COMPARISON OF THE MILLIPORE DIGITAL TOTAL COUNT SYSTEM AND STANDARD MEMBRANE FILTRATION PROCEDURE TO ENUMERATE MICROORGANISMS IN WATER SAMPLES FROM COSMETIC/PHARMACEUTICAL ENVIRONMENTS

A study was performed to compare the Millipore Digital Total Count System with a standard membrane filtration procedure in enumerating the number of microorganisms present in several types of water samples (e.g., Hot/Cold Deionized, Tap, and RO/Ultra Filtration). Water samples were collected over a 4 month period. Statistical data analysis demonstrated an overall correlation of greater than 82% between the two test methodologies. The linearity of the microbial counts between both test methods was compared by artificially contaminating sterile water samples with Pseudomonas aeruginosa. The linearity of the microbial counts between both methods was found to be greater than 96%. The Millipore Digital Total Count System was found to be comparable to the standard membrane filtration method in determining the number of microorganisms in a water sample. In conclusion, the Millipore Digital Total Count System was able to provide a 24 h enumeration of microorganisms present in a water sample. This rapid enumeration allows for a faster quality evaluation of water samples from an industrial water system that is used in the manufacture of cosmetic/pharmaceutical products.     

Phlorizin increases the permeability of intestinal mucosal membrane to sodium

We reported previously that when jejunal transmural glucose transport was inhibited by phlorizin the ratio of Na:glucose transport increased from 2.0:1 (in controls) to 3.3:1. To elucidate the mechanism of this increased ratio of Na:glucose transport, in the present study we have investigated the effect of phlorizin on Na uptake by brush border membrane vesicles and by everted sacs of hamster jejunum. In experiments on membrane vesicles the following observations were made. The time course of Na uptake showed that the control vesicles were in complete equilibrium with a Na-containing (100 mM) medium between 30 and 90 min incubation. In these periods of incubation, the vesicles incubated with phlorizin presumably also equilibrated with the medium, but lost their intravesicular Na during Millipore filtration and washing, and consequently the residual Na content was lower than that of controls. This effect of phlorizin was concentration dependent, and appeared to be unrelated to Na-coupled glucose transport, because it was also observed in the absence of glucose. This loss of Na during Millipore filtration and washing was also observed (i) when vesicles were equilibrated in a Na-containing solution in the absence of phlorizin and then exposed to a similar solution containing phlorizin, or (ii) when vesicles were equilibrated in a Na-containing solution in the presence of phlorizin and then washed repeatedly following Millipore filtration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Improvement of the preliminary classification of Corynebacterium diphtheriae v. gravis

In this study the previously published preliminary scheme for the subdivision of toxigenic and nontoxigenic Corynebacterium diphtheriae, classified with cultivar gravis, is made more precise. 3 groups remain in this scheme: I, II and III; each of them contains toxigenic C. diphtheriae (subgroup a) and nontoxigenic precursors of C. diphtheriae (subgroup b). For the first time nontoxigenic analogs of C. diphtheriae, phagovar OPQSTg, have been introduced into group I and newly discovered toxigenic C. diphtheriae, phagovar K, with their nontoxigenic precursors converted by phages 5 tox+, 6 tox+ and W tox+ have been introduced into group III. Group IV has been provisionally excluded from the scheme because this group comprises a small number of strains (3 strains). This classification can already be used in research practice for a finer differentiation of strains classified with cultivar gravis and for correct epidemic orientation.     

Study of the Effect of Different Techniques on Diversity of Freshwater Hyphomycetes in the River Nile (Upper Egypt)

Four different techniques were applied, lead mapping of Eucalptus rostrata; randomly leaf sampling; Millipore filtration; spores in foam, for the study of aquatic hyphomycets communities in the River Nile. Triscelophorus monlosporus, Anguilospora longissima, Flagellospora curvula and Tetracladium marchalianum were the dominant species in all the techniques used. Aquatic hyphomycetes diversity was high by using leaf mapping (23 species) and randomly leaf sampling (30 species) but it was low by using Millipore filtration (11 species) and spores in foam (8 species) techniques.     

