Entry Of Small Molecules Research Articles

Kwaśna modyfikacja gruzińskiego heulandytu naturalnego

Acid treatment is a powerful tool for improving the performance of natural zeolites, and the purpose of our work was to study chemical composition, structure and properties of acid-treated heulandite from the Tedzami-Dzegvi deposit. Samples of heulandite-containing tuff from the Rkoni plot with zeolite phase content up to 90%, consisting of heulandite and chabazite in a ratio of 8:1, and having chemical composition described by empirical formula |Na0.25K0.06Ca0.19Mg0.15)|[AlSi3.6O9.2].3H2O were treated with hydrochloric acid solutions with concentration up to 2.0 N. It was established that acid treatment leads to significant dealumination (the molar ratio of Si/Al increases from 3.6 to 9.5) and decationization (the total charge per aluminium atom decreases from 1 to 0.68), sodium and magnesium are mainly leached, calcium and potassium does not take part in the decationization process. Powder X-ray diffraction patterns show that hydrochloric acid solutions with a concentration up to 2.0 N do not lead to amorphization of the zeolite microporous crystal structure, but can gradually dissolve it. The adsorption of water vapor indicates the availability of micropores for the entry of small polar molecules, benzene adsorption shows a slight increase of hydrophobicity of the surface as a result of acid treatment. Nitrogen adsorption-desorption isotherms show acid-mediated sharp increase of adsorption in micropores and of the surface area, as well as changes in the mesoporous system, leading to the prevalence of pores with a diameter of 3 – 10 nm. The concentration of dilute solutions of hydrochloric acid is determined, which provides availability of micropores for large ions and nonpolar molecules, but at which dealumination is insignificant and ion-exchange capacity remains at a sufficient level. Materials obtained by acid treatment of heulandite can be used as adsorbents, ion exchangers, and carriers of biologically active substances and metal ions.

Ag nanocubes/cationic cellulose nanofibers/polyacrylamide hydrogel as a SERS platform for in situ separation, rapid enrichment and sensitive detection of anticancer drugs in plasma

Surface Enhanced Raman Spectroscopy (SERS) is always challenging for the detection of complex samples due to the pre-processing issues. Therefore, there is a need to develop a high-performance SERS platform that can avoid pre-processing problems and simultaneously enrich target molecules. In this work, we encapsulated cationic cellulose nanofibers (CCNF) stabilized Ag NCs in polyacrylamide hydrogels via photo-crosslinking to construct a high-performance SERS platform for rapid separation, concentration, and enrichment without pretreatment. Ag NCs/CCNF/PAAM hydrogels have good swelling and shrinking properties, which are favorable for enriching the substances to be measured and improving the sensitivity of the SERS platform. The limit of detection (LOD) was as low as 9.33 × 10−11 M for the detection of the dye molecule R6G. The hydrogel SERS platform has excellent selectivity. Its pores can be adjusted to allow the entry of small hydrophilic molecules and the exclusion of large molecules and hydrophobic substances in complex samples. Therefore, we performed the SERS assay for hydrophilic anticancer drugs in the plasma environment, where the hydrogel allowed doxorubicin hydrochloride (DOX) and 6-thioguanine (6-TG) to enter into the interior of the hydrogel, while hydrophobic substances such as plasma proteins and lipids in plasma were excluded from the hydrogel. As a validation, we performed spiking experiments on plasma from five lung cancer patients, and the assay was sensitive and reliable, with recoveries ranging from 89.0% to 111.0%. Based on the above results, Ag NCs/CCNF/PAAM hydrogels are promising for the real-time detection of drugs in clinical blood.

