Extremozymes — biocatalysts with unique properties from extremophilic microorganisms

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Does glycosylation of melanoma cells influence their interactions with fibronectin?

Cell surface integrins, especially those binding to fibronectin (FN), participate in processes of tumor cell invasion and metastasis. Changes in glycosylation of cell surface adhesion proteins are often associated with malignant transformation of cells. In this study we examined the influence of swainsonine (SW) on adhesion, wound healing and haptotactic migration on FN, comparing the responses of different human melanoma cell lines: primary WM35 and metastatic WM9, WM239 and A375. We also examined the role of alpha subunits in adhesion to FN. All of the antibodies inhibited adhesion to FN but with different efficiencies depending on the cell line. Adhesion was mediated mainly by integrin alpha(5)beta(1) (WM9, A375), alpha(3)beta(1) (WM35, A375, WM239). Scratch wound repair was significantly faster on FN-coated wells than on plastic for all cells except for WM9. A375 and WM9 had the greatest migration ability, both expressing the highest level of alpha(5)beta(1) integrin. It seems very likely that adhesion to FN can be accomplished by many different integrins, but for effective migration alpha(5)beta(1) integrin is responsible. Only A375 and WM239 cell lines reacted to SW treatment. In the presence of SW WM239 and A375 cells had 70% and 40% increased adhesion to FN, and their migration was decreased 40% and 50%, respectively. Interestingly, although most of the cell lines share a common profile of integrins, each line interacted with FN differently. They differed mainly in the repertoire of integrins used for adhesion, and in the manner in which glycosylation affected these processes. The influence of SW was observed in two metastatic cell lines indicating the contribution of glycosylation status to the progression of melanoma. The lack of reaction to SW in WM9 cells may suggest that there is a threshold in the expression level of the highly branched N-glycans that may influence the adhesion and migration properties of the cell.     

An Empirical Potential Study of the Interaction of L—;Lysine—;L—;Alanine—;L—;Alanine Tripeptide with Four models of B—;DNA with different Compositions

Abstract

The empirical potential including the intra- and intermolecular energy terms was used to study the interaction of L-Lysine-L-Alanine-L-Alanine Tripeptide with four models of B-DNA with different compositions. On the basis of a detailed search of the respective potential energy surface, it was found that the peptide is preferentially bounded to the AT-rich sequences. Analysis of the different energy contributions indicated that the electrostatic term is responsible for this preference. The results agree with the experimental data on the selectivity of some DNA—binding proteins and polypeptides to AT—;rich DNA.     

Is the interspecific variation of body size of land snails correlated with rainfall in Israel and Palestine?

The hypothesis that body size of land snail species increases with aridity in Israel and Palestine because large snails lose relatively less water due to their lower surface to volume ratio has been investigated. Data on rainfall amplitudes of 84 land snail species in Israel and Palestine and on their body sizes were used to test for interspecific correlations between body size and rainfall. Four methods, means of body sizes in rainfall categories, the midpoint method, the across-species method, and a phylogenetically controlled analysis (CAIC) showed that there is no significant correlation between body size of land snail species and their rainfall amplitude in Israel and Palestine. The lack of an interspecific correlation between body size and rainfall amplitude may be the result of conflicting selective forces on body size.     

Glial reactivity in dogs with visceral leishmaniasis: correlation with T lymphocyte infiltration and with cerebrospinal fluid anti-<Emphasis Type="Italic">Leishmania</Emphasis> antibody titres

Visceral leishmaniasis is a multisystemic zoonotic disease that can manifest with several symptoms, including neurological disorders. Because glial cells are extensively associated with the immune response within the brain, we evaluated the morphology of astrocytes and microglia of dogs naturally infected with Leishmania chagasi. We used immunohistochemical and lectin-histochemical techniques for morphological analyses and we also examined the glial correlation with lymphocyte infiltration of the brain and with the presence of anti-Leishmania antibodies within the cerebrospinal fluid of the dogs. Although we did not detect a shared morphological pattern in the astrocytes or microglia in the brain tissue, these cells were more intensely labelled in infected dogs than in the control group. The density of microglia was increased in the ependymal/subependymal area, thus demonstrating a strong correlation with the presence of T lymphocytes and with cerebrospinal fluid antibody titres. Thus, our results indicate a pro-inflammatory state in the brains of dogs naturally infected with L. chagasi and strongly suggest that microglia and astrocytes are involved in the pathogenesis of the neurological disorders of visceral leishmaniasis in dogs.     

