Active specific immunotherapy with Newcastle-diseasevirus-modified autologous tumor cells following resection of liver metastases in colorectal cancer

Summary A group of 23 colorectal cancer patients were treated by a new type of active specific immunotherapy (ASI) following complete surgical resection of liver metastases (R0 resection). For ASI treatment we used a vaccine consisting of 1 × 10⁷ autologous, irradiated (200 Gy) metastases-derived tumor cells incubated with 32 hemagglutination units (HU) of Newcastle disease virus (NDV). The adjuvant vaccine therapy was started 2 weeks after surgery and was repeated five times at 14-days intervals followed by one boost 3 months later. The delayed-type hypersensitivity (DTH) skin reactions to the vaccine were measured as well as the DTH reactions to a challenge test of 1 × 10⁷ non-virus-modified autologous tumor cells from liver metastases or 1 × 10⁷ autologous normal liver cells. In addition 32 HU NDV alone and a standard antigen test (Merieux test) were applied pre- and post-vaccination. The vaccination was well tolerated. In 13 of 23 patients an increasing reactivity against the vaccine was observed during the vaccination procedure. Nine patients (40%) experienced an increased DTH reactivity against autologous tumor cells following vaccination, while 17% or fewer showed an increased reactivity to Merieux test antigens, NDV, or normal liver cells. The increased antitumor response was not correlated to responsiveness to NDV alone, autologous liver cells, enzymes and culture medium used for vaccine preparation or standard antigens (Merieux test). After a follow-up of at least 18 months 61% of the vaccinated patients developed tumor recurrence in comparison to 87% of a matched control groups from the same institution that had been only surgically treated. The results of this phase II trial are encouraging and should stimulate further prospective randomized studies.     

Molecular cloning of a pea mRNA encoding an early light induced,nuclear coded chloroplast protein

Summary cDNA clones were isolated for a chloroplast protein, the mRNA of which is induced to maximum levels within 2–4 h after onset of illumination in five day old, etiolated pea seedlings. The cDNA library was constructed from poly(A)⁺-mRNA which was isolated from 4 h illuminated seedlings. The extremely short induction period of the early light induced protein(ELIP)-mRNA established the basis of our screening procedure. Colony hybridization experiments were performed with³²P-labelled cDNA probes, synthesized from RNA of seedlings which had been exposed to different programs of illumination. Plasmid DNAs were isolated from colonies showing strong hybridization signals exclusively with cDNA corresponding to the 4 h-mRNA. Hybrid released translation of preselected plasmids p 17/C2 and p17/C4 revealed a peptide of M_r 24 000. After posttranslational importin vitro, the processed product of M_r 17 000 appears in the chloroplast. Using these clones, the expression of the ELIP-mRNA was investigated by DOT-hybridization. The ELIP-mRNA reaches maximum levels within 2–4 hours after onset of illumination. Our results correspond precisely to thein vivo characteristics and indicate positive identification of the sought clones.     

Thin-layer chromatography of auxin and inhibitors in Nicotiana glauca,N. langsdorffii and three of their tumor-forming hybrids

Summary Auxin and auxin-inhibitors from acidic ether extracts of normal Nicotiana stem tissues of N. glauca and N. langsdorffii and their tumor-producing 4n, 3n, and 2n-hybrids were separated by thin-layer chromatography. The growth substances were eluted and subjected to an Avena curvature test. A considerably higher amount of IAA was found in the tumor-forming 2n-hybrid (GL) than in the other plant material. The 4n-hybrid (GGLL) showed a small but significant increase in extractable IAA in comparison to its parents, whereas the 3n-hybrid (LLG) showed no difference from the langsdorffii parent. Inhibitory substances appeared at different Rf's but generally in low quantities. The inhibitor at Rf 0.5-0.6 (chromatography in n-butanol water-ammonia, 10: 10: 1, upper phase) seems to be identical with the inhibitor of Bennett-Clark and Kefford (1953). The results indicate that the potential for massive tumor production in the 2n- and 4n-hybrids of N. glauca x N. langsdorffii plants is coupled with increased IAA and inhibitor levels, whereas the 3n-hybrid, which forms tumors of much smaller size and in a later stage of development, does not differ considerably in its extractable IAA content from its N. langsdorffii parent.     

