Comparison of the Antimicrobial Adhesion Potential of Human Body Fluid Glycoconjugates Using Fucose-Binding Lectin (PA-IIL) of Pseudomonas aeruginosa and Ulex europaeus Lectin (UEA-I)

Pseudomonas aeruginosa produces a fucose-binding lectin (PA-IIL) which strongly binds to human cells. This lectin was shown to be highly sensitive to inhibition by fucose-bearing human milk glycoproteins. Since the glycans of these glycoproteins mimic human cell receptors, they may function as decoys in blocking lectin-dependent pathogen adhesion to the host cells. Human saliva and seminal fluid also contain such compounds, and body fluids of individuals who are “secretors” express additional fucosylated (alpha 1,2) residues. The latter are selectively detected by Ulex europaeus lectin UEA-I. The aim of the present research was to compare the PA-IIL and UEA-I interactions with human salivas and seminal fluids of “secretors” and “nonsecretors” with those obtained with the respective milks. Using hemagglutination inhibition and Western blot analyses, we showed that PA-IIL interactions with the saliva and seminal fluid glycoproteins were somewhat weaker than those obtained with the milk and that “nonsecretor” body fluids were not less efficient than those of “secretors” in PA-IIL blocking. UEA-I, which interacted only with the “secretors” glycoproteins, was most sensitive to those of the seminal fluids.     

Potential Toxicological Problems Associated with Antioxidants in Plastics and Rubber Consumables

Antioxidants and stabilisers are of critical importance in the manufacture and use of polymer artifacts. Most conventional additives are readily leached from plastics or rubbers by foodstuffs or body fluids with consequent danger associated with the potential toxicity in the body. Strategy for the development of biologically “safe” stabilisation systems for polymers are discussed and it is concluded that the attachment of the appropriate reactive functional group to the polymer backbone by “reactive processing” provides the best means of immobilising the functional group to leaching fluids without sacrificing its protective action.     

The development of a “microtitre” fluctuation test for the detection of indirect mutagens, and its use in the evaluation of mixed enzyme induction of the liver

The “Microtitre” Fluctuation test recently introduced for the detection of direct mutagens has been adapted for the detection of indirect mutagens through the incorporation of an “S9-mix” metabolic system. It compares favourably with Greens' original method for the detection of a range of chemical mutagens.

The technique has been employed in the evaluation of mixed enzyme induction using phenobarbitone and β-naphthoflavone (benzoflavone). as a safe substitute for Aroclor-1254. The post-mitochondrial preparations from rats induced with the combined inducers had a similar “metabolic competence” to those derived from Aroclor induced animals. Such a combination would therefore provide a useful alternative to Aroclor-1254 for routine screening.

It was found that the level of “S9” present in the metabolic system greatly affected the quantitative mutagenic response. This varied considerably from chemical to chemical and underlined the need for such preliminary investigations in routine screening.     

Glucuronic acid conjugates

The methods of assay in body fluids of 1-β-alkyl, 1-β-phenyl and 1-β-acyl glucuronic acids (“glucuronide conjugates”) have been reviewed. Most of the 78 references cited (from the literature of the period 1990–1997) concern the glucuronide conjugates of drug metabolites, and these have been considered, for reasons of accessibility, within sections of individual drug classes such as analgesics, anti-cancer agents and opioids. Other glucuronide conjugates are considered under “miscellaneous compounds”. A few gas chromatography and capillary electrophoresis methods are described, but the major technique of assay (62 citations) is reversed-phase high-performance liquid chromatography.     

Method for Treating and Processing Whole Chick Embryos for Autoradiography, Immunocytochemistry and other Techniques

A method is presented for In situ treatment of whole chick embryos with drugs and immunocytochemical and fixative reagents that resembles conditions “in ovo.” The chick embryo is placed in a “shell-less” culture system where it is contained by an agar ring allowing for treatment in vivo. The conceptus (embryo + membranes) is then mounted on a microporous membrane and inserted into a filter device connected to a three-way stopcock that permits fluids to be changed using syringes. The embryo is then processed in toto or after embedding and sectioning for light or electron microscopy. The proposed handling system decreases technical artifacts and changes in the topographic microanatomy produced by conventional manipulation of chick embryos. This method is useful also for directly observing and recording changes in the embryo during drug treatments and allows processing with dangerous reagents without their direct contact with the operator. It is simple, inexpensive and requires only minimal technical training.     

