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The afp gene encoding the antifungal protein (AFP) of Aspergillus giganteus has a prototypical alkaline gene expression pattern, which suggests that the gene might be under the control of the ambient pH-dependent zinc-finger transcription factor PacC. This notion is corroborated by the presence in the upstream region of afp of two putative PacC binding sites, afpP1 and afpP2, which are specifically recognised by the PacC protein of A. nidulans in vitro. However, in this report we provide several lines of evidence to show that pH-dependent up-regulation of afp is not mediated by transcriptional activation through PacC. (1) The temporal expression pattern of the A. giganteus pacC gene does not parallel the accumulation of the afp mRNA during cultivation. (2) Inactivation of afpP1 and afpP2 did not reduce promoter activity under alkaline conditions, as determined from the relative wild-type and mutant afp::lacZ reporter activities in A. nidulans. (3) Reporter activities in acidity- and alkalinity-mimicking mutant strains are inconsistent with a positive role for PacC in afp expression. (4) In A. giganteus, the pH-dependent increase in afp mRNA and AFP levels can be completely prevented by the calcineurin inhibitor FK506, suggesting that the calcineurin signalling pathway might control the in vivo activation of the afp promoter by alkaline pH.

We report here on the characterization and immunohistochemical localization in human tissues of calcitonin receptor-like receptor (CRLR) which was recently found to mediate the effects of both calcitonin gene-related peptide (CGRP) and adrenomedullin (ADM). Western blot analysis using antibodies raised against the first extracellular loop and the carboxy-terminal part of hCRLR, respectively, detected two major bands corresponding to about 70 and 60 kDa in membrane preparations of cultured endothelial cells and numerous organs including lung, heart ventricle and kidney. Immunohistochemical analysis of the cardiovascular system revealed CRLR-like immunoreactivity (CRLR-LI) in the endothelium of all blood vessels including large and small arteries, veins and capillaries, and in heart muscle cells and endocardium. The lung showed intense staining over the alveolar capillaries. Within the digestive tract, staining was observed over the cells lining the excretory ducts of the parotid gland, over the epithelium of the fundic glands of stomach, endocrine cells of the duodenum and ileum and some myenteric ganglia. The kidney presented staining of the juxtaglomerular arteries, the glomerular capillaries and chief cells of the collecting duct. Within the endocrine organs, a strong CRLR-LI signal was observed over the Langerhans islets, and weak immunoreactivity in the Leydig cells of testis. Spleen showed intense staining in trabecular veins and sinuses. Macrophages displayed a variable immunoreactivity. Our data demonstrate a wide distribution of CRLR throughout the human body and suggest CRLR to be involved in the mediation of a variety of actions in addition to vascular control.

Osteoporosis is one of the most common diseases of the elderly. This leads to the hypothesis that the ageing of the organism is reflected as a cytogerontological effect in a specific loss of bone cell function. Three underlying pathogenetic mechanisms need to be considered: (1) cellular aging in general, (2) impairment of the systemic stimulation of bone formation by e.g. decreasing hormone levels, and (3) lower cellular effectiveness of cytokines and growth factors. Cellular aging consists of replicative and postmitotic senescence. While the replicative senescence limits only the number of cell cycles, the postmitotic aging is influenced by endo- and exogenous factors. These lead to genetic alterations known as delayed persistent genomic instability and to an increasing impairment of specific cellular functions. In the postmitotic phase, osteopenia caused by the decrease of systemically available sexual hormones is a major field of research. Osteopenia caused by a decreased activity of locally effective cytokines and growth factors is becoming increasingly understood. New therapeutic strategies, which modulate the local osteoblast activity, e.g. in bone defect healing, are under development. In conclusion, cellular senescence is considered to be one element in the development of bone loss. Potential therapeutic targets may open up an additional path in the treatment of local and systemic osteopenias.

