Intracellular trafficking and subcellular distribution of a large array of HPMA copolymers

The basic physicochemical properties that determine the distribution and fate of synthetic macromolecules in living cells were characterized using fluorescently labeled HPMA (N-(2-hydroxypropyl)methacrylamide) copolymers. Twelve different classes of water-soluble copolymers were created by incorporating eight different functionalized comonomers. These comonomers possessed functional groups with positive or negative charges or contained short hydrophobic peptides. The copolymers were fractionated to create parallel "ladders" consisting of 10 fractions of narrow polydispersity with molecular weights ranging from 10 to 200 kDa. The intracellular distributions were characterized for copolymer solutions microinjected into the cytoplasm of cultured ovarian carcinoma cells. Even the highest molecular weight HPMA copolymers were shown to quickly and evenly diffuse throughout the cytoplasm and remain excluded from membrane-bound organelles, regardless of composition. The exceptions were the strongly cationic copolymers, which demonstrated a pronounced localization to microtubules. For all copolymers, nuclear entry was consistent with passive transport through the nuclear pore complex (NPC). Nuclear uptake was shown to be largely dictated by the molecular weight of the copolymers, however, detailed kinetic analyses showed that nuclear import rates were moderately, but significantly, affected by differences in comonomer composition. HPMA copolymers containing amide-terminated phenylalanine-glycine (FG) sequences, analogous to those found in the NPC channel protein, demonstrated a potential to regulate import to the nuclear compartment. Kinetic analyses showed that 15 kDa copolymers containing GGFG, but not those containing GGLFG, peptide pendant groups altered the size-exclusion characteristics of NPC-mediated nuclear import.     

Ants accelerate succession from mountain grassland towards spruce forest

Question: What is the role of mound‐building ants (Lasius flavus) in successional changes of a grassland ecosystem towards a spruce forest? Location: Slovenské Rudohorie Mountains, Slovakia; ca. 950 m a.s.l. near the Obrubovanec point (1020 m a.s.l.; 48°41′N, 19°39′E). Methods: Both chronosequence data along a successional gradient and temporal data from long‐term permanent plots were collected on ants, spruce establishment, and vegetation structure, together with additional data on spruce growth. Results: There are more spruce seedlings on ant mounds (4.72 m^?2) than in the surrounding vegetation (0.81 m^?2). Spruce seedlings grow faster on these mounds compared to surrounding areas. The first colonization wave of seedlings was rapid and probably occurred when grazing prevailed over mowing. Ant colony presence, mound volume, and plant species composition change along the successional gradient. Mounds become bigger when partly shaded but shrink in closed forest, when ant colonies disappear. Shade‐tolerant acidophylic species replace grassland plants both on the mounds and in surrounding areas. Conclusions: The massive occurrence of Lasius flavus anthills contributes to a runaway feedback process that accelerates succession towards forest. The effect of ants as ecosystem engineers is scale‐dependent: although they stabilize the system at the scale of an individual mound, they may destabilize the whole grassland system over a longer time scale if combined with changes in mowing regime.     

Multiplex PCR for detection of three exfoliative toxin serotype genes inStaphylococcus aureus

Rapid and specific detection of exfoliative toxin (ET)-producing Staphylococcus aureus strains by multiplex polymerase chain reaction (PCR) was used for identification of exfoliative toxin genes in a diverse set of 115 clinical S. aureus strains isolated in 14 Czech cities between 1998 and 2004. Fifty-nine wild-type ET-positive isolates of which 40 strains were the causative agents of toxic epidermolysis in neonates were classified into 4 PCR types. The genes coding for ETA, ETB or ETD were not detected in any of non-ET-producing isolates. The PCR method using the multiplex and specific primer set was shown to be reliable in rapid identification of the exfoliative toxin producing S. aureus and can be used as a convenient tool for hospital epidermolytic infection control.     

Synthesis of α-carboxyphosphinopeptides derived from norleucine

In the present study, we describe in detail the synthesis of a relatively rare class of phosphorus compounds, α-carboxyphosphinopeptides. We prepared several norleucine-derived α-carboxyphosphinic pseudopeptides of the general formula Nle-Ψ[PO(OH)]-Gly. These compounds could have important applications as transition state-mimicking inhibitors for methionine or leucine aminopeptidases or other enzymes. For the preparation of the key α-carboxyphosphinate protected precursors, we investigated, compared and improved two different synthetic methods described in literature: the Arbuzov reaction of a silylated N-protected phosphinic acid with a bromoacetate ester and the nucleophilic addition of a mixed O-methyl S-phenyl N-protected phosphonic acid or a methyl N-protected phosphonochloridate with tert-butyl lithioacetate. We also prepared two N-Fmoc protected synthons, Fmoc-Nle-Ψ[PO(OH)]-Gly-COOH and Fmoc-Nle-Ψ[PO(OAd)]-Gly-COOH, and demonstrated that these precursors are suitable building blocks for the solid-phase synthesis of α-carboxyphosphinopeptides.     