A novel 111‐nucleotide duplication in the G gene of human metapneumovirus

In 2017, novel human metapneumovirus (HMPV) A2b subgroup strains with a 111‐nucleotide duplication in the G gene was detected by the present team. These strains were related to previously identified HMPV A2b strains with a 180‐nucleotide duplication; however, they appeared to be different strains, produced by an independent duplication event. The recent evolution of HMPV suggests that careful monitoring of this virus is required.

     

Nafamostat mesylate, a serine protease inhibitor, demonstrates novel antimicrobial properties and effectiveness in Chlamydia-induced arthritis

Introduction

Effective treatment of reactive arthritis would ideally achieve both control of inflammation and eradication of persisting arthritogenic pathogens. We use a model of experimental Chlamydia trachomatis-induced arthritis (CtIA) to evaluate the effectiveness of nafamostat mesilate (NM), a serine protease inhibitor with complement-modifying effects and anticoagulant properties. To date clinical use of NM has largely been in Asia and has been primarily confined to inflammatory states such as pancreatitis.

Methods

In vitro studies examined inhibition of Chlamydia proliferation using fibroblast cell lines as targets and phase contrast microscopy. In vivo studies used an established protocol, experimental CtIA, induced in Lewis rats by injection of synoviocyte-packaged C. trachomatis. NM was dissolved in water and administered by daily intraperitoneal injection at a dose of 10 mg/kg beginning the day prior to the administration of Chlamydia. Readouts in vivo included (i) joint swelling, (ii) histopathology scoring of severity of arthritis, (iii) host clearance of the pathogen (by ELISA, the IDEIA PCE Chlamydia).

Results

NM exerted a dose-dependent inhibition of chlamydial proliferation in vitro. Without NM, the mean number of inclusion bodies (IB) per well was 17,886 (± 1415). At 5 μg/mL NM, there were 8,490 (± 756) IB, at 25 μg/mL NM there were 35 IB and at 50 μg/mL NM no IB was observed. Chlamydial antigens in each well along the concentration gradient were assayed by ELISA, demonstrating that at 25 μg/mL NM inhibition of Chlamydia was almost complete. In the experimental arthritis model, joint swelling was significantly reduced with NM treatment: average joint width for the NM-treated animals was 8.55 mm (s.d. ± 0.6578, n = 10) versus 11.18 mm (s.d. ± 0.5672, n = 10) in controls (P < 0.001). Histopathology scoring indicated that NM resulted in a marked attenuation of the inflammatory infiltration and joint damage: mean pathology score in NM-treated animals was 10.9 (± 2.45, n = 11) versus 15.9 (± 1.45, n = 10) in controls (P < 0.0001). With respect to persistence of Chlamydia within the synovial tissues, NM treatment was accompanied by a reduction in the microbial load in the joint: mean optical density (O.D.) for ELISA with NM treatment was 0.05 (± 0.02, n = 4) versus 0.18 (± 0.05, n = 4) in controls (P < 0.001).

Conclusions

NM is a protease inhibitor not previously recognized to possess antimicrobial properties. The present study demonstrates for the first time that NM exerts significant impact on C. trachomatis-induced arthritis and suggests that such approaches may prove clinically useful in chronic reactive arthritis.     

Horizontal and vertical movement of Pseudomonas fluorescens toward exudate of Macrophomina phaseolina in soil: influence of motility and soil properties

