Isolation and Properties of Flagella of Trypanosomatids*

SYNOPSIS. A procedure is described for the isolation of flagella of Crithidia fasciculata. Herpetomonas samuelpessoai and Leishmania tarentolae in a highly purified state and giving reasonably good yield. The 3 types of flagella give a similar electrophoretic pattern of proteins. It is shown that H. samuelpessoai and, to a lesser extent, C. fasciculata flagella confer protection against Trypanosoma cruzi infection.     

Method for Staining Bacterial Flagella

The following method of staining bacterial flagella is ecommended for use on smears made from suspensions of 10 to 16-tour agar slant cultures, incubated 30 minutes at 37°C before spreadng on thoroly cleaned and named slides:

Cover with fixative (100 cc. of 1/4 sat. aqu. solution picric acid, with 5 g. tannic acid and 7.5 g. ferrous sulfate).

Wash with tap water, dry and cover with Fontana spirochaete stain; heat to steaming and allow to act for 1 to 2 minutes. Wash in ap water. The stain is prepared as follows: To 25 cc. 2% AgNO₃ add dilute ammonia till the precipitate which forms redissolves; then add more AgNO₃ till a faint turbidity results. A clear solution is useess.     

Archaeal communities associated with shallow to deep subseafloor sediments of the New Caledonia Basin

The distribution of the archaeal communities in deep subseafloor sediments [0–36 m below the seafloor (mbsf)] from the New Caledonia and Fairway Basins was investigated using DNA- and RNA-derived 16S rRNA clone libraries, functional genes and denaturing gradient gel electrophoresis (DGGE). A new method, Co-Migration DGGE (CM-DGGE), was developed to access selectively the active archaeal diversity. Prokaryotic cell abundances at the open-ocean sites were on average ∼3.5 times lower than at a site under terrestrial influence. The sediment surface archaeal community (0–1.5 mbsf) was characterized by active Marine Group 1 (MG-1) Archaea that co-occurred with ammonia monooxygenase gene ( amoA ) sequences affiliated to a group of uncultured sedimentary Crenarchaeota . However, the anoxic subsurface methane-poor sediments (below 1.5 mbsf) were dominated by less active archaeal communities, such as the Thermoplasmatales , Marine Benthic Group D and other lineages probably involved in the methane cycle ( Methanosarcinales , ANME-2 and DSAG/MBG-B). Moreover, the archaeal diversity of some sediment layers was restricted to only one lineage (Uncultured Euryarchaeota , DHVE6, MBG-B, MG-1 and SAGMEG). Sequences forming two clusters within the Thermococcales order were also present in these cold subseafloor sediments, suggesting that these uncultured putative thermophilic archaeal communities might have originated from a different environment. This study shows a transition between surface and subsurface sediment archaeal communities.     

A Method for the Staining of Bacterial Flagella

A modification of the Loeffler method of staining bacterial flagella is proposed. The chief points of the modification are: The cultures are inoculated into distilled water after two successive daily transfers on agar slants, and the distilled water cultures are incubated at optimum temperature for from 48 to 72 hours. The mordant (tannic acid, ferrous sulphate, basic fuchsin) is allowed to stand 18 to 24 hours before use, and then cleared by centrifuging or filtering. An anilin water fuchsin is used as a stain. No heat is used for either mordanting or staining; but both mordant and stain are allowed to act on the preparation for 15 minutes. The writer finds the method admirably adapted for use in class work, where nearly 100 per cent success has been obtained except in the case of some three or four species of bacteria that are especially difficult to stain.     

Nanomaterials for ocular drug delivery

Efficient drug delivery to the eye remains a challenging task for pharmaceutical scientists. Due to the various anatomical barriers and the clearance mechanisms prevailing in the eye, conventional drug delivery systems, such as eye drop solutions, suffer from low bioavailability. More invasive methods, such as intravitreal injections and implants, cause adverse effects in the eye. Recently, an increasing number of scientists have turned to nanomaterial-based drug delivery systems to address the challenges faced by conventional methods. This paper highlights recent applications of various nanomaterials, such as polymeric micelles, hydrogels, liposomes, niosomes, dendrimers, and cyclodextrins as ocular drug delivery systems to enhance the bioavailability of ocular therapeutic agents.     