Artificial Siderophores with a Trihydroxamate‐DOTAM Scaffold Deliver Iron and Antibiotic Cargo into the Bacterial Pathogen Escherichia coli

AbstractInfections with multidrug‐resistant Gram‐negative bacteria constitute a silent pandemic threat that is increasing globally. A major technical and scientific hurdle hampering the development of efficient antibiotics against Gram‐negative species is the low permeability of their outer membrane that prevents the entry of most small molecules into the cells. This can be overcome by targeting active iron transport systems of the pathogens in a Trojan‐Horse strategy that makes use of drug‐loaded artificial siderophores. While we utilized catechols as iron‐binding motifs in previous work, this study reports the design, synthesis and characterization of siderophores with a DOTAM scaffold that was substituted with three hydroxamate arms allowing for a hexacoordination of iron. Their iron‐chelating capabilities were shown colorimetrically, and the ability of compound 1 to deliver iron into Escherichia coli in a chelation‐specific manner was proven by a growth recovery assay. A covalent siderophore‐ciprofloxacin conjugate exerted antibiotic effects against E. coli, albeit it was less potent than the free drug. The study qualifies artificial DOTAM siderophores with hydroxamate binders as scaffolds for bacterial Trojan Horses. This contribution for honoring my mentor Helmut Schwarz echoes two motifs of my work with him: Hydroxylamin, the topic of my first paper ever, and the fascinating properties of iron ions, studied in the gas phase during my Ph.D. Thesis, became a core subject of our current chemical biology research on antiinfectives.

Selective Binding and Elution of Aptamers for Pesticides Based on Sol-Gel-Coated Nanoporous Anodized Aluminum Oxide Membrane.

Sol-gel-based mesopores allow the entry of target small molecules retained in their cavity and aptamers to bind to target molecules. Herein, sol-gel-based materials are applied to screen-selective aptamers for small molecules, such as pesticides. To enhance the efficiency of aptamer screening using a sol-gel, it is necessary to increase the binding surface. In this study, we applied the sol-gel to an anodized aluminum oxide (AAO) membrane, and the morphological features were observed via electron microscopy after spin coating. The binding and elution processes were conducted and confirmed by fluorescence microscopy and polymerase chain reaction. The sol-gel coating on the AAO membrane formed a hollow nanocolumn structure. A diazinon-binding aptamer was bound to the diazinon-containing sol-gel-coated AAO membrane, and the bound aptamer was effectively retrieved from the sol-gel matrix by thermal elution. As a proof of concept, a sol-gel-coated AAO disc was mounted on the edge of a pipette tip, and the feasibility of the prepared platform for the systematic evolution of ligands by exponential enrichment (SELEX) of the aptamer binding was also confirmed. The proposed approach will be applied to an automated SELEX cycle using an automated dispenser, such as a pipetting robot, in the near future.

HIV Tat-mediated induction of autophagy regulates the disruption of ZO-1 in brain endothelial cells

ABSTRACT The blood-brain barrier (BBB) is a tight barrier that is critical for preventing the entry of pathogens and small molecules into the brain. HIV protein Tat (Tat) is known to disrupt the tight junctions of the BBB. Autophagy is an intracellular process that involves degradation and recycling of damaged organelles to the lysosome and has recently been implicated in the BBB disruption. The role of autophagy in Tat-mediated BBB disruption, however, remains elusive. Herein we hypothesized that Tat induces endothelial autophagy resulting in decreased expression of the tight junction protein ZO-1 leading to breach of the BBB. In this study, we demonstrated that exposure of human brain microvessel endothelial cells (HBMECs) to Tat resulted in induction of autophagy in a dose- and time-dependent manner, with upregulation of BECN1/Beclin 1, ATG5 and MAP1LC3B proteins and a concomitant downregulation of the tight junction protein ZO-1 ultimately leading to increased endothelial cell monolayer paracellular permeability in an in vitro BBB model. Pharmacological and genetic inhibition of autophagy resulted in the abrogation of Tat-mediated induction of MAP1LC3B with a concomitant restoration of tight junction proteins, thereby underscoring the role of autophagy in Tat-mediated breach of the BBB. Additionally, our data also demonstrated that Tat-mediated induction of autophagy involved PELI1/K63-linked ubiquitination of BECN1. Increased autophagy and decreased ZO-1 was further recapitulated in microvessels isolated from the brains of HIV Tg26 mice as well as the frontal cortex of HIV-infected autopsied brains. Overall, our findings identify autophagy as an important mechanism underlying Tat-mediated disruption of the BBB.