The role of motility and cell surface hydrophobicity in transport and dispersal of Pseudomonas fluorescens strains LAM1-hydrophilic, LAM2-hydrophobic and LAM(NM) (non-motile mutant of LAM2) under different soil conditions was studied. Maximum adhesion was recorded for LAM2 in clay loam (70%), followed by sandy loam (68%) and sandy soil (40%). Vertical migration of P fluorescens isolates in soils was recorded at 5 and 25 cm flow of wafer or M. phaseolina exudate. In all the treatments, LAM1 exhibited maximum migration followed, by LAM2 and LAM(NM). The rate of migration of such isolates was lowered in water irrigated soils compared to those irrigated with M. phaseolina exudate. In sandy soil, cells of LAM1 migrated up to 13 cm in comparison to LAM2 (11 cm) and LAN(NM) (9 cm) at 5 cm flow of fungal exudate. Population of LAM1, LAM2 and LAM(NM) was 5.7, 5.68 and 5.61 log cfu g(-1) soil at 1 cm depth, but it decreased to 2.56, 2.21 and 1.99 log cfu during migration up to 11 cm in sandy soil at 5 cm flow of fungal exudate. Greater motility was observed in sandy soil irrigated with water or fungal exudate, followed by sandy loam and clay loam. In general, filtration coefficient (lambda) of P. fluorescens was higher in soils irrigated with 5 cm of water or exudate than with 25 cm of irrigation. The horizontal movement of P. fluorescens strains in sandy soil adjusted at different psi m showed marked reduction with decrease in psi m. The non-motile LAN(NM) did not show chemotactic response and migrated up to a maximum of 3 mm in saturated soils (0 kPa). After 96 h, LAM1 and LAM2 migrated upto 35 and 29 mm respectively in sandy soil. Motile isolates had significantly greater colonization of M. phaseolina sclerotia over the non-motile mutant.     

Non-Muscle Myosin II Regulates Neuronal Actin Dynamics by Interacting with Guanine Nucleotide Exchange Factors

Background

Non-muscle myosin II (NM II) regulates a wide range of cellular functions, including neuronal differentiation, which requires precise spatio-temporal activation of Rho GTPases. The molecular mechanism underlying the NM II-mediated activation of Rho GTPases is poorly understood. The present study explored the possibility that NM II regulates neuronal differentiation, particularly morphological changes in growth cones and the distal axon, through guanine nucleotide exchange factors (GEFs) of the Dbl family.

Principal Findings

NM II colocalized with GEFs, such as βPIX, kalirin and intersectin, in growth cones. Inactivation of NM II by blebbistatin (BBS) led to the increased formation of short and thick filopodial actin structures at the periphery of growth cones. In line with these observations, FRET analysis revealed enhanced Cdc42 activity in BBS-treated growth cones. BBS treatment also induced aberrant targeting of various GEFs to the distal axon where GEFs were seldom observed under physiological conditions. As a result, numerous protrusions and branches were generated on the shaft of the distal axon. The disruption of the NM II–GEF interactions by overexpression of the DH domains of βPIX or Tiam1, or by βPIX depletion with specific siRNAs inhibited growth cone formation and induced slender axons concomitant with multiple branches in cultured hippocampal neurons. Finally, stimulation with nerve growth factor induced transient dissociation of the NM II–GEF complex, which was closely correlated with the kinetics of Cdc42 and Rac1 activation.

Conclusion

Our results suggest that NM II maintains proper morphology of neuronal growth cones and the distal axon by regulating actin dynamics through the GEF–Rho GTPase signaling pathway.     

Electron microscopic examination of subcellular fractions. I. The preparation of representative samples from suspensions of particles

A method is described for preparing, by filtration on Millipore filters, very thin (about 10 µ) pellicles of packed particles. These pellicles can be embedded in Epon for electron microscopic examination. They are also suitable for cytochemical assays. The method was used with various particulate fractions from rat liver. Its main advantages over the usual centrifugal packing techniques are that it produces heterogeneity solely in the direction perpendicular to the surface of the pellicle and that sections covering the whole depth of the pellicle can be photographed in a single field. It thus answers the essential criterion of random sampling and can be used for accurate quantitative evaluations.     

Comparison of gauze swabs and membrane filters for isolation of Campylobacter spp. from surface water

The epidemiology of Campylobacter jejuni indicates that waterborne transmission is important; the organism has been isolated from seawater, fresh water, and estuarine sites. Membrane filtration, with and without use of an enrichment broth, has been the most common method for isolating C. jejuni from water. We evaluated two methods for isolating C. jejuni from water: membrane filtration and gauze filtration. The membrane filters evaluated included 0.22- and 0.45-micron-pore Millipore filters (Millipore Corp., Bedford, Mass.), 0.2- and 0.4-micron-pore Nuclepore filters (Nucleopore Corp., Pleasanton, Calif.), and a 0.45-micron-pore Zetapor filters (AMF Cuno, Meridian, Conn.). The gauze filters included both Moore and Spira swabs. Of the membrane filters evaluated, the 0.45-micron-pore Millipore and Zetapor filters were the most sensitive for recovery of C. jejuni from seeded waters. The 0.45-micron-pore Millipore filter placed in Oosterom broth was better for recovery of C. jejuni from seeded stationary surface waters than either the Spira or Moore swab. However, the 0.45-micron-pore Millipore filter placed on a plate or in enrichment broth was equivalent to the Spira gauze swab when used to examine water from Atlanta area streams. C. jejuni organisms were isolated from 9 of 24 surface water samples representing 5 of 12 streams.     