Therapeutic effects of PG201, an ethanol extract from herbs, through cartilage protection on collagenase-induced arthritis in rabbits

In order to assess the therapeutic effects of PG201 (an ethanol extract from herbs) on osteoarthritis, we investigated whether PG201 could suppress the disease progression of collagenase-induced arthritis (CNIA) in rabbits. The right knees of rabbits were injected intra-articularly with collagenase, and the rabbits were orally treated with distilled water (DW), PG201 (200 mg/kg) or diclofenac (DCF, 10 mg/kg) once a day for 8 weeks. Oral administration of PG201 significantly suppressed the stiffness and bone space narrowing. Cartilage erosion and GAG release (p<0.01) were considerably reduced in the knee joints. As well, the mRNA expression of matrix degradation enzymes including MMP-1, -3, and -13 was decreased. On the contrary, the concentrations of TIMP-2 in the synovial fluids were considerably amplified in the PG201 treated group (p<0.01), but not in the DCF treated group. The pathologic inflammatory molecules involved in cartilage destruction such as IL-1beta, PGE2, and NO were also diminished by PG201. Taken together, these results indicate that PG201 has therapeutic effects on CNIA through the prominent protection of cartilage. PG201 indeed has great potential as a form of treatment for osteoarthritis.     

Acetylation of Lysine 201 Inhibits the DNA-Binding Ability of PhoP to Regulate Salmonella Virulence

The two-component system PhoP-PhoQ is highly conserved in bacteria and regulates virulence in response to various signals for bacteria within the mammalian host. Here, we demonstrate that PhoP could be acetylated by Pat and deacetylated by deacetylase CobB enzymatically in vitro and in vivo in Salmonella Typhimurium. Specifically, the conserved lysine residue 201(K201) in winged helix–turn–helix motif at C-terminal DNA-binding domain of PhoP could be acetylated, and its acetylation level decreases dramatically when bacteria encounter low magnesium, acid stress or phagocytosis of macrophages. PhoP has a decreased acetylation and increased DNA-binding ability in the deletion mutant of pat. However, acetylation of K201 does not counteract PhoP phosphorylation, which is essential for PhoP activity. In addition, acetylation of K201 (mimicked by glutamine substitute) in S. Typhimurium causes significantly attenuated intestinal inflammation as well as systemic infection in mouse model, suggesting that deacetylation of PhoP K201 is essential for Salmonella pathogenesis. Therefore, we propose that the reversible acetylation of PhoP K201 may ensure Salmonella promptly respond to different stresses in host cells. These findings suggest that reversible lysine acetylation in the DNA-binding domain, as a novel regulatory mechanism of gene expression, is involved in bacterial virulence across microorganisms.     

Clinical blood values of the northern fur seal, Callorhinus ursinus. III. Comparison of eye fluid and serum values

The levels of 13 components in the serum and eye fluids of the northern fur seal, Callorhinus ursinus, are compared. The wide variations observed would appear to limit the usefulness of eye fluid values as a substitute for serum values.     

Improved Microscopic Detection of Bacteriuria

A simple method is described which permits the microscopic detection of bacteria in sediments of urine and other fluids, including bacteria that have eluded detection by conventional means. The method introduces increased centrifugal force and stepwise chemical fixation and then conventional staining. It is rapid, economical, and suitable for use in a physician's office. Use of this method immediately reveals those bacteria reported as “significant” by the conventional laboratory culture. More importantly, it immediately reveals the presence of bacteria, living or dead, which are missed by the conventional culture and by the conventional Gram staining procedure. These bacteria usually can be grown in special media and they appear to be related to systemic disease as evidenced by the clinical response to appropriate antibiotics.     

