Identification of actin from hepatoma Morris 5123 cells

The hepatoma Morris 5123 tumor growth is accompanied by changes in actin content and polymerization (Malicka-B?aszkiewicz et al. (1995) Mat. Med. Pol., 27, 115-118; Nowak et al. (1995) J. Exp. Cancer Res. 14, 37-40). Presently actin isoforms from cytosol and cytoskeleton fractions were separated by SDS/PAGE and identified with antibodies directed against different actin isoforms. Actin isolated from the cytosol by affinity chromatography on DNase I bound to agarose shows the presence of only one protein spot on 2D gel electrophoresis corresponding to the mobility of the rabbit a skeletal muscle actin (Mr 43,000) and isoelectric point equal to 5.3. It interacts only with monoclonal anti beta actin isoform antibodies, posing the question of differential affinity of actin isoforms to DNase I.     

Cholecystokinin, acting through the A receptor subtype, stimulates the proliferative activity of adrenocortical cells and thymocytes in the rat

Cholecystokinin (CCK) is a multifunctional regulatory peptide, which acts through two main subtypes of receptors, named CCK-A and CCK-B. Evidence indicates that CCK modulates cell proliferation in various tissues in a paracrine manner, and proofs are available of the presence of CCK in both adrenal glands and thymus. Hence, we have investigated the possible mitogenic action of this peptide on these two tissues, by evaluating the /1000 of metaphase-arrested cells after vincristin injection (mitotic index). The systemic administration of CCK (three subcutaneous injections of 20 nmol/kg, 28, 16 and 4 h before the sacrifice) increased the mitotic index in both the outer adrenal and thymus cortexes of immature (20-day-old) rats and the enucleated adrenal gland of adult (2-month-old) animals at day 5 and 8 of regeneration. The simultaneous administration of equimolar doses of a selective CCK-A receptor antagonist blocked the effect of CCK, while a CCK-B antagonist was ineffective. These findings indicate that CCK exerts a marked CCK-A-mediated proliferogenic effect on both adrenal cortex and thymus in the rat, the physiological relevance of which, however, remains to be demonstrated. In fact, the administration of the CCK-A antagonist alone was ineffective, thereby casting doubts on the role played by endogenous CCK in the maintenance and stimulation of adrenal and thymus growth.     

Peroxisomal localization of sulfite oxidase separates it from chloroplast-based sulfur assimilation

Recently, we isolated the sulfite oxidase (SO) gene from Arabidopsis thaliana and characterized the purified SO protein. The purpose of the present study was to determine the subcellular localization of this novel plant enzyme. Immunogold electron-microscopic analysis showed the gold labels nearly exclusively in the peroxisomes. To verify this finding, green fluorescent protein was fused to full-length plant SO including the putative peroxisomal targeting signal 1 (PTS1) 'SNL' and expressed in tobacco leaves. Our results showed a punctate fluorescence pattern resembling that of peroxisomes. Co-labelling with MitoTracker-Red excluded that the observed fluorescence was due to mitochondrial sorting. By investigation of deleted or mutated PTS1, no functional peroxisomal targeting signal 2 (PTS2) could be detected in plant SO. This conclusion is supported by expression studies in Pichia pastoris mutants with defined defects either in PTS1- or PTS2-mediated peroxisomal import.     

Designing better probes: effect of probe size, mismatch position and number on hybridization in DNA oligonucleotide microarrays

DNA microarrays represent a powerful technology whose use has been hampered by the uncertainty of whether the same principles, established on a scale typical for membrane hybridizations, apply when using the smaller, rigid support of microarrays. Our goal was to understand how the number and position of base pair mismatches, probe length and their G+C content affect the intensity and specificity of the hybridization signal. One set of oligonucleotides (50-mers) based on three regions of the Bacillus thuringiensis cry1Aa1 gene possessing 30%, 42%, and 56% G+C content, a second set with similar G+C content (37% to 40%) but different lengths (30 to 100 bases), and finally amplicon probes (101 to 3000 base pairs) with G+C contents of 37% to 39%, were used. Probes with mismatches distributed over their entire length were the most specific, while those with mismatches grouped at either the 3' or 5'-end were the least specific. Hybridizations done at 8 to 13 degrees C below the calculated T(m) of perfectly matched probes, as compared to the widely used lower temperatures of 20 to 25 degrees C, enhanced probe discrimination. Longer probes produced higher fluorescent hybridization signals than shorter ones. These results should help to optimize the design of oligonucleotide-based DNA microarrays.     

Histopathological effects of trypanorhynch metacestodes in the digestive gland of a novel host, Carcinus maenas (Decapoda)

The green crab Carcinus maenas was introduced to Australian temperate waters in the late 1800s, has since become established, and is now considered to be a pest. We undertook an extensive parasite survey to find potential natural enemies of C. maenas and found it to be infected in Australia by 2 species of larval trypanorhynch tapeworm, Trimacracanthus aetobatidis and Dollfusiella martini. We describe the gross pathology and histopathology of the parasites' new host (C. maenas) and note that the plerocercoid larvae are located in the lumen of the digestive gland tubules. The presence of D. martini in C. maenas with low population numbers suggests that either D. martini has an impact (direct or indirect) on the survival of C. maenas, or that the parasite may be an indicator of high predation pressure. If the former were true, this would contribute to the control of this introduced pest species.     

