Rapid expression screening of Caenorhabditis elegans homeobox open reading frames using a two-step polymerase chain reaction promoter-gfp reporter construction technique.

In this paper a description is given of the expression pattern of the Caenorhabditis elegans homeobox gene ceh-38 using GFP reporter constructs, which were generated using a two-step polymerase chain reaction (PCR) procedure. This method allows fast analysis of genes of interest by looking at their expression in vivo using their putative promoter region to control the expression of a reporter gene. In this case the method was applied to screen C. elegans homeobox-containing genes to identify those that are expressed in the head and nervous system. The C. elegans genome project has made rapid progress, and more than 79 megabases of genomic data with several thousand open reading frames are available. This information can be used to design primers from putative promoter regions, which are amplified using long-range PCR. The long-range PCR product is then directly joined to the vector in a long-range Fill-in PCR. Since many genome projects are advancing rapidly, this approach should also be applicable for other model systems, and the method lends itself to automation, since no gel-purification steps are necessary. ceh-38 is a member of the ONECUT class of homeobox genes. Expression of ceh-38 starts during embryogenesis. In larvae and adults, expression was seen in many different types of tissues, such as the pharynx, gut, hypodermis and many nerve cells.

Influence of cardiopulmonary resuscitation on levels of tumour markers.

Tumour markers (TM), including alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen (CA) 15-3 and prostate-specific antigen (PSA), are serum markers for malignant diseases. Previous studies investigating the effect of acute and chronic inflammation, cardiopulmonary bypass surgery and cardiopulmonary resuscitation (CPR) on levels of TM showed conflicting results. Cardiopulmonary resuscitation (CPR) may result in a profound inflammatory response, and is frequently associated with severe tissue hypoperfusion. The present study investigated whether AFP, CEA, CA 15-3 and PSA are influenced by CPR. Alpha-fetoprotein (AFP), CEA, CA 15-3 and PSA (only in male patients) were assessed immediately after hospital admission, 6 h, 12 h and 2 days after prolonged CPR in eight male and 12 female patients. Serum levels of AFP, CEA, CA 15-3 did not change significantly after CPR. Prostate-specific antigen (PSA) levels increased significantly with a highest level in the study period 48 h after CPR (3.3 +/- 3.1 and 28.3 +/- 30.5 ng/mL for baseline and 48 h levels, respectively; P < 0.001). Alpha-fetoprotein (AFP), CEA, CA 15-3 and PSA (in men) values above the normal range were observed in 0%, 13.8%, 3.8% and 46.9% of all measurements respectively. At least one value above the normal range were observed in 0%, 20%, 5% and 75% of all patients for AFP, CEA, CA 15-3 and PSA (in men) respectively. Baseline values of AFP, CEA, CA 15-3 and PSA (in men) were above the normal range in 0%, 15%, 5% and 10% of all patients respectively. Levels for all markers did not differ significantly between survivors and non-survivors. In conclusion, prolonged CPR does not influence AFP, CEA, CA 15-3 serum levels, but is frequently associated with increases of PSA. Thus, in contrast to PSA, interpretation of AFP, CEA, CA 15-3 serum levels is not influenced by recent CPR.

Exon duplication from a fork head to a homeodomain protein.

The evolution of complex organisms such as animals requires a large expansion of the number of genes controlling developmental events. In addition, it is thought that domains are shuffled between genes to further increase the complexity and generate new types of genes and functions. Working with the Caenorhabditis elegans homeobox gene ceh-43, the orthologue of fly Distal-less (Dll), we observed sequence similarity to the C. elegans gene fkh-1. Now, with the complete genomic sequence available, we examined this similarity in detail. The region of similarity is confined essentially to one exon in the carboxy terminus of the two genes. Based on the gene structure, we think that an exon of fkh-1 was duplicated to the carboxy terminus of ceh-43, where it was incorporated as the last exon. This duplication event seems to have happened recently since the similarity on the nucleotide level is higher than the sequence similarity between fkh-1 of C. elegans and C. briggsae. Potentially the duplication event was mediated via a short region of sequence similarity between the two open reading frames of the genes. This duplication event clear shows that a part of a gene can successfully be juxtaposed to another gene. These events may perhaps not be rare.

The Caenorhabditis elegans distal-less ortholog ceh-43 is required for development of the anterior hypodermis.

