A phase II pilot trial of 13-cis retinoic acid and interferon-alpha in patients with advanced pancreatic carcinoma.

BACKGROUND: Advanced unresectable pancreatic adenocarcinoma has a dismal prognosis. The authors previously have shown that retinoic acid (RA) and interferon-alpha (IFN-alpha) inhibit growth and induce differentiation in human pancreatic carcinoma cells in vitro and in vivo. The purpose of this trial was to examine the feasibility and tolerability of a combination therapy of 13-cis RA and IFN-alpha in patients with advanced unresectable pancreatic carcinoma. METHODS: Twenty-two patients (median age, 62 years) with histologically confirmed, unresectable pancreatic adenocarcinoma classified as International Union Against Cancer Stage III (5 patients) or IV (17 patients) were included. Patients received 1 mg/kg body weight 13-cis RA orally and 6 million IU IFN-alpha subcutaneously daily. Restaging by ultrasound, computed tomography scan, and chest X-ray was performed every 2 months. RESULTS: No complete remission and 1 partial remission (PR) (4.5%) were observed. Fourteen patients (63.6%) demonstrated stable disease with a median duration of 5.0 months (range, 2.3-17.7+ months). Toxicity mainly was related to IFN-alpha and predominantly was hematologic (no toxicity was World Health Organization [WHO] Grade 4 and 13.6% were WHO Grade 3). Nonhematologic toxicities did not exceed Grade 2 (skin and oral mucosa) and mainly were related to 13-cis RA. The median survival of the patients with Stage III disease was 8.7 months (range, 6.8-23.9+ months) and was 7.4 months for patients with Stage IV disease (range, 0.9-19.2+ months), resulting in a median overall survival of 7.7 months (range, 0.9-23.9+ months). CONCLUSIONS: Combination therapy with 13-cis RA and IFN-alpha is feasible and well tolerated in patients with advanced pancreatic carcinoma. Based on the median survival rates observed in this study this combination should be investigated further in Phase III trials.

[Vulvar involvement as a rare extra-intestinal manifestation of Crohn disease]. / Vulvabeteiligung als seltene extraintestinale Manifestation bei Morbus Crohn.

BACKGROUND: Crohn's disease may involve all parts of the gastrointestinal tract. Extraintestinal manifestations with and without continuity to the intestine are described. The most common complications are the involvement of bone (articulations), liver and eyes. Crohn's disease of the vulva is rare and only a few cases have been reported in the literature. CASE REPORT: The 45-year-old woman has a 15-year history of a marked chronic-inflammatory bowel disease. The diagnosis of Crohn's disease was never confirmed histologically. In 1984, she developed a painful vulvar swelling and erythema. A fungal infection was suspected and she was treated with topical antimycotic medication, but there wasn't any improvement. In July 1995, biopsies of the vulva showed a granulomatous inflammation consistent with Crohn's disease. Oral metronidazol therapy (20 mg/kg/day) was started. After 2 months, the erythema had healed completely but the vulvar swelling remained. CONCLUSIONS: The treatment of this extraintestinal manifestation is very difficult. Systemic or topical application of steroids is without response in most cases. Surgical excision showed good results in a few cases. A long-term treatment with oral metronidazol 20 mg/kg/day could be a chance for healing without adverse effects.

Sterile UGA nonsense mutants of fission yeast.

Eight sterile mutants, which regain their fertility upon reactivation of an inactivated UGA suppressor allele of the serine tRNA gene sup3, are shown to carry UGA nonsense alleles of two established ste genes, ste1 (one mutant) and ste6 (two mutants), and of two novel genes, ste9 (four mutants) and ste10 (one leaky mutant of ras1-/ste5-like cell morphology). The mutant alleles of ste1 and ste9 lead to a defect in both conjugation and meiosis, whereas those of ste6 and ste10 affect mating only. Two of the four genes map to chromosome I, ste1 in the left arm 6 cM distal of ura1, and ste9 in the right arm 3 cM distal of ade2. The ste10 and ste6 genes are located in the right arms of chromosomes II and III, respectively, the former 4 cM distal of trp1 and the latter 1 cM proximal or distal of trp3.