Comparison of gauze swabs and membrane filters for isolation of Campylobacter spp. from surface water.

The epidemiology of Campylobacter jejuni indicates that waterborne transmission is important; the organism has been isolated from seawater, fresh water, and estuarine sites. Membrane filtration, with and without use of an enrichment broth, has been the most common method for isolating C. jejuni from water. We evaluated two methods for isolating C. jejuni from water: membrane filtration and gauze filtration. The membrane filters evaluated included 0.22- and 0.45-micron-pore Millipore filters (Millipore Corp., Bedford, Mass.), 0.2- and 0.4-micron-pore Nuclepore filters (Nucleopore Corp., Pleasanton, Calif.), and a 0.45-micron-pore Zetapor filters (AMF Cuno, Meridian, Conn.). The gauze filters included both Moore and Spira swabs. Of the membrane filters evaluated, the 0.45-micron-pore Millipore and Zetapor filters were the most sensitive for recovery of C. jejuni from seeded waters. The 0.45-micron-pore Millipore filter placed in Oosterom broth was better for recovery of C. jejuni from seeded stationary surface waters than either the Spira or Moore swab. However, the 0.45-micron-pore Millipore filter placed on a plate or in enrichment broth was equivalent to the Spira gauze swab when used to examine water from Atlanta area streams. C. jejuni organisms were isolated from 9 of 24 surface water samples representing 5 of 12 streams.     

Molecular Characterization of Acanthamoeba spp. Occurring in Water Bodies and Patients in Poland and Redefinition of Polish T16 Genotype

Acanthamoeba genus is divided into 20 genotypes (T1–T20) on the basis of the gene encoding 18S rRNA sequence. Using of at least 2 kbp gene fragments is strongly recommended to identify new genotypes and 5% difference is commonly used as a criterion of new genotypes, however, this value is questionable. In this paper, Polish Acanthamoeba strains described earlier on the basis of ~850 bp Ami fragment of 18S rRNA gene as T4, T11 and a new T16 genotype, have been analyzed using near‐complete sequence of the gene. This analysis was needed because the Ami fragment does not reveal full variability within 18S rRNA gene. Phylogenetic analysis based on Ami fragment is biased by artifacts in the construction of the tree, so the fragment should not be used for identification of new putative Acanthamoeba genotypes. The analysis confirmed that the Polish sequences represent T4 and T11 genotypes and that the strains described earlier as T16 genotype are in fact a new subgroup of the T20 genotype and that this genotype should be divided into two subgroups: T20a (two strains described by [J. Eukaryot. Microbiol. 62 (2015) 69]) and T20b (11 Polish strains described in this study). The T20b subgroup was isolated from both clinical samples and water bodies used by people as bathing places and there is a risk of infection for humans during contact with water.     

苜蓿、草木樨、锦鸡儿根瘤菌的表型多样性分析

选用48株分离自新疆和内蒙的苜蓿属、草木樨属、锦鸡儿属植物的根瘤菌菌株, 进行了营养利用、抗生素抗性、耐逆性和酶活性测定等133个表型性状分析。发现分离自同种寄主植物的根瘤菌由于地理来源的不同而存在着较大的多样性。通过数值分类,各已知种分别聚群,41株未知菌在85%的相似水平上分为3个不同于已知种的群。群1的菌株主要分自苜蓿,群2的菌株主要分自草木樨,群3的菌株分自锦鸡儿,XJ96 333(寄主为Melilotus)作为1个单株在84%的相似水平上和群1聚在一起。NM 020(寄主为Caragana)在88%的相似水平上和R.leguminosarum聚在一起; NM 183、NM 218(寄主为Caragana)在86%的相似水平上和S.fredii聚在一起; 在67%的相似水平上, XJ96 482(寄主为Medicago sativa)和其它所有供试菌株聚群。群1、2、3的所有菌株能在含2.0%(340 mM)NaCl的YMA培养基上、pH 9.0的YMA培养基、40℃的高温下生长; 群3的所有菌株还能在含3.0%(510 mM)NaCl的YMA培养基上生长;群1中90%的菌株和群2、3的所有菌株能在10℃的低温下生长。 表明群1、2、3的菌株具有较强的耐盐碱、耐高低温的特性。和已知种S.meliloti具有相同寄主的群1、群2在85%相似水平上未和S.meliloti聚群,表现了这两群菌在表型上的多样性。     

