Target cell specificity of a bacteriocin molecule: a C-terminal signal directs lysostaphin to the cell wall of Staphylococcus aureus.

Microbial organisms secrete antibiotics that cause the selective destruction of specific target cells. Although the mode of action is known for many antibiotics, the mechanisms by which these molecules are directed specifically to their target cells hitherto have not been described. Staphylococcus simulans secretes lysostaphin, a bacteriolytic enzyme that cleaves staphylococcal peptidoglycans in general but that is directed specifically to Staphylococcus aureus target cells. The sequence element sufficient for the binding of the bacteriocin as well as of hybrid indicator proteins to the cell wall of S.aureus consisted of 92 C-terminal lysostaphin residues. Targeting to the cell wall of S.aureus occurred either when the hybrid indicator molecules were added externally to the bacteria or when they were synthesized and exported from their cytoplasm by an N-terminal leader peptide. A lysostaphin molecule lacking the C-terminal targeting signal was enzymatically active but had lost its ability to distinguish between S.aureus and S.simulans cells, indicating that this domain functions to confer target cell specificity to the bacteriolytic molecule.     

Ein Beitrag zur Nomenklatur sphinctozoider Schwämme (Porifera)

The sphinctozoid sponge generaFania Senowbari-Daryan 1990 andSpica Termier &Termier 1977 are preoccupied.Fania is replaced byFanthalamia nom. nov. andSpica by the younger synonymFistulispongia Termier &Termier 1977. The invalid subfamily name FaniinaeSenowbari-Daryan 1990 is replaced by Fanthalamiinae n. subfam. The invalid family and subfamily names SpicidaeTermier &Termier 1977 and SpicinaeSenowbari-Daryan 1990 respectively are replaced by FistulispongiidaeTermier atTermier 1977 and FistulispongiinaeSenowbari-Daryan 1990. The generaWaagenium de Laubenfels 1957 andCatubria Merla 1931 were previously overlooked.Waagenium DeLaubenfels 1957 is a younger synonym ofColospongia Laube 1865. The position ofCatubria Merla 1931 is uncertain. Most probablyCatubria is an alga.     

Automated monitoring of cell concentration and viability using an image analysis system

In order to automate measurements of cell concentration and viability in a suspended animal cell culture, we have developed anin situ microscopic image analysis system with an effective cell recognition algorithm. With a small amount of sample, this system can measure the cell density rapidly and aseptically. In addition, it can measure a cell size histogram including cell debris small particle distribution. These small particles have been found to be related to the viability of the mouse-mouse hybridoma STK1 cell line. By using cell debris small particle density as an indicator of cell viability, the developed system provides non-destructive viability monitoring without trypan blue staining.     

Determinants of the quantity of the stable SecY complex in the Escherichia coli cell.

While SecY in wild-type Escherichia coli cells is stable and is complexed with other proteins within the membrane, moderately overexpressed and presumably uncomplexed SecY was degraded with a half-life of 2 min. The fact that the amount of stable SecY is strictly regulated by the degradation of excess SecY was demonstrated by competitive entry of the SecY+ protein and a SecY-LacZ alpha fusion protein into the stable pool. Simultaneous overexpression of SecE led to complete stabilization of excess SecY. Overproduced SecD and SecF did not affect the stability of SecY, but plasmids carrying ORF12 located within the secD-secF operon partially stabilized this protein. In contrast, mutational reduction of the SecE content (but not the ORF12 content) led to the appearance of two populations of newly synthesized SecY molecules, one that was immediately degraded and one that was completely stable. Thus, the E. coli cell is equipped with a system that eliminates SecY unless it is complexed with SecE, a limiting partner of SecY. Our observations implied that in wild-type cells, SecY and SecE rapidly associate with each other and remain complexed.     

Phloretin as an Antagonist of Prostaglandin F2α Receptor in Cultured Rat Astrocytes

Abstract: The effect of phloretin on prostaglandin (PG) F_2α-induced phosphoinositide hydrolysis and elevation of intracellular Ca²⁺ concentration was examined in cultured rat astrocytes. Phloretin inhibited PGF_2α (1 μ M )-induced phosphoinositide hydrolysis in a concentration-dependent manner with an IC₅₀ value of 16 μ M . The inhibitory action of phloretin was specific for PGs. The addition of increasing concentrations of phloretin caused progressive shifts of the dose-response curves of PGF_2α to the right. In digitoninpermeabilized astrocytes, phloretin (100 μ M ) inhibited the stimulation induced by PGF_2α (1 μ M ) plus GTPγS (50 μ M ) without affecting that induced by GTPγS alone. PGF_2α at 1 μ M transiently increased astrocytic intracellular Ca²⁺ concentration in 39% of the cells tested. The response was completely blocked by 100 μ M phloretin and the calcium response recovered again after washing out phloretin. These results suggest that phloretin is an antagonist of PGF_2α receptor linked to phospholipase C in astrocytes.     

Recruitment of epidermal growth factor and transferrin receptors into coated pits in vitro: differing biochemical requirements.

