Lack Of Cell Wall Research Articles

Data on nucleoid-associated proteins isolated from Mycoplasma Gallisepticum in different growth phases.

Mycoplasma gallisepticum (MG) is one of the smallest free-living and self-replicating organisms, it is characterized by lack of cell wall and reduced genome size. As a result of genome reduction, MG has a limited variety of DNA-binding proteins and transcription factors. To investigate the dynamic changes of the proteomic profile of MG nucleoid, that may assist in revealing its mechanisms of functioning, regulation of chromosome organization and stress adaptation, a quantitative proteomic study was performed on MG nucleoids obtained from the cell culture in logarithmic and stationary phases of synchronous growth. MG cells were grown on a liquid medium with a 9 h starvation period. Nucleoids were obtained from the cell culture at the 26th and the 50th hour (logarithmic and stationary growth phases respectively) by sucrose density gradient centrifugation. LC-MS analysis was carried out on an Ultimate 3000 RSLCnano HPLC system connected to a Fusion Lumos mass spectrometer, controlled by XCalibur software (Thermo Fisher Scientific) via a nanoelectrospray source (Thermo Fisher Scientific). For comprehensive peptide library generation one sample from each biological replicate was run in DDA mode. Then, all the samples were run in a single LC-MS DIA run. Identification of DDA files and DIA quantitation was performed with MaxQuant and Skyline software, correspondingly. All raw data generated from IDA and DDA acquisitions are presented in the PRIDE database with identifier PXD019077.

Can Amplicephalus funzaensis Linnavuori 1968 (Hemiptera: Cicadellidae) transmit phytoplasmas to strawberry?

Phytoplasmas are plant pathogenic bacteria of the class Mollicutes that lack cell walls, are restricted to the phloem of their plant hosts, are difficult to culture, and are transmitted by insect vectors. Phytoplasmas from 16SrI and 16SrVII groups have been associated with diseases in urban trees in the Bogota plateau and with potato and strawberry crops in Cundinamarca,Colombia. The objective of this work was to evaluate if the vector Amplicephalus funzaensis (Hemipera: Cicadellidae) could transmit phytoplasmas to Fragaria x ananassa under semi-controlled conditions. A transmission assay on F. x ananassa var. Monterrey was performed with A. funzaensis from a population naturally infected with phytoplasmas, whose host was Cenchrus clandestinus. Seven months after herbivory by these insects, the plants did not show symptoms associated to phytoplasmas, even though more than one third of the insects used carried phytoplasmas. In total, 120 A. funzaensis individuals were tested for the presence of phytoplasmas by molecular methods; of these, 46 (38%) were positive for phytoplasmas, showing the existence of insect populations with a high number of individuals that are a potential source of inoculum for the pathogen transmission. Additionally, for the molecular identification of A. funzaensis, a DNA barcode was generated from the cyitochrome c oxidase (COI) gene.

Ureaplasma urealyticum disseminated multifocal abscesses in an immunocompromised adult patient: a case report

BackgroundUreaplasma urealyticum is a fastidious bacteria which lacks a cell wall. Extragenital infections are rare in immunocompetent adults. There are few literature reports of perinephric abscess. We present a case of non-resolving multifocal “culture-negative” abscesses in a hypogammaglobulinemic adult female due to U. urealyticum.Case presentation66-year-old female with a one-week history of fever, malaise and new right hip and leg pain. Past medical history was notable for chronic pancytopenia secondary to in remission B cell follicular lymphoma, ESRD on intermittent hemodialysis with bilateral nephrostomy tubes and Crohn’s. CT abdomen/pelvis revealed a small left perinephric hematoma and proximal right femur fluid collection. Persistent right thigh pain led to additional ultrasound with anterior thigh collection and CT revealed an irregular rim-enhancing fluid collection in the left posterior pararenal space. Antimicrobial therapy included ertapenem and vancomycin followed by meropenem, trimethoprim-sulfamethoxazole, daptomycin and metronidazole in setting of persistent culture-negative results and clinical deterioration. Following detection of U. urealyticum by 16S rDNA PCR in both left pararenal and right trochanteric bursa abscesses doxycycline was started. Despite this, the patient died four days later.ConclusionsDisseminated infection by U. urealyticum has been documented in immunocompromised adult patients with few reports of perinephric abscess. We propose that ascending genitourinary route led to perinephric abscess. The multiple disseminated fluid collections make it highly suspicious for hematogenous spread given the lack of radiographic enhancement to suggest contiguous spread. Diagnosis and treatment of U. urealyticum-disseminated infection is extremely challenging as culture is laborious and not routinely performed. Furthermore, the lack of cell wall renders beta-lactams and vancomycin ineffective and therefore requirement for “atypical” coverage. Early diagnosis and treatment are key to prevent further complications and death.