Characterization of a partially fertile ras1-like ste10-UGA nonsense mutant of fission yeast.

Mutants which carry a leaky UGA nonsense mutation in the fertility gene ste10 are characterized by a deformed cell morphology which resembles that described in the literature for sterile ras1- (ste5) and ral1 to ral4 mutant cells. Although frequent conjugation attempts are observed in combinations of two ste10 mutant strains of opposite heterothallic mating type, zygotes and asci are formed only rarely and the fertility of such crosses remains low (not more than 1% of the fertility of comparable crosses of two ste+ wild-type strains). The fertility is considerably increased, however, in combinations of the ste10 mutant with ste+ wild-type strains (up to 10% if the h- partner, and more than 30% if the h+ partner, carries the ste10 mutation.

Pheromone production and response in sterile mutants of fission yeast.

Genetically heterothallic strains of various sterile mutants were assayed for residual production of the corresponding mating pheromone as well as responsiveness towards the opposite pheromone. No sexual activities were detected in ste11 strains (previously referred to as aff1 or steX, which we show are allelic), whilst the production of M factor was unaffected by ste1 to ste10 mutations. P factor production was still possible in class I ste mutants (ste5, ste6 and ste10), which also allow meiosis in diploid strains. With the exception of the leaky ste10-F23 mutant, no changes in cell morphology were induced by exposure to the opposite pheromone in the ste mutant strains.

[Diagnostic aspects of neurological diseases of cattle. A retrospective study]. / Diagnostische Aspekte neurologischer Krankheiten des rindes. Eine retrospektive Studie.

188 bovines with neurological disease were analysed in a retrospective study. We found that only few diseases can be diagnosed on the basis of neurologic signs only. However, with a few additional clinical data such as signalment, history, extraneural findings, hematological findings and cerebrospinal fluid analysis, a diagnosis can be made in many cases. A history of neurologic signs occurring since birth, is highly suggestive of a congenital malformation. High fever and mucosal changes are typical for malignant catarrhal fever. Lesions of peripheral nerves and spinal cord compression can be diagnosed based on the neurologic signs. Cranial nerve dysfunction and central vestibular signs in combination with other findings as age, season and cerebrospinal fluid, are indicative of listeriosis. Tetanus is characterized by pronounced extensor rigidity. Systemic muscle diseases were also included, because motor neurons and muscle cells are a functional unit. A suspicion of white muscle disease can be confirmed by increased creatine phosphokinase activity in serum. Suppurative bacterial meningoencephalitis can be diagnosed by the presence of a polymorphonuclear pleocytosis in the cerebrospinal fluid. The clinical differentiation between sporadic non suppurative meningoencephalitis and metabolic diseases of the central nervous system remains difficult.

Inactivation of nonsense suppressor transfer RNA genes in Schizosaccharomyces pombe. Intergenic conversion and hot spots of mutation.

Intergenic conversion is a mechanism for the concerted evolution of repeated DNA sequences. A new approach for the isolation of intergenic convertants of serine tRNA genes in the yeast Schizosaccharomyces pombe is described. Contrary to a previous scheme, the intergenic conversion events studied in this case need not result in functional tRNA genes. The procedure utilizes crosses of strains that are homozygous for an active UGA suppressor tRNA gene, and the resulting progeny spores are screened for loss of suppressor activity. In this way, intergenic convertants of a tRNA gene are identified that inherit varying stretches of DNA sequence from either of two other tRNA genes. The information transferred between genes includes anticodon and intron sequences. Two of the three tRNA genes involved in these information transfers are located on different chromosomes. The results indicate that intergenic conversion is a conservative process. No infidelity is observed in the nucleotide sequence transfers. This provides further evidence for the hypothesis that intergenic conversion and allelic conversion are the result of the same molecular mechanism. The screening procedure for intergenic revertants also yields spontaneous mutations that inactivate the suppressor tRNA gene. Point mutations and insertions of A occur at various sites at low frequency. In contrast, A insertions at one specific site occur with high frequency in each of the three tRNA genes. This new type of mutation hot spot is found also in vegetative cells.

