The Properties of Water in the Pressure–Temperature Landscape

Water represents the major component of most food systems. During thermal or high-pressure processing, physical and chemical properties of water are changed. The p–T diagram represents an obvious presentation of isoproperty lines and their pressure and temperature dependencies. In this work, 15 different properties of pure water are shown as isoproperty lines in the pressure–temperature landscape. By using functional relationships from the “International Association for the Properties of Water and Steam” and databases from the “National Institute of Standards and Technology,” highest accuracy is guaranteed. Applying the generated graphs, a compact overview is given and a wide range of thermal and high-pressure processes can easily be compared. The different pressure and temperature dependencies of all properties showed the complexity of medium conditions during thermal and high-pressure processing. An extended understanding of pressure–temperature dependencies will improve process concepts as well as industrial applications at high temperature and high isostatic pressure.     

Fano Resonance in a Gear-Shaped Nanocavity of the Metal–Insulator–Metal Waveguide

The filter function of the metal–insulator–metal (MIM) waveguide with a gear-shaped nanocavity is investigated using the finite-difference time-domain method. Since the gear breaks the symmetric distribution of the resonance, Fano resonance occurs in the gear-shaped nanocavity. Fano resonance strongly depends on the structural parameters of the gear. Compared to the MIM waveguide with a disk-shaped nanocavity, the MIM waveguide with a gear-shaped nanocavity allows for a much more sensitive detection of small refractive index changes of the filled media inside the nanocavity, which reveals a potential sensor application of the MIM waveguide with a gear-shaped nanocavity.     

The Antigenic Properties of β-Lactoglobulin Examined with Mouse IgE Antibody

The effects of enzymic or chemical fragmentations and of chemical modifications on the antigenic properties of bovine β-Mactoglobulin were examined using specific mouse IgE antibody. The antigenic reactivity of β-Mactoglobulin derivatives was represented in terms of their ability to neutralize specific IgE antibodies assayed by passive cutaneous anaphylaxis in rat. The tryptic, chymotryptic or peptic hydrolysate free of native β-Mactoglobulin had no antigenic reactivity but the fragments obtained after CNBr cleavage retained the ability to bind the antibody. Modification of the sulfhydryl group, arginine or tryptophan residues and amino groups had no effect on antigenic reactivity but a little decrease in the reactivity was observed on the cleavage of the two disulfide bridges. These results suggest that one sulfhydryl group, two arginine and tryptophan residues and most of the amino groups are out of antigenic sites in β-Mactoglobulin and that the antigenicity depends on the conformation maintained by the disulfide bridges.     

The α-Subunit Regulates Stability of the Metal Ion at the Ligand-associated Metal Ion-binding Site in β3 Integrins

The aspartate in the prototypical integrin-binding motif Arg-Gly-Asp binds the integrin βA domain of the β-subunit through a divalent cation at the metal ion-dependent adhesion site (MIDAS). An auxiliary metal ion at a ligand-associated metal ion-binding site (LIMBS) stabilizes the metal ion at MIDAS. LIMBS contacts distinct residues in the α-subunits of the two β₃ integrins α_IIbβ₃ and α_Vβ₃, but a potential role of this interaction on stability of the metal ion at LIMBS in β₃ integrins has not been explored. Equilibrium molecular dynamics simulations of fully hydrated β₃ integrin ectodomains revealed strikingly different conformations of LIMBS in unliganded α_IIbβ₃ versus α_Vβ₃, the result of stronger interactions of LIMBS with α_V, which reduce stability of the LIMBS metal ion in α_Vβ₃. Replacing the α_IIb-LIMBS interface residue Phe¹⁹¹ in α_IIb (equivalent to Trp¹⁷⁹ in α_V) with Trp strengthened this interface and destabilized the metal ion at LIMBS in α_IIbβ₃; a Trp¹⁷⁹ to Phe mutation in α_V produced the opposite but weaker effect. Consistently, an F191/W substitution in cellular α_IIbβ₃ and a W179/F substitution in α_Vβ₃ reduced and increased, respectively, the apparent affinity of Mn²⁺ to the integrin. These findings offer an explanation for the variable occupancy of the metal ion at LIMBS in α_Vβ₃ structures in the absence of ligand and provide new insights into the mechanisms of integrin regulation.     