Elastic energy storage in β-sheets with application to F<Subscript>1</Subscript>-ATPase

We present a methodology for obtaining the elastic properties of protein motifs. We combine the use of interpolated structures (IS), molecular dynamics (MD) and collective coordinates to deduce the elastic properties of the -sheet in F₁ ATPase. We find that about 3.5 kcal/mol (6 k_BT at room temperature) of elastic energy is stored in the -sheet as the -subunit undergoes its hinge bending motion, in good agreement with the finite element model of Wang and Oster [Nature (1998) 396:279–282]. The technique should be useful for -sheets in other proteins and aid in the construction of phenomenological models for molecular motors that are computationally prohibitive for MD alone.     

iPAD or PADi — ‘tablets’ with therapeutic disease potential?

The PAD enzymes.

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Plasma visfatin,associated with a genetic polymorphism −1535C > T,is correlated with C-reactive protein in Chinese Han patients with traumatic brain injury

Visfatin is a newly identified pro-inflammatory adipokine and a genetic polymorphism −1535 C > T located in the visfatin gene promoter has been suggested to be associated with the regulation of visfatin expression in some inflammatory illness. However, there were some conflicting results regarding whether this variant is functional or not. This study aimed to examine the relations of the −1535 C > T single nucleotide polymorphism (SNP) of visfatin gene to the plasma visfatin and C-reactive protein concentrations in traumatic brain injury (TBI). 318 Chinese Han patients with TBI were recruited in this study. Plasma visfatin and C-reactive protein levels were significantly different between the genotypes in the SNP-1535 C > T even after adjustment for age, sex and body mass index. The genotype C–C had the highest plasma visfatin and C-reactive protein concentrations. The plasma visfatin and C-reactive protein concentrations between the variant genotypes C–T and T–T did not differ significantly. Plasma visfatin level was significantly associated with plasma C-reactive protein level using multivariate linear regression. Thus, the SNP-1535 C > T of visfatin gene seemed to be potentially involved in the inflammatory component of TBI through a decreased production of visfatin.     

Laccase-induced C–N coupling of substituted <Emphasis Type="Italic">p</Emphasis>-hydroquinones with <Emphasis Type="Italic">p</Emphasis>-aminobenzoic acid in comparison with known chemical routes

Magnetotactic bacteria are difficult to grow under defined conditions in culture, which has presented a major obstacle to commercial application of magnetosomes. We studied the relationships among the cell growth, magnetosome formation, dissolved oxygen concentration (DO), and the ability to supply oxygen to the cells. Mass culture of Magnetospirillum gryphiswaldense MSR-1 for the production of magnetosomes was established in a 42-L fermentor under the following conditions: (1) sterile air was the sole gas supplied in the fermentor, and DO could be regulated at any level below 10% saturation by cascading the stir rate to DO, (2) to resolve the paradoxical situation that the cell growth requires higher DO whereas magnetosome formation requires low DO below the detectable range of regular oxygen electrode, DO was controlled to optimal level using the change of cell growth rate, rather than reading from the highly sensitive oxygen electrode, as the signal for determining appropriate DO, and (3) timing and rate of supplying the substrates were determined by measuring cell density and Na-lactate concentration. Under these conditions, cell density (OD565) of strain MSR-1 reached 7.24 after 60-h culture in a 42-L fermentor, and cell yield (dry weight) was 2.17 g/L, the highest yield so far being reported. The yield of magnetosomes (dry weight) was 41.7 mg/L and 16.7 mg/L/day, which were 2.8 and 2.7 times higher than the previously reported yields.     