Interactions between Mycoplasma pulmonis and immune systems in the mealworm beetle, Tenebrio molitor

Mycoplasmas, the smallest self-replicating organisms, are unique in that they lack cell walls but possess distinctive plasma membranes containing sterol acquired from their growth environment. Although mycoplasmas are known to be successful pathogens in a wide range of animal hosts, including humans, the molecular basis for their virulence and interaction with the host immune systems remains largely unknown. This study was conducted to elucidate the biochemical relationship between mycoplasma and the insect immune system. We investigated defense reactions of Tenebrio molitor that were activated in response to infection with Mycoplasma pulmonis. The results revealed that T. molitor larvae were more resistant to mycoplasma infection than normal bacteria equipped with cell walls. Intruding M. pulmonis cells were effectively killed by toxins generated from activation of the proPO cascade in hemolymph, but not by cellular reactions or antimicrobial peptides. It was determined that these different anti-mycoplasma effects of T. molitor immune components were primarily attributable to surface molecules of M. pulmonis such as phospholipids occurring in the outer leaflet of the membrane lipid bilayer. While phosphatidylcholine, a phospholipid derived from the growth environment, contributed to the resistance of M. pulmonis against antimicrobial peptides produced by T. molitor, phosphatidylglycerol was responsible for triggering activation of the proPO cascade.

Effects of medium composition on the growth and lipid production of microplasmodia of Physarum polycephalum.

Physarum polycephalum is a plasmodial slime mold. One of the trophic stages in the life cycle of this organism is a plasmodium. In submerged culture, plasmodia are fragmented into microplasmodia. The latter both lack cell walls and are capable of rapid growth. There has been limited information on the effects of medium composition on the growth and lipid accumulation of microplasmodia. In this study, optimization of medium components by response surface methodology showed that tryptone and yeast extract concentrations had the most significant effects on lipid and biomass production; significant synergistic interactions between glucose and tryptone concentration on these responses were also recorded. The optimal medium was composed of 20 g/L of glucose, 6.59 g/L of tryptone, and 3.0 g/L of yeast extract. This medium yielded 13.86 g/L of dry biomass and 1.97 g/L of lipids. These amounts are threefold higher than those of the American Type Culture Collection (ATCC) medium. In addition, biomass and lipid production reached maximal values between only 4 and 5 days. Fatty acid compositions analysis by gas chromatography-mass spectrometer (GC-MS) revealed that P. polycephalum lipids consisted mainly of oleic acid (40.5%), linoleic acid (10%), and octadecynoic (15.8%). This is the first report on the fatty acid composition of P. polycephalum microplasmodia. These results suggest that the biomass of microplasmodia could be used as a source of material for direct conversion into biodiesel because of the absence of cell walls or it could also be used as a supplemental source of beneficial fatty acids for humans, albeit with some further evaluation needed.