Biomimetic drug-delivery systems for the management of brain diseases.

Acting as a double-edged sword, the blood-brain barrier (BBB) is essential for maintaining brain homeostasis by restricting the entry of small molecules and most macromolecules from blood. However, it also largely limits the brain delivery of most drugs. Even if a drug can penetrate the BBB, its accumulation in the intracerebral pathological regions is relatively low. Thus, an optimal drug-delivery system (DDS) for the management of brain diseases needs to display BBB permeability, lesion-targeting capability, and acceptable safety. Biomimetic DDSs, developed by directly utilizing or mimicking the biological structures and processes, provide promising approaches for overcoming the barriers to brain drug delivery. The present review summarizes the biological properties and biomedical applications of the biomimetic DDSs including the cell membrane-based DDS, lipoprotein-based DDS, exosome-based DDS, virus-based DDS, protein template-based DDS and peptide template-based DDS for the management of brain diseases.

Directing Traffic: Halogen-Bond-Mediated Membrane Transport.

The plasma membrane regulates the transport of molecules into the cell. Small hydrophobic molecules can diffuse directly across the lipid bilayer. However, larger molecules require specific transporters for their entry into the cell. Regulating the cellular entry of small molecules and proteins is a challenging task. The introduction of halogen, particularly iodine, to small molecules and proteins is emerging to be a promising strategy to improve the cellular uptake. Recent studies reveal that a simple substitution of hydrogen atom with iodine not only increases the cellular uptake, but also regulates the membrane transport. The strong halogen-bond-forming ability of iodine atoms plays a crucial role in the transport and the introduction of iodine may provide an efficient strategy for studying membrane activity and cellular functions and improving the delivery of therapeutic agents. This Concept article does not provide a comprehensive picture of membrane transport but highlights halogen-substitution as a novel strategy for understanding and regulating the cell-membrane traffic.

Unveiling the Modulating Role of Extracellular pH in Permeation and Accumulation of Small Molecules in Subcellular Compartments of Gram-negative Escherichia coli using Nonlinear Spectroscopy.

Quantitative evaluation of small molecule permeation and accumulation in Gram-negative bacteria is important for drug development against these bacteria. While these measurements are commonly performed at physiological pH, Escherichia coli and many other Enterobacteriaceae infect human gastrointestinal and urinary tracts, where they encounter different pH conditions. To understand how external pH affects permeation and accumulation of small molecules in E. coli cells, we apply second harmonic generation (SHG) spectroscopy using SHG-active antimicrobial compound malachite green as the probe molecule. Using SHG, we quantify periplasmic and cytoplasmic accumulations separately in live E. coli cells, which was never done before. Compartment-wise measurements reveal accumulation of the probe molecule in cytoplasm at physiological and alkaline pH, while entrapment in periplasm at weakly acidic pH and retention in external solution at highly acidic pH. Behind such disparity in localizations, up to 2 orders of magnitude reduction in permeability across the inner membrane at weakly acidic pH and outer membrane at highly acidic pH are found to play key roles. Our results unequivocally demonstrate the control of external pH over entry and compartment-wise distribution of small molecules in E. coli cells, which is a vital information and should be taken into account in antibiotic screening against E. coli and other Enterobacteriaceae members. In addition, our results demonstrate the ability of malachite green as an excellent SHG-indicator of changes of individual cell membrane and periplasm properties of live E. coli cells in response to external pH change from acidic to alkaline. This finding, too, has great importance, as there is barely any other molecular probe that can provide similar information.