A Simple, Rapid Method for Demonstrating Bacterial Flagella

We developed a simple, rapid method for demonstrating flagellation of bacteria using the fluorescent protein stain NanoOrange (Molecular Probes, Eugene, Oreg.). The NanoOrange reagent binds to hydrophobic regions of proteins, which results in substantial enhancement of fluorescence. Unbound reagent is essentially nonfluorescent. NanoOrange fluorescently stained bacterial cell bodies, as well as flagella and other appendages, which could be directly observed by epifluorescence microscopy. Detection of flagella was further improved by using a charge-coupled device camera for image capture and processing. The reliability of the method was tested by using 37 pure cultures of marine bacteria. Detection of flagella on the isolates by NanoOrange staining was compared to detection by transmission electron microscopy (TEM). For 36 of 37 cultures, the two methods yielded the same results. In one case, flagella were detected by TEM but not by NanoOrange, although the difference may be attributable to differences between the culture preparations. NanoOrange staining is rapid (10 to 15 min) and does not require fixation or dehydration, so live samples can be stained. Since NanoOrange is a general protein stain and works directly in seawater, it may also prove to be useful for staining other proteinaceous material that is of interest to aquatic microbial ecologists.     

Nanomaterials for cancer therapy and imaging

A variety of organic and inorganic nanomaterials with dimensions below several hundred nanometers are recently emerging as promising tools for cancer therapeutic and diagnostic applications due to their unique characteristics of passive tumor targeting. A wide range of nanomedicine platforms such as polymeric micelles, liposomes, dendrimers, and polymeric nanoparticles have been extensively explored for targeted delivery of anti-cancer agents, because they can accumulate in the solid tumor site via leaky tumor vascular structures, thereby selectively delivering therapeutic payloads into the desired tumor tissue. In recent years, nanoscale delivery vehicles for small interfering RNA (siRNA) have been also developed as effective therapeutic approaches to treat cancer. Furthermore, rationally designed multi-functional surface modification of these nanomaterials with cancer targeting moieties, protective polymers, and imaging agents can lead to fabrication versatile theragnostic nanosystems that allow simultaneous cancer therapy and diagnosis. This review highlights the current state and future prospects of diverse biomedical nanomaterials for cancer therapy and imaging.     

Hot-Alkaline DNA Extraction Method for Deep-Subseafloor Archaeal Communities

A prerequisite for DNA-based microbial community analysis is even and effective cell disruption for DNA extraction. With a commonly used DNA extraction kit, roughly two-thirds of subseafloor sediment microbial cells remain intact on average (i.e., the cells are not disrupted), indicating that microbial community analyses may be biased at the DNA extraction step, prior to subsequent molecular analyses. To address this issue, we standardized a new DNA extraction method using alkaline treatment and heating. Upon treatment with 1 M NaOH at 98°C for 20 min, over 98% of microbial cells in subseafloor sediment samples collected at different depths were disrupted. However, DNA integrity tests showed that such strong alkaline and heat treatment also cleaved DNA molecules into short fragments that could not be amplified by PCR. Subsequently, we optimized the alkaline and temperature conditions to minimize DNA fragmentation and retain high cell disruption efficiency. The best conditions produced a cell disruption rate of 50 to 80% in subseafloor sediment samples from various depths and retained sufficient DNA integrity for amplification of the complete 16S rRNA gene (i.e., ∼1,500 bp). The optimized method also yielded higher DNA concentrations in all samples tested compared with extractions using a conventional kit-based approach. Comparative molecular analysis using real-time PCR and pyrosequencing of bacterial and archaeal 16S rRNA genes showed that the new method produced an increase in archaeal DNA and its diversity, suggesting that it provides better analytical coverage of subseafloor microbial communities than conventional methods.     

Prediction of Bacterial and Archaeal Allergenicity with AllPred Program

Nowadays, allergic disorders have become one of the most important social problems in the world. This can be related to the advent of new allergenic agents in the environment, as well as an increasing density of human contact with known allergens, including various proteins. Thus, the development of computer programs designed for the prediction of allergenic properties of proteins becomes one of the urgent tasks of modern bioinformatics. Previously we developed a web accessible Allpred Program (http://www-bionet.sscc.ru/ psd/cgi-bin/programs/Allpred/allpred.cgi) that allows users to assess the allergenicity of proteins by taking into account the characteristics of their spatial structure. In this paper, using AllPred, we predicted the allergenicity of proteins from 462 archaea and bacteria species for which a complete genome was available. The segregation of considered proteins on archaea and bacteria has shown that allergens are predicted more often among archaea than among bacteria. The division of these proteins into groups according to their intracellular localization has revealed that the majority of allergenic proteins were among the secreted proteins. The application of methods for predicting the level of gene expression of microorganisms based on DNA sequence analysis showed a statistically significant relationship between the expression level of the proteins and their allergenicity. This analysis has revealed that potentially allergenic proteins were more common among highly expressed proteins. Sorting microorganisms into the pathogenic and nonpathogenic groups has shown that pathogens can potentially be more allergenic because of a statistically significant greater number of allergens predicted among their proteins.     

