Calcium-alginate beads for the oral delivery of transforming growth factor-β1 (TGF-β1): stabilization of TGF-β1 by the addition of polyacrylic acid within acid-treated beads

The susceptibility of the gastrointestinal tract to the toxic effects of chemotherapeutic drugs remains a complication in chemotherapy. Recent studies have suggested that transforming growth factor-β₁ (TGF-β₁) can be used as a cytoprotectant against cell cycle specific drugs. This work describes the use of alginate beads as a potential oral delivery system for TGF-β₁ designed to release the drug in the lumen of the small intestine. TGF-β₁ encapsulation and extent of release from alginate beads approached 100% as determined by ¹²⁵I-labelled TGF-β₁. However, when assayed by ELISA and a growth inhibition assay, nearly all immunoreactivity and bioactivity was lost, apparently due to a very high affinity of the alginate for TGF-β₁. This limitation was overcome by two novel methods: (1) incorporation of selected polyanions within the alginate beads to ‘shield’ TGF-β₁ from interaction with alginate and (2) exposure of the alginate beads containing TGF-β₁ to 0.1 N HCl (acid treatment) to simultaneously reduce the molecular weight of the alginate and its ability to interact with TGF- β₁. If the beads were only acid treated, just 8% of the immunoreactivity ofTGF-β₁ was retained. If polyacrylic acid (90 kDa) was added to the beads, 50% of the immunoreactivity of TGF-β₁ was retained. However, when TGF-β₁ was released from acid-treated beads also containing polyacrylic acid, more than 80% of the TGF-β₁ remained immunoreactive and bioactive. The retained TGF-β₁ activity after release from the beads was found to continue to increase with increasing concentrations of polyacrylic acid, until a concentration was reached where beads would not form. The dramatic increase in retained TGF-β₁ activity is attributed to the ability of polyacrylic acid to shield TGF-β₁ from interaction with lower molecular fragments of alginate.     

Cystic fibrosis α2-macroglobulin protease interaction in vitro

α₂-Macroglobulin was purified from plasma of five cystic fibrosis patients and five normal controls. SDS gel electrophoresis of native az-macroglobulin from cystic fibrosis patients and normal donors showed identical subunit molecular weights, as did trypsin cleavage products. Cystic fibrosis and control α₂-macroglobulins were indistinguishable by isoelectric focusing and exhibited appropriate shifts in isoelectric point following binding of trypsin. The trypsinbinding capacities of control and cystic fibrosis α₂-macroglobulins did not differ, nor did the esterolytic activity of the trypsin-α₂-macroglobulin complexes.     

The small acid soluble proteins (SASP α and SASP β) of Bacillus weihenstephanensis and Bacillus mycoides group 2 are the most distinct among the Bacillus cereus group

The Bacillus cereus group includes Bacillus anthracis, B. cereus, Bacillus thuringiensis, Bacillus mycoides and Bacillus weihenstephanensis. The small acid soluble spore protein (SASP) β has been previously demonstrated to be among the biomarkers differentiating B. anthracis and B. cereus; SASP β of B. cereus most commonly exhibits one or two amino acid substitutions when compared to B. anthracis. SASP α is conserved in sequence among these two species. Neither SASP α nor β for B. thuringiensis, B. mycoides and B. weihenstephanensis have been previously characterized as taxonomic discriminators. In the current work molecular weight (MW) variation of these SASPs were determined by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for representative strains of the 5 species within the B. cereus group. The measured MWs also correlate with calculated MWs of translated amino acid sequences generated from whole genome sequencing projects. SASP α and β demonstrated consistent MW among B. cereus, B. thuringiensis, and B. mycoides strains (group 1). However B. mycoides (group 2) and B. weihenstephanensis SASP α and β were quite distinct making them unique among the B. cereus group. Limited sequence changes were observed in SASP α (at most 3 substitutions and 2 deletions) indicating it is a more conserved protein than SASP β (up to 6 substitutions and a deletion). Another even more conserved SASP, SASP α–β type, was described here for the first time.     