Internodal elongation and orientation of cellulose microfibrils and microtubules in deepwater rice

Excised stem sections of deepwater rice (Oryza sativa L.) containing the highest internode were used to study the induction of rapid internodal elongation by gibberellin (GA). It has been shown before that this growth response is based on enhanced cell division in the intercalary meristem and on increased cell elongation. In both GA-treated and control stem sections, the basal 5-mm region of the highest internode grows at the fastest rate. During 24 h of GA treatment, the internodal elongation zone expands from 15 to 35 mm. Gibberellin does not promote elongation of internodes from which the intercalary meristem has been excised. The orientation of cellulose microfibrils (CMFs) is a determining factor in cell growth. Elongation is favored when CMFs are oriented transversely to the direction of growth while elongation is limited when CMFs are oriented in the oblique or longitudinal direction. The orientation of CMFs in parenchymal cells of GA-treated and control internodes is transverse throughout the internode, indicating that CMFs do not restrict elongation of these cells. Changes in CMF orientation were observed in epidermal cells, however. In the basal 5-mm zone of the internode, which includes the intercalary meristem, CMFs of the epidermal cell walls are transversely oriented in both GA-treated and control stem sections. In slowly growing control internodes, CMF orientation changes to the oblique as cells are displaced from this basal 5-mm zone to the region above it. In GA-treated rapidly growing internodes, the reorientation of CMFs from the transverse to the oblique is more gradual and extends over the 35-mm length of the elongation zone. The CMFs of older epidermal cells are obliquely oriented in control and GA-treated internodes. The orientation of the CMFs parallels that of the cortical microtubules. This is consistent with the hypothesis that cortical microtubules determine the direction of CMF deposition. We conclude that GA acts on cells that have transversely oriented CMFs but does not promote growth of cells whose CMFs are already obliquely oriented at the start of GA treatment.     

Biosynthetic pathways of Vibrio succinogenes growing with fumarate as terminal electron acceptor and sole carbon source

1. With fumarate as the terminal electron acceptor and either H₂ or formate as donor, Vibrio succinogenes could grow anaerobically in a mineral medium using fumarate as the sole carbon source. Both the growth rate and the cell yield were increased when glutamate was also present in the medium.
2. Glutamate was incorporated only into the amino acids of the glutamate family (glutamate, glutamine, proline and arginine) of the protein. The residual cell constituents were synthesized from fumarate.
3. Pyruvate and phosphoenolpyruvate, as the central intermediates of most of the cell constituents, were formed through the action of malic enzyme and phosphoenolpyruvate synthetase. Fructose-1,6-bisphosphate aldolase was present in the bacterium suggesting that this enzyme is involved in carbohydrate synthesis.
4. In the absence of added glutamate the amino acids of the glutamate family were synthesized from fumarate via citrate. The enzymes involved in glutamate synthesis were present.
5. During growth in the presence of glutamate, net reducing equivalents were needed for cell synthesis. Glutamate and not H₂ or formate was used as the source of these reducing equivalents. For this purpose part of the glutamate was oxidized to yield succinate and CO₂.
6. The α-ketoglutarate dehydrogenase involved in this reaction was found to use ferredoxin as the electron acceptor. The ferredoxin of the bacterium was reoxidized by means of a NADP-ferredoxin oxidoreductase. Enzymes catalyzing the reduction of NAD, NADP or ferredoxin by H₂ or formate were not detected in the bacterium.

     

Effect of sulphite on adenine nucleotides of the green alga Trebouxia

The amounts of AMP, ADP and ATP were determined after sulfite incubation in photo-organotrophically cultivated cells of the green alga Trebouxia. D     

Rat model of fractionated (2 Gy/day) 60 Gy irradiation of the liver: long-term effects

The liver is considered a radiosensitive organ. However, in rats, high single-dose irradiation (HDI) showed only mild effects. Consequences of fractionated irradiation (FI) in such an animal model have not been studied so far. Rats were exposed to selective liver FI (total dose 60 Gy, 2 Gy/day) or HDI (25 Gy) and were killed three months after the end of irradiation. To study acute effects, HDI-treated rats were additionally killed at several time points between 1 and 48 h. Three months after irradiation, no differences between FI and HDI treatment were found for macroscopically detectable small “scars” on the liver surface and for an increased number of neutrophil granulocytes distributed in the portal fields and through the liver parenchyma. As well, no changes in HE-stained tissues or clear signs of fibrosis were found around the portal vessels. Differences were seen for the number of bile ducts being increased in FI- but not in HDI-treated livers. Serum levels indicative of liver damage were determined for alkaline phosphatase (AP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma-glutamyltransferase (γGT) and lactate dehydrogenase (LDH). A significant increase of AP was detected only after FI while HDI led to the significant increases of AST and LDH serum levels. By performing RT-PCR, we detected up-regulation of matrix metalloproteinases, MMP-2, MMP-9, MMP-14, and of their inhibitors, TIMP-1, TIMP-2 and TIMP-3, shortly after HDI, but not at 3 month after FI or HDI. Overall, we saw punctual differences after FI and HDI, and a diffuse formation of small scars at the liver surface. Lack of “provisional clot”-formation and absence of recruitment of mononuclear phagocytes could be one explanation for scar formation as incomplete repair response to irradiation.     

