Intracellular localization of prostatic binding protein (PBP) in rat prostate by light and electron microscopic immunocytochemistry

Summary Extra- and intracellular distribution of Prostatic Binding Protein (PBP) was studied in the different genital organs of the male rat by immunocytochemistry at the light and electron microscopic levels. PBP was extracted from cytosols of rat ventral prostate and used for immunization of rabbits. The specificity of the antiserum raised was tested by western blotting and immunoelectrophoresis. From the different fixatives tested for optimal structural and antigenic preservation of the ventral prostate a mixture containing 2.5% paraformaldehyde, 0.5% glutaraldehyde and 0.5% CaCl₂ in cacodylate buffer, 0.05 M, pH 7.3 was selected. Using the immunofluorescence technique and the unlabeled antibody enzyme method PBP-immunoreactivity was detected at the light microscopic level in the luminal secretions of the ventral prostate. No reaction was observed with the seminal vesicle, the coagulating gland, the dorsal and lateral prostates, the epididymis and the testis. Intracellular secretory granules reacting with PBP antiserum were exclusively found in the secretory cells of the ventral prostate. Insufficiently fixed cells showed a diffuse generalized reaction of the cytoplasm indicating a leakage of the antigen from the secretory granules. Such artifacts were common in tissue sections processed with the preembedding-staining procedure. At the ultrastructural level therefore mostly the postembedding staining method was performed using both the unlabeled antibody enzyme method and the ferritin-labeled immunoglobulin technique in osmicated, Epon-embedded tissue. Labeling with either method was intense in the secretory granules and the condensing vacuoles, while the labeling density of the rough endoplasmic reticulum and the Golgi cisternae was in the background range. Castration experiments showed that secretory material displaying PBP immunoreactivity was retained within the acinar lumen of the gland for several days after castration, but was absent from most secretory cells already by four days after castration. Immunocytochemistry of PBP therefore is a very sensitive method for analysing the secretory activity and its androgen dependence of the prostate of the rat.Supported by a grant from the Deutsche Forschungsgemeinschaft (Au 48/7-6) and a grant from the Cystic Fibrosis Foundation (G 261 A)     

Detection of a novel plasma serine protease during purification of vitamin K-dependent coagulation factors.

A novel serine protease (PHBSP) was purified from human plasma by two chromatographic steps with a final yield of 1.6 mg/l plasma. The protease consists of two disulfide-bridged chains of about 50 and 30 kDa with the light chain containing the active site of the enzyme. NH2-terminal sequence analysis revealed identity to the deduced amino acid sequence of HGFA-like mRNA. The activity of PHBSP is strongly dependent on Ca2+ ions and is efficiently inhibited by alpha2-antiplasmin and aprotinin. Possible functions of PHBSP in the hemostatic system are discussed.     

The N-terminus promotes oligomerization of the Escherichia coli initiator protein DnaA

Initiation of chromosome replication in Escherichia coli is governed by the interaction of the initiator protein DnaA with the replication origin oriC. Here we present evidence that homo-oligomerization of DnaA via its N-terminus (amino acid residues 1-86) is also essential for initiation. Results from solid-phase protein-binding assays indicate that residues 1-86 (or 1-77) of DnaA are necessary and sufficient for self interaction. Using a 'one-hybrid-system' we found that the DnaA N-terminus can functionally replace the dimerization domain of coliphage lambda cl repressor: a lambdacl-DnaA chimeric protein inhibits lambda plasmid replication as efficiently as lambdacI repressor. DnaA derivatives with deletions in the N-terminus are incapable of supporting chromosome replication from oriC, and, conversely, overexpression of the DnaA N-terminus inhibits initiation in vivo. Together, these results indicate that (i) oligomerization of DnaA N-termini is essential for protein function during initiation, and (ii) oligomerization does not require intramolecular cross-talk with the nucleotide-binding domain III or the DNA-binding domain IV. We propose that E. coli DnaA is composed of largely independent domains - or modules - each contributing a partial, though essential, function to the proper functioning of the 'holoprotein'.     

Increased antiviral activity of cyclic urea HIV protease inhibitors by modifying the P1/P1' substituents.