The importance of amniotic fluids for the establishment of maternal behaviour in experienced and inexperienced ewes

Maternal behaviour at parturition was studied in multiparous and primiparous ewes whose lambs had been washed to remove amniotic fluids from their coats. In multiparous ewes, washing of the newborn with soap or water decreased licking of the neonate but did not significantly affect other parameters of maternal behaviour (aggressive behaviour, maternal bleats, or acceptance at udder). By contrast, in primiparous ewes, washing of the neonate with only water dramatically reduced licking behaviour and acceptance at udder, while the incidence of aggressive behaviour towards the neonate was increased (P<0·05 in all cases). It is concluded that for inexperienced ewes amniotic fluid on the lamb's coat is necessary for the normal development of maternal behaviour at parturition. For experienced ewes amniotic fluids also play a facilitatory role, but other sensory information can substitute for this cue.     

Caractères et évolution des enzymes chitinolytiques chez les vertébrés infèrieurs

The activity of chitinases extracted from various organs of different fish, amphibians and reptiles was estimated as a function of pH by using “native” chitin as substrate. Three types of chitinase activity were recorded, suggesting the existence of three different chitinase types: Type 1: (optimum pH; 4.5, no activity at pH 1.0) was found in various organs, such as intestine, pyloric caeca, pancreas, liver, spleen, etc.); Type IIa: (optimum pH; 3.0, weak activity at pH 1.0) was obtained from the gastric mucosa of fish and one species of urodele; Type IIb: (optimum pH; 3.0, strong activity at pH 1.0) was found in the gastric mucosa of reptiles and batrachian anura. Chitinase activity appears to be adapted to the pH of the digestive fluids. A tentative scheme is presented of chitinase evolution among lower vertebrates.     

Polyester Sheeting (Melinex O), a Tissue-Culture Support Easily Separable from Epoxy Resins after Flat-Face Embedding

Pieces of Melinex O polyester films (0.003 in. thick) can be used as substitute for glass in flat-face embedding in epoxy resins for electron microscopy. Melinex sheeting can be obtained from Imperial Chemical Industries. It can be sterilized by alcohol or by autoclaving. It is transparent and it is resistant to customary cytological processing fluids. It separates readily from the resin block wihout the use of water and without cooling.     

Hyphenated liquid chromatographic techniques in forensic toxicology

The prerequisite of applicability of hyphenated methods in forensic analysis is the achievement of a stage of “final maturity”. In the field of liquid chromatography, HPLC coupled with diode array detection (DAD) seems to fulfill this criterion, whilst the combination with atmospheric pressure ionization mass spectrometry (HPLC–API-MS) is still in a development stage. HPLC–DAD is broadly used as identification tool in forensic and in emergency toxicology. Two main approaches were observed; development of retention index scales for intra-laboratory exchange of data and establishing of databases only for intra-laboratory use. Using these approaches, several databases were established for toxicological relevant substances (illicit and therapeutic drugs and their metabolites, environmental poisons etc.) in biological fluids. Also, complete HPLC–DAD identification systems are commercially available. Further possibility of progress depends on the on-line combination (“triple hyphenation”) with other detection methods, preferably API-MS. HPLC–API-MS, both in electrospray (ESI) and atmospheric pressure chemical ionization (APCI) options, underwent dramatic development in the last decade and is reaching its final shape. The method was broadly applied for various groups of toxicologically relevant substances, a lot of them unaccessible for other techniques, including GC–MS. Particularly important was application of HPLC–API-MS for detection and quantitation of active, polar metabolites of various drugs and for analysis of macromolecules. APCI seems to be more useful for analysis of less polar compounds, whereas ESI is particularly valuable for determination of polar, large molecules (e.g., toxic peptides, polar metabolites etc.) Up to now, HPLC–API-MS has been mainly applied for dedicated analyses, but the introduction of APCI or ESI in systematic toxicological screening may be expected in the near future.     

The Application of Dyes in the Cancer Problem

Three fundamental requirements for the problem of developing a differential stain for cancer are discussed: I. the choice of a technic for the microscopic preparation of tissues; II. an analysis of the biological properties peculiar to cancer; and III. various groups of dyes adaptable to such peculiar properties of cancer tissue. Under I the disadvantages of intravitam staining are pointed out and the use of cell suspensions, frozen sections, and fixed material favored. Under II three characteristics of cancer tissue offering possibilities for differential staining are discussed, the cytological structure known as the “plastin reaction”, the histogenic cycle of cancer tissue, and the viability of cancer tissue under anaerobic conditions. Under III modifications of the Giemsa stain are suggested for application to the plastin reaction, specific tissue stains advocated for the use of indicating end points in histogenic cycles, and the vital dyes, congo red and trypan blue, suggested as indicators for the survival of malignant tissues because of the failure of these dyes to permeate living cancer cells.

