed in the gut, but was much weaker after immunization.

Cloning and expression of 32 kDa ferritin from Galleria mellonella.

We have sequenced a cDNA clone encoding 32-kDa ferritin subunit in the Wax Moth, Galleria mellonella. The 32-kDa ferritin subunit cDNA was obtained from PCR using identical primer designed from highly conserved regions of insect ferritins. RACE PCR was used to obtain the complete protein coding sequence. The 32-kDa ferritin subunit encoded a 232 amino acid polypeptide, containing a 19 leader peptide. The iron-responsive element (IRE) sequence with a predicted stem-loop structure was present in the 5'-untranslated region of the wax moth 32-kDa ferritin subunit mRNA. The 32-kDa sequence alignment had 78 and 69% identity with Manduca sexta and Calpodes ethlius (G), respectively. The G. mellonella ferritin subunits showed minimal identity with each other (19%). The glycosylation site (Asn-X-Ser/Thr) was found in the 32-kDa subunit but not in the 26-kDa subunit. Northern blot analysis showed that the mRNA expression of the 32-kDa ferritin was detected in the fat body and midgut. The fat body expression increased after 6 h and the mRNA in midgut dramatically increased about 3-fold the expression level at 12 h after iron feeding. Western blot revealed that a protein level of the 32-kDa subunit is abundant in midgut after 12 and 24 h iron feeding.

Purification and characterization of a 7-kDa protein from Clonorchis sinensis adult worms.

A 7-kDa protein was purified from extracts of adult Clonorchis sinensis by a combination of ammonium sulfate precipitation, anion exchange chromatography, cation exchange chromatography, gel-filtration chromatography, and reversed-phase FPLC. The 7-kDa protein exists in the excretory-secretory products of adult C. sinensis, but not in extracts of adult Paragonimus westermani. Also, the 7-kDa protein reacted with the sera of patients with clonorchiasis but not with paragonimiasis or normal human sera. To observe the localization of the 7-kDa protein in the tissue of adult C. sinensis, an immunogold labeling method was followed using anti-7-kDa antibody. The gold particles were observed in the basal layer below the tegumental syncytium, in the interstitial matrix of the parenchyma, and in the content of the uterus. The 7-kDa cDNA was obtained through reverse transcription-polymerase chain reaction using a primer designed from N-terminal sequence analysis. Rapid amplification of cDNA ends (5'-RACE) was used to obtain the complete protein coding sequence. The sequence encodes a 90-amino acid polypeptide. The deduced amino acid sequence of the 7-kDa protein revealed no homology with proteins of different organisms reported so far. These results suggest that the 7-kDa protein is a fluid antigen and may be valuable as a tool for the immunodiagnosis of clonorchiasis.

Frequent detection of reviraemia in ducks persistently infected with avian leukosis retroviruses.

Ducks intraembryonally infected with avian leukosis viruses of subgroup C (ALV-C) were followed for a long period (up to 6.8 years), and the viraemia and production of virus-neutralizing antibodies were measured. In three independent experiments comprising ducks inoculated with uncloned and/or molecularly cloned ALV-C, we found that after the elimination of primary post-hatching viraemia, reviraemia could be detected in 60-70% of infected animals. Based on the course of viraemia, the individual ducks were assigned to four different groups: Group I (no reviraemia), Group II (one transient reviraemic period), Group III (one persistent reviraemic period), Group IV (fluctuating reviraemia). In comparison to sera from ducks included in Group I and/or II, a significant decrease in neutralizing activity of sera from animals comprised in Group III and/or IV was observed. Two out of four reviraemic viruses were not neutralized by antiserum against ALV-C, instead their infectivity was enhanced. Long-term follow-up of the cell-associated virus revealed that its rescuability by cocultivation with chicken embryo fibroblasts fluctuated in about 50% of animals. In the reviraemic phase of infection, integrated proviruses could be detected by Southern blotting in a majority of tissues examined. Our data document that many features recognized in lentiviruses are valid also for oncoviruses transmitted to heterologous hosts and substantiate further the suitability of ALV-C-infected ducks as a model for studying persistent retroviral infection.

High rate of morphological reversion in tumor cell line H-19 associated with permanent transcriptional suppression of the LTR, v-src, LTR provirus.