A series of alkyl substituted P1/P1' analogs was prepared in an attempt to increase translation of the 3-aminoindazole class of HIV protease inhibitors. Increasing the lipophilicity of the P1/P1' residues dramatically improved translation of enzyme activity to antiviral activity in the whole cell assay.     

Invertase activity and cell growth in lentil epicotyls

The activity of invertase and its relation to growth were studied in the epicotyls of lentil seedlings incubated in the presence and absence of gibberellic acid (GA₃).

Invertase activity per epicotyl increases relatively more rapidly than does length, reaches a maximum during most active elongation, and declines upon cessation of growth.

GA₃ enhances both growth and increase in invertase activity, without altering the kinetics of the 2 processes. If GA₃ is added during incubation invertase activity increases more rapidly than does elongation rate.

Incubation of the seedlings in solutions of polyethyleneglycol inhibits the increase of both growth and invertase activity, the latter actually undergoing a decline, but causes no great change in the relative effect of GA₃. In presence of polyethyleneglycol GA₃ has however a relatively greater effect on invertase activity than on growth.

Sugars in the incubation medium have no significant effect on growth and invertase activity in the epicotyl, except inhibition at relatively high concentrations.

Cycloheximide, actinomycin D, and 5-fluorodeoxyuridine (FUDR) inhibit both growth and the increase in invertase activity. Added during incubation cycloheximide causes complete inhibition of growth and a decrease in invertase activity with no appreciable lag phase. With actinomycin D and FUDR the inhibition occurs after lag periods of 2 to 3 and of at least 10 hours, respectively. Thus the increase in enzyme activity is very probably based on de-novo synthesis, and the enzyme is in a state of turnover during growth.

The enzyme is present in soluble form in the cytoplasm, not firmly bound to any cell structures.

     

Low diversity but high differentiation: the population genetics of Aglaope infausta (Zygaenidae: Lepidoptera)

Aim Aglaope infausta is a thermophilous Zygaenid of Atlanto-Mediterranean origin, distributed in Portugal, Spain, France and north-western Italy reaching its north-eastern distribution limit in western Germany. The local, regional and inter-regional genetic structure of this species is studied in this analysis. Location and methods The allozymes of 456 individuals from 12 populations (11 from western Germany and one from southern Portugal) were studied by electrophoresis. Results Six of the 19 loci analysed were monomorphic. Genetic differentiation between populations was high (F_ST: 0.404), while the mean genetic diversity was low (H_e: 3.4%). Most (96.5%) of the genetic variance between populations was between the Portuguese and the German samples, but also the differentiation within Germany was considerable (F_SR: 0.101). In Germany, A. infausta occurs in two major regions (middle Rhine and Nahe) that are geographically separated, and 55.5% of the genetic variance was found between these two regions. The populations of both areas do not differ in their genetic diversity, but those of the middle Rhine have significantly higher genetic distances among them than the Nahe populations (0.020 and 0.015, respectively). F_ST was also higher in the middle Rhine region than in the Nahe region (0.089 and 0.045, respectively). Main conclusions Aglaope infausta shows a very low level of genetic heterogeneity for a lepidopteran species. However, this genetic poverty is not affecting the species’ viability. During the ice ages, differentiation into two genetic lineages occurred, most probably in a south-western and a south-eastern differentiation centre in Iberia. The gap in the distribution range in Germany is clearly reflected in the genetic structure. This differentiation must have developed relatively quickly because western Germany most probably was colonized during the climatic optimum 6000 years ago.     

Ontogeny of the Marmorkrebs (marbled crayfish): a parthenogenetic crayfish with unknown origin and phylogenetic position

Development, growth, and egg production of the Marmorkrebs (marbled crayfish), a crayfish with parthenogenetic reproduction, uncertain geographic origin, and taxonomic position, was studied under laboratory conditions. Length and weight increments strongly depended on temperature being highest at 30 degrees C, and lowest at 15 degrees C. At 25 degrees C, cephalothorax length and weight increased by 17.5 mm and 1700 mg, respectively, in the course of 150 d, whereas at 15 degrees C these parameters increased by only 7 mm and 100 mg during the same period of time. Photoperiod slightly affected growth at 25 degrees C. During growth experiments, mortality was lower at 20 degrees C compared to higher (25 degrees , 30 degrees C) or lower temperatures (15 degrees C), and lower under short-day than under long-day conditions. Females matured early (at an age of 141-255 d, a cephalothorax length of 14-21.5 mm, and a weight of 0.63-2 g) compared to other crayfish species. Reproductive females with a cephalothorax length of between 25-35 mm produced large clutches (up to 416 eggs) and brooding periods varied between 22 and 42 d. In order to establish a staging scheme for Marmorkrebs embryos, embryos were photographed, externally visible ontogenetic events charted, and dissected embryos stained with a nuclear dye. These experiments indicate that their development is virtually identical to that of other crayfish. In conclusion, these results and others show that the Marmorkrebs may be taken as a representative valid model organism for future developmental studies on Crustacea.     