Mutations preventing expression of sup3 tRNASer nonsense suppressors of Schizosaccharomyces pombe.

Suppression of nonsense codons in Schizosaccharomyces pombe by sup3-e tRNASerUGA or sup3-i tRNASerUAA is reduced or abolished by mutations within the suppressor locus. Twenty-five suppressor-inactive sup3-e genes and thirteen mutant sup3-i genes were isolated from S. pombe genomic clone banks by colony hybridization. Sequence analysis of these revertant alleles corroborates genetic evidence for mutational hotspots within the sup3 tRNA gene. Fifteen types of point mutations or insertions were found. Many of these replace bases which are highly or completely conserved in eucaryotic tRNA genes. Transcription of the altered sup3 genes in a Saccharomyces cerevisiae extract enabled the identification of mutations which affect the rate of 5'-end maturation or splicing of the tRNA precursors or both. A total of seven mutations were found which alter transcriptional efficiencies. Of these, five are located outside the internal transcription control regions.

Mutations affecting excision of the intron from a eukaryotic dimeric tRNA precursor.

The nucleotide sequences of a Schizosaccharomyces pombe opal suppressor serine tRNA gene (sup9-e) and of 12 in vivo-generated mutant genes, which have lost the ability to suppress UGA mutations, have been determined. Analysis of the expression of these genes in Saccharomyces cerevisiae in vitro and in vivo systems has revealed defects in tRNA gene transcription and precursor tRNA processing. Single base changes in the D-loop, the intron and the extra arm affect the efficiency of splicing of the tRNA precursors while an anti-codon stem mutation may affect the accuracy of this process. Two mutations which occur in the intervening sequence of the sup9-e gene allow an alternate tRNA base pairing configuration. Transcription of the sup9-e gene and of the adjacent tRNAMet gene (located 7 bp downstream) is essentially abolished in vivo by a G----A19 mutation in the tRNASer gene, suggesting that tRNAMet may be derived solely via processing of the tRNASer-tRNAMet dimeric precursor.

The genetic fine structure of nonsense suppressors in Schizosaccharomyces pombe : II. sup8 and sup10.

Meiotic fine-structure maps of two efficient UGA suppressors of Schizosaccharomyces pombe which are known (sup8-e) or inferred (sup10-e) to code for two leucine tRNAs carrying the mutant anticodon U(*)CA (Kohli et al. 1979, 1980a, b; Wetzel et al. 1979; Mao et al. 1981) are presented. In both cases, the recombination frequencies given by the primary site of the anticodon mutation fitwell into the map defined by the sites of a number of inactivating secondary mutations. This contrasts the corresponding situation found in the serine tRNA genes sup3 and sup9 where the anticodon site exhibits a specific marker effect which strongly increases recombination frequencies in crosses with all revertant sites, due to a decrease in the efficiency of excision repair of base-pair mismatches whenever the anticodon site is included in hybrid-DNA (Hofer et al. 1979; Munz and Leupold 1979; Thuriaux et al. 1980). A pronounced specific marker effect which leads to a several fold increase of the recombination frequencies over those expected is observed, however, at one of the secondary inactivating sites mapping in the leucine tRNA gene sup8.

Putative frameshift suppressors in Schizosaccharomyces pombe.

Nine genetically distinct suppressors of ICR-170-induced ade6 and ade7 mutations have been identified in Schizosaccharomyces pombe. The nine suppressors of ICR-170-induced and spontaneous origin have been assigned to the three chromosomes by haploidization and meiotic analysis. They do not suppress missense or nonsense mutations and are therefore likely to be frameshift suppressors. Based on the spectrum of suppression, the nine suppressors fall into two mutually exclusive groups. Group I comprises the two dominant suppressors sufl and suf11. Group II consists of the seven dominant suppressors suf2 through suf8. The suppressors of both groups are inefficient and all lead to a marked reduction of growth rate. Within suppressor groups, combinations of suppressors lead to drastic reductions of growth rates and to an increased efficiency of suppression. Freely segregating modifiers of suppression increasing and decreasing the efficiency of supression have been found for all the suppressors. The two omnipotent suppressors sup1 and sup2 increase the efficiency of suppression of some frameshift suppressors. The suf5 locus is unstable and reverts at very high frequency both meiotically and mitotically.