A theoretical study on the reaction mechanism of O<Subscript>2</Subscript> with C<Subscript>4</Subscript>H<Subscript>9</Subscript>• radical

Ab initio calculations have been performed using the complete basis set model (CBS-QB3) to study the reaction mechanism of butane radical (C₄H₉•) with oxygen (O₂). On the calculated potential energy surface, the addition of O₂ to C₄H₉• forms three intermediates barrierlessly, which can undergo subsequent isomerization or decomposition reaction leading to various products: HOO• + C₄H₈, C₂H₅• + CH₂CHOOH, OH• + C₃H₇CHO, OH• + cycle-C₄H₈O, CH₃• + CH₃CHCHOOH, CH₂OOH• + C₃H₆. Five pathways are supposed in this study. After taking into account the reaction barrier and enthalpy, the most possible reaction pathway is C₄H₉• + O₂ → IM1 → TS5 → IM3 → TS6 → IM4 → TS7 → OH• + cycle-C₄H₈O.     

G protein β<Subscript>1</Subscript>γ<Subscript>2</Subscript> subunits purification and their interaction with adenylyl cyclase

A preliminary study on the interaction of G protein (guanine triphosphate binding pro- tein) β1γ2 subunits and their coupled components in cell signal transduction was conducted in vitro. The insect cell lines, Sf9 (Spodoptera frugiperda) and H5 (Trichoplusia ni) were used to express the recombinant protein Gβ1γ2. The cell membrane containing Gβ1γ2 was isolated through affinity chromatography column with Ni-NTA agarose by FPLC method, and the highly purified protein was obtained. The adenylyl cyclase 2 (AC2) activity assay showed that the purified Gβ1γ2 could significantly stimulate AC2 activity. The interaction of β1γ2 subunits of G protein with the cytoplasmic tail of various mammalian adenylyl cyclases was monitored by BIAcore technology using NTA sensor chip, which relies on the phenomenon of surface plasmon resonance (SPR). The experiments showed the direct binding of Gβ1γ2 to the cytoplasmic tail C2 domain of AC2. The specific binding domain of AC2 with Gβ1γ2 was the same as AC2 activity domain which was stimulated by β1γ2.     

What’s wrong with a little sex?

In many species, most (or all) offspring are produced by sexual means. However, theory suggests that selection should often favour the evolution of species in which a small fraction of offspring are produced sexually, and the rest are produced asexually. Here, we present the analysis of a model that may help to resolve this paradox. We show that, when heterozygote advantage is in force, members of species in which sex is rare will tend to produce poorly adapted offspring when they mate. This problem should be less severe in species where most offspring are produced by sexual means. As a consequence, once the rate of sexual reproduction becomes sufficiently rare, the benefits of sex may vanish, leading to the evolution of obligate asexuality. Substantial benefits of sexual reproduction may tend to accrue only if a large proportion of offspring are produced sexually. We suggest that similar findings are likely in the case of epistatic interactions between loci.     

Computation of multicenter overlap integrals with Slater-type orbitals using Ψ<Superscript>α</Superscript>-ETOs

Multicenter overlap integrals appearing in the evaluation of multicenter–multielectron integrals of central and noncentral interaction potentials are calculated using complete orthonormal sets of -ETOs (=1, 0, –1, –2, ...). The final results are expressed in terms of two-center overlap integrals between STOs. The convergence of the series is tested by calculating concrete cases for arbitrary quantum numbers, screening constants and location of STOs.     

Histopathology findings in common marmosets (<Emphasis Type="Italic">Callithrix jacchus</Emphasis> Linnaeus, 1758) with chronic weight loss associated with bile tract obstruction by infestation with <Emphasis Type="Italic">Platynosomum</Emphasis> (Loos, 1907)

Chronic weight loss in marmosets is often associated with wasting marmoset syndrome (WMS), an important disease that occurs in callitrichid colonies around the world. Even though its etiology is very difficult to determine, particular variables, such as weight loss, diarrhea and alopecia, associated or not with infestation in the pancreatic ducts with Trichospirura leptossoma (Nematoda: Thelazioidea), seem to be linked with the syndrome. This study investigated the histopathology of the lungs, duodenum, liver, gallbladder, extrahepatic bile ducts and pancreatic ducts of six common marmosets (Callithrix jacchus) suffering from severe non-diarrheic weight loss. Three individuals died naturally and the other three were euthanized. Microscopic findings showed the presence of adult flukes (Platynosomum) in the liver. These flukes, which provoke common infection in cats, were also observed inside the gallbladder as well as in the intra and extrahepatic bile ducts in common marmosets. Portal fibrosis was observed in two animals, which developed chronic fibrosing hepatopathy (biliary pattern, grade 3). The disease progresses without diarrhea and without pancreatic lesions or infestation. With the progression, the animals presented with ascending cholangitis, cholestasis and portal fibrosis, sometimes culminating in secondary biliary cirrhosis. Therefore, this infirmity, associated with chronic weight loss in common marmosets, could be another etiological factor linked with WMS.     

