Investigation of the Accompaniment of Calcium During Active Calcium Transport from Human Erythrocyte Ghosts

To determine whether a cell metabolite was involved in active calcium transport, the cell contents of human erythrocytes were subjected to high dilutions and the resultant ghosts were checked for their ability to actively transport calcium. It was found that the diluted erythrocyte ghosts did retain their capacity to actively transport calcium and that the characteristics of this transport process appeared to be unaltered by the high dilutions. Calcium analysis of the cell membrane and cell supernatant indicated that almost all of the calcium was lost from the cell solution rather than the cell membrane as active calcium transport proceeded. Therefore it appeared that calcium was able to cross the cell membrane without the aid of a cell metabolite. Investigations with layered erythrocytes indicated that the active transport of calcium was not assisted by centrifugation. Neither inorganic phosphate, pyrophosphate, nor an adenine nucleotide appeared to accompany calcium across the membrane as indicated by total phosphate and inorganic phosphate analysis and 260-nm readings of the deproteinized supernatant.     

Shoulder muscle load and muscle fatigue among industrial sewing-machine operators

Physiological responses to physical work were assessed for 29 female industrial sewing-machine operators during an 8-h working day under ordinary working conditions. During sewing-machine work, the average (left and right) static load in the trapezius muscle was 9% of the maximal electromyogram (EMG) amplitude (% EMG_max), while the average mean load was 15% EMG_max, and the average peak load was 23% EMG_max. The static load level was unrelated to the muscle strength of the sewing-machine operators, which for the group as a whole was within the normal range. The load levels remained unchanged during the working day, while changes in the EMG mean power frequency and zero crossing frequency rate occurred, both indicating the development of muscle fatigue in left and right trapezius muscle during the working day. In line with this, the rating of perceived exertion in the shoulder and neck region increased during. the working day. Dividing the group of sewing-machine operators into two groups, those with the highest frequency and those with the lowest frequency of shoulder/neck troubles showed that the former group had significantly lower muscle strength, despite the fact that no differences in the surface EMG during sewing were found between the two groups. It was concluded that industrial sewing-machine work involves a pattern of shoulder muscle activity which induces fatiguing processes in the shoulder and neck regions. Furthermore, since the static shoulder muscle load was independent of muscle strength, factors other than working posture may be of significance for the static shoulder muscle load.     

Characterization of 4-Hydroxyphenylacetate 3-Hydroxylase (HpaB) of Escherichia coli as a Reduced Flavin Adenine Dinucleotide-Utilizing Monooxygenase

4-Hydroxyphenylacetate 3-hydroxylase (HpaB and HpaC) of Escherichia coli W has been reported as a two-component flavin adenine dinucleotide (FAD)-dependent monooxygenase that attacks a broad spectrum of phenolic compounds. However, the function of each component in catalysis is unclear. The large component (HpaB) was demonstrated here to be a reduced FAD (FADH₂)-utilizing monooxygenase. When an E. coli flavin reductase (Fre) having no apparent homology with HpaC was used to generate FADH₂ in vitro, HpaB was able to use FADH₂ and O₂ for the oxidation of 4-hydroxyphenylacetate. HpaB also used chemically produced FADH₂ for 4-hydroxyphenylacetate oxidation, further demonstrating that HpaB is an FADH₂-utilizing monooxygenase. FADH₂ generated by Fre was rapidly oxidized by O₂ to form H₂O₂ in the absence of HpaB. When HpaB was included in the reaction mixture without 4-hydroxyphenylacetate, HpaB bound FADH₂ and transitorily protected it from rapid autoxidation by O₂. When 4-hydroxyphenylacetate was also present, HpaB effectively competed with O₂ for FADH₂ utilization, leading to 4-hydroxyphenylacetate oxidation. With sufficient amounts of HpaB in the reaction mixture, FADH₂ produced by Fre was mainly used by HpaB for the oxidation of 4-hydroxyphenylacetate. At low HpaB concentrations, most FADH₂ was autoxidized by O₂, causing uncoupling. However, the coupling of the two enzymes' activities was increased by lowering FAD concentrations in the reaction mixture. A database search revealed that HpaB had sequence similarities to several proteins and gene products involved in biosynthesis and biodegradation in both bacteria and archaea. This is the first report of an FADH₂-utilizing monooxygenase that uses FADH₂ as a substrate rather than as a cofactor.     

