Intracellular membrane trafficking pathways in bone-resorbing osteoclasts revealed by cloning and subcellular localization studies of small GTP-binding rab proteins

A variety of intracellular membrane trafficking pathways are involved in establishing the polarization of resorbing osteoclasts and regulating bone resorption activities. Small GTP-binding proteins of rab family have been implicated as key regulators of membrane trafficking in mammalian cells. Here we used a RT-PCR-based cloning method and confocal laser scanning microscopy to explore the expression array and subcellular localization of rab proteins in osteoclasts. Rab1B, rab4B, rab5C, rab7, rab9, rab11B, and rab35 were identified from rat osteoclasts in this study. Rab5C may be associated with early endosomes, while rab11B is localized at perinuclear recycling compartments and may function in the ruffled border membrane turnover and osteoclast motility. Interestingly, late endosomal rabs, rab7, and rab9, were found to localize at the ruffled border membrane indicating a late endosomal nature of this specialized plasma membrane domain in resorbing osteoclasts. This also suggests that late endocytotic pathways may play an important role in the secretion of lysosomal enzymes, such as cathepsin K, during bone resorption.     

The core structure of the lipopolysaccharide from the causative agent of plague, Yersinia pestis

The rough-type lipopolysaccharide (LPS) of the plague pathogen, Yersinia pestis, was studied after mild-acid and strong-alkaline degradations by chemical analyses, NMR spectroscopy and electrospray-ionization mass spectrometry, and the following structure of the core region was determined:where L-alpha-D-Hep stands for L-glycero-alpha-D-manno-heptose, Sug1 for either 3-deoxy-alpha-D-manno-oct-2-ulosonic acid (alpha-Kdo) or D-glycero-alpha-D-talo-oct-2-ulosonic acid (alpha-Ko), and Sug2 for either beta-D-galactose or D-glycero-alpha-D-manno-heptose. A minority of the LPS molecules lacks GlcNAc.     

Sulfur distribution in bacteriorhodopsin from multiple wavelength anomalous diffraction near the sulfur K-edge with synchrotron x-ray radiation.

Bacteriorhodopsin contains nine sulfur atoms from the nine methionine residues. The distribution of these sulfur atoms in the projected density map was determined from x-ray diffraction experiments using multiple wavelength anomalous diffraction (MAD) at the sulfur K-edge (5.02 A) with synchrotron radiation. The experiments were performed with uniaxial samples of oriented purple membranes at room temperature and 86% relative humidity. For such samples only the real part f' (lambda) of the resonant scattering amplitude of sulfur contributes to the observed scattering intensity. The sulfur density was determined from the difference in diffraction intensities detected at two wavelengths near the sulfur K-edge that were approximately 0.004 A apart. The measured change in f' between these two wavelengths corresponds to 6 electron units. This shows that large anomalous dispersion effects occur near the sulfur K-edge. The in-plane positions of the sulfur atoms of Met32, Met56, and Met209 were determined unambiguously. The difference density from Met20, Met60, Met118, and Met145 is concentrated in the interior of the seven alpha-helical bundle, overlaps strongly in the projected density map, and cannot be resolved at the resolution of these experiments (8.2 A). This method of localizing individual sulfur atoms can be applied to other two-dimensional protein crystals and is promising in conjunction with the site-directed introduction of sulfur atoms by the use of cysteine mutants.     

Identification,Purification, and Characterization of Transpeptidase and Glycosyltransferase Domains of Streptococcus pneumoniae Penicillin-Binding Protein 1a