An Archaeal Homolog of Proteasome Assembly Factor Functions as a Proteasome Activator

Assembly of the eukaryotic 20S proteasome is an ordered process involving several proteins operating as proteasome assembly factors including PAC1-PAC2 but archaeal 20S proteasome subunits can spontaneously assemble into an active cylindrical architecture. Recent bioinformatic analysis identified archaeal PAC1-PAC2 homologs PbaA and PbaB. However, it remains unclear whether such assembly factor-like proteins play an indispensable role in orchestration of proteasome subunits in archaea. We revealed that PbaB forms a homotetramer and exerts a dual function as an ATP-independent proteasome activator and a molecular chaperone through its tentacle-like C-terminal segments. Our findings provide insights into molecular evolution relationships between proteasome activators and assembly factors.     

Archaeal ApbC/Nbp35 Homologs Function as Iron-Sulfur Cluster Carrier Proteins

Iron-sulfur clusters may have been the earliest catalytic cofactors on earth, and most modern organisms use them extensively. Although members of the Archaea produce numerous iron-sulfur proteins, the major cluster assembly proteins found in the Bacteria and Eukarya are not universally conserved in archaea. Free-living archaea do have homologs of the bacterial apbC and eukaryotic NBP35 genes that encode iron-sulfur cluster carrier proteins. This study exploits the genetic system of Salmonella enterica to examine the in vivo functionality of apbC/NBP35 homologs from three archaea: Methanococcus maripaludis, Methanocaldococcus jannaschii, and Sulfolobus solfataricus. All three archaeal homologs could correct the tricarballylate growth defect of an S. enterica apbC mutant. Additional genetic studies showed that the conserved Walker box serine and the Cys-X-X-Cys motif of the M. maripaludis MMP0704 protein were both required for function in vivo but that the amino-terminal ferredoxin domain was not. MMP0704 protein and an MMP0704 variant protein missing the N-terminal ferredoxin domain were purified, and the Fe-S clusters were chemically reconstituted. Both proteins bound equimolar concentrations of Fe and S and had UV-visible spectra similar to those of known [4Fe-4S] cluster-containing proteins. This family of dimeric iron-sulfur carrier proteins evolved before the archaeal and eukaryal lineages diverged, representing an ancient mode of cluster assembly.Members of the Archaea produce many proteins that require iron-sulfur cluster cofactors, including redox proteins, aconitase-like dehydratases, radical S-adenosylmethionine enzymes, and RNA polymerase (9, 13, 18, 32). Methanogenic archaea are obligate anaerobes, and many heterotrophic archaea grow anaerobically, indicating that oxidative stress has not limited the proliferation of iron-sulfur proteins in these lineages. Archaea must have a mechanism to assemble Fe-S clusters, but many members lack homologs of the known bacterial and eukaryotic Nif or Isc systems, suggesting that an alternative system is present (see Table S1 in the supplemental material) (14, 15).Some euryarchaea have homologs of the bacterial genes iscS (encoding cysteine desulfurase) and iscU (encoding a scaffold protein). Yet many other archaea, including the euryarchaea Pyrococcus furiosus, Methanocaldococcus jannaschii, and Methanococcus maripaludis, plus most crenarchaea, either lack a homologous cysteine desulfurase gene or have no homologs of A-type or U-type scaffold genes. Due to their sulfide-rich environments, it is reasonable that the anaerobic archaea may use an inorganic sulfur source to assemble Fe-S clusters. Most archaeal genome sequences do carry homologs of the sufBC genes that are part of the alternative Suf system for Fe-S cluster biosynthesis (36). Biochemical studies have shown that SufC is an ABC-type ATPase and that SufB is a persulfide acceptor that may act as a site for Fe-S cluster assembly (20). The SufB and SufC proteins interact, and SufB stimulates the ATPase activity of SufC. We hypothesize that the Archaea share a common mechanism for Fe-S cluster biosynthesis, supplemented with genes acquired by horizontal gene transfer in some lineages.A screen for Salmonella enterica bacteria defective in thiamine biosynthesis identified lesions in the apbC locus (28) that compromised Fe-S metabolism (33). An abpC mutant cannot grow with tricarballylate as a carbon and energy source, which may be due to a defect in assembling or repairing [4Fe-4S] clusters in the membrane-bound TcuB protein (21, 22). ApbC is a 40-kDa cytoplasmic protein with Walker A and B nucleotide-binding domains and two conserved carboxy-terminal cysteine residues separated by two amino acids (Cys-X-X-Cys). Mutational analyses have shown that ApbC proteins with directed changes in the Cys-X-X-Cys or Walker A motifs are not active in vivo (6). Suppressor analysis allowed the conclusion that a degree of functional redundancy between ApbC and the Fe-S scaffold protein IscU exists (4, 38). Although purified ApbC does not contain iron or sulfur, biochemical studies showed that ApbC can bind an Fe-S cluster and rapidly transfer it to an apoprotein (5).It is thought that in eukaryotes, Fe-S clusters are assembled by the mitochondrial iron-sulfur cluster (ISC) system (23). The clusters are transported into the cytosol and delivered by the cytosolic iron-sulfur protein assembly system. Two components of this system, Nbp35 and Cfd1, are homologs of bacterial ApbC (Fig. ​(Fig.1)1) and act as intermediate Fe-S cluster-trafficking proteins in the cytosol (16, 27, 30). Electron paramagnetic resonance, Mössbauer, and absorbance spectra of the Saccharomyces cerevisiae, human, and Arabidopsis Nbp35 holoproteins suggest that these holoproteins form dimers with stable amino-terminal [4Fe-4S] clusters and a shared carboxy-terminal [4Fe-4S] cluster (10, 34).Open in a separate windowFIG. 1.A protein sequence alignment of bacterial, archaeal, and eukaryotic ApbC/Nbp35 homologs was constructed using the ClustalW program (version 1.83) (37). The sequence of the S. enterica serovar Typhimurium protein (ApbC; RefSeq accession no. NP_461098.1) is shown without the amino-terminal domain that is not homologous to the amino-terminal domains of the archaeal and eukaryotic proteins. The archaeal homologs are from S. solfataricus (SSO0460; accession no. NP_341994.1), P. furiosus (PF1145; accession no. NP_578874.1), Methanosarcina acetivorans (MA4246; accession no. NP_619111.1), M. jannaschii (MJ0283; accession no. NP_247256.1), and M. maripaludis (MMP0704; accession no. NP_987824.1). The two paralogs from S. cerevisiae are Nbp35 (accession no. NP_011424.1) and Cfd1 (accession no. NP_012263.1). Conserved amino acid residues are shown in white on a black background. Similar residues are shown in black on a gray background. The four conserved amino-terminal cysteine residues shared by the MMP0704 and Nbp35p proteins are boxed. Asterisks above the sequences indicate MMP0704 residues replaced by mutagenesis in this study. A vertical bar indicates the N termini of the truncated proteins MJ0283(19-290) and MMP0704(20-289).Archaeal homologs of bacterial ApbC and eukaryotic Nbp35 are underannotated as nucleotide-binding proteins or misannotated as cobyrinic acid a,c-diamide synthases in sequence databases. The hallmarks of the Nbp35 sequences are an amino-terminal ferredoxin-like domain, an ATP-binding motif, and two conserved carboxy-terminal cysteine residues that are believed to bind an Fe-S cluster. The amino-terminal ferredoxin-like domain is absent in the ApbC family of proteins. The ApbC and Nbp35 proteins belong to a large superfamily of P-loop-containing nucleoside triphosphate hydrolases that also includes the bacterial MinD and CooC proteins. The M. maripaludis MMP0704 protein shows approximately 40% amino acid identity to both the S. enterica ApbC and S. cerevisiae Nbp35 proteins (Fig. ​(Fig.1).1). However, the MMP0704 protein also shows 30% sequence identity to two paralogous proteins from M. maripaludis. The genome sequence of M. maripaludis encodes at least nine paralogs, although only the MMP0704 protein contains the conserved cysteine residues found in most ApbC/Nbp35 proteins.The experiments described herein identified the first archaeal proteins that form functional Fe-S carrier proteins. The apbC/NBP35 homologs from M. maripaludis (MMP0704), M. jannaschii (MJ0283), and Sulfolobus solfataricus (SSO0460) allowed an S. enterica strain with an apbC null mutation to grow on tricarballylate. Genetic studies showed that the Walker A box and at least one cysteine residue from the Cys-X-X-Cys motif were required for in vivo functionality. The unique amino-terminal ferredoxin-like domains of the MMP0704 and MJ0283 proteins were not required. Purified MMP0704 proteins bound Fe-S clusters. Orthologs of ApbC/Npb35 proteins were found in all of the available genomes of free-living archaea, identifying this protein family as an ancient part of the Fe-S assembly system that evolved before the divergence of Archaea and Eukarya.     