Transporter-Mediated Nuclear Entry of Jasmonoyl-Isoleucine Is Essential for Jasmonate Signaling

To control gene expression by directly responding to hormone concentrations, both animal and plant cells have exploited comparable mechanisms to sense small-molecule hormones in nucleus. Whether nuclear entry of these hormones is actively transported or passively diffused, as conventionally postulated, through the nuclear pore complex, remains enigmatic. Here, we identified and characterized a jasmonate transporter in Arabidopsis thaliana, AtJAT1/AtABCG16, which exhibits an unexpected dual localization at the nuclear envelope and plasma membrane. We show that AtJAT1/AtABCG16 controls the cytoplasmic and nuclear partition of jasmonate phytohormones by mediating both cellular efflux of jasmonic acid (JA) and nuclear influx of jasmonoyl-isoleucine (JA-Ile), and is essential for maintaining a critical nuclear JA-Ile concentration to activate JA signaling. These results illustrate that transporter-mediated nuclear entry of small hormone molecules is a new mechanism to regulate nuclear hormone signaling. Our findings provide an avenue to develop pharmaceutical agents targeting the nuclear entry of small molecules.

Structural Insights into Outer Membrane Permeability of Acinetobacter baumannii

Bacterial resistance against antibiotics is an increasing global health problem. In Gram-negative bacteria the low permeability of the outer membrane (OM) is a major factor contributing to resistance, making it important to understand channel-mediated small-molecule passage of the OM. Acinetobacter baumannii has five Occ (OM carboxylate channel) proteins, which collectively are of major importancefor the entry of small molecules. To improve our understanding of the OM permeability of A.baumannii, we present here the X-ray crystal structures of four Occ proteins, renamed OccAB1 to OccAB4. In addition we have carried out a biochemical and biophysical characterization using electrophysiology and liposome swelling experiments, providing information on substrate specificities. We identify OccAB1 as having the largest pore of the Occ proteins with corresponding high rates of small-molecule uptake, and we suggest that the future design of efficient antibiotics should focus on scaffolds that can permeate efficiently through the OccAB1 channel.

Small-Molecule Transport by CarO, an Abundant Eight-Stranded β-Barrel Outer Membrane Protein from Acinetobacter baumannii

Outer membrane (OM) β-barrel proteins composed of 12–18 β-strands mediate cellular entry of small molecules in Gram-negative bacteria. Small OM proteins with barrels of 10 strands or less are not known to transport small molecules. CarO (carbapenem-associated outer membrane protein) from Acinetobacter baumannii is a small OM protein that has been implicated in the uptake of ornithine and carbapenem antibiotics. Here we report crystal structures of three isoforms of CarO. The structures are very similar and show a monomeric eight-stranded barrel lacking an open channel. CarO has a substantial extracellular domain resembling a glove that contains all the divergent residues between the different isoforms. Liposome swelling experiments demonstrate that full-length CarO and a “loop-less” truncation mutant mediate small-molecule uptake at low levels but that they are unlikely to mediate passage of carbapenem antibiotics. These results are confirmed by biased molecular dynamics simulations that allowed us to quantitatively model the transport of selected small molecules.

Molecular water in nominally unhydrated carbonated hydroxylapatite: The key to a better understanding of bone mineral