The angle of approach thruout has been an attempt to avoid unconscious pitfalls inherent in certain microscopic technics, and to substitute analytical methods for the blind trial and error method of routinely applying dye after dye in endless succession.     

Control of hypocotyl elongation in Arabidopsis thaliana by photoreceptor interaction

In order to test the interaction of different phytochromes and blue-light receptors, etiolated seedlings of wild-type Arabidopsis thaliana (L.) Heynh., a phytochrome (phy) B-overexpressor line (ABO), and the photoreceptor mutants phyA-201, phyB-5, hy4-2.23n, fha-1, phyA-201/phyB-5, and phyA-201/hy4-2.23n were exposed to red and far-red light pulses after various preirradiations. The responsiveness to the inductive red pulses is primarily mediated by phyB which is rather stable in its far-red-absorbing form as demonstrated by a very slow loss of reversibility. Without preirradiation the red pulses had an impact on hypocotyl elongation only in PHYA mutants but not in the wild type. This indicates a suppression of phyB function by the presence of phyA. Preirradiation with either far-red or blue light resulted in an inhibition of hypocotyl elongation by red pulses in the wild type. Responsiveness amplification by far-red light is mediated by phyA and disappears slowly in the dark. The extent of responsiveness amplification by blue light was identical in the wild type and in the absence of phyA, or the cryptochromes cryl (hy4-2.23n) or cry2 (fha-1). Therefore, we conclude that stimulation of phyB by blue light preirradiation is either mediated by an additional still-unidentified blue-light-absorbing pigment or that phyA, cry1 and cry2 substitute for each other completely. Both blue and red preirradiation established responsiveness to red pulses in phyA-201/phyB-5 double mutants. These results demonstrate that inhibition of hypocotyl elongation by red pulses is not only mediated by phyB but also by a phytochrome(s) other than phyA and phyB. Received: 21 July 1998 / Accepted: 7 December 1998     

Interactive signal transfer between host and pathogen during successful infection of barley leaves by Blumeria graminis and Bipolaris sorokiniana

Using ion-selective microprobes, interactive signalling between barley and Blumeria graminis or Bipolaris sorokiniana has been investigated. The question was raised whether a biotrophically growing fungus manipulates the electrical driving forces (membrane potential, transmembrane pH), required for H⁺ cotransport of energy-rich compounds. Electrodes were positioned in the substomatal cavity of open stomata or on the leaf surface, and pH was measured continuously up to several days during fungal development. We demonstrate that surface and apoplastic fluids are electrically coupled and respond in a similar manner to stimuli. Apoplastic pH, monitored from the moment of inoculation with conidia, reveals several phases: 2–4 h after inoculation of the barley leaf with either fungus, the host displays rapid transient responses after its first contact with the fungal cell wall; apoplastic pH and pCa increases, cytoplasmic pH and pCa decreases. About 1 day after inoculation, the apoplastic pH increases by up to 2 pH units, which is thought to reflect a resistance response against the intruder. Whereas barley leaf cells possess a membrane potential of −152±5 mV, hyphae of B. graminis yield −251±8 mV, indicative of a substantial driving force advantage for the fungus. Although the resting membrane potential of barley remains constant during the first days after inoculation, leaves infected with B. sorokiniana get confronted with an energy problem, indicated by a retarded repolarization following a “light-off” stimulus. Five days after inoculation, apoplastic pH has increased to 5.97±0.47 (n=11) and does no longer respond to “light-off” when measured within lesions. In contrast, it stays at near normal values outside the lesions and responds to “light-off”.It is concluded that biotrophically growing fungi do not manipulate the cotransport driving forces since (i) any change in apoplastic pH would be experienced by both partners; (ii) the resting membrane potential is not changed. It is suggested that measured pH changes reflect defence responses of the host against the fungus rather than fungal action to increase compatibility.     