Transport of neutral, cationic and anionic amino acids by systems B, b, XAG, and ASC in swine small intestine

Amino acid influx across the brush border membrane of the intact pig ileal epithelium was studied. It was examine whether in addition to system B, systems ASC and b^o,+ were involved in transport of bipolar amino acids. The kinetics of interactions between lysine and leucine demonstrates that system b^o,+ is present and accessible also to -glutamine. -aspartate (K_1/2 0.3 mM) and -glutamate (K_i 0.5 mM) share a high affinity transporter with a maximum rate of 1.3 μmol cm⁻² h⁻¹, while only -glutamate with a K_1/2 of 14.4 mM uses a low affinity transporter with a maximum rate of 2.7 μmol cm⁻² h⁻¹, system ASC, against which serine has a K_i of 1.6 mM. In the presence of 100 mM lysine, -glutamine (A), leucine (B), and methionine (C) fulfilled the criteria of the ABC test for transport by one and the same transporter. However, serine inhibits not only transport of -glutamate but also of glutamine (K_i 0.5 mM), and -glutamate inhibits part of the transport of glutamine. The test does, therefore, only indicate that the three bipolar amino acids have similar affinities for transport by systems B and ASC. Further study of the function of system B must be carried out under full inhibition by lysine and glutamate.     

Steady-state sodium absorption and chloride secretion of colon and coprodeum,and plasma levels of osmoregulatory hormones in hens in relation to sodium intake

Summary The plasma levels of four osmoregulatory hormones and their target ion-transport systems in the lower intestines of the domestic fowl were determined in order to elucidate their interrelationship and their setpoints in relation to NaCl intake. White Plymouth Rock hens were adapted to six intake levels of NaCl (0.20±0.02–24.7±1.9 mmoles Na⁺·kg bw^–1·day^–1) for 6 weeks. The Na⁺ absorption and the Cl^– secretion of colon and coprodeum were characterized in vitro by the effects of hexoses, amino acids, amiloride, and theophylline on the short-circuit current (SCC) and electrical potential difference (PD). The NaCl-conserving system of the adult chicken is set at low intake levels of NaCl as the 80% range (quantitized by non-linear, logistic regression analyses) of the change in the plasma [ALDO], the amiloride-inhibitable Na⁺ absorption of coprodeum and colon ( SCC), occurred from 0.18 to 2.3, from 0.9 to 4.3, and from 1.2 to 7.3 mmoles Na⁺·kg bw^–1·day^–1, respectively. These results demonstrate that the amiloride-inhibitable Na⁺ absorption of coprodcum is more closely linked to plasma [ALDO] than that of colon. The aminoacid-Na⁺ coabsorption of colon increased over exactly the same range of Na⁺ intake as the colonic amiloride-inhibitable Na⁺ absorption decreased, whereas the hexose-Na⁺ coabsorption increased at higher levels of Na⁺ intake, from 2 to 11 mmoles Na⁺·kg bw^–1·day^–1. Both these Na⁺ absorption types had reached their maximums at 24.7 mmoles Na⁺·kg bw^–1·day^–1, whereas the plasma [AVT] and plasma [PRL], although significantly increased, apparently had not; their 80% range of change occurred from 9.9 to 99 mmoles Na⁺·kg bw^–1·day^–1, and the main changes in plasma osmolity were predicted to occur from 5.4 to 107 mmoles Na⁺·kg bw^–1·day^–1. These results suggest that these colonic and hormonal variables conserve osmotically-free water and operate at high NaCl intake. The theophylline-induced colonic Cl^– secretion did not change with NaCl intake, whereas the stimulation of SCC in coprodeum decreased with increasing NaCl intake: The main change occurred between 0 and 3.2 mmoles Na⁺·kg bw^–1·day^–1. Thus, all ion-transport capacity disappears in coprodeum with increased dietary NaCl intake, whereas colon maintains its ion-transport capacity (although the nature of the Na⁺ transport changes). It is suggested that hormones defending the extracellular volume and composition are regulated close to zero input and output of both NaCl and water, regardless of whether they are NaCl conserving or free-water conserving. Therefore, changes in their stable plasma concentrations occur at the extremes of tolerable range of NaCl intake.Abbreviation AA aminoacids - ALDO aldosterone - AMI amiloride - AVT arginine vasotocin - bw body weight - CS corticosterone - HEX hexoses - INDO indomethacin - PD potential difference - PRL prolactin - R resistance - SCC short-circuit current - SD standard deviation - SEM standard error of mean - THEO theophylline     

Cloning of Choriocarcinoma Cells Shows That Invasion Correlates with Expression and Activation of Gelatinase A

Implantation and placental development are dependent upon trophoblast invasion of the endometrium. While the villous trophoblast does not display invasive behavior, the extravillous cytotrophoblast is highly invasive. By cloning BeWo choriocarcinoma cells, we have isolated two distinct clones that share similarities with villous and extravillous cytotrophoblasts. When cultured at the surface of a type I collagen gel, BeWo MC-1 cells were not invasive, whereas BeWo MC-2 cells rapidly invaded this matrix. When injected subcutaneously in nude mice, BeWo MC-1 cells developed a localized tumor and BeWo MC-2 cells developed larger tumors with micrometastases. Gelatinase A expression and minute amounts of gelatinase B were detected in the parental cell line and in both clones. However, the parental and the BeWo MC-2 cells secreted 5- to 10-fold more gelatinase A than the BeWo MC-1 cells. Laminin and matrigel stimulated the production of gelatinase A in BeWo MC-2 cells. Type I collagen promoted the conversion of the 72-kDa progelatinase A in an active enzyme only in parental BeWo and in BeWo MC-2 cells. These clones provide an interesting model for studying the complex mechanisms regulating implantation as well as the controlled invasiveness during implantation compared to tumor invasion.     

