Targeting of frog prodermorphin to the regulated secretory pathway by fusion to proenkephalin.

We have investigated the sorting and processing of the amphibian precursor prepro-dermorphin in mammalian cells. Dermorphin, a D-alanine-containing peptide with potent opioid activity, has been isolated from the skin of the frog Phyllomedusa sauvagei. The maturation of this peptide from the precursor involves several posttranslational steps. Recombinant vaccinia viruses were used to infect AtT-20, PC12, and HeLa cells to study the sorting and processing of prepro-dermorphin. While this precursor was not processed in any of the examined cell lines, AtT-20 cells were able to process approximately 40% of a chimeric precursor consisting of the first 241 amino acids of prepro-enkephalin fused to a carboxy-terminal part of pro-dermorphin. By immunogold-EM, we could show that the chimeric protein, but not pro-dermorphin, was sorted to dense-core secretion granules. The processing products could be released upon stimulation by 8-Br-cAMP. We conclude that the pro-enkephalin part of the fusion protein contains the information for targeting to the regulated pathway of secretion, while this sorting information is missing in pro-dermorphin. This indicates that sorting mechanisms may differ between amphibian and mammalian cells.

Epithelioid sarcoma: a clinicopathologic review of 55 cases.

OBJECTIVE: To identify any clinical and pathologic features of treatment modalities that are predictive of outcome in patients with epithelioid sarcoma, a rare slow-growing soft tissue tumor most commonly occurring in the distal extremities of young adults. DESIGN: We reviewed the institutional files for cases of epithelioid sarcoma for the period 1956 to 1991 and analyzed the effect of various factors on survival. MATERIAL AND METHODS: Fifty-five cases of epithelioid sarcoma were found, and the relevant clinical, pathologic, treatment, follow-up, and outcome features were assessed. RESULTS: All tumors were treated initially by operative resection. The recurrence rate progressively decreased with increasing aggressiveness of the initial operation; however, no difference was noted in metastatic rate. Overall, the recurrence rate was 38% and the metastatic rate was 47%. At the end of a mean follow-up of 102 months, 69% of patients had no evidence of disease, 27% had died of the disease, and 4% were alive with disease. Increasing tumor size, necrosis of more than 30%, and vascular invasion correlated significantly with a worse prognosis. CONCLUSION: Epithelioid sarcoma should be considered a malignant neoplasm with a significant potential for aggressive behavior, and close follow-up of affected patients should be maintained for many years. Initial treatment should be aggressive in an attempt to prevent recurrence.

Genomic 3' terminal sequence comparison of three isolates of rabbit haemorrhagic disease virus.

Comparison of sequence data is necessary in older to investigate virus origins, identify features common to virulent strains, and characterize genomic organization within virus families. A virulent caliciviral disease of rabbits recently emerged in China. We have sequenced 1100 bases from the 3' ends of two independent European isolates of this virus, and compared these with previously determined calicivirus sequences. Rabbit caliciviruses were closely related, despite the different countries in which isolation was made. This supports the rapid spread of a new virus across Europe. The capsid protein sequences of these rabbit viruses differ markedly from those determined for feline calicivirus, but a hypothetical 3' open reading frame is relatively well conserved between the caliciviruses of these two different hosts and argues for a functional role.

Haemorrhagic disease of lagomorphs: evidence for a calicivirus.

Studies on the aetiological agents of rabbit haemorrhagic disease (RHD) and European brown hare syndrome show that the viruses responsible for these infections can be placed in the family Caliciviridae. Established members of this group are vesicular exanthema virus (prototype), San Miguel sea lion virus and feline calcivirus. The human hepatitis E virus and the Norwalk agent may soon be included. The RHD virus genome consists of a positive stranded RNA molecule composed of 7437 nucleotides. A major subgenomic RNA of 2.2 kb, colinear with the 3' end of the genomic RNA, can also be recovered from infected liver tissue, and both RNAs are enclosed within viral capsids formed by a single major protein of approximately 60 kDa. Electron microscopic examination of organ suspensions from diseased animals shows two types of particle; 35-40 nm complete virions have the regularly arranged cup-shaped depressions typical of calcivirus morphology, and 23-25 nm smooth particles resulting from degradation of the outer surface structures of the complete virions.

Ewing's sarcoma.

Ewing's sarcoma is the second most common primary malignant bone tumor of children. Tremendous strides in the multidisciplinary treatment of Ewing's sarcoma have been made over the past 25 years. Aggressive chemotherapy has increased the 5-year survival rates from 10% to over 70%. The role of surgery for local control has gained importance. These advances in the diagnosis, staging, and treatment of Ewing's sarcoma are discussed in detail.