Evaluation of Five Membrane Filtration Methods for Recovery of Cryptosporidium and Giardia Isolates from Water Samples

We evaluated the efficiency of five membrane filters for recovery of Cryptosporidium parvum oocysts and Giardia lamblia cysts. These filters included the Pall Life Sciences Envirochek (EC) standard filtration and Envirochek high-volume (EC-HV) membrane filters, the Millipore flatbed membrane filter, the Sartorius flatbed membrane filter (SMF), and the Filta-Max (FM) depth filter. Distilled and surface water samples were spiked with 10 oocysts and 10 cysts/liter. We also evaluated the recovery efficiency of the EC and EC-HV filters after a 5-s backwash postfiltration. The backwashing was not applied to the other filtration methods because of the design of the filters. Oocysts and cysts were visualized by using a fluorescent monoclonal antibody staining technique. For distilled water, the highest percent recovery for both the oocysts and cysts was obtained with the FM depth filter. However, when a 5-s backwash was applied, the EC-HV membrane filter (EC-HV-R) was superior to other filters for recovery of both oocysts (n = 53 ± 15.4 per 10 liters) and cysts (n = 59 ± 11.5 per 10 liters). This was followed by results of the FM depth filter (oocysts, 28.2 ± 8, P = 0.015; cysts, 49.8 ± 12.2, P = 0.4260), and SMF (oocysts, 16.2 ± 2.8, P = 0.0079; cysts, 35.2 ± 3, P = 0.0079). Similar results were obtained with surface water samples. Giardia cysts were recovered at higher rates than were Cryptosporidium oocysts with all five filters, regardless of backwashing. Although the time differences for completion of filtration process were not significantly different among the procedures, the EC-HV filtration with 5-s backwash was less labor demanding.     

The metastasis suppressor NM23-H1 possesses 3'-5' exonuclease activity

NM23-H1 belongs to a family of eight gene products in humans that have been implicated in cellular differentiation and development, as well as oncogenesis and tumor metastasis. We have defined NM23-H1 biochemically as a 3'-5' exonuclease by virtue of its ability in stoichiometric amounts to excise single nucleotides in a stepwise manner from the 3' terminus of DNA. The activity is dependent upon the presence of Mg(2+), is most pronounced with single-stranded substrates or mismatched bases at the 3' terminus of double-stranded substrates, and is inhibited by both ATP and the incorporation of cordycepin, a 2'-deoxyadenosine analogue, into the 3'-terminal position. The 3'-5' exonuclease activity was assigned to NM23-H1 by virtue of: 1) precise coelution of enzymatic activity with wild-type and mutant forms of NM23-H1 protein during purification by hydroxylapatite and gel filtration column high performance liquid chromatography and 2) significantly diminished activity exhibited by purified recombinant mutant forms of the proteins. Lysine 12 appears to play an important role in the catalytic mechanism, as evidenced by the significant reduction in 3'-5' exonuclease activity resulting from a Lys(12) to glutamine substitution within the protein. 3'-5' Exonucleases are believed to play an important role in DNA repair, a logical candidate function underlying the putative antimetastatic and oncogenic activities of NM23-H1.     

An analysis of factors influencing the isolation rate of herpes simplex virus.

