Increased expression of a high molecular weight matrix component in rat hepatocellular carcinoma

The urea extract of the glycoproteins from the extracellular matrix of rat liver has been compared with that of Morris hepatoma 7777. A high molecular weight glycoprotein present in Morris hepatoma 7777 was not found in the extract of liver matrix. Under reducing conditions in SDS-gel electrophoresis this component gave two glycoprotein bands with Mr 53 k and 56 k. The indirect immunofluorescence staining with a monospecific antiserum directed against the component showed its abundant presence in Morris hepatoma 7777 as well as in the less malignant Morris hepatoma 9121 in form of extracellular network structures. The antigen also densely filled some cumuli of cells. In contrast the liver tissue showed only very weak staining of the extracellular areas. The overall distribution of the component could be correlated with the distribution of several hydrolases in the tumor matrix, notably beta-D-glucuronidase.     

Enhancement of α-amylase production by integrating and amplifying the α-amylase gene of Bacillus amyloliquefaciens in the genome of Bacillus subtilis

Summary The -amylase gene of Bacillus amyloliquefaciens was integrated into the genome of Bacillus subtilis by homologous recombination. In the first transformation step, several strains were obtained carrying the -amylase gene as two randomly located copies. These strains produced -amylase in the quantities comparable with that of the multicopy plasmid pKTH10, carrying the same -amylase gene. With the plasmid system, however, the rate of the -amylase synthesis was faster and the production phase shorter than those of the chromosomally encoded -amylase. The two chromosomal gene copies were further multiplied either by amplification using increasing antibiotic concentration as the selective pressure or by performing a second transformation step, identical to the first integration procedure. Both methods resulted in integration strains carrying up to eight -amylase gene copies per one genome and producing up to eightfold higher -amylase activity than the parental strains. Six out of seven transformants, studied in more detail, were stable after growth of 42 h even without antibiotic selection. The number of the DNA and mRNA copies of the -amylase gene was quantitavely determined by sandwich hybridization techniques, directly from culture medium.     

Proteases in the human full-term placenta

Summary Aminopeptidase A, not yet defined aminopeptidases and endopeptidases, dipeptidyl peptidase I, II and IV, -glutamyl transferase and oxytocinase were investigated in the normal human full-term placenta using qualitative (catalytic) cytochemistry, isoelectric focusing, immunocytochemistry and kinetic fluorometry. Aminopeptidase A could be visualized cytochemically in the smooth muscle cells of the chorionic plate, stem villi and basal plate blood vessels. Aminopeptidases were found in connective tissue fibres of the chorionic plate, villous stroma, basal plate and paraplacenta. Dipeptidyl peptidase IV was detected at the same sites as the aminopeptidases and, in addition, in amniotic epithelial cells, fibroblasts of the villous stroma, endothelium of chorinic plate and villous blood vessels as well as in the basophilic cytotrophoblast cells (x-cells) of the basal plate and paraplacenta, and it possibly also occurred in some domains of the plasma membrane of the syncytiotrophoblast and cytotrophoblast cells. The x-cells surrounded the fetus in the form of a dipeptidyl peptidase IV-positive shell at the border to the mother. The enzyme represented the first specific marker for x-cells. Dipeptidyl peptidase I and II were primarily found in Hofbauer cells (macrophages) of the villous stroma, but also in the syncytiotrophoblast, other villous stromal cells and cells of the chorionic and basal plate. -Glutamyl transferase was present in some connective tissue elements of the chorionic plate. Oxytocinase and endopeptidases were not detected. Isoclectric focusing of proteases revealed different molecular forms of dipeptidyl peptidase IV in the paraplacenta and villous tree, while the aminopeptidases shared the same pattern in both regions. Immunocytochemical staining of dipeptidyl peptidase IV in the villous tree resembled the pattern obtained by catalytic cytochemistry except for the blood vessel endothelium and the x-cells of the basal plate. Fluorometrically, all proteases were more active in the villous tree than in the paraplacenta. The kinetic measurements revealed the highest hydrolysis rates for dipeptidyl peptidase IV followed by the aminopeptidases. In contrast to eatalytic cytochemistry all proteases were detectable when using fluorometry.Supported by the German Research Foundation (Sfb 174)A preliminary account of this work was presented at a Symposion on Progress in General, Applied and Diagnostic Histochemistry (Smolenice, Czechoslovakia on March 24–28, 1986).     

