The SLC4 family of HCO3 − transporters

The SLC4 family consists of ten genes. All appear to encode integral membrane proteins with very similar hydropathy plots-consistent with the presence of 10-14 transmembrane segments. At least eight SLC4 members encode proteins that transport HCO(3)(-) (or a related species, such as CO(3)(2-)) across the plasma membrane. Functionally, these eight proteins fall into two major groups: three Cl-HCO(3) exchangers (AE1-3) and five Na(+)-coupled HCO(3)(-) transporters (NBCe1, NBCe2, NBCn1, NDCBE, NCBE). Two of the Na(+)-coupled HCO(3)(- )transporters (NBCe1, NBCe2) are electrogenic; the other three Na(+)-coupled HCO(3)(-) transporters and all three AEs are electroneutral. At least NDCBE transports Cl(-) in addition to Na(+) and HCO(3)(-). Whether NCBE transports Cl(-)-in addition to Na(+) and HCO(3)(-)-is unsettled. In addition, two other SLC4 members (AE4 and BTR1) do not yet have a firmly established function; on the basis of homology, they fall between the two major groups. A characteristic of many, though not all, SLC4 members is inhibition by 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS). SLC4 gene products play important roles in the carriage of CO(2) by erythrocytes, the absorption or secretion of H(+) or HCO(3)(-) by several epithelia, as well as the regulation of cell volume and intracellular pH.     

The nature of ‘species-specific’ amino acid residues

Amino acid sequences for the first 40 N-terminal residues of three rat κ-type Bence-Jones (B.-J.) proteins and for the first 23 residues of pooled rat immunoglobulin light chains were determined. Comparison of these sequences with those of human and mouse κ chains indicates that it is difficult to find any amino acid residue which is absolutely ‘species-specific’ or ‘phylogenetically-associated’. Therefore, the ‘species-specific’ or ‘phylogenetically-associated’ residues cannot be used as a strong argument in support of either the somatic mutation or the somatic recombination hypothesis regarding the genetic control of antibody variability.     

The synthesis of the sodium salt of β-oxybutyric acid from the acetoacetic ester

Summary A method is described for preparing the sodium salt of -oxybutyric acid from acetoacetic ester. The acetoacetic ester is reduced by aluminium amalgam in moist sulphuric acid, and the -oxybutyric acid thus obtained is then saponified to form the sodium salt of -oxybutyric acid. The yield is approximately 30%.(Presented by Active Member AMN SSSR S. E. Severin) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 51, No. 6, pp. 104–105, June, 1961     

Integrated omics analysis of sweat reveals an aberrant amino acid metabolism pathway in Vogt–Koyanagi–Harada disease

Vogt–Koyanagi–Harada (VKH) disease is an autoimmune disease leading to visual impairment. Its pathogenic mechanisms remain poorly understood. Our purpose was to investigate the distinctive protein and metabolic profiles of sweat in patients with VKH disease. In the present study, proteomics and metabolomics analysis was performed on 60 sweat samples (30 VKH patients and 30 normal controls) using liquid chromatography tandem mass spectrometry. Parallel reaction monitoring (PRM) analysis was used to validate the results of our omics analysis. In total, we were able to detect 716 proteins and 175 metabolites. Among them, 116 proteins (99 decreased and 17 increased) were observed to be significantly different in VKH patients when compared to controls. Twenty-one differentially expressed metabolites were identified in VKH patients, of which 18 included choline, L-tryptophan, betaine and L-serine were reduced, while the rest were increased. Our multi-omics strategy reveals an important role for the amino acid metabolic pathway in the pathogenesis of VKH disease. Significant differences in proteins and metabolites were identified in the sweat of VKH patients and, to some extent, an aberrant amino acid metabolism pathway may be a pathogenic factor in the pathogenesis of VKH disease.     

Carboxyl terminal amino acid sequence of guinea pig γ1G

The immunoglobulin γ₁G has been isolated from the serum of immunised guinea pigs relatively pure form in quantities large enough to allow a start to be made on amino acid sequence studies. Limited proteolysis with pepsin produces a crystallizable C-terminal pFc′ fragment containing about 114 amino acid residues. Cyanogen bromide cleavage of this fragment yields the C-terminal octadecapeptide of teh γ₁ chain. The isolation, analysis and amino acid sequence of this peptide is reported. It differs from guinea pig γ₂G at positions 7(Ser), 10(Ile) and 14(Pro), numbering from the C-terminal.     