Essential Mycoplasma Glycolipid Synthase Adheres to the Cell Membrane by Means of an Amphipathic Helix

Because of the lack of cell wall, Micoplasma species require a fine control of membrane fluidity and integrity. mg517 is an essential gene of Mycoplasma genitalium responsible for the biosynthesis of membrane glycoglycerolipids. It encodes for a unique glycosyltransferase (MG517) with processive activity, transferring activated glycosyl donors to either nude diacylglycerol or already glycosylated diacylglycerol. This dual activity, asserted to different enzymes in other species, is sensitive to and regulated by the presence of anionic lipid vesicles in vitro. We present here a computational model of the C-terminus domain of MG517 that complements a previous structural model of the N-terminus domain. By means of sequence analysis, molecular dynamics and metadynamics simulations, we have identified a short α-helix at the apical C-terminus of MG517 with clear amphipathic character. Binding to a membrane model is thermodynamically favored which suggests that this structural element guides the adhesion of MG517 to the cell membrane. We have experimentally verified that truncation of part of this helix causes a substantial reduction of glycoglycerolipids synthesis. The model proposes that MG517 recognizes and binds the diacylglycerol substrate embedded in the membrane by means of this α-helix at the C-terminus together with a previously identified binding pocket at the N-terminus.

Comparative genomics of Mycoplasma: Insights on genome reduction and identification of potential antibacterial targets

Background: Mycoplasmas are cell wall-deficient bacteria which cause respiratory and urogenital infections in human. Mycoplasmas are resistant to many of the available antibiotics because of their high mutation rates and lack of cell wall. Various studies have identified the emergence of treatment-resistant Mycoplasma isolates. Novel drug target identification has become significant for the development of successful antibacterial treatments. Computational genomic analysis plays a significant role in facilitating the identification of potential drug targets in many bacterial pathogens. Methods: In the present study, 12 Mycoplasma genomes were subjected to comparative genomic analysis to reinforce the understanding of their genomic organization and to identify potential drug targets. The distributions of genes under the Clusters of Orthologous Groups of proteins (COG) functional categories were analyzed for all the 12 Mycoplasma genomes. Genes from each functional category that are conserved across all the Mycoplasma genomes were extracted to identify the backbone genome of Mycoplasma species. The genes in the backbone genome were subjected to similarity search against a database of essential genes to validate their essentiality. Essentiality of these genes was further analyzed based on their function and subcellular localization. Results: The 12 Mycoplasma genomes under study were found to exhibit marked similarities in COG functional category distributions. An overall loss of genes in various functional categories has been observed in all the 12 Mycoplasma genomes. In all the 12 Mycoplasma genomes under study, a maximum reduction in the genes involved in the secondary metabolites biosynthesis, transport, and catabolism (Q) is observed. Conclusion: Comparative genomic studies have identified a set of 170 genes which are commonly present all the 12 Mycoplasma genomes. Further analysis of these genes has identified a set of 158 core essential genes which serve as a promising cluster of novel antibacterial targets.

Enlargement of Deinococcus grandis spheroplasts requires  Mg2+ or Ca2.

While the cell wall strictly controls cell size and morphology in bacteria, spheroplasts lack cell walls and can become enlarged in growth medium under optimal conditions. Optimal conditions depend on the bacterial species. We frequently observed extreme enlargement of spheroplasts of the radiation-resistant bacterium Deinococcus grandis in Difco Marine Broth 2216, but not in TGY broth (a commonly used growth medium for Deinococcus). Thorough investigation of media components showed that the presence of Mg2+ or Ca2+ promoted extreme spheroplast enlargement, synthesizing the outer membrane. Our findings strongly suggest that Mg2+ or Ca2+ enlarges spheroplasts, which could change the lipid composition of the spheroplast membrane.

Aloe Emodin Reduces Phthiodiolone Dimycocerosate Potentiating Vancomycin Susceptibility on Mycobacteria.

Treatment of tuberculosis still represent a major public health issue. The emergence of multi-and extensively-drug resistant (MDR and XDR) Mycobacterium tuberculosis clinical strains further pinpoint the urgent need for new anti-tuberculous drugs. We previously showed that vancomycin can target mycobacteria lacking cell wall integrity, especially those lacking related phthiocerol and phthiodolone dimycocerosates, PDIM A and PDIM B, respectively. As aloe emodin was previously hypothesized to be able to target the synthesis of mycobacterial cell wall lipids, we tested its ability to potentiate glycopeptides antimycobacterial activity. The aloe emodin with the vancomycin induced a combination effect beyond simple addition, close to synergism, at a concentration lower to reported IC50 cytotoxic value, on M. bovis BCG and on H37Rv M. tuberculosis. Interestingly, out of six MDR and pre-XDR clinical strains, one showed a strong synergic susceptibility to the drug combination. Mycobacterial cell wall lipid analyses highlighted a selective reduction of PDIM B by aloe emodin.