Effect of Cyanotoxins on the Hypothalamic–Pituitary–Gonadal Axis in Male Adult Mouse

Background

Microcystins LR (MC-LR) are hepatotoxic cyanotoxins that have been shown to induce reproductive toxicity, and Hypothalamic–Pituitary–Gonadal Axis (HPG) is responsible for the control of reproductive functions. However, few studies have been performed to evaluate the effects of MC-LR on HPG axis. This study aimed to investigate the MC-LR-induced toxicity in the reproductive system of mouse and focus on the HPG axis.

Methods

Adult male C57BL/6 mice were exposed to various concentrations of MC-LR (0, 3.75, 7.50, 15.00 and 30.00 µg/kg body weight per day) for 1 to 14 days, and it was found that exposure to different concentrations of MC-LR significantly disturbed sperm production in the mice testes in a dose- and time-dependent manner. To elucidate the associated possible mechanisms, the serum levels of testosterone, follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were assessed. Meanwhile, PCR assays were employed to detect alterations in a series of genes involved in HPG axis, such as FSH, LH, gonadotropin-releasing hormone (GnRH) and their complement receptors. Furthermore, the effect of MC-LR on the viability and testosterone production of Leydig cells were tested in vitro. Results: MC-LR significantly impaired the spermatogenesis of mice possibly through the direct or indirect inhibition of GnRH synthesis at the hypothalamic level, which resulted in reduction of serum levels of LH that lead to suppression of testosterone production in the testis of mice.

Conclusions

MC-LR may be a GnRH toxin that would disrupt the reproductive system of mice.     

Expression of the Cloned Hemagglutinin–Neuraminidase Gene of the Newcastle Disease Virus in Mouse Myeloma Cells

Vaccination with autologous cancer cells expressing a potent foreign antigen is promising for immunotherapy of tumors. A construct was obtained to transfect cancer cells with the hemagglutinin–neuraminidase (HN) gene of the Newcastle disease virus (NDV). Specific primers were designed, and the HN cDNA was amplified from RNA isolated from the allantoic fluid of NDV-infected embryonated chicken eggs. The amplified fragment was cloned in pCR2.1, sequenced, and recloned in expression vector pCDNA3.1/Zeo(+). The resulting construct was used to transfect mouse myeloma cells SP2/0. Production of HN was checked by ELISA and by a neuraminidase activity assay. Cell agglutination on ice was proposed as a test for surface HN.     

The Distribution of Tissue Insulin Receptors in the Mouse by Whole – Body Autoradiography

Abstract

The distribution of insulin binding sites in the mouse was investigated by in vivo whole – body autoradiography. Male mice were injected intravenousely with ¹²⁵ I – insulin in the absence of and, in the presence of, excess unlabeled insulin. Three, 6, 15, 30 and 60 minutes after injection, the animals were perfused and subjected to autoradiographic procedures.

Specific insulin binding was observed in the choroid plexus, liver, gastrointestinal tract, spleen, pancreas, deferent duct, and Harderian gland.

In the liver and spleen, the distribution of binding sites was heterogeneous. In the liver, the density of the binding was higher around the branches of the portal vein than around the central vein. In the spleen, the marginal zone exhibited a higher density than the white and red pulp.

The kidney cortex, and the thyroid gland showed a high degree of insulin binding, but the binding was nonspecific. The binding of insulin to other tissues and organs, including the skeletal muscle and fat, was weak, and most of the binding was nonspecific.     

Changes in the Electron Transfer Symmetry in the Photosystem I Reaction Centers upon Removal of Iron–Sulfur Clusters

Biochemistry (Moscow) - In photosynthetic reaction centers of intact photosystem I (PSI) complexes from cyanobacteria, electron transfer at room temperature occurs along two symmetrical branches of...     