[Changes in the distal end of the radius from the aspect of the injured person]. / Zlomeniny distálního konce rádia z hlediska zranených.

Based on an evaluation of a group of patients treated on account of fractures of the wrist. Colles type, we assume that the decisive factor is the type of fracture. We recommend therefore to divide these fractures into two groups. The first one includes all types where the fracture line does not involve the radiocarpal or radioulnar joint. The second one includes all multifragmental intraarticular fractures where an active surgical procedure can improve hitherto achieved therapeutic results.

[Injuries of the ligaments of the talar joint]. / Poranení vazu hlezenného kloubu.

Based on experience with the treatment of 404 patients with injuries of the soft parts of the talus the authors maintain that markedly better results are achieved in patients where sufficiently long immobilization with a plaster bandage was used or where early reconstruction of the injured ligaments was performed. A very important negative factor is treatment of these injuries without immobilization or inadequate immobilization, as a high percentage of the patients develop instability of the talus with all its consequences. The authors consider arthrography the exact diagnostic method, the results of which are decisive for the therapeutic procedure.

[Antioxidants and their gastrointestinal absorption and interferences of their effects]. / Antioxidanty a jejich gastrointestinálni absorpce a interference jejich ucinku.

Antioxidants and trace elements are using by hundreds millions of people. Effective are especially mixtures of antioxidants. Usually is declared only the composition of the tablets, but nowadays it is not satisfactory. Substantial is how much of the antioxidants is absorbed and where, how it increases the antioxidant capacity in the blood, which effect it has, the stability of them and who, how much, which and when they are to be used. It is also very important which antioxidants during the detoxication of free radicals react first and therefore they are soon exhausted and whether at all or how quickly can they be reduced back to an active component. In aging the antioxidant capacity decreases, it is influenced by the season, all of factors are to be taken in account. The absorption and the effect are influenced by the state of gastrointestinal tract, including the microbiological flora, pH, the size of the molecules, sometimes by partial oxygen tension in the blood. Free radicals are generated mostly after a load and therefore it is suitable to have the antioxidants capacity on a high level, it is possible to increase it during the load and it is recommended the administration of them after the load. Some authors recommend low doses of antioxidants five times a day. In some diseases the antioxidants are effluenced from the tissues to the blood and then there is a defficit in tissues of them. Important are the interferences during the absorption, their metabolism in organism; it may decrease their level or increase their effectivness, the metabolism can infuence to which tissues are the antioxidants deposited, and how long will stay the increased level of antioxidant capacity. The speed of elimination by urine and stool is also important. It is useful to know from which and how much of isomers the antioxidant is composed, because the single isomer may have a different effect. The origin of antioxidants is important, as natural antioxidants are usually more effective than the sythetic ones. The toxicity of the substances should not to be neglected. Storing of antioxidants sometimes deteriorate them, or sometimes they are contaminated by anabolic steroids. Some substances like phytates can bind them and so decrease their bioavavilability. Lipid soluble substances need lipids in the diet, some antioxidants are differently absorbed from different sources of nutrition. Genetic equipment is important as well. It is apparent that the administration of antioxidants and trace elements is not simple and that the informations of commercial preparates is usually not sufficient, probably in the future at least may be mentioned total antioxidant capacity.

The receptor-destroying enzyme of influenza C virus is required for entry into target cells.

The hemagglutinin-esterase (HE) protein of influenza C viruses possesses an acetylesterase activity, which appears essential for replication, as determined by reduced infectivity after inhibition of the viral enzyme [Vlasak et al., J. Virol. 63, 2056-2062 (1989)]. Analysis revealed the absence of virus-specific RNA and protein synthesis in infected cells after inhibition of the receptor-destroying enzyme. In addition, hemolytic activity was reduced after incubation of influenza C/JJ/50 virus with diisopropyl-fluorophosphate or 3,4-dichloro-isocoumarin. Further analysis revealed that inhibition of hemolysis depends on virus and erythrocyte concentrations. It is suggested that an active receptor-destroying enzyme is required for entry of influenza C virus into target cells at a step prior to fusion of the viral and cellular membrane. Our data indicate that cleavage of receptors bound to the HE protein is a prerequisite for the low pH-triggered conformational change required for fusion.

Nucleotide sequence of cloned cDNAs coding for preprosecapin, a major product of queen-bee venom glands.