Stability assessment of chitosan–sodium hexametaphosphate capsules

The assessment of the stability of capsules based on chitosan-sodium hexametaphosphate complex formation has been carried out using two independent methods - compression and osmotic swelling, and the influence of the preparation variables was evaluated. The formulation containing 1.5% core polymer (chitosan) and 1.5% oligophosphate, in the absence of salt or at low ionic strength (0.15% NaCl) was found to provide the best membrane resistance. A higher concentration of crosslinker (2.25%) produced stable capsules only in absence of electrolyte. Mannitol, a porogen added to the preparation solutions, did not affect the stability of the obtained membranes. At elevated polyol (1%) and crosslinker levels (2.25%), and at 0% salt, membranes with decreased elasticity were obtained, having lower compression and osmotic bursting values and lower deformation at the breaking points. A significant influence of salt amount on the capsule stability was also found. This was attributed to changes in the membrane formation process resulting in membranes with different thickness and structure. Membrane compression stability was found to be dependent on the pH of both oligophosphate and chitosan solutions, as well as on the reaction time. The bursting force values decreased for capsule diameters below 1.6 mm. The increased membrane/capsule volume ratio for the small capsules decreased the capsule deformation freedom and caused capsule rupture at low force values. The capsules made at low salt amounts showed very good storage stability over time and at elevated temperatures. The results demonstrated that the capsules could be formulated with controlled properties for various biomedical applications.     

The anticancer efficacy of pixantrone-loaded liposomes decorated with sialic acid–octadecylamine conjugate

Based on the knowledge that sialic acid is a critical element for tumor development and its receptors are highly expressed on the tumor-associated macrophages (TAMs) which play important roles in the growth and metastasis of tumors, we synthesized a sialic acid–octadecylamine conjugate (SA–ODA) and anchored it on the surface of pixantrone (Pix)-loaded liposomes, to achieve an improved anticancer effect. Four Pix formulations (Pix-S, Pix-CL, Pix-PL and Pix-SAL represent solution, conventional liposome, stealth liposome, and SA–ODA modified liposome, respectively) were developed, and various parameters, including drug loading, stability, in vitro release, cytotoxicity and pharmacokinetics, were evaluated. The tumor growth inhibition and toxicity studies were performed in S180-bearing Kunming mice. Pix-S exhibited a strong toxicity to the immune system, accelerated the growth of tumors and reduced the lifespan of mice. In contrast, Pix-SAL displayed the strongest anticancer and life-prolonging effects among all of the formulations in this study. More importantly, injection of Pix-SAL induced a phenomenon whereby the cancerous tissues were “shed” from mice, after which the wound healed. We speculate that this special efficacy may be partly due to the killing of TAMs by Pix-SAL. This study suggests that SA–ODA modified liposomes may serve as an effective intravenous delivery vehicle for Pix.     

Can–Evo–Ens: Classifier stacking based evolutionary ensemble system for prediction of human breast cancer using amino acid sequences

The diagnostic of human breast cancer is an intricate process and specific indicators may produce negative results. In order to avoid misleading results, accurate and reliable diagnostic system for breast cancer is indispensable. Recently, several interesting machine-learning (ML) approaches are proposed for prediction of breast cancer. To this end, we developed a novel classifier stacking based evolutionary ensemble system “Can–Evo–Ens” for predicting amino acid sequences associated with breast cancer. In this paper, first, we selected four diverse-type of ML algorithms of Naïve Bayes, K-Nearest Neighbor, Support Vector Machines, and Random Forest as base-level classifiers. These classifiers are trained individually in different feature spaces using physicochemical properties of amino acids. In order to exploit the decision spaces, the preliminary predictions of base-level classifiers are stacked. Genetic programming (GP) is then employed to develop a meta-classifier that optimal combine the predictions of the base classifiers. The most suitable threshold value of the best-evolved predictor is computed using Particle Swarm Optimization technique. Our experiments have demonstrated the robustness of Can–Evo–Ens system for independent validation dataset. The proposed system has achieved the highest value of Area Under Curve (AUC) of ROC Curve of 99.95% for cancer prediction. The comparative results revealed that proposed approach is better than individual ML approaches and conventional ensemble approaches of AdaBoostM1, Bagging, GentleBoost, and Random Subspace. It is expected that the proposed novel system would have a major impact on the fields of Biomedical, Genomics, Proteomics, Bioinformatics, and Drug Development.     