The Conserved DNA Binding Protein WhiA Influences Chromosome Segregation in Bacillus subtilis.

The DNA binding protein WhiA is conserved in Gram-positive bacteria and is present in the genetically simple cell wall-lacking mycoplasmas. The protein shows homology to eukaryotic homing endonucleases but lacks nuclease activity. WhiA was first characterized in streptomycetes, where it regulates the expression of key differentiation genes, including the cell division gene ftsZ, which is essential for sporulation. For Bacillus subtilis, it was shown that WhiA is essential when certain cell division genes are deleted. However, in B. subtilis, WhiA is not required for sporulation, and it does not seem to function as a transcription factor, despite its DNA binding activity. The exact function of B. subtilis WhiA remains elusive. We noticed that whiA mutants show an increased space between their nucleoids, and here, we describe the results of fluorescence microscopy, genetic, and transcriptional experiments to further investigate this phenomenon. It appeared that the deletion of whiA is synthetic lethal when either the DNA replication and segregation regulator ParB or the DNA replication inhibitor YabA is absent. However, WhiA does not seem to affect replication initiation. We found that a ΔwhiA mutant is highly sensitive for DNA-damaging agents. Further tests revealed that the deletion of parAB induces the SOS response, including the cell division inhibitor YneA. When yneA was inactivated, the viability of the synthetic lethal ΔwhiA ΔparAB mutant was restored. However, the nucleoid segregation phenotype remained. These findings underline the importance of WhiA for cell division and indicate that the protein also plays a role in DNA segregation.IMPORTANCE The conserved WhiA protein family can be found in most Gram-positive bacteria, including the genetically simple cell wall-lacking mycoplasmas, and these proteins play a role in cell division. WhiA has some homology with eukaryotic homing endonucleases but lacks nuclease activity. Because of its DNA binding activity, it is assumed that the protein functions as a transcription factor, but this is not the case in the model system B. subtilis The function of this protein in B. subtilis remains unclear. We noticed that a whiA mutant has a mild chromosome segregation defect. Further studies of this phenomenon provided new support for a functional role of WhiA in cell division and indicated that the protein is required for normal chromosome segregation.

Screening of Isochrysis Strains and Utilization of a Two-Stage Outdoor Cultivation Strategy for Algal Biomass and Lipid Production.

Isochrysis is a genus of marine algae without cell wall and capable of accumulating lipids. In this study, the lipid production potential of Isochrysis was assessed by comparing 15 Isochrysis strains with respect to their growth rate, lipid production, and fatty acid profiles. Three best strains were selected (lipid productivity, 103.0~121.7mgL-1day-1) and their lipid-producing capacities were further examined under different controlled parameters, e.g., growth phase, medium nutrient, and light intensity in laboratory cultures. Furthermore, the three Isochrysis strains were monitored in outdoor panel photobioreactors with various initial cell densities and optical paths, and the strain CS177 demonstrated the superior potential for outdoor cultivation. A two-stage semi-continuous strategy for CS177 was subsequently developed, where high productivities of biomass (1.1gL-1day-1) and lipid (0.35gL-1day-1) were achieved. This is a comprehensive study to evaluate the lipid-producing capability of Isochrysis strains under both indoor and outdoor conditions. Results of the present work lay a solid foundation for the physiological and biochemical responses of Isochrysis to various conditions, shedding light on the future utilization of this cell wall-lacking marine alga for biofuel production.

Antimycoplasmal Activities of Compounds from Solanum aculeastrum and Piliostigma thonningii against Strains from the Mycoplasma mycoides Cluster.