Attempts were made to improve the rate of isolation of herpes simplex virus (HSV) from clinical specimens by minimizing loss of virus infectivity during transportation and employing the most sensitive cells for isolation. Basical analyses using standard strains of type 1 and type 2 HSV indicated that virus titer decrease was marked even at low temperatures in environments free of proteinous stabilizer such as normal serum or tissue extract, negating the generally held concept that HSV is stable in distilled water. YLE (Earle-lactalbumin HYDROLYSATE-YEAST EXTRACT) medium containing 20% inactivated calf serum was determined to be a transport medium of choice, because degradation of suspended virus during storage and freeze-thawing was negligible and loss of virus during Millipore filtration was minimal. Special coating of the membrane could also be obviated by the use of this solution. In a cell susceptibility test using clinical specimens, secondary rabbit kidney (SRK) cells were the most sensitive, showing a quick development of cytopathic effect. Vero and RK-13 cells were the second best, whereas monkey kidney, HeLa and L cells were far less sensitive. A total of 136 specimens from suspected cases, sent by dermatologists, were tested using SRK cells, and 99 strains of type 1 and 15 strains of type 2 HSV were isolated. Excluding one case from which vaccinia virus was isolated, the isolation rate of HSV was 84.4%.     

Cytoskeletal and Cytocontractile Protein Composition of Smooth Muscle Cells in Developing and Obstructed Rabbit Bladder

The differentiation patterns of smooth muscle cells (SMC) in rabbit bladder during development and in the hypertrophic response to partial outflow obstruction induced in adult animals were evaluated by biochemical and immunochemical techniques and by using a panel of monoclonal antibodies specific for desmin, vimentin, α-actin of smooth muscle (SM) type, SM myosin, and nonmuscle (NM) myosin isoforms. Desmin and SM α-actin were homogeneously distributed in SMC of developing, adult, and obstructed bladders. Conversely, marked changes in the ratio and antigenicity of SM myosin isoforms were observed by SDS electrophoresis and Western blotting, respectively. In particular, the 205 K (SM1) isoform was down-regulated with development whereas the 200 K (SM2) isoform was up-regulated around 7 days after birth and down-regulated in the obstructed bladder. Vimentin was expressed in SMC of the fetal bladder and declined markedly during postnatal, physiological hypertrophy of SMC, which occurs concomitantly with diminution of DNA synthesis. This polypeptide became detectable, however, in SMC of obstructed bladders. The 196 K (NM) myosin isoform recognized by NM-A9 antibody, present only in endothelium of blood vessels and in mucosa of normal fetal and adult bladders, became expressed in detrusor muscle, when SMC underwent a process of pathological hypertrophy. The reexpression of vimentin and the de novo appearance of NM myosin isoform in hypertrophic bladders can be reversed when the tissue mass is reduced, such as in bladders after 1-month recovery from partial obstruction. Thus, a specific NM myosin isoform can be used as a marker of SMC hypertrophy in obstructed bladder. In addition, the combined use of anti-vimentin and NM-A9 antibodies can distinguish between SMC which are in the physiological or in the pathological condition of adaptive bladder hypertrophy.     

Structure and serological studies of the O-polysaccharide of Proteus penneri 75 Epitopes and subgroups of Proteus serogroup O73

The O-specific polysaccharide of the lipopolysaccharide of Proteus penneri strain 75 consists of tetrasaccharide-ribitol phosphate repeating units and resembles ribitol teichoic acids of Gram-positive bacteria. The following structure of the polysaccharide was elucidated by chemical methods and 1H and 13C NMR spectroscopy: [structure in text] where Rib-ol is ribitol. Serological studies with polyclonal antisera showed that the same structure of the O-polysaccharide occurred in two strains: P. penneri 75 and 128. A similar structure has been established earlier for the O-polysaccharide of P. penneri 103 [Drzewiecka, D., et al., Carbohydr. Res. 337 (2002) 1535-1540]. On the basis of complex serological investigations with use of two polyclonal P. penneri 75 and 103 O-antisera, five strains could be classified into Proteus O73 serogroup: P. penneri 48, 75, 90, 103 and 128, two of which (P. penneri 75 and 128) should be subdivided into subgroup 73a, 73b and three others (P. penneri 48, 90 and 103) into subgroup 73a, 73c. Epitopes responsible for the cross-reactivity of P. penneri O73 strains and a related strain of P. mirabilis O20 were tentatively defined.