Gene conversion in nonsense suppressors of Schizosaccharomyces pombe : II. Specific marker effects.

Gene conversion and postmeiotic segregation patterns have been analysed at 14 mutant sites of sup3, sup8 and sup9 including 5 alleles with a strong marker effect on recombination frequencies in two-factor crosses. The total frequency of gene conversion and postmeiotic segregation tetrads is fairly constant within each gene, but may vary from one gene to another. About 97% of the conversion events are coconversions spanning the whole sup gene. Postmeiotic segregations are usually quite rare. None of the marker-effect alleles has an increased rate of hybrid DNA formation at the allele considered, as judged from the frequency of gene conversion and postmeiotic segregation in one-factor crosses. At least two of them, sup3-e and sup9-e, are associated with a high frequency of postmeiotic segregation indicating a poor repair of the corresponding base-pair mismatches. This is also observed in a two-factor cross and can account for the marker effect on recombination frequencies. The properties of a third marker effect allele, sup3-e,r10, are best explained by a higher probability of single site conversions as opposed to coconversions in two-factor crosses involving the mutant site r10.

Reversion of nonsense mutants induced by 4-nitroquinoline-1-oxide in Schizosaccharomyces pombe.

We have studied the reversion of 8 nonsense alleles located in 7 different genes of Schizosaccharomyces pombe using 4-nitroquinoline-1-oxide (NQO) as a mutagenic agent. The nonsense mutants of S. pombe have been classified according to their suppressibility by defined opal and ochre suppressors into a class of efficiently suppressed opal and a class of inefficiency suppressed ochre mutants. The UGA alleles tested all revert consistently with NQO, in agreement with the high specificity of this mutagen for G-residues reported for bacteria and yeast. The UAA alleles show a lack or a low level of reversion with NQO. This low level of reversion is due to the low level of non-G-specific transversions at A sites of the UAA triplet. Within each class of nonsense mutants the extent of induction is site-dependent. We conclude that NQO acts predominantly on G-residues in S. pombe.

The genetic fine structure of nonsense suppressors in Schizosaccharomyces pombe : I. sup3 and sup9.

Meiotic and mitotic fine-structure maps of two efficient UGA suppressors of Schizosaccharomyces pombe which are known (sup3-e) or inferred (sup9-e) to code for two serine tRNAs carrying the mutant anticodon U*CA (Kohli et al. 1979a, b, Rafalski et al. 1979) are presented. Maps based on spontaneous meiotic, spontaneous mitotic and MMS induced mitotic recombination between the primary site of the anticodon mutation and a number of inactivating second-site mutations are similar. Specific marker effects, which drastically increase the frequency of spontaneous meiotic and mitotic recombination in crosses involving one or the other of four exceptional sites (including the anticodon sites of both sup3-e and sup9-e), disappear when mapping is based on MMS induced mitotic recombination. The meiotic marker effect characterizing the anticodon site of one of the two efficient UGA suppressors (sup3-e) also disappears upon further mutation to an inefficient UAA suppressor allele (sup3-i), as shown by its absence in a fine-structure map based on meiotic recombination between the anticodon mutation of this ochre suppressor allele and a new set of inactivating second-site mutations derived from it.

Genetic analysis of antisuppressor mutants in the fission yeast Schizosaccharomyces pombe.

Fourteen unlinked sin genes could be mutated to recessive antisuppressor alleles preventing the expression of suppressors in the fission yeast Schizosaccharomyces pombe. cyh1 alleles, resistant to the ribosomal inhibitor cycloheximide, also have some antisuppressor effect. The genetical and physiological characterization of these mutants is consistent with the hypothesis that they affect components of the messenger RNA translation machinery such as tRNA modifying enzymes or ribosomal proteins.