Inhibition of bFGF/EGF-dependent endothelial cell proliferation by the hyaluronan-binding protease from human plasma

Recently we identified a plasma serine protease with a high affinity to glycosaminoglycans like heparin or hyaluronic acid, termed hyaluronan-binding protease (HABP). Since glycosaminoglycans are found on cell surfaces and in the extracellular matrix a physiological role of this plasma protease in a pericellular environment was postulated. Here we studied the influence of HABP on the regulation of endothelial cell growth. We found that HABP efficiently prevented the basic fibroblast growth factor/epidermal growth factor (bFGF/EGF)-dependent proliferation of human umbilical vein endothelial cells. Proteolytic cleavage of adhesion molecules was found to be involved, but was not solely responsible for the anti-proliferative activity. Pre-treatment of growth factor-supplemented cell culture medium with HABP indicated that no direct contact between the active protease and cells was required for growth inhibition. In vitro studies revealed a growth factor-directed activity of HABP, resulting in complexation and partial hydrolysis and, thus, inactivation of basic fibroblast growth factor, a potent mitogen for endothelial cells. Heparin and heparan sulfate fully protected bFGF from complexation and cleavage by HABP, although these glycosaminoglycans are known to enhance the proteolytic activity of HABP. This finding suggested that free circulating bFGF rather than bFGF bound to heparan sulfate proteoglycans would be a physiologic substrate. In conclusion, down-regulation of bFGF-dependent endothelial cell growth represents an important mechanism through which HABP could control cell growth in physiologic or pathologic processes like angiogenesis, wound healing or tumor development.     

Synthesis and evaluation of indenopyrazoles as cyclin-dependent kinase inhibitors. Part 4: Heterocycles at C3

New indeno[1,2-c]pyrazol-4-one cyclin dependent kinase inhibitors have been disclosed. The most promising compounds are nanomolar enzyme inhibitors with excellent activity against tumor cells. The most advanced compound retains cell culture activity even in the presence of human serum proteins. The most advanced compound did not kill the normal fibroblast line AG1523.     

Population dependent Fourier decomposition of fitness landscapes over recombination spaces: Evolvability of complex characters

The effect of recombination on genotypes can be represented in the form of P-structures, i.e., a map from the set of pairs of genotypes to the power set of genotypes. The interpretation is that the P-structure maps the pair of parental genotypes to the set of recombinant genotypes which result from the recombination of the parental genotypes. A recombination fitness landscape is then a function from the genotypes in a P-structure to the real numbers. In previous papers we have shown that the eigenfunctions of (a matrix associated with) the P-structure provide a basis for the Fourier decomposition of arbitrary recombination landscapes. Here we generalize this framework to include the effect of genotype frequencies, assuming linkage equilibrium. We find that the autocorrelation of the eigenfunctions of the population-weighted P-structure is independent of the population composition. As a consequence we can directly compare the performance of mutation and recombination operators by comparing the autocorrelations on the finite set of elementary landscapes. This comparison suggests that point mutation is a superior search strategy on landscapes with a low order and a moderate order of interaction p < n/3 (n is the number of loci). For more complex landscapes 1-point recombination is superior to both mutation and uniform recombination, but only if the distance among the interacting loci (defining length) is minimal. Furthermore we find that the autocorrelation on any landscape is increasing as the distribution of genotypes becomes more extreme, i.e., if the population occupies a location close to the boundary of the frequency simplex. Landscapes are smoother the more biased the distribution of genotype frequencies is. We suggest that this result explains the paradox that there is little epistatic interaction for quantitative traits detected in natural populations if one uses variance decomposition methods while there is evidence for strong interactions in molecular mapping studies for quantitative trait loci.