The biochemical requirements for epidermal growth factor (EGF) and transferrin receptor-mediated endocytosis were compared using perforated human A431 cells. Morphological studies showed that horseradish peroxidase (HRP)-conjugated EGF and gold-labeled antitransferrin (Tfn) receptor antibodies were colocalized during endocytosis in vitro. The sequestration of both ligands into deeply invaginated coated pits required ATP hydrolysis and cytosolic factors and was inhibited by GTP gamma S, indicating mechanistic similarities. Importantly, several differences in the biochemical requirements for sequestration of EGF and Tfn were also detected. These included differing requirements for soluble AP (clathrin assembly protein) complexes, differing cytosolic requirements, and differing sensitivities to the tyrosine kinase inhibitor, genistein. The biochemical differences detected between EGF and Tfn sequestration most likely reflect specific requirements for the recruitment of EGF-receptors (R) into coated pits. This assay provides a novel means to identify the molecular bases for these biochemical distinctions and to elucidate the mechanisms involved in ligand-induced recruitment of EGF-R into coated pits.     

Nitroprusside and Cyclic GMP Stimulate Na+-Ca2+ Exchange Activity in Neuronal Preparations and Cultured Rat Astrocytes

Abstract: The effects of nitric oxide (NO)-generating agents on ⁴⁵Ca²⁺ uptake in rat brain slices and cultured rat astrocytes were studied in the presence of monensin, which is considered to drive the Na⁺-Ca²⁺ exchanger in the reverse mode. Sodium nitroprusside (SNP) at >10 µ M increased monensin-stimulated Ca²⁺ uptake in the slices, although it did not affect high K⁺-stimulated Ca²⁺ uptake. Another NO donor, 3-morpholinosydnonimine, was effective. The effect of SNP was antagonized by hemoglobin (50 µ M ), a NO scavenger, and mimicked by 8-bromo-cyclic GMP (100 µ M ). In rat brain synaptosomes, SNP increased monensin-stimulated Ca²⁺ uptake, but it did not affect high K⁺-stimulated Ca²⁺ uptake. 8-Bromocyclic GMP, but not SNP, increased Na⁺-dependent Ca²⁺ uptake significantly in synaptic membrane vesicles in the absence of monensin. In cultured rat astrocytes, SNP and 8-bromo-cyclic GMP increased Ca²⁺ uptake in the presence of ouabain and monensin, which were required for the Ca²⁺ uptake in the cells. These findings suggest that NO stimulates the Na⁺-Ca²⁺ exchanger in neuronal preparations and astrocytes in a cyclic GMP-dependent mechanism.     

Does Paramecium sense gravity?

In order to get an insight into the cellular mechanisms for the integration of the effects of gravity, we investigated the gravitactic behaviour in Paramecium. There are two main categories for the model of the mechanism of gravitaxis; one is derived on the basis of the mechanistic properties of the cell (physical model) and the other of the physiological properties including cellular gravireception (physiological model). In this review article, we criticized the physical models and introduced a new physiological model. Physical models postulated so far can be divided into two; one explaining the negative gravitactic orientation of the cell in terms of the static torque generated by the structural properties of the cell (gravity-buoyancy model by Verworn, 1889 and drag-gravity model by Roberts, 1970), and the other explaining it in terms of the dynamic torque generated by the helical swimming of the cell (propulsion-gravity model by Winet and Jahn, 1974 and lifting-force model by Nowakowska and Grebecki, 1977). Among those we excluded the possibility of dynamic-torque models because of their incorrect theoretical assumptions. According to the passive orientation of Ni(2+)-immobilized cells, the physical effect of the static torque should be inevitable for the gravitactic orientation. Downward orientation of the immobilized cells in the course of floating up in the hyper-density medium demonstrated the gravitactic orientation is not resulted by the nonuniform distribution of cellular mass (gravity-buoyancy model) but by the fore-aft asymmetry of the cell (drag-gravity model). A new model explaining the gravitactic behaviour is derived on the basis of the cellular gravity sensation through mechanoreceptor channels of the cell membrane. Paramecium is known to have depolarizing receptor channels in the anterior and hyperpolarizing receptors in the posterior of the cell. The uneven distribution of the receptor may lead to the bidirectional changes of the membrane potential by the selective deformation of the anterior and posterior cell membrane responding to the orientation of the cell in the gravity field; i.e. negative- and positive-going shift of the potential due to the upward and downward orientation, respectively. The orientation dependent changes in membrane potential with respect to gravity, in combination with the close coupling of the membrane potential and the ciliary locomotor activity, may allow the changes in swimming direction along with those in the helical nature of the swimming path; upward shift of axis of helix by decreasing the pitch angle due to hyperpolarization in the upward-orienting cell, and also the upward shift by increasing the pitch angle due to depolarization in the downward-orienting cell. Computer simulation of the model demonstrated that the cell can swim upward along the "super-helical" trajectory consisting of a small helix winding helically an axis parallel to the gravity vector, after which the model was named as "Super-helix model". Three-dimensional recording of the trajectories of the swimming cells demonstrated that about a quarter of the cell population drew super-helical trajectory under the unbounded, thermal convection-free conditions. In addition, quantitative analysis of the orientation rate of the swimming cell indicated that gravity-dependent orientation of the swimming trajectory could not be explained solely by the physical static torque but complementarily by the physiological mechanism as proposed in the super-helix model.