Application of a GM1 ganglioside β-galactosidase microassay method to diagnosis of GM1 gangliosidosis

The enzymatic diagnosis of G_M1 gangliosidosis, including the diagnosis of heterozygosity, requires a microassay of G_M1 ganglioside β-galactosidase activity in lymphocytes and cultured skin fibroblasts. We have adopted high-performance liquid chromatography (HPLC) to the assay of this enzyme and can measure the activity in crude samples fluorometrically. Reaction conditions were examined to determine those optimal for the assay of G_M1 ganglioside β-galactosidase activity in lymphocyte and skin fibroblast homogenates. Under these optimal conditions, reduced enzymatic activities could be detected in lymphocytes and cultured skin fibroblasts from three patients with G_M1 gangliosidosis. Thus, this assay can be used for the diagnosis, rather than the usual assays employing radioactive or artificial substrates.     

Conformational state and receptor recognition of the C-terminal domain of human α2-macroglobulin after dissociation into half-molecules

Background: Dissociation of native human α₂-macroglobulin (α₂M) by sodium thiocyanate generates stable half-molecules with intact thiol esters. Significant conformational changes occur by the dissociation, which are similar to those occurring by transformation from native to methylamine-treated α₂-macroglobulin. Methods: The conformational state of the receptor-binding domain of the half-molecules was investigated by receptor binding and clearance studies, and by use of a panel of 11 monoclonal antibodies (mAbs) specific for the 18-kDa C-terminal receptor-binding fragment of α₂-macroglobulin. Results: The half-molecules simultaneously express epitopes specific for native, as well as epitopes specific for transformed α₂-macroglobulin. While it is possible to immunochemically discriminate between the different forms of tetrameric protein, the half-molecules retain a conformational state with no observed conformational changes in the C-terminal domain following cleavage of thiol esters or bait regions. The in vivo clearance rate in mice was consequently significantly slower for the half-molecules than for the tetrameric receptor-recognized forms of α₂-macroglobulin. Furthermore, half-molecules demonstrate lower affinity for binding to mouse macrophages than methylamine-treated tetrameric α₂-macroglobulin in competition studies. Conclusions: It is suggested that contact zones are functionally important for mediating conformational switches, which result in trapping and exposure of the receptor-binding sites.     

Selective monitoring of vitamin D2 and D3 supplementation with a highly specific 25-hydroxyvitamin D3 immunoassay with negligible cross-reactivity to 25-hydroxyvitamin D2

Background

The effects of vitamin D₂ and D₃ supplementation on circulating concentrations of 25(OH)D₃ require reliable analytical tools for specific determination of 25(OH)D₃ and 25(OH)D₂. We have developed a highly specific 25-OH Vitamin D3 ELISA with negligible cross-reactivity towards 25(OH)D₂.

Methods

25(OH)D₃ concentrations were measured in several study participants; 1) 641 healthy men and women; 2) 39 postmenopausal women receiving 400-800 IU vitamin D₃ daily for 4 months; 3) 45 men and women with hip fracture receiving 1000 IU vitamin D₂ daily for 3 months.

Results

This 25-OH Vitamin D3 ELISA had minimal cross-reactivity to 25(OH)D₂, (0.7%), and demonstrated a high correlation (r² = 0.93) with 25(OH)D₃ determined by HPLC. 25(OH)D₃ increased by 14% in subjects receiving vitamin D₃ for 4 months (p < 0.01), whereas there was no significant change in 25(OH)D₃ levels in those receiving vitamin D₂.

Conclusions

We report that 25(OH)D₃ ELISA was used for evaluation of 25(OH)D₃ concentrations in subjects receiving vitamin D₂ and D₃ supplementation. The increase of 25(OH)D₃ in circulation with vitamin D3 supplementation and lack of increase with vitamin D₂ supplementation suggest that this assay has sufficient sensitivity and specificity to be used as a reliable measurement of nutritional vitamin D₃ status in humans.     