Nucleotide and deduced amino acid sequence of the 3′ end of the BYMV-MI genome

Summary We have cloned and sequenced cDNA transcribed from the 3 1239 nucleotides of the genomic RNA of a Western Australian isolate (MI) of bean yellow mosaic potyvirus (BYMV). This sequence contains 246 nucleotides of the NIb (replicase) gene and 819 nucleotides representing the entire coding region of the viral coat protein gene, followed by a 3 non-coding region of 174 nucleotides. The coding region of the coat protein gene is identical in length (273 amino acids) to that already reported for other isolates of this virus. The sequence identities obtained for BYMV-MI and published sequences of BYMV isolates range between 85% and 92% for the coding region of the coat protein and 90% to 98% for the 3 non-coding region. Likewise, the region of the NIb gene sequenced shows 99% and 97% sequence identity in the deduced amino acid and the nucleotide sequences, respectively.     

The glutamate transporter excitatory amino acid carrier 1 associates with the actin-binding protein α-adducin

Excitatory amino acid carrier 1 (EAAC1) belongs to the family of the Na⁺-dependent glutamate carriers. Although the association between defective EAAC1 function and neurologic disease has been repeatedly studied, EAAC1 regulation is not yet fully understood. We have reported that in C6 glioma cells both the activity and membrane targeting of EAAC1 require the integrity of actin cytoskeleton. Here we show that, in the same model, EAAC1 partially co-localizes with actin filaments at the level of cell processes. Moreover, perinuclear spots in which EAAC1 co-localizes with the actin binding protein α-adducin are observed in some cells and, consistently, faint co-immunoprecipitation bands between EAAC1 and α-adducin are detected. Co-localization and partial co-immunoprecipitation of EAAC1 and adducin are still detectable after cell treatment with phorbol esters, a condition that leads to a protein kinase C (PKC)-dependent increase of EAAC1 expression on the membrane and to the phosphorylation of adducin. A co-immunoprecipitation band was also detected in protein extracts of rat hippocampus. The amount of adducin co-immunoprecipitated with EAAC1 increases after the treatment of C6 cells with retinoic acid, a differentiating agent that induces EAAC1 overexpression in this cell model. Moreover, in clones of C6 cells transfected with a hemagglutinin (HA)-tagged adducin, the bands of EAAC1 immunoprecipitated by an anti-HA antiserum were proportional to EAAC1 expression. These results suggest the existence of a pool of EAAC1 transporters associated with the actin binding protein α-adducin in a PKC-insensitive manner.     

Bioactive enzyme–metal composites: The entrapment of acid phosphatase within gold and silver

This paper is concerned with the entrapment of an enzyme within an aggregated metallic matrix and the development of a bioactive enzyme–metal composite. Whereas the use of organic polymers and metal oxides for the preparation of enzymatically active materials is well developed, the third principle enzyme–material combination, namely protein–metal bulk, has not yet been reported. A new methodology for the entrapment of organic molecules and polymers within metals has been employed for the preparation of bioactive acid phosphatase@gold and acid phosphatase@silver, according to which room temperature reduction of the metal cation is carried out in the presence of the enzyme to be entrapped. Protectability of the entrapped enzyme against harsh conditions is shown: the acidic enzyme is kept alive under basic conditions.     

On the structure–function of MHC class II molecules and how single amino acid polymorphisms could alter intracellular trafficking

Classical HLA class II molecules are highly polymorphic heterodimeric transmembrane proteins encoded by a polygenic cluster on chromosome 6. Polymorphic residues in the membrane-distal domains ensure that a large collection of microbial peptides can be bound in the human population. Still, the HLA-DR, -DP and -DQ isotypes show a high degree of conservation in their overall tertiary and quaternary structures, in line with their common function in T cell receptor activation. Interestingly, the primary structure of the intracellular domains are highly divergent between isotypes and they also show allotypic variations. The functional impact of these differences remains to be fully appreciated. Here, we address the role of the MHC class II cytoplasmic tails in intracellular trafficking. First, the emphasis will be on the interplay between the cytoplasmic domains of classical human MHC class II molecules and those of the invariant chain chaperone (CD74) isoforms. Then, we will examine the importance of the highly conserved β-chain cytoplasmic lysine residue in the ubiquitin-driven trafficking of MHC class II molecules. These considerations should help understand the potential functional impact of sequence variations that may arise in the cytoplasmic tails and transmembrane domains of MHC class II molecules.     

N- and C-terminal amino acid sequences of a γ-heavy chain disease protein YOK

The N- and C-terminal peptides of a γ-heavy chain disease protein YOK were isolated by cyanogen bromide cleavage of the protein followed by reduction, carboxymethylation and gelfiltration, and their sequences were determined by the conventional methods. Protein YOK consisted of Fc-fragments of IgG₁ and had heterogeneous N-termini, serine, threonine and histidine, which corresponded to positions 215, 223 and 224 respectively, from the N-terminal end of the gamma chain of immunoglobulin EU. A new amino acid substitution was found in position 431, in which YOK contained glycine in contrast to alanine in corresponding position of known human γ₁-chains, suggesting one structural correlation with a genetic marker.These results were compared with those of other γ-heavy chain disease proteins and discussed for the genetic implications of the sequence of this protein.     