Untersuchungen über die Wuchs- und Hemmstoffe der Wurzelspitze und der Plumula vonVicia faba

Zusammenfassung Wurzelspitzen und Plumulastückchen vonVicia faba-Keimlingen wurden auf ihren Wuchs- und Hemmstoffgehalt untersucht. Durch die Wuchsstoff-Präparate, die mittels der Agar-Abfangmethode aus Wurzelspitzen gewonnen worden waren, konnte im Haferkrümmungstest (mit unter- und überoptimaler IES-Zugabe zu den Präparaten) neben Wuchsstoff ein Hemmstoff nachgewiesen werden, der vermutlich nicht durch Wuchsstoffverdrängung von den plasmatischen Wirkorten angreift.Die Ergebnisse der Abfangversuche an Plumulastückchen zeigen diesen Hemmstoff nicht, lassen aber auf die Anwesenheit eines von IES verschiedenen Wuchsstoffes schließen.Die in Wurzelspitzen und Plumulae vonVicia faba-Keimlingen vorliegenden Wuchs- und Hemmstoffe wurden papierchromatographisch untersucht. Zur Auswertung der Ergebnisse wurden der Haferkrümmungstest, der Zylinder-Zuwachstest und der Wurzelspitzen-Zuwachstest herangezogen.Übereinstimmend konnte sowohl in Wurzelspitzen als auch in Plumulastückchen bei Chromatographie mit n-Butanol—Aqua bidest.—Ammoniak außer IES ein weiterer Wuchsstoff mit demR _f von 0,65 bis 0,75 nachgewiesen werden. Außerdem ist die Anwesenheit eines Wuchsstoffes mit einemR _f von 0,85–0,9 in beiden Organen zu vermuten. In Wurzelspitzen liegt bei einemRf von 0,1 ein weiterer Wuchsstoff vor, der als accelerator (nachBennet-Clark undKefford 1953) gedeutet wurde und der, im Gegensatz zur IES, eine stark wachstumsfördernde Wirkung auf Wurzelspitzen ausübt.Deutliche Hemmwirkungen auf Koleoptilzylinder wurden bei Plumula-und Wurzelspitzen-Extrakten durch Zonen mit demR _f von 0,45 erzielt. Es dürfte sich hierbei um den vonBennet-Clark undKefford (1953) beschriebenen inhibitor handeln. AufFaba-Wurzelspitzen wirkt dieser Stoff jedoch nicht hemmend.Als weiterer in Wurzelspitzen gebildeter Hemmstoff war eine Substanz mit demR _f von 0,65–0,75 zu vermuten.Mit 8 TextabbildungenTeil einer Dissertation der Mathematisch-Naturwissenschaftlichen Fakultät der Universität Hamburg.     

A comparative method for finding and folding RNA secondary structures within protein-coding regions

Existing computational methods for RNA secondary-structure prediction tacitly assume RNA to only encode functional RNA structures. However, experimental studies have revealed that some RNA sequences, e.g. compact viral genomes, can simultaneously encode functional RNA structures as well as proteins, and evidence is accumulating that this phenomenon may also be found in Eukaryotes. We here present the first comparative method, called RNA-DECODER, which explicitly takes the known protein-coding context of an RNA-sequence alignment into account in order to predict evolutionarily conserved secondary-structure elements, which may span both coding and non-coding regions. RNA-DECODER employs a stochastic context-free grammar together with a set of carefully devised phylogenetic substitution-models, which can disentangle and evaluate the different kinds of overlapping evolutionary constraints which arise. We show that RNA-DECODER's parameters can be automatically trained to successfully fold known secondary structures within the HCV genome. We scan the genomes of HCV and polio virus for conserved secondary-structure elements, and analyze performance as a function of available evolutionary information. On known secondary structures, RNA-DECODER shows a sensitivity similar to the programs MFOLD, PFOLD and RNAALIFOLD. When scanning the entire genomes of HCV and polio virus for structure elements, RNA-DECODER's results indicate a markedly higher specificity than MFOLD, PFOLD and RNAALIFOLD.