Acetylsalicylic acid regulates MMP-2 activity and inhibits colorectal invasion of murine B16F0 melanoma cells in C57BL/6J mice: Effects of prostaglandin F2α

Epidemiological studies indicate that acetylsalicylic acid may reduce the risk of mortality due to colon cancers. Metastasis is the major cause of cancer death. Matrix metalloproteinases (MMPs) play important roles in tumor invasion regulation, and prostaglandin F₂α (PGF₂α) is a key stimulator of MMP production. Thus, we investigated whether acetylsalicylic acid regulated MMP activity and the invasion of cancer cells and whether PGF₂α attenuated acetylsalicylic acid-inhibited invasion of cancer cells. Gelatin-based zymography assays showed that acetylsalicylic acid inhibited the MMP-2 activity of B16F0 melanoma cells. Matrigel-based chemoinvasion assays showed that acetylsalicylic acid inhibited the invasion of B16F0 cells. Acetylsalicylic acid can inhibit PGF₂α synthesis and PGF₂α is a key stimulator of MMP-2 production. Our data showed that PGF₂α treatment attenuated the acetylsalicylic acid-inhibited invasion of B16F0 cells. In animal experiments, acetylsalicylic acid reduced colorectal metastasis of B16F0 cells in C57BL/6J mice by 44%. Our results suggest that PGF₂α is a therapeutic target for metastasis inhibition and acetylsalicylic acid may possess anti-metastasis ability.     

The contribution of α1-antitrypsin to the total antitrypsin activity of human serum. a study of two phenotype pi oo individuals

Comparison of the levels of α₁AT, α₂-M, inter α-AT, C₁ inactivator and antiplasmin and global antitrypsin activity in a group of normal phenotype PI MM individuals, a group of normal individuals with phenotypes with intermediate α₁AT activities and two α₂-AT-deficient persons show that α₁AT contributes more than 90% of the total antitrypsin activity of normal plasma.AT III and fast reacting antiplasmin are shown to contribute to the remaining activity. It can be assumed that due to test conditions the antitrypsin activity of α₂-M is not assessed. C₁ inactivator and inter α₁-AT do not contribute to a perceptible extent to the overall antitrypsin activity estimated according to the method of Eriksson (Eriksson, S. (1965) Acta Med. Scand. 177, 1).     

Biochemical assay for amyloid β deposits to distinguish Alzheimer's disease from other dementias

Biochemical markers for Alzheimer's disease (AD) are of great value for precise diagnosis and in studies of the pathogenetic processes of this disease. A new biochemical assay allowing to differentiate AD from other forms of dementia is described. The assay is based on the extraction of amyloid β (Aβ) from milligram amounts of brain tissue by using 20% acetonitrile in 0.1% trifluoroacetic acid and its detection by Western blotting. The presence of the 4 kDa Aβ was demonstrated in all cases of AD (n=8) that were diagnosed by the independent histopathological examination of the postmortem tissues. No Aβ was found in tissue extracts from seven out of eight cases of other forms of dementia. In contrast to other biochemical techniques of Aβ detection in brain, the developed assay is simple; it does not require any special equipment and allows detection of Aβ using milligram amounts of brain tissue.     

Menthol Enhances an Antiproliferative Activity of 1α,25-Dihydroxyvitamin D3 in LNCaP Cells

1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃], the most active form of vitamin D₃, and its analogues have therapeutic benefits for prostate cancer treatment. However, the development of hypercalcemia is an obstacle to clinical applications of 1α,25(OH)₂D₃ for cancer therapy. In this study, we provide evidence that menthol, a key component of peppermint oil, increases an anti-proliferation activity of 1α,25(OH)₂D₃ in LNCaP prostate cancer cells. We found that menthol per se does not exhibit antiproliferative activity, but it is able to enhance 1α,25(OH)₂D₃-mediated growth inhibition in LNCaP cells. Fluorometric assays using Fura-2 showed that 1α,25(OH)₂D₃ does not induce acute Ca²⁺ response, whereas menthol evokes an increase in [Ca²⁺]_i, which suggests that cross-talks of menthol-induced Ca²⁺ signaling with 1α,25(OH)₂D₃-mediated growth inhibition pathways. In addition, Western blot analysis revealed that 1α,25(OH)₂D₃ and menthol cooperatively modulate the expression of bcl-2 and p21 which provides the insight into the molecular mechanisms underlying the enhanced 1α,25(OH)₂D₃-mediated growth inhibition by menthol. Thus, our findings suggest that menthol may be a useful natural compound to enhance therapeutic effects of 1α,25(OH)₂D₃.     

