The structure of the endospore cell wall peptidoglycan of Bacillus subtilis has been examined. Spore peptidoglycan was produced by the development of a method based on chemical permeabilization of the spore coats and enzymatic hydrolysis of the peptidoglycan. The resulting muropeptides which were >97% pure were analyzed by reverse-phase high-performance liquid chromatography, amino acid analysis, and mass spectrometry. This revealed that 49% of the muramic acid residues in the glycan backbone were present in the ␦-lactam form which occurred predominantly every second muramic acid. The glycosidic bonds adjacent to the muramic acid ␦-lactam residues were resistant to the action of muramidases. Of the muramic acid residues, 25.7 and 23.3% were substituted with a tetrapeptide and a single L-alanine, respectively. Only 2% of the muramic acids had tripeptide side chains and may constitute the primordial cell wall, the remainder of the peptidoglycan being spore cortex. The spore peptidoglycan is very loosely cross-linked at only 2.9% of the muramic acid residues, a figure approximately 11-fold less than that of the vegetative cell wall. The peptidoglycan from strain AA110 (dacB) had fivefold-greater cross-linking (14.4%) than the wild type and an altered ratio of muramic acid substituents having 37.0, 46.3, and 12.3% ␦-lactam, tetrapeptide, and single L-alanine, respectively. This suggests a role for the DacB protein (penicillin-binding protein 5*) in cortex biosynthesis. The sporulationspecific putative peptidoglycan hydrolase CwlD plays a pivotal role in the establishment of the mature spore cortex structure since strain AA107 (cwlD) has spore peptidoglycan which is completely devoid of muramic acid ␦-lactam residues. Despite this drastic change in peptidoglycan structure, the spores are still stable but are unable to germinate. The role of ␦-lactam and other spore peptidoglycan structural features in the maintenance of dormancy, heat resistance, and germination is discussed. Dormant bacterial endospores formed by the genera Bacillus and Clostridium are the most resistant living structures known and are able to survive thousands if not millions of years (9). During the quiescent state, spores exhibit high-level resistance to many treatments, including heat, UV light, desiccation, and the action of deleterious chemicals. As a result of their resistance properties, spores are able to survive many food preservation and pasteurization procedures and thus cause huge problems to the food industry (11).Endospores are characterized by a relatively dehydrated protoplast encased in integument layers (20). The most prominent of the integuments are the spore coat layers which determine the physical properties of the spore surface and are responsible for resistance to enzymatic assault (56). However, the spore coats are not involved in the maintenance of dormancy and heat resistance (56). Between the spore coats and the protoplast membrane is a thick layer of bacterial peptidoglycan, consisting of two sublayers. Innermost is the ...

Recommendations about structuring proteomic biomarker studies should increase the probability that such markers will be clinically useful.

Indazolium trans-[tetrachlorobis(1H-indazole)ruthenate(III)] (KP1019) shows particular promise as an antitumour agent against colorectal cancer. It is known that KP1019 reacts with human serum proteins, whereby the major amount binds to albumin (present in large excess) and a smaller amount to transferrin. It has been hypothesised that transferrin-mediated uptake by transferrin receptor expressing tumour cells may in part explain the apparent tumour selectivity of this compound. Circular dichroism spectroscopy and electrospray ionisation mass spectrometry studies demonstrate that two equivalents of KP1019 bind specifically to human apotransferrin, while additional amounts of the ruthenium complex bind unspecifically. Uptake studies in the transferrin receptor-expressing human colon carcinoma cell line SW480 revealed a higher cellular accumulation of KP1019 in comparison to a KP1019-transferrin adduct (2:1), while the uptake of a KP1019-Fe(III)-transferrin conjugate (1:0.3:1) significantly exceeded that of KP1019, suggesting that iron binding is necessary to obtain a protein conformation which favours recognition by the transferrin receptors on the cell surface. Our study showed that KP1019 is transported into the cell by both transferrin-independent and transferrin-dependent mechanisms. Transferrin-mediated uptake is more efficient when transferrin is saturated with iron to a physiological degree (y30%). Cell fractionation experiments demonstrated that after a 2 h treatment of human colon cancer cells with 10 mM KP1019 on average 55% of the intracellular ruthenium is located in the cellular nucleus, while 45% remain in the cytosol and other cellular components.