Novel Properties of a Mouse γ-Aminobutyric Acid Transporter (GAT4)

We expressed the mouse gamma-aminobutyric acid (GABA) transporter GAT4 (homologous to rat/ human GAT-3) in Xenopus laevis oocytes and examined its functional and pharmacological properties by using electrophysiological and tracer uptake methods. In the coupled mode of transport (Na+/ Cl-/GABA cotransport), there was tight coupling between charge flux and GABA flux across the plasma membrane (2 charges/GABA). Transport was highly temperature-dependent with a temperature coefficient (Q10) of 4.3. The GAT4 turnover rate (1.5 s(-l); -50 mV, 21 degrees C) and temperature dependence suggest physiological turnover rates of 15-20 s(-1). No uncoupled current was observed in the presence of Na+. In the absence of external Na+, GAT4 exhibited two distinct uncoupled currents. (i) A Cl- leak current (ICl(leak)) was observed when Na+ was replaced with choline or tetraethylammonium. The reversal potential of (ICl(leak)) followed the Cl- Nernst potential. (ii) A Li+ leak current (ILi(leak)) was observed when Na+ was replaced with Li+. Both leak currents were inhibited by Na+, and both were temperature-independent (Q10 approximately 1). The two leak modes appeared not to coexist, as Li+ inhibited (ICl(leak)). The results suggest the existence of cation- and anion-selective channel-like pathways in GAT4. Flufenamic acid inhibited GAT4 Na+/Cl-/GABA cotransport, ILi(leak), and ICl(leak), (Ki approximately 30 microM), and the voltage-induced presteady-state charge movements (Ki approximately 440 microM). Flufenamic acid exhibited little or no selectivity for GAT1, GAT2, or GAT3. Sodium and GABA concentration jicroumps revealed that slow Na+ binding to the transporter is followed by rapid GABA-induced translocation of the ligands across the plasma membrane. Thus, Na+ binding and associated conformational changes constitute the rate-limiting steps in the transport cycle.     

Comparative Analysis of the Recombinant α-Glucosidases from the Thermotoga neapolitana and Thermotoga maritima Maltodextrin Utilization Gene Clusters

Abstract

The hyperthermophilic bacterium Thermotoga maritima contains an amylolytic gene cluster with two adjacent α-glucosidase genes, aglB and aglA. We have now identified a similar pair of α-glucosidase genes on a 5,451 bp fragment of T. neapolitana genomic DNA. Like in T. maritima, aglA of T. neapolitana is located downstream of aglB. The deduced AglB primary structure allows its assignment to glycoside hydrolase family 13 (GHF13), whereas AglA belongs to GHF4. The aglB gene of T. neapolitana and the corresponding gene from T. maritima were expressed in E. coli, and the recombinant enzymes were characterized. Both enzymes hydrolyzed cyclomaltodextrins and linear maltooligosaccharides to yield glucose and maltose. Evidence from the hydrolysis of non-natural oligosaccharides and the pseudo-tetrasaccharide acarbose suggests that linear malto-oligosaccharides are progressively degraded by T. neapolitana and T. maritima AglB from the reducing end, which is highly uncommon for α-glucosidases. AglB, in contrast to the cofactor-dependent (NAD⁺, Mn²⁺) α-glucosidase AglA, does not cleave maltose. The recent elucidation of the crystal structure of T. miritima AglA indicates that AglA and AglB employ different catalytic mechanisms for glycosidic bond cleavage. Possible reasons for the presence of two α-glucosidase genes in the same amylolytic gene cluster of Thermotoga species are discussed.     

The Role of the Transport System in the Control of the Source–Sink Relations in Pinus sylvestris

Morphophysiological correlations were studied in medium-aged (20- to 60-year-old) Scots pine trees under the northern taiga conditions. Under various ecological conditions, pine trees developed a well-balanced structure, with close linear relationships between needle and root weight and their cross-section areas in all components of the continuous transport network (the coefficient of determination was between 0.88 and 0.999). When the annual cycle of soluble and insoluble carbohydrate contents was followed in various pine tissues, the total concentrations of soluble and insoluble carbohydrates were maintained at constant and tissue-specific levels, except in the growth period. The maximum level of carbohydrates was observed in all tissues at the beginning of rapid growth, and the minimum, at growth cessation. The qualitative composition and amount of carbohydrates matched the phenological phases of development and were not affected by the ecological growth conditions pertinent to the particular environment. The authors conclude that assimilate synthesis and partitioning are related to structural development, and the state of sink centers determines the attracting capacity, whereas the transport network, from roots to needles, and its conducting capacity are essential for the realization of systemic relationships and the control over growth and development in Pinus sylvestris L.     