Total mRNA from queen-bee venom glands was transcribed into cDNA and cloned into the PstI and ClaI site of pBR322. The nucleotide sequence of two clones (pUM9/24 and pBMC1) with inserts derived from mRNAs for preprosecapin is presented. The two inserts, which are identical in their overlapping regions, have a coding region encompassing 77 triplets. The cloned cDNAs differ in the length of the 3'-untranslated region, which is about twice as long in clone pUM9/24. The postulated amino acid sequence of preprosecapin starts with a signal peptide containing an estimated 32 residues, followed by a pro part which terminates with a single arginine. The end-product secapin is located at the COOH end of the precursor. Activation of prosecapin must, therefore, differ from the processing of promelittin synthesized in the same gland. Cell-free translation of total venom gland mRNA in the presence of radioactive histidine yields only two major products, the smaller of which is presumably preprosecapin. Edman degradation of total translation products yields peaks of radioactive tyrosine and histidine at the 4th and 10th cycles respectively. This confirms the sequence of preprosecapin deduced from the cDNA clones. The content of secapin in queen-bee venom must be higher than it is in worker bee venom.

cDNA cloning and expression of secreted Xenopus laevis dipeptidyl aminopeptidase IV.

From a Xenopus laevis skin library a cDNA coding for dipeptidyl aminopeptidase IV (DPP IV) was isolated. The ORF codes for a protein with sequence similarity to DPP-IV-like proteins, including mammalian DPP IV and X. laevis fibroblast activation factor. In contrast to the membrane-bound mammalian enzymes, mature X. laevis DPP IV is a soluble secreted polypeptide. The frog enzyme possesses a cleavable signal sequence; the mature protein starts at Thr30 of the polypeptide predicted from the cDNA sequence. Expression of the cloned cDNA by recombinant vaccinia virus resulted in the formation of a protein with the expected molecular mass and substrate specificity. Recombinant DPP IV was present in high concentration in the supernatant of infected cells and exhibited enzymatic activity towards the synthetic substrate alanyl-prolyl-p-nitroanilide.

Knee arthrodesis in the treatment of failed total knee replacement.

Twenty-six primary arthrodeses and 6 repeat arthrodeses were done for a total of 32 arthrodesis procedures in 26 patients, with followup from 2 to 10 years (mean, 4 years). The indications for arthrodesis were septic failure in 18 patients and aseptic loosening in 8 patients. Thirteen arthrodeses were done using external fixators, and 12 arthrodeses were done using a closed fluted intramedullary nail for fixation (1 arthrodesis was done with a custom proximal and distal interlocked nail). In addition, 6 repeat arthrodeses with intramedullary nail fixation were done for treatment of nonunions after external fixation. Five (38%) of the 13 patients who underwent arthrodesis with an external fixator had clinical and radiographic union at a mean of 5 months. All 13 patients with primary intramedullary nail arthrodesis achieved union. The knees of the 6 patients with nonunions after external fixation that were treated with repeat intramedullary nail arthrodesis achieved union. Patients with septic failure had staged debridements before intramedullary nail arthrodesis. Intramedullary nail arthrodesis can be done safely in patients with sepsis as a staged procedure. Knee arthrodesis using intramedullary nail fixation gives a much higher union rate than does external fixation and is associated with fewer complications.

The influenza C virus glycoprotein (HE) exhibits receptor-binding (hemagglutinin) and receptor-destroying (esterase) activities.

A cDNA copy of RNA segment 4 of influenza C/Cal/78 virus was cloned into an SV40 vector and expressed in CV-1 cells. The gene product expressed from the SV40 recombinant virus was immunoprecipitated by monoclonal antibodies directed against the influenza C virus glycoprotein. Cells infected with the recombinant virus also exhibited C virus-specific hemagglutinin and O-acetylesterase activity. This suggests that the same C virus protein is associated with receptor-binding as well as receptor-destroying activity. The latter viral activity was measured using as substrates bovine submaxillary mucin or a low molecular weight compound p-nitrophenylacetate. In analogy to the parainfluenza virus HN protein, the influenza C virus glycoprotein was termed HE, because it possesses hemagglutinin and esterase (receptor-destroying) activity.

Human and bovine coronaviruses recognize sialic acid-containing receptors similar to those of influenza C viruses.

