Isolation and partial characterization of basic proteinases from stem bromelain

Crude bromelain extracts from pineapple stems (Ananas comosus) were fractionated by two-step FPLC-cation-exchange chromatography. At least eight basic proteolytically active components were detected. The two main components F4 and F5 together with the most active proteinase fraction F9 were characterized by SDS-PAGE, mass spectroscopy, multizonal cathodal electrophoresis, partial amino acid sequence, and monosaccharide composition analysis. F9 amounts to about 2% of the total protein and has a 15 times higher specific activity against the substratel-pyroglutamyl-l-phenylanalyl-l-leucine-p-nitroanilide (PFLNA) than the main component F4. The molecular masses of F4, F5, and F9 were determined to 24,397, 24,472, and 23,427, respectively, by mass spectroscopy. Partial N-terminal amino acid sequence analysis (20 amino acids) revealed that F9 differs from the determined sequence of F4 and F5 by an exchange at position 10 (tyrosineserine) and position 20 (asparagine glycine). F4 and F5 contained fucose, N-acetylglucosamine, xylose, and mannose in ratio of 1.02.01.02.0, but only 50% of the proteins seem to be glycosylated, whereas F9 was found to be unglycosylated. Polyclonal antibodies (IgG) against F9 detected F4 and F5 with tenfold reduced reactivity. ThepH optimum of F4 and F5 was betweenpH4.0 and 4.5 and for F9 close to neutralpH. The kinetic parameters for PFLNA hydrolysis were similar for F4 (K _m 2.30 mM,k _cat 0.87 sec^–1 and F5 (K _m 2.42 mM,k _cat 0.68 sec^–1), and differed greatly from F9 (K _m 0.40 mM,k _cat 3.94 sec^–1).Dedicated to H. Tschesche, Bielefeld, Germany, on behalf of his 60th anniversary.     

Tight junction biogenesis during early development

The tight junction (TJ) is an essential component of the differentiated epithelial cell required for polarised transport and intercellular integrity and signalling. Whilst much can be learnt about how the TJ is constructed and maintained and how it functions using a wide range of cellular systems, the mechanisms of TJ biogenesis within developmental models must be studied to gain insight into this process as an integral part of epithelial differentiation. Here, we review TJ biogenesis in the early mammalian embryo, mainly considering the mouse but also including the human and other species, and, briefly, within the amphibian embryo. We relate TJ biogenesis to inherent mechanisms of cell differentiation and biosynthesis occurring during cleavage of the egg and the formation of the first epithelium. We also evaluate a wide range of exogenous cues, including cell-cell interactions, protein kinase C signalling, gap junctional communication, Na⁺/K⁺-ATPase and cellular energy status, that may contribute to TJ biogenesis in the embryo and how these may shape the pattern of early morphogenesis.     

Bee diseases as factors in the life and behavior of the honeybee Colony

Summary The honeybee has prospered through the years of recorded history by following a plan of life that contributes to the survival of the colony rather than to the individual. The colony is capable of air-conditioning its hive to meet changing weather and to store up food sources for adverse periods. Since it contributes to the production of fruits, seeds, vegetables and pasture crops, and produces honey, a delectable natural sweet, man has been interested in its production and care.The honeybee is subject to many diseases which affect both the adults and the developing young. Several of the diseases have caused the death or destruction of thousands of colonies annually. Fortunately, none of these diseases are transmitted to other animals. Through the natural laws of survival, certain strains of bees have developed resistance to some of the diseases and man has assisted in this by selective breeding and cultivation of the hardier strains.The most noteworthy contribution to the control of bee diseases has been the use of sulfathiazole, terramycin and other therapeutics in the food of bees in the spring and fall. By their use, at least one disease which had been considered as incurable for centuries can be prevented or controlled without the destruction of colonies or valuable equipment. The use of chemicals and antibiotics as therapeutic agents in disease control has measurably strengthened the position of the honeybee in our general economy and will save the beekeeping industry many thousands of dollars annually.     