Semiquantitative Performance and Mechanism Evaluation of Carbon Nanomaterials as Cathode Coatings for Microbial Fouling Reduction

In this study, we examine bacterial attachment and survival on a titanium (Ti) cathode coated with various carbon nanomaterials (CNM): pristine carbon nanotubes (CNT), oxidized carbon nanotubes (O-CNT), oxidized-annealed carbon nanotubes (OA-CNT), carbon black (CB), and reduced graphene oxide (rGO). The carbon nanomaterials were dispersed in an isopropyl alcohol-Nafion solution and were then used to dip-coat a Ti substrate. Pseudomonas fluorescens was selected as the representative bacterium for environmental biofouling. Experiments in the absence of an electric potential indicate that increased nanoscale surface roughness and decreased hydrophobicity of the CNM coating decreased bacterial adhesion. The loss of bacterial viability on the noncharged CNM coatings ranged from 22% for CB to 67% for OA-CNT and was dependent on the CNM dimensions and surface chemistry. For electrochemical experiments, the total density and percentage of inactivation of the adherent bacteria were analyzed semiquantitatively as functions of electrode potential, current density, and hydrogen peroxide generation. Electrode potential and hydrogen peroxide generation were the dominant factors with regard to short-term (3-h) bacterial attachment and inactivation, respectively. Extended-time electrochemical experiments (12 h) indicated that in all cases, the density of total deposited bacteria increased almost linearly with time and that the rate of bacterial adhesion was decreased 8- to 10-fold when an electric potential was applied. In summary, this study provides a fundamental rationale for the selection of CNM as cathode coatings and electric potential to reduce microbial fouling.     

New Selective Grass Herbicides and Their Hydrolytic Properties as Pro-herbicides

New derivatives of pyridinyloxyphenoxypropionic acid were synthesizd, which have a keto group in their ester-alkyl structures. Some acylmethyl esters, e.g., acetylmethyl ester, hydroxyacetylmethyl ester and diazoacetylmethyl ester, were found to show outstanding herbicidal activity against grass weeds. A study of the hydrolysis rate of esters with regard to their pro-herbicide action is also discussed.     

A New Bithiazole Derivative with Intercalative Properties

Abstract

In the course of studies related to new molecules with intercalative properties, we have been led to design and synthesize a bithiazole derivative, namely the 2-phenyl-6- [2′-(4′-(ethoxy-carbony1)thiazoly1)] thiazolo[3,2-b] [1,2,4]triazole (PETT). Its interaction with calf thymus DNA was studied using thermal denaturation and viscometry. Our results set in evidence that PETT acts as an intercalator, giving Δ Tm, elongation and unwinding of DNA comparable to the values obtained for daunorubicin. The discrepancy between the data presented herein and those precedently obtained for bleomycin and bleomycin models provide evidence that these bithiazole derivatives interact differently with DNA.     

New Inhibitor of Phosphodiesterase with Anti-bronchoconstrictor Properties

IN earlier work on the synthesis of isosteres of purine and in particular of theophylline (I), a series of triazolopyrimidines was found which could protect guinea-pigs against histamine bronchospasm. One compound (II) was also found to be active in certain cases of human asthma but it was found to be of no practical value because it had a tendency to cause nausea and vomiting. But because there is at present no adequate treatment for asthma, we undertook to prepare related substances which retain the activity but which are free of the defect: Thus we produced 3-acetamido-6-methyl-8-n-propyl-s-triazolo [4,3a] pyrazine (III; R=CO.Me;I.C.I.58,301 (ref. 3)). This substance is only in the early stages of clinical study in man, but we report here the chemical, biological and biochemical properties that led to its selection.