Release of cotyledonary shoots of pea (<Emphasis Type="Italic">Pisum sativum</Emphasis>) seedlings from inhibition as related to endogenous zeatin and ethylene and exogenous IAA and benzyladenine

This paper deals with apical dominance using a dicotylar model obtained after decapitation of pea seedlings with two shoots — one dominant and the other inhibited. When the dominant shoot was decapitated the inhibited one is released from inhibition and after 24 to 72 h begins to grow. However, the levels of trans-zeatin and production of ethylene increase within 4 and 6 hours respectively after release from inhibition, and within an interval of 72 h the levels of both phytohormones begin gradually to decrease. This indicates that also in this model, the release from apical dominance is associated with an increase in the level of cytokinin zeatin and, thereafter, also with an increased production of ethylene. If indolyl-3-acetic acid (IAA) is applied on the decapitated main stem after decapitation of the dominant shoot, the growth of the initially inhibited one is very strongly retarded; if, however, IAA is applied on the decapitated dominant shoot, this inhibition is significantly weaker. This means that the inhibiting effect of IAA on the inhibited shoot originates to a greater degree from the main stem rather than from the dominant shoot. The effect of benzyladenine (BA) is transferred equally from the decapitated main stem and from the decapitated dominant shoot because the initially inhibited shoot begins to grow as well as also other shoots from serial cotyledonary buds.     

Karyological and taxonomic observations onDracocephalum foetidum Bunge andKoenigia islandica L.

Chromosome numbers are reported for two Mongolian species,Dracocephalum foetidum Bunge (2n=12) andKoenigia islandica L. (2n=14). The relationship ofD. foetidum toD. moldavica L. (2n=10) and some patterns of phenotypic variation inK. islandica are briefly discussed. The following new combinations are proposed:K. cyanadra (Diels) Měsí?ek etSoják,K. forrestii (Diels) Měsí?ek etSoják,K. hubertii (Lingelsh.) Měsí?ek etSoják, andK. nummularifolia (Meisn.) Měsí?ek etSoják.     

A new phage typing scheme forProteus mirabilis andProteus vulgaris strains

A new bacteriophage typing set, composed of 22 phages, was established as a tool for differentiation ofProteus strains. All the phages were tailed and included 4 morphological types (A1, A2, B1 and C1). They were classified into the familiesMyoviridae, Siphoviridae andPodoviridae. From the set, 19 phages had double-stranded DNA and 3 were single-stranded DNA phages.     

The Paleozoic evolution of the gastropod larval shell: larval armor and tight coiling as a result of predation‐driven heterochronic character displacement

Early and middle Paleozoic gastropod protoconchs generally differ strongly from their corresponding adult morphologies, that is, most known protoconchs are smooth and openly coiled, whereas the majority of adult shells are ornamented and tightly coiled. In contrast, larval and adult shells of late Paleozoic gastropods with planktotrophic larval development (Caenogastropoda, Neritimorpha) commonly resemble each other in shape and principle ornamentation. This is surprising because habitat and mode of life of planktonic larvae and benthic adults differ strongly from each other. Generally, late Paleozoic to Recent protoconchs are tightly coiled. This modern type of larval shell resembles the adult shell morphology and was obviously predisplaced onto the larval stage during the middle Paleozoic. The oldest known planktonic‐armored (strongly ornamented) larval shells are known from the late Paleozoic. However, smooth larval shells are also common among the studied late Paleozoic gastropods. The appearance of larval armor at the beginning of the late Paleozoic could reflect an increase of predation pressure in the plankton. Although there are counter examples in which larval and adult shell morphology differ strongly from each other, there is statistical evidence for a heterochronic predisplacement of adult characters onto the larval stage. Larval and adult shells are built in the same way, by accretionary secretion at the mantle edge. It is likely that the same underlying gene expression is responsible for that. If so, similarities of larval and adult shell may be explained by gene sharing, whereas differences may be due to different (planktic vs. benthic life) epigenetic patterns.     

Absence of MMACHC in peripheral retinal cells does not lead to an ocular phenotype in mice

Combined methylmalonic aciduria with homocystinuria (cblC type) is a rare disease caused by mutations in the MMACHC gene. MMACHC encodes an enzyme crucial for intracellular vitamin B₁₂ metabolism, leading to the accumulation of toxic metabolites e.g. methylmalonic acid (MMA) and homocysteine (Hcy), and secondary disturbances in folate and one-carbon metabolism when not fully functional. Patients with cblC deficiency often present in the neonatal or early childhood period with a severe multisystem pathology, which comprises a broad spectrum of treatment-resistant ophthalmological phenotypes, including retinal degeneration, impaired vision, and vascular changes. To examine the potential function of MMACHC in the retina and how its loss may impact disease, we performed gene expression studies in human and mouse, which showed that local expression of MMACHC in the retina and retinal pigment epithelium is relatively stable over time. To study whether functional MMACHC is required for retinal function and tissue integrity, we generated a transgenic mouse lacking Mmachc expression in cells of the peripheral retina. Characterization of this mouse revealed accumulation of cblC disease related metabolites, including MMA and the folate-dependent purine synthesis intermediates AICA-riboside and SAICA-riboside in the retina. Nevertheless, fundus appearance, morphology, vasculature, and cellular composition of the retina, as well as ocular function, remained normal in mice up to 6 or 12 months of age. Our data indicates that peripheral retinal neurons do not require intrinsic expression of Mmachc for survival and function and questions whether a local MMACHC deficiency is responsible for the retinal phenotypes in patients.