Resistance to gram-negative organisms due to high-level expression of plasmid-encoded ampC β-lactamase blaCMY-4 promoted by insertion sequence ISEcp1

A Klebsiella pneumoniae strain, KU6500, which showed resistance to extended-spectrum β-lactams and produced the plasmid-encoded AmpC β-lactamase CMY-4, was identified from clinical isolates in Japan. The aim of this study was to identify the mechanism of the high-level expression of bla _CMY-4. Sequence analysis indicated that the promoter element of Citrobacter freundii was conserved, but the insertion sequence ISEcp1 coding with the putative promoter element, was inserted into the AmpR binding site. We determined the influence of the promoter on bla _CMY-4 expression and β-lactam resistance. Two recombinant plasmids containing the entire bla _CMY-4 gene, with or without the ISEcp1-mediated promoter sequences, were constructed and named pMWampC and pMWISEcp1, respectively. Escherichia coli DH5α (pMWISEcp1) was resistant to almost all β-lactams tested and E. coli DH5α (pMWampC) was susceptible to all, except for cephalothin. In addition, the activity of each promoter was measured by subcloning the element into a promoterless luciferase plasmid pGL3-Basic vector. The expression of the putative promoter of ISEcp1 was 18.9-fold higher than that of C. freundii. These results suggest that the putative promoter element of ISEcp1 is necessary for the high-level expression of bla _CMY-4 to confer resistance to extended-spectrum cephalosporins.     

β-N-Acetylhexosaminadases in human cerebrospinal fluid and serum of patients with multiple sclerosis

β-N-Acetylhexosaminidase activity and isoenzyme have been investigated in normal human cerebrospinal fluid and that of patients with multiple sclerosis. β-N-acetylhexosaminidase activity in normal cerebrospinal fluids has been resolved into five components. The major component was in a form that eluted from DEAE cellulose at the same salt concentration as hexosaminidase As, the isoenzyme previously identified in human serum. Cerebrospinal fluid from patients exhibited a different isoenzyme profile, showing a remarkable increase in a form having a pI which was more acidic than that of As. These changes have a potential use in the diagnosis and further biochemical characterization of multiple sclerosis.     

The β3 subunit of the Na,K-ATPase mediates variable nociceptive sensitivity in the formalin test

It is widely appreciated that there is significant inter-individual variability in pain sensitivity, yet only a handful of contributing genetic variants have been identified. Computational genetic mapping and quantitative trait locus analysis suggested that variation within the gene coding for the β₃ subunit of the Na⁺,K⁺-ATPase pump (Atp1b3) contributes to inter-strain differences in the early phase formalin pain behavior. Significant strain differences in Atp1b3 gene expression, β₃ protein expression, and biophysical properties of the Na⁺,K⁺ pump in dorsal root ganglia neurons from resistant (A/J) and sensitive (C57BL/6J) mouse strains supported the genetic prediction. Furthermore, in vivo siRNA knockdown of the β₃ subunit produced strain-specific changes in the early phase pain response, completely rescuing the strain difference. These findings indicate that the β₃ subunit of the Na⁺,K⁺-ATPase is a novel determinant of nociceptive sensitivity and further supports the notion that pain variability genes can have very selective effects on individual pain modalities.     

Participation of peripheral tachykinin NK1 receptors in the carrageenan-induced inflammation of the rat temporomandibular joint

Temporomandibular disorders represent one of the major challenges in dentistry therapeutics. This study was undertaken to evaluate the time course of carrageenan-induced inflammation in the rat temporomandibular joint (TMJ) and to investigate the role of tachykinin NK₁ receptors. Inflammation was induced by a single intra-articular (i.art.) injection of carrageenan into the left TMJ (control group received sterile saline). Inflammatory parameters such as plasma extravasation, leukocyte influx and mechanical allodynia (measured as the head-withdrawal force threshold) and TNFα and IL-1β concentrations were measured in the TMJ lavages at selected time-points. The carrageenan-induced responses were also evaluated after treatment with the NK₁ receptor antagonist SR140333. The i.art. injection of carrageenan into the TMJ caused a time-dependent plasma extravasation associated with mechanical allodynia, and a marked neutrophil accumulation between 4 and 24 h. Treatment with SR140333 substantially inhibited the increase in plasma extravasation and leukocyte influx at 4 and 24 h, as well as the production of TNFα and IL-1β into the joint cavity, but failed to affect changes in head-withdrawal threshold. The results obtained from the present TMJ-arthritis model provide, for the first time, information regarding the time course of this experimental inflammatory process. In addition, our data show that peripheral NK₁ receptors mediate the production of both TNFα and IL-1β in the TMJ as well as some of the inflammatory signs, such as plasma extravasation and leukocyte influx, but not the nociceptive component.     