Despite numerous analytical studies, the exact nature of the mineral component of bone is not yet totally defined, even though it is recognized as a type of carbonated hydroxylapatite. The present study addresses the hydration state of bone mineral through Raman spectroscopic and thermogravimetric analysis of 56 samples of carbonated apatite containing from 1 to 17 wt% CO 3 , synthesized in H 2 O or D 2 O. Focus is on the relation between the concentration of molecular water (as distinguished from hydroxyl ions) and the concentration of carbonate in the apatite. Raman spectra confirm the presence of molecular water as part of the crystalline structure in all the aqueously precipitated carbonated apatites. TGA results quantitatively document that, regardless of the concentration of carbonate in the structure, all hydroxylapatites contain ~3 wt% of structurally incorporated water in addition to multiple wt% adsorbed water. We spectroscopically confirmed that natural bone mineral also contains structurally incorporated molecular H 2 O based on independent analyses of bone by means of spectral stripping (subtracting the spectrum of collagen from that of bone) and chemical stripping (chemically removing the collagen content of bone prior to analysis). Taken together, the above data support a model in which water molecules densely populate the apatite channels regardless of the abundance of hydroxyl vacancies. We hypothesize that water molecules keep the apatite channels stable even when 80% of the hydroxyl sites are vacant (typical in bone), hinder carbonate ions from substituting for hydroxyl ions in the channels, and help regulate chemical access to the channels (e.g., ion exchange, entry of small molecules). Our results show that bone apatite is not a “flawed hydroxylapatite,” but instead a definable mineralogical entity, a combined hydrated-hydroxylated calcium phosphate phase of the form Ca 10−x [(PO 4 ) 6−x (CO 3 ) x ](OH) 2−x ·nH 2 O, where n ~ 1.5. Water is therefore not an accidental, but rather an essential, component of bone mineral and other natural and synthetic low-temperature carbonated apatite phases.

143 Porous three-dimensional DNA crystals as biomolecular containers for catalysis

The major goal of DNA nanotechnology has been the design and manufacture of artificial DNA structures for technological uses. The porous three-dimensional DNA crystals have been proposed as macromolecular scaffolds for host–guest structure determination, as molecular sieves and as molecular containers for catalysis. By using fluorescence dequenching technique, we have demonstrated that a protein enzyme adsorbed in a designed three-dimensional DNA crystal is capable of performing catalysis. The axially distinct aperture sizes in the crystal design allowed us to improve the enclosing of the enzyme with a protective protein-based “coating” cross-linked over the crystal surface. This coating allows entry and exit of small molecules through the crystal while restricting enzymes inside the crystal. This enzyme-enclosed DNA crystal is capable of performing multiple cycles of catalysis and it retained its enzymatic activity over numerous days after being protein-coated. The concepts of the enzyme-enclosed DNA crystal and the unique protein coating technique provide possibilities to the development of enzyme replacement therapies and biodegradable solid-state catalysts and biosensors.

Inducible and reversible breaching of the blood brain barrier by RNAi

Sequence-specific knockdown of gene expression is a goal that has been long sought by both basic and clinical investigators. In this regard, the discovery of RNA interference (RNAi) in Caenorhabditis elegans was immediately recognized as a potential breakthrough for studying gene function (Fire et al, 1998). These findings demonstrated that double-stranded (ds)RNAs are triggers for sequence-specific, post-transcriptional gene silencing via targeted degradation of messenger RNAs harbouring a complementary sequence to one of the two strands. Initially, it was thought that such post-transcriptional regulation of gene expression could not be achieved in mammalian systems due to the strong induction of interferon by dsRNAs. This potential restriction was short lived with the demonstration that endonuclease processed dsRNAs of 21–25 nucleotides in length, designated small interfering RNAs (siRNAs), were able to elicit sequence-specific degradation of mRNAs in mammalian cells without triggering interferon responses (Elbashir et al, 2001). These findings provided a huge impetus to develop RNAi as a therapeutic modality. The dream to selectively block the expression of deleterious proteins and treat formerly non-drugable diseases led to the rapid establishment of new biotech companies and branches of major pharmaceutical companies devoted to RNAi therapeutics.