An X‐linked Myh11‐CreERT2 mouse line resulting from Y to X chromosome‐translocation of the Cre allele

The Myh11‐CreER^T2 mouse line (Cre⁺) has gained increasing application because of its high lineage specificity relative to other Cre drivers targeting smooth muscle cells (SMCs). This Cre allele, however, was initially inserted into the Y chromosome (X/Y^Cre+), which excluded its application in female mice. Our group established a Cre⁺ colony from male ancestors. Surprisingly, genotype screening identified female carriers that stably transmitted the Cre allele to the following generations. Crossbreeding experiments revealed a pattern of X‐linked inheritance for the transgene (k > 1000), indicating that these female carries acquired the Cre allele through a mechanism of Y to X chromosome translocation. Further characterization demonstrated that in hemizygous X/X^Cre+ mice Cre activity was restricted to a subset arterial SMCs, with Cre expression in arteries decreased by 50% compared to X/Y^Cre+ mice. This mosaicism, however, diminished in homozygous X^Cre+/X^Cre+ mice. In a model of aortic aneurysm induced by a SMC‐specific Tgfbr1 deletion, the homozygous X^Cre+/X^Cre+ Cre driver unmasked the aortic phenotype that is otherwise subclinical when driven by the hemizygous X/X^Cre+ Cre line. In conclusion, the Cre allele carried by this female mouse line is located on the X chromosome and subjected to X‐inactivation. The homozygous X^Cre+/X^Cre+ mice produce uniform Cre activity in arterial SMCs.     

Calcium domains associated with individual channels can account for anomalous voltage relations of CA-dependent responses.

Computer-assisted modeling of calcium influx through voltage-activated membrane channels predicted that buffer-limited elevation of cytoplasmic free calcium ion concentration occurs within microscopic hemispherical "domains" centered upon the active Ca channels. With increasing depolarization, the number of activated channels, and hence the number of Ca domains, should increase; the single-channel current should, however, decrease, thereby decreasing Ca2+ accumulation in each domain relative to the macroscopic current. Such voltage dependence of the microscopic distribution of Ca2+ may influence relations between total Ca2+ entry and Ca-dependent processes. Ca-mediated inactivation of Ca channels in Aplysia neurons exhibits behavior consistent with the calcium domain hypothesis.     

Phosphorylation modulates keratin structure.

Bovine hoof keratin was shown to be a substrate for cAMP-dependent protein kinase using [gamma-32P]ATP. Natural-abundance cross-polarization (CP) MAS 13C NMR was used to examine the effect of phosphorylation on keratin structure. When short contact times were used, phosphorylation was shown to increase the number of residues in the motionally restricted portions of the protein; i.e., a portion(s) of the protein became more rigid upon phosphorylation. Circular dichroism (CD) spectra showed a spectral shape characteristic of alpha helix for this keratin. Phosphorylation of the keratin by cAMP-dependent protein kinase resulted in a CD spectrum with the same shape but of greater apparent intensity. This may have been the result of an increase in the alpha-helical content of the protein. These data showed that the structure of keratin changed significantly upon phosphorylation by cAMP-dependent protein kinase. The region of the keratin molecule most likely to be altering its structure was the end of the molecule, which was involved in the formation of, and intracellular attachment of, intermediate filaments. Therefore, these data suggested that cAMP-dependent phosphorylation may produce significant changes in the intracellular organization of intermediate filaments. When the keratin was phosphorylated using cold ATP, magic-angle spinning (MAS) 31P nuclear magnetic resonance (NMR) revealed two resonances arising from the phosphorylation sites on the keratin. The more shielded resonance was shown to arise from cAMP-dependent protein kinase phosphorylation. Static 31P NMR measurements suggested that at least two classes of cAMP-dependent sites existed with the same isotropic 31P chemical shift; one was considerably motionally restricted with respect to the other.     

Assembly of keratin onto PtK1 cytoskeletons: evidence for an intermediate filament organizing center

Purified keratin, solubilized in 8 M of urea, was added to Triton X-100-extracted PtK1 cells in 5 mM PIPES buffer. The buffer conditions induced assembly of keratin filaments which appear to associate with nuclei of extracted cells. These keratin fibers extend beyond the original margin of the cells and frequently form bridges between adjacent cells. Electron microscopy shows that keratin filaments associate closely with the surface of the nucleus. We suggest that the site of association between keratin and the nucleus may represent an intermediate filament organizing center.     