Infections caused by Mycoplasma species belonging to the ‘mycoides cluster’ negatively affect the agricultural sector through losses in livestock productivity. These Mycoplasma strains are resistant to many conventional antibiotics due to the total lack of cell wall. Therefore, there is an urgent need to develop new antimicrobial agents from alternative sources such as medicinal plants to curb the resistance threat. Recent studies on extracts from Solanum aculeastrum and Piliostigma thonningii revealed interesting antimycoplasmal activities hence the motivation to investigate the antimycoplasmal activities of constituent compounds. The CH2Cl2/MeOH extracts from the berries of S. aculeastrum yielded a new β-sitosterol derivative (1) along with six known ones including; lupeol (2), two long-chain fatty alcohols namely undecyl alcohol (3) and lauryl alcohol (4); two long-chain fatty acids namely; myristic acid (5) and nervonic acid (6) as well as a glycosidic steroidal alkaloid; (25R)-3β-O-α-L-rhamnopyranosyl-(1→2)-O-[α-L-rhamnopyranosyl-(1→4)]-β-D-glucopyranosyloxy-22α-N-spirosol-5-ene (7) from the MeOH extracts. A new furan diglycoside, (2,5-D-diglucopyranosyloxy-furan) (8) was also characterized from the CH2Cl2/MeOH extract of stem bark of P. thonningii. The structures of the compounds were determined on the basis of spectroscopic evidence and comparison with literature data. Compounds 1, 3, 4, 7, and 8 isolated in sufficient yields were tested against the growth of two Mycoplasma mycoides subsp. mycoides (Mmm), two M. mycoides. capri (Mmc), and one M. capricolum capricolum (Mcc) using broth dilution methods, while the minimum inhibitory concentration (MIC) was determined by serial dilution. The inhibition of Mycoplasma in vitro growth was determined by the use of both flow cytometry (FCM) and color change units (CCU) methods. Compounds 4 and 7 showed moderate activity against the growth of Mmm and Mmc but were inactive against the growth of Mcc. The lowest MIC value was 50 μg/ml for compound 7 against Mmm. The rest of the compounds showed minimal or no activity against the strains of Mycoplasma mycoides tested. This is the first report on the use of combined FCM and CCU to determine inhibition of in vitro growth of Mycoplasma mycoides. The activity of these compounds against other bacterial strains should be tested and their safety profiles determined.

EPS Characterization of a Cell Wall-Lacking Archaeon <i>Ferroplasma</i> <i>acidiphilum</i>

We studied the surface properties of F. acidiphilum DSM 28986 by attenuated total reflection-Fourier transformed infra-red (ATR-FTIR) spectroscopy and microbial adhesion to hydrocarbon (MATH) techniques. In addition, extracellular polymeric substances (EPS) were extracted and characterized by conventional colorimetric analysis and fluorescence lectin-binding analysis (FLBA). Results showed that: 1) cells selectively adhered to mineral surfaces and showed maximum attachment to pyrite of approx. 50% within 30 min; 2) EPS synthesis by F. acidiphilum DSM 28986 was influenced by growth substrates; and 3) tightly-bound EPS (capsular EPS) were composed of carbohydrates and proteins. In contrast, loosely-bound EPS (colloidal EPS) were mainly characterized as carbohydrates. Monosaccharides like glucose, fucose, arabinose, galactose, mannose, and sialic acid were detected in the EPS of F. acidiphilum DSM 28986. This study provides first insight into surface characterization of the cell wall-lacking archaeon F. acidiphilum and facilitates the understanding of interactions of this organism with other acidophiles and metal sulfides.

Effect Of Sparging In Microalgae (Dunaliella Tertiolecta) Cuture Using Bubble Column

Microalgae as a photosynthetic microorganism that contain chlorophyll has high potential to produce novel high value compounds that can be used in food, pharmaceutical and cosmetic industries. With lack of rigid cell wall, microalgae susceptible to have hydrodynamic stress by increasing aeration rate. Increasing gas flow rate will increase the productivity to optimum condition, but after this condition the productivity will decrease due to cell disruption. In this research has been focused on effect of sparging on microalgae cell damage. Sparging experiment was carried out by varying gas flow rate between 0.59 to 5.13 L.min-1 with a sparger made from needle inserted to a piece of silicon. The needle was used with diameter 0.4 to 1.2 mm and 1-9 needles. With this variables resulted gas entrance velocity between 2.56-104 m.s-1. The cell death rate constant (kd) was calculated by loss of cell viability in time using Coulter counter and Flowcytometer. From the experiments resulted gas entrance velocity was main cause for cell damagae. The small bubble was more detrimental than bigger size of bubble. Bubble rising as like in animal cell culture was no effect to detrimental cell.Keywords : D. tertiolecta, cell death rate constant, gas entrance velocity