Production of diphtheria toxin CRM228 in B. subtilis

The gene coding for a nontoxic diphtheria toxin (DT), tox228, was isolated from lysogenic Corynebacterium diphtheriae and cloned into pBR322. A mature form of the tox228 gene, lacking its signal sequence, was expressed in Bacillus subtilis using a B. amyloliquefaciens alpha-amylase secretion vector. To test the possibility of producing partially deleted DT molecules, which could be used for cell-directed toxin conjugates, a truncated form lacking 151 amino acids from the C-terminus of the DT was generated by oligonucleotide mutagenesis. Both the truncated and intact DT were efficiently secreted into the culture medium. During prolonged cultivation, the truncated form was less stable than the intact DT molecule.     

Cleavage of the O antigen 4,5,12 of Salmonella typhimurium by hydrofluoric acid

The effect of hydrofluoric acid (aqueous 48% HF) upon different lipopolysaccharides (LPS) was studied, employing conditions (48 h at + 4°C) that are commonly used to dephosphorylate LPS. From the LPS of Salmonella typhimurium having the O antigen 4,5,12 almost all of the O-antigenic sugars (Abe, Gal, Glc, Man, Rha) were liberated in dialysable form, whereas the saccharide chains of Salmonella LPS with O antigen 6,7 (Man, Glc, GlcNAc) were resistant to HF. The lability towards HF was shown to be due to the presence of the deoxysugar L-rhamnose in the saccharide backbone of the O antigen 4,5,12, since only Rha was found as the terminal sugar in the corresponding dialysable material. Hydrofluoric acid can thus be used to specifically cleave Rha-containing polysaccharides.     

Female-biased natal dispersal in the Siberian flying squirrel

Natal dispersal is usually sex biased in birds and mammals.Female-biased natal dispersal is the prevailing pattern in birdsbut is rare among mammals. Hypotheses explaining sex bias indispersal include the mate-defense mating hypothesis, whichpredicts male-biased dispersal, the resource-defense hypothesispredicting female-biased dispersal, and the competition hypothesis,which predicts that if dispersal is caused by competition forresources between sexes, then the subdominant sex will disperse.We studied natal dispersal of Siberian flying squirrels Pteromysvolans using radio telemetry in Southern Finland in 1996–2004.Of 86 juveniles that survived over the dispersal period, almostall young females dispersed from the natal site, whereas almost40% of males were philopatric. Dispersal was farther for femalesthan males. Females began dispersal on average 2 weeks earlierthan males and were lighter in mass at the onset of dispersalthan later dispersing males. No mate- or resource-defense matingsystem could be found among males, but females seemed to defendnest and apparently food resources, in contrast to the expectationof dispersal bias in resource-defense systems. Competition forresources between sexes does not explain female bias either:in the flying squirrel, the female seems to be the dominantsex. We propose that young females are subordinate to theirmothers and have to disperse to find a vacant, suitable sitefor reproduction.     

Increasing frequency of low summer precipitation synchronizes dynamics and compromises metapopulation stability in the Glanville fritillary butterfly

Climate change is known to shift species'' geographical ranges, phenologies and abundances, but less is known about other population dynamic consequences. Here, we analyse spatio-temporal dynamics of the Glanville fritillary butterfly (Melitaea cinxia) in a network of 4000 dry meadows during 21 years. The results demonstrate two strong, related patterns: the amplitude of year-to-year fluctuations in the size of the metapopulation as a whole has increased, though there is no long-term trend in average abundance; and there is a highly significant increase in the level of spatial synchrony in population dynamics. The increased synchrony cannot be explained by increasing within-year spatial correlation in precipitation, the key environmental driver of population change, or in per capita growth rate. On the other hand, the frequency of drought during a critical life-history stage (early larval instars) has increased over the years, which is sufficient to explain the increasing amplitude and the expanding spatial synchrony in metapopulation dynamics. Increased spatial synchrony has the general effect of reducing long-term metapopulation viability even if there is no change in average metapopulation size. This study demonstrates how temporal changes in weather conditions can lead to striking changes in spatio-temporal population dynamics.     

1H, 13C and 15N resonance assignments of the human filamin A tandem immunoglobulin-like domains 16–17 and 18–19

Filamins are large actin-binding and cross-linking proteins which act as linkers between the cytoskeleton and various signaling proteins. Filamin A (FLNa) is the most abundant of the three filamin isoforms found in humans. FLNa contains an N-terminal actin-binding domain and 24 immunoglobulin-like (Ig) domains. The Ig domains are responsible for the FLNa dimerization and most of the interactions that FLNa has with numerous other proteins. There are several crystal and solution structures from isolated single Ig domains of filamins in the PDB database, but only few from longer constructs. Here, we present nearly complete chemical shift assignments of FLNa tandem Ig domains 16–17 and 18–19. Chemical shift mapping between FLNa tandem Ig domain 16–17 and isolated domain 17 suggests a novel domain–domain interaction mode.