Human coronavirus OC43 and bovine coronavirus elute from agglutinated chicken erythrocytes when incubated at 37 degrees C, suggesting the presence of a receptor-destroying enzyme. Moreover, bovine coronavirus exhibits an acetylesterase activity in vitro using bovine submaxillary mucin as substrate similar to the enzymatic activity found in influenza C viruses. Furthermore, pretreatment of erythrocytes with either influenza C virus or bovine coronavirus eliminates subsequent binding and agglutination by either coronaviruses or influenza C virus, whereas binding of influenza A virus remains intact. In addition, hemagglutination by coronaviruses can be inhibited by pretreatment of erythrocytes with Arthrobacter ureafaciens or Clostridium perfringens neuraminidase or by addition of sialic acid-containing gangliosides. These results suggest that, like influenza C viruses, human coronavirus OC43 and bovine coronavirus recognize O-acetylated sialic acid or a similar derivative as cell receptor.

Identification of a coronavirus hemagglutinin-esterase with a substrate specificity different from those of influenza C virus and bovine coronavirus.

We have characterized the hemagglutinin-esterase (HE) of puffinosis virus (PV), a coronavirus closely related to mouse hepatitis virus (MHV). Analysis of the cloned gene revealed approximately 85% sequence identity to HE proteins of MHV and approximately 60% identity to the corresponding esterase of bovine coronavirus. The HE protein exhibited acetylesterase activity with synthetic substrates p-nitrophenyl acetate, alpha-naphthyl acetate, and 4-methylumbelliferyl acetate. In contrast to other viral esterases, no activity was detectable with natural substrates containing 9-O-acetylated sialic acids. Furthermore, PV esterase was unable to remove influenza C virus receptors from human erythrocytes, indicating a substrate specificity different from HEs of influenza C virus and bovine coronavirus. Solid-phase binding assays revealed that purified PV was unable to bind to sialic acid-containing glycoconjugates like bovine submaxillary mucin, mouse alpha1 macroglobulin or bovine brain extract. Because of the close relationship to MHV, possible implications on the substrate specificity of MHV esterases are suggested.

Nucleotide sequence of cloned cDNA coding for honeybee prepromelittin.

Total mRNA from venom glands of young queen bees was transcribed into cDNA and cloned into the PstI site of plasmid pBR322. The nucleotide sequence of two clones with inserts containing genetic information for prepromelittin is presented. The longer insert encompasses 374 base pairs including 52 nucleotides before the initiation codon and a 3' non-coding region of 112 base pairs. The 70 amino acids of prepromelittin represent the total coding capacity of the mRNA from which this insert is derived. Southern blot analysis with this cloned cDNA has shown that it hybridizes with a single EcoRI fragment of honeybee DNA which contains about 3000 base pairs.

The E3 protein of bovine coronavirus is a receptor-destroying enzyme with acetylesterase activity.

In addition to members of the Orthomyxoviridae and Paramyxoviridae, several coronaviruses have been shown to possess receptor-destroying activities. Purified bovine coronavirus (BCV) preparations have an esterase activity which inactivates O-acetylsialic acid-containing receptors on erythrocytes. Diisopropyl fluorophosphate (DFP) completely inhibits this receptor-destroying activity of BCV, suggesting that the viral enzyme is a serine esterase. Treatment of purified BCV with [3H]DFP and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the proteins revealed that the E3 protein was specifically phosphorylated. This finding suggests that the esterase/receptor-destroying activity of BCV is associated with the E3 protein. Furthermore, treatment of BCV with DFP dramatically reduced its infectivity in a plaque assay. It is assumed that the esterase activity of BCV is required in an early step of virus replication, possibly during virus entry or uncoating.

Conserved exon-intron organization in two different caerulein precursor genes of Xenopus laevis. Additional detection of an exon potentially coding for a new peptide.

From a genomic library of Xenopus laevis, two genes coding for different preprocaeruleins have been isolated and sequenced. These correspond to the type I and type III precursors analyzed previously at the cDNA level [Richter, K., Egger, R. and Kreil, G. (1986) J. Biol. Chem. 261, 3676-3680]. The type III gene comprises eight exons; the type I apparently contains eight exons as well, of which six have been sequenced. The genetic information for the dekapeptide caerulein is present on small exons of 45 base pairs. The two genes are highly homologous in their 5'-flanking region, the exon/intron boundaries, and long stretches of intron sequences. A possible scheme for the evolution of this small family of genes through exon and gene duplications is presented. In the type I gene, in place of one of the caerulein exons, a potential exon with conserved splice sites was discovered. If expressed in some frog cells, this exon would code for a new peptide 60% homologous to caerulein.