A Single Amino Acid Substitution Makes WNK4 Susceptible to SB 203580 and SB 202190

Regulation of the SLC12 family of membrane transporters including NCCT involves a scaffold of interacting proteins including the STE 20 kinase SPAK and the WNK kinases, WNK 1 and WNK 4, which are mutated in the hypertensive syndrome of pseudohypoaldosteronism type 2 (PHAII). WNK4 regulates NCCT by affecting forward trafficking to the surface membrane. Studies in Xenopus using kinase dead WNK4 site mutants have produced inconsistent results with regard to the necessity of kinase function for NCCT regulation. Dynamic inhibition of WNK4 by small molecules may bring clarity to this issue however WNK4 is naturally resistant to commercial MAP kinase inhibitors owing to steric constraints prohibiting entry of small molecules to the active site. Using an approach similar to that used in p38 and ERK, we show that a single substitution in WNK4 (T261G) dramatically enhances its susceptibility to the inhibitors SB 202190 and SB 203580.

What is (Still not) Known of the Mechanism by Which Electroporation Mediates Gene Transfer and Expression in Cells and Tissues

Cell membranes can be transiently permeabilized under application of electric pulses. This treatment allows hydrophilic therapeutic molecules, such as anticancer drugs and DNA, to enter into cells and tissues. This process, called electropermeabilization or electroporation, has been rapidly developed over the last decade to deliver genes to tissues and organs, but there is a general agreement that very little is known about what is really occurring during membrane electropermeabilization. It is well accepted that the entry of small molecules, such as anticancer drugs, occurs mostly through simple diffusion after the pulse while the entry of macromolecules, such as DNA, occurs through a multistep mechanism involving the electrophoretically driven interaction of the DNA molecule with the destabilized membrane during the pulse and then its passage across the membrane. Therefore, successful DNA electrotransfer into cells depends not only on cell permeabilization but also on the way plasmid DNA interacts with the plasma membrane and, once into the cytoplasm, migrates towards the nucleus. The focus of this review is to describe the different aspects of what is known of the mechanism of membrane permeabilization and associated gene transfer and, by doing so, what are the actual limits of the DNA delivery into cells.

Structure of a conserved hypothetical protein SA1388 from S. aureus reveals a capped hexameric toroid with two PII domain lids and a dinuclear metal center

BackgroundThe protein encoded by the SA1388 gene from Staphylococcus aureus was chosen for structure determination to elucidate its domain organization and confirm our earlier remote homology based prediction that it housed a nitrogen regulatory PII protein-like domain. SA1388 was predicted to contain a central PII-like domain and two flanking regions, which together belong to the NIF3-like protein family. Proteins like SA1388 remain a poorly studied group and their structural characterization could guide future investigations aimed at understanding their function.ResultsThe structure of SA1388 has been solved to 2.0Å resolution by single wavelength anomalous dispersion phasing method using selenium anomalous signals. It reveals a canonical NIF3-like fold containing two domains with a PII-like domain inserted in the middle of the polypeptide. The N and C terminal halves of the NIF3-like domains are involved in dimerization, while the PII domain forms trimeric contacts with symmetry related monomers. Overall, the NIF3-like domains of SA1388 are organized as a hexameric toroid similar to its homologs, E. coli ybgI and the hypothetical protein SP1609 from Streptococcus pneumoniae. The openings on either side of the toroid are partially covered by trimeric "lids" formed by the PII domains. The junction of the two NIF3 domains has two zinc ions bound at what appears to be a histidine rich active site. A well-defined electron density corresponding to an endogenously bound ligand of unknown identity is observed in close proximity to the metal site.ConclusionSA1388 is the third member of the NIF3-like family of proteins to be structurally characterized, the other two also being hypothetical proteins of unknown function. The structure of SA1388 confirms our earlier prediction that the inserted domain that separates the two NIF3 domains adopts a PII-like fold and reveals an overall capped toroidal arrangement for the protein hexamer. The six PII-like domains form two trimeric "lids" that cap the central cavity of the toroid on either side and provide only small openings to allow regulated entry of small molecules into the occluded chamber. The presence of the electron density of the bound ligand may provide important clues on the likely function of NIF3-like proteins.