Linear model of colon cancer initiation

Cancer results if regulatory mechanisms of cell birth and death are disrupted. Colorectal tumorigenesis is initiated by somatic or inherited mutations in the APC tumor suppressor gene pathway. Several additional genetic hits in other tumor suppressor genes and oncogenes drive the progression from polyps to malignant, invasive cancer. The majority of colorectal cancers present chromosomal instability, CIN, which is caused by mutations in genes that are required to maintain chromosomal stability. A major question in cancer genetics is whether CIN is an early event and thus a driving force of tumor progression. We present a new mathematical model of colon cancer initiation assuming a linear flow from stem cells to differentiated cells to apoptosis. We study the consequences of mutations in different cell types and calculate the conditions for CIN to precede APC inactivation. We find that early emergence of CIN is very likely in colorectal tumorigenesis.     

Three recombinant serine proteinase inhibitors expressed from the coding region of the thrombin inhibitor dipetalogastin

Dipetalogastin is a potent thrombin inhibitor from Dipetalogaster maximus. The cDNA of dipetalogastin codes for a large protein which consists of six Kazal-type domains. There are three tandem, homologous regions each including two domains. Three biologically active recombinant proteins rDI, rDII and rDIII each corresponding to one region of the dipetalogastin cDNA were expressed, purified and investigated with regard to their biological activities. rDI and rDII with molecular masses of 12,660 and 12,911 Da, respectively, proved to be potent thrombin inhibitors. The investigation of their influences on amidolytic activities of different serine proteases showed no inhibition of factor Xa (FXa) and alpha-chymotrypsin. At a large molar excess of rDI and rDII over the enzymes only low effects on the activities of trypsin and plasmin were observed. rDIII differs much from the both others. An inhibition of thrombin was found only at a molar excess of rDIII over the enzyme. Furthermore, an inhibition of trypsin and low effects on plasmin were detected at a molar excess of inhibitor over these enzymes. These results indicate that rDIII is active against thrombin, trypsin and plasmin, and finally possesses no specificity for only one serine proteinase.     

Reaction trajectory of pyrophosphoryl transfer catalyzed by 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase

6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the Mg(2+)-dependent pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP). The reaction follows a bi-bi mechanism with ATP as the first substrate and AMP and HP pyrophosphate (HPPP) as the two products. HPPK is a key enzyme in the folate biosynthetic pathway and is essential for microorganisms but absent from mammals. For the HPPK-catalyzed pyrophosphoryl transfer, a reaction coordinate is constructed on the basis of the thermodynamic and transient kinetic data we reported previously, and the reaction trajectory is mapped out with five three-dimensional structures of the enzyme at various liganded states. The five structures are apo-HPPK (ligand-free enzyme), HPPK.MgATP(analog) (binary complex of HPPK with its first substrate) and HPPK.MgATP(analog).HP (ternary complex of HPPK with both substrates), which we reported previously, and HPPK.AMP.HPPP (ternary complex of HPPK with both product molecules) and HPPK.HPPP (binary complex of HPPK with one product), which we present in this study.     

Induction of tumor cell apoptosis in vivo increases tumor antigen cross-presentation,cross-priming rather than cross-tolerizing host tumor-specific CD8 T cells

Cross-presentation of cell-bound Ags from established, solid tumors to CD8 cells is efficient and likely to have a role in determining host response to tumor. A number of investigators have predicted that when tumor Ags are derived from apoptotic cells either no response, due to Ag "sequestration," or CD8 cross-tolerance would ensue. Because the crucial issue of whether this happens in vivo has never been addressed, we induced apoptosis of established hemagglutinin (HA)-transfected AB1 tumors in BALB/c mice using the apoptosis-inducing reagent gemcitabine. This shrank the tumor by approximately 80%. This induction of apoptosis increased cross-presentation of HA to CD8 cells yet neither gross deletion nor functional tolerance of HA-specific CD8 cells were observed, based on tetramer analysis, proliferation of specific CD8 T cells, and in vivo CTL activity. Interestingly, apoptosis primed the host for a strong antitumor response to a second, virus-generated HA-specific signal in that administration of an HA-expressing virus after gemcitabine administration markedly decreased tumor growth compared with viral administration without gemcitabine. Thus tumor cell apoptosis in vivo neither sequesters tumor Ags nor cross-tolerizes tumor-specific CD8 cells. This observation has fundamental consequences for the development of tumor immunotherapy protocols and for understanding T cell reactivity to tumors and the in vivo immune responses to apoptotic cells.