Resistance to β-lactam antibiotics in Streptococcus pneumoniae is due to alteration of penicillin-binding proteins (PBPs). S. pneumoniae PBP 1a belongs to the class A high-molecular-mass PBPs, which harbor transpeptidase (TP) and glycosyltransferase (GT) activities. The GT active site represents a new potential target for the generation of novel nonpenicillin antibiotics. The 683-amino-acid extracellular region of PBP 1a (PBP 1a*) was expressed in Escherichia coli as a GST fusion protein. The GST-PBP 1a* soluble protein was purified, and its domain organization was revealed by limited proteolysis. A protease-resistant fragment spanning Ser 264 to Arg 653 exhibited a reactivity profile against both β-lactams and substrate analogues similar to that of the parent protein. This protein fragment represents the TP domain. The GT domain (Ser 37 to Lys 263) was expressed as a recombinant GST fusion protein. Protection by moenomycin of the GT domain against trypsin degradation was interpreted as an interaction between the GT domain and the moenomycin.The synthesis of the bacterial cell wall requires cytoplasmic and periplasmic enzymes. The final steps of peptidoglycan biosynthesis occur outside the cytoplasmic membrane, and they are catalyzed by membrane-bound penicillin-binding proteins (PBPs). PBPs play essential roles in cell division and morphology (6, 20, 31). Based upon their molecular sizes and amino acid sequence similarities, PBPs can be classified into two groups (6): low-molecular-weight (low-M_r) PBPs, which act as d,d-carboxypeptidases, and high-molecular-weight (high-M_r) PBPs, which carry transpeptidase (TP) and glycosyltransferase (GT) activities. The high-M_r group can be further divided into bifunctional enzymes with TP and GT activities (class A) and monofunctional TP enzymes (class B).β-Lactam antibiotics bind with high affinity specifically to d,d-carboxypeptidase and TP domains because of their structural similarity to the natural substrates, the stem peptides. This binding results in the formation of a covalent acyl-PBP enzyme complex, leading to the inactivation of PBPs.High-M_r PBPs are multidomain proteins (6). The three-dimensional structure of Streptococcus pneumoniae PBP 2x (class B high-M_r PBP) illustrates this domain organization (25). The only non-penicillin-binding domain of known function is the GT domain, corresponding to the N-terminal region of class A PBPs. This GT activity, clearly identified in Escherichia coli PBP 1b, is difficult to measure (23, 29, 31–35). It is insensitive to penicillin but sensitive to moenomycin, an antibiotic which is not used for human therapy (23, 29, 32, 33).S. pneumoniae is one of the major human pathogens of the upper respiratory tract, causing pneumonia, meningitis, and ear infections. Six PBPs have been identified in S. pneumoniae: high-M_r PBPs 1a, 1b, 2a, 2x, and 2b and low-M_r PBP 3 (8). PBPs 1a, 1b, and 2a belong to class A, while PBPs 2x and 2b are monofunctional class B proteins. Deletion of pbp2x and pbp2b in S. pneumoniae is lethal for the bacteria, while the deletion of pbp1a is tolerated (11), probably due to compensation by PBP 1b. This has been observed for E. coli class A PBP 1a, whose deletion can be compensated for by PBP 1b (36). In clinical isolates of resistant pneumococci, pbp1a, pbp2x, and pbp2b genes were shown to present a mosaic organization, encoding PBPs with reduced affinity for β-lactam antibiotics (2, 5, 15, 18). The specific resistance to ceftriaxone and cefotaxime of S. pneumoniae from the hospital environment is mediated by modification of PBP 2x and PBP 1a (22). Furthermore, gene transfer of pbp1a, pbp2x, and pbp2b from resistant strains conferred penicillin resistance on sensitive S. pneumoniae strains under laboratory conditions (2–4, 14, 15, 27, 30).The effort to overcome resistance to antibiotics in S. pneumoniae might therefore benefit from a detailed understanding of the molecular basis of TP and GT activities. The GT domain represents a new potential target for novel nonpenicillin antibiotics. Here, we delineate the GT and TP domains of S. pneumoniae PBP 1a* (a water-soluble form of PBP 1a) by limited proteolytic digestion and expression of recombinant domains. The TP activity of PBP 1a* and that of the isolated TP domain were compared. We also present evidence for an interaction between the isolated GT domain and moenomycin.     

Secoiridoid glucosides from Lonicera periclymenum

The stems of Lonicera periclymenum have been investigated for secoiridoid glycosides. In addition to two well-known glucosides, secologanin and morroniside, two rare secoiridoids, secoxyloganin and secologanoside, have been isolated and characterized by chemical and spectroscopic means. Secologanoside has been isolated for the first time as a genuine, non-derivatized compound.     

Brownian motion of the wormlike chain and segmental diffusion of DNA

The dynamics of the wormlike chain model for a polymer in solution is investigated in the case of free torsional and no longitudinal variations. A Langevin equation is derived and solved for circularly closed chains, neglecting hydrodynamic interactions. The local diffusion behavior of particular segments is described, and it is found that the mean-square displacements are proportional to t^3/4 at short times. Also, the equilibrium correlation functions for the closed chain are derived from the dynamic model in both the discrete and wormlike cases.     

Influence of Male Songs on Female Mating Behavior in the Grasshopper Chorthippus biguttulus (Orthoptera: Acrididae)

In males of the grasshopper Chorthippus biguttulus the relationship between song and mating success was investigated in three isolated field populations of individually marked animals within their natural habitat. In a population with muted males (forewings removed), females mated on average 1.7 days later than in a population with intact males. But approximately 14 days after the imaginal moult, roughly corresponding with the time of the first oviposition, 100% of females in both populations had mated. In a further test population, females with a choice between singing and mute males mated almost exclusively (16 from 17 observed copulations) with the intact, singing males. The chance encounter frequency of a male and female was equally high for all populations (on average one encounter every 1.2 h). Different encounter probabilities cannot therefore have caused either mating delay in the population with muted males or preferential selection of intact partners.