Toward the de novo design of a catalytically active helix bundle: a substrate-accessible carboxylate-bridged dinuclear metal center.

De novo design of proteins provides an attractive approach to uncover the essential features required for their functions. Previously, we described the design and crystal structure determination of a di-Zn(II) complex of "due-ferri-1" (DF1), a protein patterned after the diiron-dimanganese class of redox-active proteins [Lombardi, A.; Summa, C.; Geremia, S.; Randaccio, L.; Pavone, V.; DeGrado, W. F. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 6298-6305]. The overall structure of DF1, which contains a carboxylate-bridged dinuclear metal site, agrees well with the intended design. However, access to this dimetal site is blocked by a pair of hydrophobic leucine residues (L13 and L13'), which prevent facile entry of metal ions and small molecules. We have now taken the next step in the eventual construction of a catalytically active metalloenzyme by engineering an active site cavity into DF1 through the replacement of these two leucine residues with smaller residues. The crystal structure of the dimanganous form of L13A-DF1 indeed shows a substrate access channel to the dimetal center. In the crystal structure, water molecules and a ligating dimethyl sulfoxide molecule, which forms a monatomic bridge between the metal ions, occupy the cavity. Furthermore, the diferric form of a derivative of L13A-DF1, DF2, is shown to bind azide, acetate, and small aromatic molecules.

Water penetration and binding to ferric myoglobin.

Flash photolysis investigations of horse heart metmyoglobin bound with NO (Mb(3+)NO) reveal the kinetics of water entry and binding to the heme iron. Photodissociation of NO leaves the sample in the dehydrated Mb(3+) (5-coordinate) state. After NO photolysis and escape, a water molecule enters the heme pocket and binds to the heme iron, forming the 6-coordinate aquometMb state (Mb(3+)H2O). At longer times, NO displaces the H2O ligand to reestablish equilibrium. At 293 K, we determine a value k(w) approximately 5.7 x 10(6) s(-1) for the rate of H2O binding and estimate the H2O dissociation constant as 60 mM. The Arrhenius barrier height H(w) = 42 +/- 3 kJ/mol determined for H2O binding is identical to the barrier for CO escape after photolysis of Mb(2+)CO, within experimental uncertainty, consistent with a common mechanism for entry and exit of small molecules from the heme pocket. We propose that both processes are gated by displacement of His-64 from the heme pocket. We also observe that the bimolecular NO rebinding rate is enhanced by 3 orders of magnitude both for the H64L mutant, which does not bind water, and for the H64G mutant, where the bound water is no longer stabilized by hydrogen bonding with His-64. These results emphasize the importance of the hydrogen bond in stabilizing H2O binding and thus preventing NO scavenging by ferric heme proteins at physiological NO concentrations.

The outer parts of the mycobacterial envelope as permeability barriers.

The permeability of mycobacteria to substances in their environment is controlled by the properties of their envelopes. Two special features are important: an outer lipid barrier based on a monolayer of characteristic mycolic acids and a capsule-like coat of polysaccharide and protein. The mycolate layer prevents entry of small hydrophilic molecules, which obtain access to the cell by way of pore-forming proteins resembling porins of Gram-negative bacteria. More lipophilic molecules can diffuse through the lipid layer. The capsule probably impedes access by macromolecules; in intracellular pathogenic species it forms the electron-transparent zone that separates the bacterium from the membrane of the host phagosome. The structure of the outer lipid barrier seems common to all mycobacteria, fast- and slow-growing, but the capsule is more abundant in slow-growing species, a group which includes all the important mycobacterial pathogens. Mycobacteria secrete proteins into their environment, which are likely to be important in the pathogenesis of mycobacterial diseases. Knowledge of how these proteins, and the polysaccharides of the capsule, cross the outer lipid barrier is minimal at present. It is likely that proper knowledge of mycobacterial permeability will enable new approaches to treatment of mycobacterial disease.