Treatment of Helicobacter pylori Infection: A Review of the World Literature

Background. None of the currently used anti- Helicobacter pylori drug regimens cures the infection 100%, and cure results still vary considerably. The present article reviews the effectiveness of currently used antimicrobial regimens, aimed to cure H. pylori infection.
Methods. Data collection started from the beginning of the anti- H. pylori -therapy era until May 1995. No attempt at formal metanalysis has been made, because many studies have been published only in abstract form. Attempts were made to exclude duplicates of studies by comparison to previously reported ones; the authors of suspected duplicates were contacted. After amalgamation of the number of included patients and the number of successfully treated patients, the mean values of eradication rates and the 95% confidence intervals were calculated.
Results. A total of 237 treatment arms were analyzed. Bismuth triple therapy continues to reach high eradication rates worldwide (78–89%). Side effects leading to diminished patient compliance and the marked decline of eradication efficacy in cases of metronidazole resistance are considered to be the major drawbacks of this therapy. Proton pump inhibitor (PPI) dual therapy is better tolerated with fewer side effects than is bismuth triple therapy. The mean eradication rates vary from 55 to 75%, and the extremes lie between 24 and 93%. PPI triple therapies have been shown to be very effective against H. pylori (eradication rates, 80–89%). Quadruple therapy leads to a mean eradication rate of 96%.
Conclusion. Based on efficacy, PPI triple or bismuth triple therapy are recommended as first-line treatment for H. pylori infection. Quadruple therapy could serve as second-line treatment for eradication of initial failures and in case of metronidazole resistance.     

Differential Roles of the Pseudomonas aeruginosa PA14 rpoN Gene in Pathogenicity in Plants, Nematodes, Insects, and Mice

We cloned the rpoN (ntrA, glnF) gene encoding the alternate sigma factor sigma(54) from the opportunistic multihost pathogen Pseudomonas aeruginosa strain PA14. A marker exchange protocol was used to construct the PA14 rpoN insertional mutation rpoN::Gen(r). PA14 rpoN::Gen(r) synthesized reduced levels of pyocyanin and displayed a variety of phenotypes typical of rpoN mutants, including a lack of motility and the failure to grow on nitrate, glutamate, or histidine as the sole nitrogen source. Compared to wild-type PA14, rpoN::Gen(r) was ca. 100-fold less virulent in a mouse thermal injury model and was significantly impaired in its ability to kill the nematode Caenorhabditis elegans. In an Arabidopsis thaliana leaf infectivity assay, although rpoN::Gen(r) exhibited significantly reduced attachment to trichomes, stomata, and the epidermal cell surface, did not attach perpendicularly to or perforate mesophyll cell walls, and proliferated less rapidly in Arabidopsis leaves, it nevertheless elicited similar disease symptoms to wild-type P. aeruginosa PA14 at later stages of infection. rpoN::Gen(r) was not impaired in virulence in a Galleria mellonella (greater wax moth) pathogenicity model. These data indicate that rpoN does not regulate the expression of any genes that encode virulence factors universally required for P. aeruginosa pathogenicity in diverse hosts.     

Elimination of a bacterial pore-forming toxin by sequential endocytosis and exocytosis

Staphylococcus aureus α-toxin is the archetype of bacterial pore forming toxins and a key virulence factor secreted by the majority of clinical isolates of S. aureus. Toxin monomers bind to target cells and oligomerize to form small β-barrel pores in the plasma membrane. Many nucleated cells are able to repair a limited number of lesions by unknown, calcium-independent mechanisms. Here we show that cells can internalize α-toxin, that uptake is essential for cellular survival, and that pore-complexes are not proteolytically degraded, but returned to the extracellular milieu in the context of exosome-like structures, which we term toxosomes.     

Binding characteristics of the Lactobacillus brevis ATCC 8287 surface layer to extracellular matrix proteins

Self-assembling proteins that form crystalline surface layers on many microorganisms can be involved in bacterial-host adhesion via specific interactions with components of the extracellular matrix. Here, we describe the interaction of the Lactobacillus brevis ATCC 8287 surface-layer protein SlpA with fibronectin, laminin, fibrinogen and collagen using surface plasmon resonance. SlpA was found to interact with high affinity to fibronectin and laminin, with a respective binding constant of 89.8 and 26.7 nM. The interaction of SlpA with collagen and fibrinogen was found to be of much lower affinity, with respective binding constants of 31.8 and 26.1 microM. The serine protease inhibitor benzamidine greatly reduced the affinity of SlpA for fibronectin, whereas the affinity for laminin remained unaffected. No protease activity of the purified SlpA protein could be detected. These data suggest that L. brevis may interact with host cells directly through high affinity interactions with laminin and fibronectin predominantly, involving distinct regions of the SlpA protein.