Development of γG, γA, γM, β1C/β1A, C′1 esterase inhibitor, ceruloplasmin, transferrin, hemopexin, haptoglobin, fibrinogen, plasminogen, α1-antitrypsin, orosomucoid, β-lipoprotein, α2-macroglobulin, and prealbumin in the human conceptus

The synthesis of γG, γA, γM, β_1C/β_1A, C′1 esterase inhibitor, ceruloplasmin, transferrin, hemopexin, haptoglobin, fibrinogen, α₁-antitrypsin, orosomucoid, β-lipoprotein, α₂-macroglobulin, and prealbumin was studied in 15 normal human embryos and fetuses of 29 days to 18 wk gestation and in the yolk sacs of four embryos from 5.5 to 11.5 wk gestation using tissue culture in ¹⁴C-labeled amino acids followed by radioimmunoelectrophoresis. The human embryo as early as 29 day gestation synthesized β_1C/β_1A, C′1 esterase inhibitor, transferrin, hemopexin, α₁-antitrypsin, β-lipoprotein, α₂-macroglobulin, and prealbumin in culture. At 32 days gestation ceruloplasmin and orosomucoid were also synthesized, but synthesis of fibrinogen was not observed before 5.5 wk. Synthesis of γM occurred as early as 10.5 wk gestation, and γG synthesis was found in cultures as early as 12 wk gestation; γA synthesis was not detected in any of the tissue cultures. With the exception of the γ-globulins, each of the proteins studied was synthesized by the liver, but additional sites of synthesis for some of these proteins were also found. Synthesis of γG and γM occurred primarily in the spleen, but other sites of synthesis were noted as well.     

Heterostructures of ε-Fe2O3 and α-Fe2O3: insights from density functional theory

Many materials used in energy devices or applications suffer from the problem of electron–hole pair recombination. One promising way to overcome this problem is the use of heterostructures in place of a single material. If an electric dipole forms at the interface, such a structure can lead to a more efficient electron–hole pair separation and thus prevent recombination. Here we model and study a heterostructure comprised of two polymorphs of Fe₂O₃. Each one of the two polymorphs, α-Fe₂O₃ and ε-Fe₂O₃, individually shows promise for applications in photoelectrochemical cells. The heterostructure of these two materials is modeled by means of density functional theory. We consider both ferromagnetic as well as anti-ferromagnetic couplings at the interface between the two systems. Both individual oxides are insulating in nature and have an anti-ferromagnetic spin arrangement in their ground state. The same properties are found also in their heterostructure. The highest occupied electronic orbitals of the combined system are localized at the interface between the two iron-oxides. The localization of charges at the interface is characterized by electrons residing close to the oxygen atoms of ε-Fe₂O₃ and electron–holes localized on the iron atoms of α-Fe₂O₃, just around the interface. The band alignment at the interface of the two oxides shows a type-III broken band-gap heterostructure. The band edges of α-Fe₂O₃ are higher in energy than those of ε-Fe₂O₃. This band alignment favours a spontaneous transfer of excited photo-electrons from the conduction band of α- to the conduction band of ε-Fe₂O₃. Similarly, photo-generated holes are transferred from the valence band of ε- to the valence band of α-Fe₂O₃. Thus, the interface favours a spontaneous separation of electrons and holes in space. The conduction band of ε-Fe₂O₃, lying close to the valence band of α-Fe₂O₃, can result in band-to-band tunneling of electrons which is a characteristic property of such type-III broken band-gap heterostructures and has potential applications in tunnel field-effect transistors.

Electron–hole pair recombination is reduced in heterostructures if used in devices in place of single material.     