2D Fluorescence spectroscopy for monitoring Dunaliella salina concentration and integrity during membrane harvesting

Dunaliella salina is able to produce simultaneously several valuable compounds (such as lipids, carotenes and functional proteins) within the biorefinery concept. However due to the lack of rigid cell wall, this microalgae can easily disrupt during harvesting, losing valuable compounds to the saline water, affecting the downstream processing. Therefore, the development of non-invasive tools able to monitor cell concentration and integrity in real-time, can assist the development of harvesting methodologies. In the present work, a monitoring approach was developed based on two-dimensional (2D) fluorescence spectroscopy. Mathematical analysis of the monitoring data involved the use of Principal Component Analysis (PCA) and Projection to Latent Structures (PLS) modelling. For green D. salina, the models developed for prediction of cell number and percentage of viability captured 90.6% and 86.3% of variance, respectively. Both models have R2 of 0.8 and 0.9, respectively for validation and training. Similar values were found for the prediction of cell number when using data from growth kinetics and harvesting combined. Orange D. salina rupture was also successfully modelled with 95% of variance captured and R2 of 0.9 for both training and validation. The combined approach using 2D fluorescence spectroscopy and the mathematical analysis proved to have the potential to monitor D. salina during cell growth and harvesting within a biorefinery concept.

Low temperature and dust favour invitro survival of Mycoplasma hyopneumoniae: time to revisit indirect transmission in pig housing.

Understanding the transmission of Mycoplasma hyopneumoniae and optimizing biosecurity practices are keys to reducing the use of antimicrobial agents to control this pathogen. Direct transmission of the pathogen between pigs is the main route of spread and its lack of cell wall may compromise its resilience outside the host. The results from our study show that M.hyopneumoniae can survive for up to several days on dry surfaces and therefore may have the potential to infect pigs by indirect transmission. Factors influencing the survival of M.hyopneumoniae outside the host are further elucidated.

Mycoplasma Clearance and Risk Analysis in a Model Bioprocess.

Mycoplasmas are a type of bacteria that lack cell walls and are occasional cell culture contaminants. In a biotechnology setting, because they can pass through 0.2 μm filters, mycoplasmas could pose a potential patient safety hazard if undetected contaminants from the production culture were not completely removed by downstream biotechnology manufacturing. In this study we investigated the ability of typical commercial monoclonal antibody purification operations to clear and kill mycoplasmas, using Acholeplasma laidlawii as a model organism. Our spike/removal studies have shown that protein A column chromatography clears about 4-5 log10 Column regeneration effectively prevents A. laidlawii column carryover between chromatography runs. Moreover, low-pH hold steps, typically implemented after protein A purification, effectively kill A. laidlawii using either pH 3.8 glycine or acetate solutions (LRV ≥5.30 and ≥4.57, respectively). Solvent/detergent treatment, used in some processes instead of low-pH hold, also completely kills highly concentrated A. laidlawii (LRV ≥5.95).LAY ABSTRACT: Biotechnology medicines need to be free from contaminating microorganisms such as mycoplasmas, a type of bacteria that can cause disease in humans (e.g., walking pneumonia). Here we show that some monoclonal antibody manufacturing steps can effectively clear and/or kill Acholeplasma laidlawii, a model mycoplasma species used in our study. This provides an additional level of safety assurance of biotechnology medicines for patients.

Intracellular Hg(0) Oxidation in Desulfovibrio desulfuricans ND132.