The growth of a vascular network inside a collagen–citric acid derivative hydrogel in rats

Three-dimensional regenerative tissue with a certain bulk cannot survive without sufficient blood perfusion in vivo, so construction of a vascular system in regenerative tissue is a key technology in tissue engineering. In order to construct such a vascular system, we attempted to create a scaffold material that induces neovascular growth from the recipient bed into the material. This material, an ion complex gel matrix (IC gel) consisting of collagen and a citric acid derivative, enabled it to associate with basic fibroblast growth factor (bFGF). The IC gel was implanted in the subfascial space of the rat rectus muscle and excised 5 days later. Cross-sections of the excised samples were stained for von Willebrand factor, and then neovascular development into the gel was observed and also quantified by image analysis. These data showed that the IC gel markedly induced growth of vascular-rich tissue into the inside of the gel by day 5, which surpassed that after implantation of Matrigel^® or gelated collagen. Further, combination with bFGF significantly enhanced the vascularization ability of IC gel. These findings suggest that IC gel functioned as a scaffold material for neovascular ingrowth and a reservoir of bFGF.     

Role of HLA-B α-3 domain amino acid position 194 in HIV disease progression

HLA class I molecules play a role in the regulation of innate immune response. Therefore, the interaction of HLA class I molecules with different activating and inhibitory receptors leads to balancing the immune response. Among the different family of receptors, NK receptors KIR3DL1/S1 and LIR1, play a major role. Aim of this study was to evaluate the role of amino acid polymorphic positions of HLA class I molecules interacting with NK receptors in HIV progression. In order to minimize the influence of viral variability, a cohort of children with a nosocomial monophyletic HIV-1 infection from the Benghazi Children Hospital has been evaluated. To assess the role of single amino acid positions, we translated all HLA alleles in the different amino acid position polymorphisms. Interestingly, the polymorphism Val 194 located in the α3-domain of HLA-B, resulted associated with LTNP (LTNP = 73.08%, FP = 34.78%; P < 0.02). When Val is present at position 194, HLA-B is known to interact with the receptor LIR1 (ILT2/LILRB1/CD85j). Therefore, we analyzed the role of the polymorphism in position 194 in HLA-B/LIR1 interaction by homology molecular modeling. The change Val to Ile at position 194 alters significantly the network of interaction between the amino acid residues of HLA-B and LIR1. In conclusion, considering the limitation of the small population evaluated, polymorphisms outside the peptide binding region of the HLA class I molecule can play a key role in HIV progression through interaction with other immune-relevant receptors.     

The cyclins: a family of widely expressed tumor antigens?

Continuous cell division is a hallmark of cancer and cell-cycle regulators therefore represent relevant target molecules for tumor therapy. Among these targets the cyclins are of particular interest as they are overexpressed in various tumor entities with little expression in normal tissue. Here we review evidence that these molecules are recognized by the immune system, summarize why cyclins A, B and D in particular appear to be interesting targets for active and passive immunotherapy, and discuss whether the entire family could be an interesting novel class of tumor antigens for cancer treatment and prevention.     

Effect of preparation conditions on properties and permeability of chitosan–sodium hexametaphosphate capsules

Capsules were obtained by interpolymer complexation between chitosan (polycation) and sodium hexametaphosphate (SMP, oligoanion). The effect of the preparation conditions on the capsule characteristics was evaluated. Specifically, the influence of variables such as pH, ionic strength, reagent concentration, and additives on the capsule permeability properties was investigated using dextran as a model permeant. The capsule membrane permeability was found to increase by decreasing the chitosan/SMP ratio as well as adding mannitol to the oligoanion recipient bath. Increasing the ionic strength or the pH of the initial chitosan solution was also found to enhance the membrane permeability, moving the membrane exclusion limit to higher values. Generally, the capsules prepared under all tested conditions had a relatively low permeability which rarely exceeded a molecular cut-off of 40 kD based on dextran standards. Furthermore, the diffusion rate showed a strong temporal dependence, indicating that the capsules prepared under various conditions exhibit different apparent pore size densities on the surface. The results indicated that, in order to obtain the desired capsule mass-transfer properties, the preparation conditions should be carefully considered and adjusted. Adding a polyol as well as low salt amount (less than 0.15%) is preferable as a means of modulating the diffusion characteristics, without disturbing the capsule mechanical stability.