In Vitro Effects of the Reduced Form of Coenzyme Q10 on Secretion Levels of TNF-α and Chemokines in Response to LPS in the Human Monocytic Cell Line THP-1

Ubiquinol-10 (QH₂), the reduced form of Coenzyme Q₁₀ (CoQ₁₀) serves as a potent antioxidant of lipid membranes. Because many antioxidants reveal potent anti-inflammatory effects, the influence of QH₂ on lipopolysaccharide (LPS)-induced pro-inflammatory cytokines and chemokines were determined in the human monocytic cell line THP-1. Stimulation of cells with LPS resulted in a distinct release of Tumour necrosis factor-alpha (TNF-α), Macrophage inflammatory protein-1 alpha (MIP-1α), Regulated upon activation, normal T cell expressed and secreted (RANTES) and Monocyte chemotattractant protein-1 (MCP-1). The LPS-induced responses were significantly decreased by pre-incubation of cells with QH₂ to 60.27 ± 9.3% (p = 0.0009), 48.13 ± 6.93% (p = 0.0007) and 74.36 ± 7.25% (p = 0.008) for TNF-α, MIP-1α and RANTES, respectively. In conclusion, our results indicate anti-inflammatory effects of the reduced form of CoQ₁₀ on various proinflammatory cytokines and chemokines in vitro.     

Elastase binding capacity of α2-macroglobulin and its association with glucocorticoid concentration in southern African black patients with hepatocellular carcinoma

Thirty-three Southern African black patients with hepatocellular carcinoma (HCC) (7 women) and 43 black control individuals (14 women), all in the age group 18–45 years, were investigated for plasma α₂-macroglobulin (α₂M) elastase binding capacity (EBC). Cortisol levels were measured in 15 (3 women) of the HCC patients and 10 (5 women) of the control subjects. A significant difference in EBC was found between the HCC patients and the control subjects (P < 0.001). A significant difference was also found in cortisol levels between the two groups (P < 0.001). A significant correlation between EBC and cortisol levels was obtained (r = 0.57; P < 0.042). The significant increase in EBC of α₂M in HCC patients could be due to an increase in circulating cortisol.     

Pentagalloylglucose, an antisecretory component of Paeoniae radix, inhibits gastric H, K-ATPase

We purified a compound with strong inhibitory effect on H⁺, K⁺-ATPase from Paeoniae radix, which has been used in Japan for the treatment of gastritis and peptic ulcers. The compound was identified as 1,2,3,4,6,-penta-o-galloyl-β- -glucose by proton nuclear magnetic resonance, carbon-13 nuclear magnetic resonance, and fast atomic bombardment mass spectrometry. The IC₅₀ of the compound for H⁺, K⁺-ATPase was 166 nmol/l. Kinetic analyses indicated that the inhibition of the enzyme by pentagalloylglucose was noncompetitive with respect to K⁺. Pentagalloylglucose had relatively weak inhibitory effects for Mg⁺-ATPase (IC₅₀: >10 μmol/l) and Na⁺, K⁺-ATPase (IC₅₀: 2.7 μmol/l). Pentagalloylglucose also inhibited the accumulation of [¹⁴C]aminopyrine in parietal cells that had been isolated from guinea pig stomach and stimulated by 10 μmol/l histamine (IC₅₀: 7.8 μmol/l) and 1 mmol/l dbc-AMP (IC₅₀: 10 μmol/l). These results suggest that pentagalloylglucose is a potent inhibitor of H⁺, K⁺-ATPase and may be responsible for inhibition of acid secretion by Paeoniae radix.     

Optical and dielectric properties of NaCoPO4 in the three phases α, β and γ

In this work, we are interested in the synthesis of monophosphate α-NaCoPO₄, β-NaCoPO₄ and γ-NaCoPO₄ compounds by mechanochemical method and their characterization by X-ray powder diffraction patterns. These compounds are crystallized in the orthorhombic, hexagonal and monoclinic system, in Pnma, P6₅ and P2₁/n space groups, respectively. The optical properties were measured by means of the UV-vis absorption spectrometry in order to deduce the absorption coefficient α and optical band gap E_g. The calculated values of the indirect band gaps (E_gi) for three samples were estimated at 4.71 eV, 4.63 eV and 3.8 for compounds α, β and γ, respectively. The Tauc model was used to determine the optical gap energy of the synthesized compounds. Then, the results of the dielectric proprieties measured by varying the frequency are described.

In this work, we are interested in the synthesis of monophosphate α-NaCoPO₄, β-NaCoPO₄ and γ-NaCoPO₄ compounds by mechanochemical method and their characterization by X-ray powder diffraction patterns.