Effect of L-azetidine 2-carboxylic acid on growth and proline metabolism in Escherichia coli

The effects of L-azetidine 2-carboxylic acid on growth and proline metabolism in a proline-requiring auxotroph of Escherichia coli are described. The homologue inhibited growth of the wild type and it, alone, did not substitute effectively for proline as a growth supplement for the mutant. In medium containing 0.05 mM proline, the addition of increasing amounts of homologue progressively inhibited growth of the wild type but stimulated growth of the mutant at homologue: proline ratios of 10 : 1 and 50 : 1. This suggested that the homologue exerted a “sparing effect” on proline in the mutant.The incorporation of L-[U-¹⁴C]proline and L-[³H]azetidine 2-carboxylic acid into hot trichloroacetic acid-insoluble material in the mutant was measured. Amino acid analysis of the insoluble material from cells incubated with radiolabeled proline alone revealed that proline was partially degraded and metabolized to other amino acids prior to incorporation into protein. The addition of unlabeled homologue to the incubation medium significantly reduced proline catabolism, suggesting that the homologue exerted a sparing effect on proline in this mutant. In medium containing unlabeled proline and radiolabeled L-azetidine 2-carboxylic acid, the homologuewas incorporated both intact and partially degraded prior to incorporation into protein. Alanine was the major L-azetidine 2-carboxylic acid catabolite.     

Iron-Alum Aceto-Carmine Staining for Chromosomes and Other Anatomical Features of Rhodophyceae

The chromosomes, certain intracellular structures and gross anatomical details of many red algae, which, as a class, have proved technically difficult material, can be demonstrated by staining with aceto-carmine after a mordant bath of iron alum. Acetic-alcohol mixtures are used as nuclear fixatives and formalin-acetic-alcohol and other similar fluids for preservation of anatomical features. The tougher more cartilaginous thalli of some species can be softened, if squashes are desired, by prolonging fixation (24-48 hr.) in acetic alcohol and subsequent washing. The fixatives are washed out of the material before the latter is transferred to 0.5-5.0% ferric ammonium sulphate, the concentration of which may be altered according to the material. Excess mordant is removed by washing and the material stained in Belling's aceto-carmine containing a trace of ferric acetate as a “ripener”. The degree of heating before covering is critical as it controls the quality of the staining. Squashing must be very thorough to spread the chromosomes which are usually very small but only slight controlled pressure is necessary when diffuse structures such as carposporophytes, nemathecia or medullary filaments are being demonstrated. Paraffin sections mounted on slides can also be stained by this method.     

Specificity of the K-channels in labyrinthine organs: effects of Rb+ and Cs+ on the conversion process in frog semicircular canals

Summary The specificity of the K-channels which are present in the ciliated membrane of ampullar receptors has been investigated by replacing K⁺ in the fluids bathing the canal with Rb⁺ and Cs⁺. Results show that, unlike Cs⁺, Rb⁺ is able to substitute K⁺ in maintaining the receptor function. These findings favour the hypothesis that the transduction channels which allow the receptor current to flow across sensory cell bodies are specific K-channels. The effects of Rb⁺ and Cs⁺ on primary sensory neuron endings were also studied.Abbreviations Adc slow ampullar potentials - Ndc slow nerve potentials     

Can Insulated Skin Temperature Act as a Substitute for Rectal Temperature when Studying Circadian Rhythms?

We measured rectal, lateral chest wall, and axillary temperature every half hour for at least 24 h while subjects were living normal life-styles and keeping a sleep/activity diary. We then used a purification method to estimate the decrease of temperature due to sleep and the increases due to sitting, standing, walking, or exercising, as well as the parameters of the cosine curve that described the “purified data.” Cosinor analysis of raw and purified data showed that the acrophases from both skin sites were much more variable and up to 8 h later than were those from the rectum (particularly if exercise had been taken), even though the acrophases from the two skin sites were similar to each other. For rectal temperature, there was an increase in the size of the masking effect as activity progressed through the sequence: sitting, standing or walking, exercising. In contrast, for both chest wall and axillary temperatures, although sitting produced masking effects similar to those for rectal temperature, masking effects due to standing or walking and exercising were much smaller, and sometimes they were even less than the masking effects due to sitting. These results indicate that our measurements of cutaneous temperature did not act as a substitute for rectal temperature, particularly when the subject was physically active rather than sedentary.