Lubiprostone activates non-CFTR-dependent respiratory epithelial chloride secretion in cystic fibrosis mice

Periciliary fluid balance is maintained by the coordination of sodium and chloride channels in the apical membranes of the airways. In the absence of the cystic fibrosis transmembrane regulator (CFTR), chloride secretion is diminished and sodium reabsorption exaggerated. ClC-2, a pH- and voltage-dependent chloride channel, is present on the apical membranes of airway epithelial cells. We hypothesized that ClC-2 agonists would provide a parallel pathway for chloride secretion. Using nasal potential difference (NPD) measurements, we quantified lubiprostone-mediated Cl(-) transport in sedated cystic fibrosis null (gut-corrected), C57Bl/6, and A/J mice during nasal perfusion of lubiprostone (a putative ClC-2 agonist). Baseline, amiloride-inhibited, chloride-free gluconate-substituted Ringer with amiloride and low-chloride Ringer plus lubiprostone (at increasing concentrations of lubiprostone) were perfused, and the NPD was continuously recorded. A clear dose-response relationship was detected in all murine strains. The magnitude of the NPD response to 20 muM lubiprostone was -5.8 +/- 2.1 mV (CF, n = 12), -8.1 +/- 2.6 mV (C57Bl/6 wild-type, n = 12), and -5.3 +/- 1.2 mV (AJ wild-type, n = 8). A cohort of ClC-2 knockout mice did not respond to 20 muM lubiprostone (n = 6, P = 0.27). In C57Bl/6 mice, inhibition of CFTR with topical application of CFTR inhibitor-172 did not abolish the lubiprostone response, thus confirming the response seen is independent of CFTR regulation. RT-PCR confirmed expression of ClC-2 mRNA in murine lung homogenate. The direct application of lubiprostone in the CF murine nasal airway restores nearly normal levels of chloride secretion in nasal epithelia.     

Behavioural evidence of colour vision in free flying stingless bees

Colour vision was first demonstrated with behavioural experiments in honeybees 100 years ago. Since that time a wealth of quality physiological data has shown a highly conserved set of trichromatic colour receptors in most bee species. Despite the subsequent wealth of behavioural research on honeybees and bumblebees, there currently is a relative dearth of data on stingless bees, which are the largest tribe of the eusocial bees comprising of more than 600 species. In our first experiment we tested Trigona cf. fuscipennis, a stingless bee species from Costa Rica in a field setting using the von Frisch method and show functional colour vision. In a second experiment with these bees, we use a simultaneous colour discrimination test designed for honeybees to enable a comparative analysis of relative colour discrimination. In a third experiment, we test in laboratory conditions Tetragonula carbonaria, an Australian stingless bee species using a similar simultaneous colour discrimination test. Both stingless bee species show relatively poorer colour discrimination compared to honeybees and bumblebees; and we discuss the value of being able to use these behavioural methods to efficiently extend our current knowledge of colour vision and discrimination in different bee species.     

Excitation energy transfer from phycobiliprotein to chlorophyll d in intact cells of Acaryochloris marina studied by time- and wavelength-resolved fluorescence spectroscopy.

The fluorescence decay spectra and the excitation energy transfer from the phycobiliproteins (PBP) to the chlorophyll-antennae of intact cells of the chlorophyll (Chl) d-dominated cyanobacterium Acaryochloris marina were investigated at 298 and 77 K by time- and wavelength-correlated single photon counting fluorescence spectroscopy. At 298 K it was found that (i) the fluorescence dynamics in A. marina is characterized by two emission peaks located at about 650 and 725 nm, (ii) the intensity of the 650 nm fluorescence depends strongly on the excitation wavelength, being high upon excitation of phycobiliprotein (PBP) at 632 nm but virtually absent upon excitation of chlorophyll at 430 nm, (iii) the 650 nm fluorescence band decayed predominantly with a lifetime of 70 +/- 20 ps, (iv) the 725 nm fluorescence, which was observed independent of the excitation wavelength, can be described by a three-exponential decay kinetics with lifetimes depending on the open or the closed state (F(0) or F(m)) of the reaction centre of Photosystem II (PS II). Based on the results of this study, it is inferred that the excitation energy transfer from phycobiliproteins to Chl d of PS II in A. marina occurs with a time constant of about 70 ps, which is about three times faster than the energy transfer from the phycobilisomes to PS II in the Chl a-containing cyanobacterium Synechococcus 6301. A similar fast PBP to Chl d excitation energy transfer was also observed at 77 K. At 77 K a small long-lived fluorescence decay component with a lifetime of 14 ns was observed in the 640-700 nm spectral range. However, it has a rather featureless spectrum, not typical for Chl a, and was only observed upon excitation at 400 nm but not upon excitation at 632 and 654 nm. Thus, this long-lived fluorescence component cannot be used as an indicator that the primary PS II donor of Acaryochloris marina contains Chl a.