Polymorphism and Linkage of the αa-Crystallin Gene in t-Haplotypes of the Mouse

Restriction fragment polymorphisms were used to order the alpha A-crystallin locus (Crya-1) relative to other genes in mouse t-chromatin and to investigate the relatedness of alpha-A-crystallin sequences among different t-haplotypes. Analysis of DNA from t-recombinant mice mapped Crya-1 to the K end of the H-2 complex and within the distal inverted region characteristic of t-haplotypes. Hybridization with Crya-1 cDNA revealed three distinct phenotypic groups among the 17 different t-haplotypes studied. A majority (9 of 17) of the t-haplotypes were classified into a novel group (Crya-1t) characterized by restriction fragments apparently unique to t-chromosomes and therefore thought to contain alpha A-crystallin sequences descended from the original t-chromosome. A second group of t-haplotypes had restriction fragment patterns indistinguishable from those observed among many common inbred strains of mice of the Crya-1a type, and a third restriction fragment pattern, observed only in the tw121 haplotype, was indistinguishable from the fragment pattern for C3H/DiSn (Crya-1b) and several other inbred strains of mice. Thus, with respect to sequences around the Crya-1 locus, different t-haplotypes show restriction fragment polymorphisms, some of which are comparable to those found in wild-type chromosomes and provide further evidence for genetic heterogeneity in DNA from the distal region of t-haplotypes.     

The Periodically Graded Metal–Insulator–Metal Gap Structure for Plasmonic Waveguides

In this paper, we explore the potential of the plasmonic metal–insulator–metal (MIM) periodically graded structure. Based on the coupled modes approach, an analytical model has been observed for the surface plasmon polariton (SPP) propagation. The band modes of SPP can be also supported by the MIM structure and we have analyzed the strong dependence of band width on structure parameters. The obtained analytical expressions allow one to easily choose the structure parameters for the desired band width.     

The Potential Role of Increasing the Release of Mouse β- Defensin-14 in the Treatment of Osteomyelitis in Mice: A Primary Study

Mammalian β-defensins are small cationic peptides that have been implicated in mediating innate immune defenses against microbial infection. Mouse β-defensin-14 (MBD-14), based on structural and functional similarities, appears to be an ortholog of human β-defensin-3 (HBD-3). Previous studies identified signaling pathway p38 mitogen-activated protein kinase (MAPK) that contributed to the expression of MBD-14 in mouse osteoblasts upon contacted with methicillin-resistance Staphylococcus aureus (MRSA) supernatant, which provided a theoretical basis as a promising therapeutic target in the treatment of intramedullary infection with MRSA in vivo. In this study, the medullary cavities of tibiae were contaminated with MRSA 10³ colony forming units and different doses of p38 MAPK agonists anisomycin were followed as group III or IV in 30 mice. Fifteen animals that received phosphate- buffered saline served as group II and 15 mice were not contaminated with MRSA and received phosphate-buffered saline served as controls (group I). Follow-up was 7 days. In day 1, day 4 and day 7 postoperatively, infection was evaluated by blood routine, microbiological and histological analyses after sacrifice. All animals of group II developed microbiological and histological signs of infection. Histological signs of infection, white blood counts and cultures of group III and IV showed significantly reduced bacterial growth compared to cultures of group II. Simultaneously, different doses of anisomycin significantly induced the expression of osteoblast-associated genes, including alkaline phosphatase, osteocalcin and collagen type I. In addition, the expression of HBD-3 in human interfacial membranes around infected periprosthetic joint by staphylococcus contaminated was evaluated, and the expression pattern changed with significant induction of HBD-3 in infected periprosthetic joint compared with aseptic loosening under inflammatory conditions. Our primary study indicated that the potential antibacterial role of increased MBD-14 in the osteomyelitis mouse model.     

The role of the abnormalities in the distal pathway of cholesterol biosynthesis in the Conradi–Hünermann–Happle syndrome

Conradi–Hünermann–Happle syndrome (CDPX2, OMIM 302960) is an inherited X-linked dominant variant of chondrodysplasia punctata (CP) caused by mutations in one gene of the distal pathway of cholesterol biosynthesis. It exhibits intense phenotypic variation and primarily affects the skin, bones and eyes. The ichthyosis following Blaschko's lines, chondrodysplasia punctata and cataracts are the typical clinical findings. The cardinal biochemical features are an increase in 8(9)-cholestenol and 8-dehydrocholesterol (8DHC), which suggest a deficiency in 3β-hydroxysteroid-Δ8,Δ7-isomerase, also called emopamil binding protein (EBP). The EBP gene is located on the short arm of the X chromosome (Xp11.22–p11.23) and encodes a 230 amino acid protein with dual function. Explaining the clinical phenotype in CDPX2 implies an understanding of both the genetics and biochemical features of this disease. CDPX2 displays an X-linked dominant pattern of inheritance, which is responsible for the distribution of lesions in some tissues. The clinical phenotype in CDPX2 results directly from impairment in cholesterol biosynthesis, and indirectly from abnormalities in the hedgehog signaling protein pathways. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.     