The composition and fine structure of the vegetative cell wall peptidoglycan from Bacillus subtilis were determined by analysis of its constituent muropeptides. The structures of 39 muropeptides, representing 97% of the total peptidoglycan, were elucidated. About 99% analyzed muropeptides in B. subtilisvegetative cell peptidoglycan have the free carboxylic group of diaminopimelic acid amidated. Anhydromuropeptides and products missing a glucosamine at the nonreducing terminus account for 0.4 and 1.5%, respectively, of the total muropeptides. These two types of muropeptides are suggested to end glycan strands. An unexpected feature of B. subtilis muropeptides was the occurrence of a glycine residue in position 5 of the peptide side chain on monomers or oligomers, which account for 2.7% of the total muropeptides. This amount is, however, dependent on the composition of the growth media. Potential attachment sites for anionic polymers to peptidoglycan occur on dominant muropeptides and account for 2.1% of the total. B. subtilis peptidoglycan is incompletely digested by lysozyme due to de-N-acetylation of glucosamine, which occurs on 17.3% of muropeptides. The cross-linking index of the polymer changes with the growth phase. It is highest in late stationary phase, with a value of 33.2 or 44% per muramic acid residue, as determined by reverse-phase high-pressure liquid chromatography or gel filtration, respectively. Analysis of the muropeptide composition of a dacA (PBP 5) mutant shows a dramatic decrease of muropeptides with tripeptide side chains and an increase or appearance of muropeptides with pentapeptide side chains in monomers or oligomers. The total muropeptides with pentapeptide side chains accounts for almost 82% in thedacA mutant. This major low-molecular-weight PBP (dd-carboxypeptidase) is suggested to play a role in peptidoglycan maturation.

Dosage of chemotherapeutic drugs is a tradeoff between efficacy and side-effects. Liposomes are nanocarriers that increase therapy efficacy and minimize side-effects by delivering otherwise difficult to administer therapeutics with improved efficiency and selectivity. Still, variabilities in liposome preparation require assessing drug encapsulation efficiency at the single liposome level, an information that, for non-fluorescent therapeutic cargos, is inaccessible due to the minute drug load per liposome. Photothermal induced resonance (PTIR) provides nanoscale compositional specificity, up to now, by leveraging an atomic force microscope (AFM) tip contacting the sample to transduce the sample’s photothermal expansion. However, on soft samples (e.g. liposomes) PTIR effectiveness is reduced due to the likelihood of tip-induced sample damage and inefficient AFM transduction. Here, individual liposomes loaded with the chemotherapeutic drug cytarabine are deposited intact from suspension via nES-GEMMA (nano-electrospray gas-phase electrophoretic mobility molecular analysis) collection and characterized at the nanoscale with the chemically-sensitive PTIR method. A new tapping-mode PTIR imaging paradigm based on heterodyne detection is shown to be better adapted to measure soft samples, yielding cytarabine distribution in individual liposomes and enabling classification of empty and drug-loaded liposomes. The measurements highlight PTIR capability to detect ≈ 103 cytarabine molecules (≈ 1.7 zmol) label-free and non-destructively.

The composition and structure of peptidoglycan (murein) extracted from the extreme thermophilic eubacterium Thermus thermophilus HB8 are presented. The structure of 29 muropeptides, accounting for more than 85% of total murein, is reported.

The peptidoglycan (murein) of Helicobacter pylori has been investigated by high-performance liquid chromatography and mass spectrometric techniques. Murein from H. pyloricorresponded to the A1γ chemotype, but the muropeptide elution patterns were substantially different from the one forEscherichia coli in that the former produced high proportions of muropeptides with a pentapeptide side chain (about 60 mol%), with Gly residues as the C-terminal amino acid (5 to 10 mol%), and with (1→6)anhydro-N-acetylmuramic acid (13 to 18 mol%). H. pylori murein also lacks murein-bound lipoprotein, trimeric muropeptides, and (l-d) cross-linked muropeptides. Cessation of growth and transition to coccoid shape triggered an increase inN-acetylglucosaminyl-N-acetylmuramyl–l-Ala–d-Glu (approximately 20 mol%), apparently at the expense of monomeric muropeptides with tri- and tetrapeptide side chains. Muropeptides with (1→6)anhydro-muramic acid and with Gly were also more abundant in resting cells.

In this work we present the characterization of PAMAM dendrimers from generation two (G2) up to ten (G10) with a focus on the G5-G10 dendrimers with matrix-assisted laser desorption/ionization linear mass spectrometry (MALDI-MS) and nanoelectrospray gas-phase electrophoretic mobility molecular analysis (nES-GEMMA). For the first time the molecular masses of high-mass dendrimers G8-G10 were determined by MALDI-MS and nES-GEMMA, techniques which are based on different physicochemical principles. Obtained experimental data allows the determination of the molecular mass (up to 580 kDa with a precision below (0.9%), of the spherical size (from 3.3 to 14.0 nm with a precision of (0.2 nm) and the calculation of their densities. Amounts in the nanogramm range were sufficient for an analysis that could be performed within several minutes. The results based on these methods for high-generation dendrimers exhibited an excellent correlation and were compared with published data using techniques based on different principles.