The bop gene codes for the membrane protein bacterio-opsin (BO), which on binding all-trans-retinal, constitutes the light-driven proton pump bacteriorhodopsin (BR) in the archaebacterium Halobacterium salinarium. This gene was cloned in a yeast multi-copy vector and expressed in Saccharomyces cerevisiae under the control of the constitutive ADH1 promoter. Both the authentic gene and a modified form lacking the precursor sequence were expressed in yeast. Both proteins are incorporated into the membrane in S. cerevisiae. The presequence is thus not required for membrane targeting and insertion of the archaebacterial protein in budding yeast, or in the fission yeast Schizosaccharomyces pombe, as has been shown previously. However, in contrast to S. pombe transformants, which take on a reddish colour when all-trans-retinal is added to the culture medium as a result of the in vivo regeneration of the pigment, S. cerevisiae cells expressing BO do not take on a red colour. The precursor of BO is processed to a protein identical in size to the mature BO found in the purple membrane of Halobacterium. The efficiency of processing in S. cerevisiae is dependent on growth phase, as well as on the composition of the medium and on the strain used. The efficiency of processing of BR is reduced in S. pombe and in a retinal-deficient strain of H. salinarium, when retinal is present in the medium.

We have shown that the second intron of the Podospora mitochondrial gene coding for cytochrome b (Cytb 12) splices autocatalytically, using in vitro transcripts generated from the T7 promoter. The reaction takes place at 37 degrees C in the presence of 50 mM TRIS-HCl pH 7.5, 60 mM MgCl2 and 1 mM GTP but shows a low efficiency even at high KCl concentrations of up to 1.2 M. Under these conditions, intron bI2 follows the conventional pathway of group I splicing, and all characteristic products, with regard to both transesterification and hydrolysis, could be identified. Moreover, the intron is capable of undergoing cyclization, thereby releasing the noncoded G and one additional nucleotide (U) from the 5' end. The 5' cleavage site is preceded by the same two nucleotides, indicating a base-pairing at the same site of the internal guide sequence (IGS) for both splicing and cyclization ("one-binding-site model"). In addition, products resulting from site-specific hydrolysis 138 nucleotides downstream of the 5' splice site were detected. Unusually, the shortened intron is also able to form a circular RNA and an alternative sequence that aligns the cyclization site to the catalytic core of the intron must be assumed.

In the alkane yeast Saccharomycopsis lipolytica (formerly: Candida lipolytica) the variability in the ascospore number is caused by the absence of a correlation between the meiotic divisions and spore wall formation. In four spored yeasts, after meiosis II, a spore wall is formed around each of the four nuclei produced by meiosis II. However, in the most frequently occurring two spored asci of S. lipolytica, the two nuclei are already enveloped by the spore wall after meiosis I due to a delay of meiosis II. This division takes place within the spore during the maturation of the ascus. In this case germination of the binucleate ascospore is not preceded by a mitosis. It follows that the cells of the new haploid clones are mononucleate. In the three spored asci, which occur rarely, only one nucleus is surrounded by a spore wall after meiosis I; the other nucleus undergoes meosis II before the onset of spore wall formation. The result is one bincleate and two mononucleate spores. In the one spored asci the two meiotic divisions occur within the young ascosphore, i.e. spore wall formation starts immediately after development of the ascus. These cytological observations were substantiated by genetic data, which in addition confirmed the prediction that binucleate spores may be heterokaryotic. This occurs when there is a postreduction of at least one of the genes by which the parents of the cross differ. This also explains the high frequency of prototrophs in the progeny of non-allelic auxotrophs since random spore isolates are made without distinguishing between mono- and binucleate spores. The possibility of analysing offspring of binucleate spores by tetrad analysis is discussed. These findings enable us to understand the life cycle of S. lipolytica in detail and we are now in a position to start concerted breeding for strain improvement especially with respect to single cell protein production.

The existence of plasmid like DNA was demonstrated in senescent mycelia of Podospora anserina (strain s) by biophysical and electronmicroscopic methods. According to their contour length of about 1.4 and 2.7 micron respectively the molecular weight for the monomer is in the range of 3.10(6).