The disposal of elemental mercury (Hg(0)) wastes in mining and manufacturing areas has caused serious soil and groundwater contamination issues. Under anoxic conditions, certain anaerobic bacteria can oxidize dissolved elemental mercury and convert the oxidized Hg to neurotoxic methylmercury. In this study, we conducted experiments with the Hg-methylating bacterium Desulfovibrio desulfuricans ND132 to elucidate the role of cellular thiols in anaerobic Hg(0) oxidation. The concentrations of cell-surface and intracellular thiols were measured, and specific fractions of D. desulfuricans ND132 were examined for Hg(0) oxidation activity and analyzed with extended X-ray absorption fine structure (EXAFS) spectroscopy. The experimental data indicate that intracellular thiol concentrations are approximately six times higher than those of the cell wall. Cells reacted with a thiol-blocking reagent were severely impaired in Hg(0) oxidation activity. Spheroplasts lacking cell walls rapidly oxidized Hg(0) to Hg(II), while cell wall fragments exhibited low reactivity toward Hg(0). EXAFS analysis of spheroplast samples revealed that multiple different forms of Hg-thiols are produced by the Hg(0) oxidation reaction and that the local coordination environment of the oxidized Hg changes with reaction time. The results of this study indicate that Hg(0) oxidation in D. desulfuricans ND132 is an intracellular process that occurs by reaction with thiol-containing molecules.

PRESENT KNOWLEDGE AND EXPERIENCE ON THE STRATEGIES EMPLOYED BY MYCOPLASMA CONTAMINATION OF THE HUMAN CELL CULTURES

Introduction: Mycoplasma species often contaminate cell cultures and other cell-derived biological substances, leading to detrimental effects on the host that include changes in growth, morphology, metabolism and protein synthesis. In cell cultures, mycoplasma are extracellular parasites, usually attached to the external surface of a cell membrane. Many researchers use a mixture of penicillin and streptomycin in the cell culture to prevent contamination. Material and methods: We prepared cell cultures of lymphocytes from peripheral blood of 12 subjects and used micronucleus, assay which is the standard method, for detection of micronuclei in binuclear lymphocytes.Results: Use of standard antibiotics does not protect cell cultures against mycoplasma contamination. Penicillin has no effect on mycoplasma since mycoplasma lack cell wall. Streptomycin inhibits about half the mycoplasma strains but is ineffective against others. In fact, mycoplasma is generally resistant to most antibiotic mixtures commonly used in cell culture. We didn’t find any mycoplasma contamination in the cell culture where penicillin-streptomycin mixture was absent, but confirmed infection in the culture containing mixture of antibiotics.Conclusion: Antibiotics and mixture of antibiotics like penicillin-streptomycin mixture does not protect the cell culture against mycoplasma contamination. Hence, contamination can spread rapidly to other cell lines through aerosol droplet dispersion.

Two Isoforms of Myosin-II Account for the Tension of the Fission Yeast Cytokinetic Ring

During cytokinesis, an actomyosin contractile ring constricts and drives or regulates division of the cell into two. Tension production is thought to be the principal function of the cytokinetic ring, but measurements of this fundamental property have not been available. Myosin-II is thought to generate tension, but a requirement was not demonstrated.Here, we developed a method to measure ring tension in fission yeast protoplasts, cells that lack cell wall in which furrowing by the contractile ring is supported by plasma membrane tension alone as demonstrated by the almost perfectly spherical lobes flanking the furrow. We use a force balance at the furrow to deduce the ring tension, after measuring the membrane tension using micropipette aspiration and accurately measuring the furrow geometry.Applying the technique to wild type and myosin-II mutant cells, we show for the first time that myosin-II is required for normal tension, and we measured the contributions of the two myosin-II isoforms present in the fission yeast ring, Myo2 and Myp2. As rings constricted, the tension increased from ∼400 to ∼800 pN. Tension was lowered by the myosin-II inhibitor blebbistatin, and tension in cells with mutations in Myo2p or Myp2p showed that the two isoforms account for all of the tension, with contributions of ∼ 65% and ∼ 40%, respectively.A molecularly detailed 3D simulation of the ring, together with the experimental tension measurements, suggest that tension is generated solely by the pulling of myosin-II on actin filaments anchored at their barbed ends to the plasma membrane. The simulations reproduced experiment, including the steady increase of ring tension as rings constricted, with best fit forces per myosin head of ∼ 1 pN for both isoforms, similar to muscle myosin II.