The highly malignant line of morphologically transformed H-19 hamster tumor cells that harbor a single LTR, v-src, LTR provirus segregates morphologically flat revertants at the rate of 1.4 to 2.4 x 10(-3)/cell/cycle. Revertants behave like almost nonmalignant cells; they keep the provirus within an unaltered junction DNA fragment. However, the provirus is methylated, permanently transcriptionally silent, and not rescuable. Using the polymerase chain reaction, we have synthesized the whole proviral structure from two revertants and established that the left-hand long terminal repeats assuring transcription remained structurally intact. Moreover, the cloned proviral DNAs from three revertants were shown to produce tumors in chickens. The unusually high reversion rate together with the finding of structural integrity of proviral transcriptional signals in revertants indicate strongly that the reversion has been mediated by cell-regulatory mechanisms.

Decrease of the chlorophyll fluorescence ratio F690/F730 during greening and development of leaves.

Using the two-wavelength chlorophyll fluorometer the fluorescence induction kinetics (Kautsky effect) were measured simultaneously in the 690 nm and 730 nm region for ten common tree species during the greening period (April to July). The chlorophyll-fluorescence ratio F690/F730 (i.e. ratio of fluorescence intensity at the two maxima near 690 and 730 nm) was calculated from the laser-induced induction kinetics (He/Ne-laser 632.8 nm) at the fluorescence maximum and the steady state. The ratio F690/F730 decreases with increasing chlorophyll content of developing leaves. Its dependence on the chlorophyll content can be fairly well expressed by a power function which has a general validity for leaves, pigment extracts and chloroplast suspensions. The ratio F690/F730 is somewhat higher at maximum (fm) than at steady-state fluorescence (fs), but there is a very good correlation between both values. The ratio F690/F730 is a good indicator of the chlorophyll content and can be used as a non-destructive measure of the chlorophyll content of leaves. It also appears to be a suitable fluorescence parameter in the future remote sensing of the physiological state of the vegetation by laser-equipped airborne systems.

The chlorophyll fluorescence ratio F690/F730 in leaves of different chlorophyll content.

The red laser-induced chlorophyll-fluorescence induction kinetics of predarkened leaf samples were registered simultaneously in the 690 and 730 nm regions i.e., in the region of the two chlorophyll fluorescence emission maxima. From the induction kinetics the chlorophyll fluorescence ratio F690/F730 was calculated. The ratio F690/F730 shows to be dependent on the chlorophyll content of leaves. It is significantly higher in needles of damaged spruces (values of 0.45-0.9) than in normal green needles of healthy trees (values of 0.35-0.5). During development and greening of maple leaves the ratio F690/F730 decreases with increasing chlorophyll content. Determination of the ratio F690/F730 can be a suitable method of monitoring changes in chlorophyll content in a non-destructive way in the same leaves during development or the yellowish-green discolouration of needles of damaged spruces in the Black Forest with the typical tree decline symptoms.

Serum levels of procaine in human after peri-oral injections.

Doses of procaine for dental purposes have been assessed previously as a result of studies of circulating blood levels of procaine after injection of the drug at body sites other than the mouth. This paper reports results of a study of circulating procaine levels in ten healthy volunteers who received 1.8 ml injections of 2% procaine hydrochloride at a defined peri-oral site. General dental practice conditions were simulated as closely as possible. In addition, procaine pharmacokinetics following peri-oral administration were studied. The serum concentration-time data were found to obey the one-compartment open model adequately with first-order absorption and elimination rates.

Salivary female sex hormone levels and gingivitis in pregnancy.

Salivary estradiol 17 beta (E2-17 beta) and progesterone (P) were determined by using radioimmunoassay techniques in 30 pregnant females in the first, second and third trimesters as well as in 10 non-pregnant controls during the luteal phase of the menstrual cycle. Plaque index (P.I.), gingival index (G.I.) and retention index (R.I.) were measured in all cases. The data obtained showed that the levels of the two hormones in saliva were significantly increasing during the 3 trimesters of pregnancy. P.I. and R.I. did not change, however, G.I. was significantly higher in the second and third trimesters when compared with controls. A correlation between both E2--17 beta and P during pregnancy and gingival changes is suggested.