Expression in Escherichia coli of cDNA Encoding Barley β-Amylase and Properties of Recombinant β-Amylase

To express the cloned β-amylase cDNA in Escherichia coli under control of the tac promoter, a plasmid pBETA92 was constructed. The plasmid consisted of 6312 bp. An extract of E. coli JM109 harboring pBETA92 had β-amylase activity that produced β-maltose from soluble starch. The enzyme production started in the logarithmic phase, increased linearly, and reached a maximum after 12 h. The recombinant barley β-amylase gave two major (pI 5.43 and 5.63) and four minor (pI 5.20, 5.36, 5.80, and 6.13) activity bands on isoelectric focusing, and their pIs didn’t change throughout the incubation. But Western blot analysis found that one β-amylase having a molecular weight of about 56,000 was synthesized. The recombinant β-amylase was purified from the cells by consecutive column chromatography. The purified enzyme gave a single band of protein on SDS–PAGE but showed heterogeneity on isoelectric focusing. The N-terminal amino acid sequence showed that the recombinant β-amylase lacked four amino acids at positions 2–5 (Glu-Val-Asn-Val) when compared with the presumed amino acid sequence of barley β-amylase. Therefore, the recombiant β-amylase consisted of 531 amino acids, and its molecular weight was calculated to be 59,169. The N-terminal amino acid sequence of the recombinant β-amylase and the nucleotide sequence of the junction position in plasmid pBETA92 indicated that GTG (Val-5 in the case of barley β-amylase) at positions 27–29 from the SD sequence (AGGA) was the translation initiation codon. The properties of the recombinant β-amylase were almost the same as those of barley β-amylase except for the pI and the K_m values for maltohexaose and maltoheptaose. The pI of recombiant barley β-amylase calculated by Genetyx Version 9 based on the presumed amino acid sequence was 5.60, but the real pIs were 5.20–6.13. Therefore, some post-translational reaction(s) might happen after protein synthesis in E. coli cells, and this modification might cause the differences in the pI and the K_m values for maltohexaose and maltoheptaose between the barley and the recombinant β-amylases.     

Ultra-Uniform Field Distribution in a Finite-Width Metal–Dielectric–Metal Waveguide

We investigate the propagation characteristics of the fundamental surface plasmon polariton (SPP) mode of a finite-width metal–dielectric–metal waveguide. By changing the refractive index or the thickness of the dielectric layer of the waveguide, the SPP mode can be transformed from a mode confined in the dielectric layer into a mode confined around the metal corners. There always exists a condition at which the mode field distribution in the dielectric layer becomes almost perfectly uniform along the direction parallel to the metal layers, and this condition is insensitive to the width of the waveguide. It is also possible to obtain an ultra-uniform field distribution by controlling the refractive index of a different dielectric placed on both sides of the waveguide. The waveguide can be used as a basic structure for the realization of nanosized photonic devices and sensors.     

Gametophyte–Sporophyte Interactions in the Pollen–Pistil System: 4. The Hormonal Status and the Mechanism of Self-Incompatibility

Following 4 and 8 h after self-incompatible pollination of Petunia hybrida plants, ethylene evolution and the contents of IAA, ABA, and cytokinins were measured in pistils and their parts (stigma, style, and ovary). The germination and initial growth of pollen tubes within the initial 4 h of the experiment were accompanied with an almost tenfold increase of the rate of ethylene production by the stigma and a twofold increase of the ABA content in the stigma and style. The inhibition of pollen tube growth in the style tissues during next 4 h coincided with a fivefold increase in the cytokinin content in the style, while high ABA content was maintained in the stigma and style. The authors conclude that phytohormones participate in the mechanism of gametophyte self-incompatibility.