Homeobox genes of the Distal-less (Dll) class are expressed in developing appendages as well as in the central nervous system in invertebrates and vertebrates. Mutant analyses in mice and Drosophila have implicated these genes in outgrowth of structures, cell adhesion, cell migration, and cell fate decisions. We have investigated the expression and function of ceh-43, the Dll ortholog from the nematode Caenorhabditis elegans, by using gfp reporter constructs and double-stranded RNA-mediated interference (RNAi). Our results show that, as in the fly, the C. elegans Dll ortholog seems to play a role in cell adhesion. An antibody against the butterfly Distal-less homeodomain stains the nervous system of C. elegans embryos (Panganiban et al. [1997] Proc Natl Acad Sci USA. 94:5162-5166). GFP expression under the control of the ceh-43 promoter looks similar, although strong expression is primarily confined to the head hypodermis and to neuronal support cells. ceh-43(RNAi) results in 100% lethality at embryonic or early larval stages. At the beginning of morphogenesis, ceh-43(RNAi) embryos start to lose cells through a hole in the head hypodermis. They either rupture anteriorly as elongation proceeds, or they elongate normally to threefold egg length with the pharynx not connected to the mouth. Elongated ceh-43(RNAi) animals die before or soon after hatching with a fluid-filled pseudocoel and large vacuoles. These phenotypes suggest a role for ceh-43 in development of adhesive properties in the head hypodermis that connects the epithelia of the skin and the digestive tract. Furthermore, possible defects in the excretory system may result at least in part from a requirement for ceh-43 in the CAN neurons where ceh-43:gfp is also expressed.

Caenorhabditis elegans has scores of hedgehog-related genes: sequence and expression analysis.

Previously, we have described novel families of genes, warthog (wrt) and groundhog (grd), in Caenorhabditis elegans. They are related to Hedgehog (Hh) through the carboxy-terminal autoprocessing domain (called Hog or Hint). A comprehensive survey revealed 10 genes with Hog/Hint modules in C. elegans. Five of these are associated with a Wart domain in wrt genes, and three with multiple copies of the Ground domain in grd genes. Both the Wart domain and the Ground domain occur also in genes encoding no Hog domain. Further, we define a new group of genes related to the grd genes, called ground-like (grl). Overall, C. elegans has more than 50 genes belonging to these gene families. Phylogenetic and sequence analysis shows that the wrt, grd, and grl genes are derived from each other. Further examination reveals a sequence motif with similarity to the core of the amino-terminal-signaling domain of Hh proteins. Our data suggest that the wrt, grd, grl, and hh genes are derived from a single ancestral gene. wrt, grd, and grl genes are also present in other nematodes, but so far not in any other phyla. Conversely, hh is not found presently in C. elegans nor other nematodes. Thus, the nematode genes could be the homologs of Hh molecules in other phyla. The membrane molecule Patched has been shown previously to be a receptor of Hh. Many Patched-related proteins are present in C. elegans, which may be targets of the hh-related genes. No Hedgehog-interacting protein (Hip) was found. We analyzed the expression patterns of eight wrt and eight grd genes. The results show that some closely related genes are expressed in the same tissues, but, overall, the expression patterns are diverse, comprising hypodermis, seam cells, the excretory cell, sheath and socket cells, and different types of neurons.

[Angiotensin converting enzyme in lung diseases]. / Das Angiotensin-Converting-Enzyme bei Lungenerkrankungen.

The aim of the study was to test the validity of ACE-determination for the differential diagnosis of pulmonary diseases. First details of the method, then the determination of the reference scale are discussed. The ACE-activity in the serum in 60 patients with sarcoidosis and 80 patients with other pulmonary diseases was measured. The sensitivity with which sarcoidosis can be established by means of the ACE-activity is to be examined. No positive correlation between the activity of the enzyme and the x-ray staging could be established, but active diseases requiring treatment could be distinguished from inactive forms to an unexpectedly high degree of reliance in our group of patients. The fact that in some cases of other pulmonary diseases--especially those connected with hepatopathy--higher ACE-activity is found as well, reduces the validity of the method. The determination of ACE comes up to all the criterias of a feasible, clinical-chemical method of analysis: practicability, reliance, high sensitivity and accuracy in a selected group, easy repetition. In some cases a typical x-ray result and corresponding clinical examination may provide the diagnosis of active sarcoidosis without further invasive methods of investigation; observation of the course of disease and adjustment of therapy are possible by means of the control of the ACE.

Rapid elimination of a high-titer spontaneous factor V antibody by extracorporeal antibody-based immunoadsorption and immunosuppression.

We report on the rapid elimination of a potent spontaneous factor V antibody of undetermined etiology by extracorporeal immunoadsorption on sepharose-bound polyclonal sheep antibodies to human immunoglobulins (Ig-Therasorb, Baxter) in combination with immunosuppressive treatment. A 68-year-old woman presented with severe hematuria. Severe factor V deficiency (< 1%) caused by an antibody to factor V (26 BU/ml) was found. Extracorporeal immunoadsorption (8.245 +/- 553 ml plasma processed per session) led to an average reduction of the antibody titer by 75% per session. The procedure was well tolerated without any side effects. Hematuria ceased after three immunoadsorptions and complete elimination of the antibody was achieved after seven sessions (day 15), followed by a rapid increase of the factor V activity to normal levels. Treatment with cyclophosphamide and prednisone was started on day 6 and continued for 2 months. The patient remains in remission at 6 months. Extracorporeal immunoadsorption is a highly effective method for eliminating antibodies to factor V (or other clotting factors) in selected cases, i.e., in patients with severe bleeding tendency, high antibody titer, and low probability of a rapid spontaneous remission.