Can a mixed damage interfere with DNA‐prottein cross‐links repair?

Some photochemical and photobiological properties of 4,5',8-trimethylpsoralen (TMP) have been studied in comparison with 1,4,6,8-tetramethyl-2H-furo[2,3-h]quinolin-2 one (FQ) and 8-methoxypsoralen (8-MOP). TMP and FQ can photobind to mammalian cell DNA in vivo , by UVA irradiation, forming DNA-protein cross-links (DPC), but only TMP shows a strong capacity of inducing interstrand cross-links (ISC). The mechanism of DPC formation was studied using the double irradiation method in Chinese hamster ovary (CHO) cells, and DPC were detected by alkaline elution. Both TMP and FQ induce covalent diadducts linking together DNA and proteins. Studying the formation of double strand breaks (DSB) in CHO cells we observed that TMP induced a low amount of DSB, similar to 8-MOP. TMP and 8-MOP induced chromosomal aberrations in CHO cells to the same extent, while FQ appeared to be more active. Our data suggest that the ISC induced by TMP could trap enzymes involved in DPC repair.     

Association of β<Subscript>2</Subscript>-microglobulin with the α<Subscript>3</Subscript> domain of H-2D<Superscript>b</Superscript> heavy chain

MHC class I molecules are heterotrimeric complexes composed of heavy chain, ₂-microglobulin (₂m) and short peptide. This trimeric complex is generated in the endoplasmic reticulum (ER), where a peptide loading complex (PLC) facilitates transport from the cytosol and binding of the peptide to the preassembled ER resident heavy chain/₂m dimers. Association of mouse MHC class I heavy chain with ₂m is characterized by allelic differences in the number and/or positions of amino acid interactions. It is unclear, however, whether all alleles follow common binding patterns with minimal contributions by allele-specific contacts, or whether essential contacts with ₂m are different for each allele. While searching for the PLC binding site in the ₃ domain of the mouse MHC class I molecule H-2D^b, we unexpectedly discovered a site critical for binding mouse, but not human, ₂m. Interestingly, amino acids in the corresponding region of another MHC class I heavy chain allele do not make contacts with the mouse ₂m. Thus, there are allelic differences in the modes of binding of ₂m to the heavy chain of MHC class I.     

Acquired Antibiotic Resistance: Are We Born with It?

The rapid emergence of antibiotic resistance (AR) is a major public health concern. Recent findings on the prevalence of food-borne antibiotic-resistant (ART) commensal bacteria in ready-to-consume food products suggested that daily food consumption likely serves as a major avenue for dissemination of ART bacteria from the food chain to human hosts. To properly assess the impact of various factors, including the food chain, on AR development in hosts, it is important to determine the baseline of ART bacteria in the human gastrointestinal (GI) tract. We thus examined the gut microbiota of 16 infant subjects, from the newborn stage to 1 year of age, who fed on breast milk and/or infant formula during the early stages of development and had no prior exposure to antibiotics. Predominant bacterial populations resistant to several antibiotics and multiple resistance genes were found in the infant GI tracts within the first week of age. Several ART population transitions were also observed in the absence of antibiotic exposure and dietary changes. Representative AR gene pools including tet(M), ermB, sul2, and bla(TEM) were detected in infant subjects. Enterococcus spp., Staphylococcus spp., Klebsiella spp., Streptococcus spp., and Escherichia coli/Shigella spp. were among the identified AR gene carriers. ART bacteria were not detected in the infant formula and infant foods examined, but small numbers of skin-associated ART bacteria were found in certain breast milk samples. The data suggest that the early development of AR in the human gut microbiota is independent of infants' exposure to antibiotics but is likely impacted by exposure to maternal and environmental microbes during and after delivery and that the ART population is significantly amplified within the host even in the absence of antibiotic selective pressure.