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•Expression of ERAP1 may depend on the experimental model and the normalizer used.•A strategy for improved selection of the best stable reference gene has been devised.•This strategy ...avoided experimental model- and normalizer-dependent conclusions.•The expression levels of RPS23, SNRPA and PGK1 housekeeping genes change during melanoma progression.
The objective of this study was to identify a normalizer or combination of normalizers for quantitative evaluation of the expression of a target gene of interest during melanoma progression. Adult melanocytes, uveal primary melanoma cells and cutaneous primary and metastatic melanoma cells were used to construct a panel of 14 experimental models reflecting cancer promotion and progression. Hypoxanthine phosphoribosyltransferase 1 (HPRT1), glucuronidase beta (GUSB), ribosomal protein S23 (RPS23), phosphoglycerate kinase 1 (PGK1) and small nuclear ribonucleoprotein progression. Adult melanocytes, uveal primary melanoma cells and cutaneous primary and metastatic melanoma cells were used to construct a panel of 14 experimental models reflecting cancer promotion and progression. Hypoxanthine phosphoribosyltransferase 1 (HPRT1), glucuronidase beta (GUSB), ribosomal protein S23 (RPS23), phosphoglycerate kinase 1 (PGK1) and small nuclear ribonucleoprotein polypeptide A (SRNPA) were chosen as candidate housekeeping genes. NormFinder software was used to identify the best reference gene or pair of reference genes from five candidate housekeeping genes, on the basis of expression stability in a given experimental model. The suitability of references was validated by normalizing the transcriptional activities of E-cadherin (CDH1), N-cadherin (CDH2) and endoplasmic reticulum aminopeptidase 1 (ERAP1) target genes. It has been shown that the relative expression of CDH2 and ERAP1 target genes in a given cell line may vary between experimental models, leading to biological misinterpretation. In view of this, we devised a strategy for improved selection of the best stable reference and for obtaining biologically consistent results. This strategy avoided experimental model- and normalizer-dependent conclusions concerning the relative expression of target gene, in the examined cell lines.
The erythrocyte binding ligand 140 (EBA-140) is a member of the Plasmodium falciparum DBL family of erythrocyte binding proteins, which are considered as prospective candidates for malaria vaccine ...development. The EBA-140 ligand is a paralogue of the well-characterized P. falciparum EBA-175 protein. They share homology of domain structure, including Region II, which consists of two homologous F1 and F2 domains and is responsible for ligand-erythrocyte receptor interaction during invasion. In this report we describe, for the first time, the glycophorin C specificity of the recombinant, baculovirus-expressed binding region (Region II) of P. falciparum EBA-140 ligand. It was found that the recombinant EBA-140 Region II binds to the endogenous and recombinant glycophorin C, but does not bind to Gerbich-type glycophorin C, neither normal nor recombinant, which lacks amino acid residues 36-63 of its polypeptide chain. Our results emphasize the crucial role of this glycophorin C region in EBA-140 ligand binding. Moreover, the EBA-140 Region II did not bind either to glycophorin D, the truncated form of glycophorin C lacking the N-glycan or to desialylated GPC. These results draw attention to the role of glycophorin C glycans in EBA-140 binding. The full identification of the EBA-140 binding site on glycophorin C molecule, consisting most likely of its glycans and peptide backbone, may help to design therapeutics or vaccines that target the erythrocyte binding merozoite ligands.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The paper deals with F-normed functions and sequence spaces. First, some general results on such spaces are presented. But most of the results in this paper concern various monotonicity properties ...and various Kadec–Klee properties of F-normed Orlicz functions and sequence spaces and their subspaces of elements with order continuous norm, when they are generated by monotone Orlicz functions on
R
+
and equipped with the classical Mazur–Orlicz F-norm. Strict monotonicity, lower (and upper) local uniform monotonicity and uniform monotonicity in the classical sense as well as their orthogonal counterparts are considered. It follows from the criteria that are presented for these properties that all the above classical monotonicity properties except for uniform monotonicity differ from their orthogonal counterparts in contrast to Köthe spaces (see Hudzik et al. in Rocky Mt J Math 30(3):933–950,
2000
). The Kadec–Klee properties that are considered in this paper correspond to various kinds of convergence: convergence locally in measure and convergence globally in measure for function spaces, uniform convergence and coordinatewise convergence in the case of sequence spaces.
We introduce a new geometric constant
C
N
J
(
p
)
(
X
)
for a Banach space
X
, called a generalized von Neumann-Jordan constant. Next, it is shown that
1
≤
C
N
J
(
p
)
(
X
)
≤
2
for any Banach space
...X
and that the right hand side inequality is sharp if and only if
X
is uniformly non-square. Moreover, a relationship between the James constant
J
(
X
)
and
C
N
J
(
p
)
(
X
)
is presented. Finally, the generalized von Neumann-Jordan constant of the Lebesgue space
L
r
(
0
,
1
)
is calculated and a relationship between
C
N
J
(
p
)
(
X
)
and the fixed point property is found.
Uveal melanoma is the most common primary intraocular malignancy in adults. The development of distant metastases is associated with a poor prognosis. Ephrine receptors (Eph) are the largest ...subpopulation of tyrosine kinase receptors. They play an important role in processes related to the formation and progression of cancer. The aim of the study was to evaluate the expression of ephrin receptors EphA1, EphA5, and EphA7 in uveal melanoma and its associations with clinicopathological parameters, overall survival, and disease-free survival. The study included 94 previously untreated patients who underwent enucleation due to uveal melanoma. High expression of EphA1 was positively correlated with a smaller tumor size, less frequent extra-scleral extension, lower mitotic activity, and more frequent vitreous hemorrhage. High expression of EphA5 was associated with less frequent chromosome 3 loss, absence of distant metastases, and more frequent vitreous hemorrhage. High expression of EphA7 was associated with a more frequent primary tumor location in the posterior pole. High EphA5 expression was associated with longer overall survival time. The above findings indicate that high expression of EphA1 and EphA5 can be considered a beneficial prognostic factor in uveal melanoma.
Abstract Plasmodium reichenowi, an ape malaria parasite is morphologically identical and genetically similar to Plasmodium falciparum, infects chimpanzees but not humans. Genomic studies revealed ...that all primate malaria parasites belong to Laverania subgenus. Laverania parasites exhibit strict host specificity, but the molecular mechanisms underlying these host restrictions remain unexplained. Plasmodium merozoites express multiple binding ligands that recognize specific receptors on erythrocytes, including micronemal proteins belonging to P. falciparum EBL family. It was shown that erythrocyte binding antigen-175 (EBA-175), erythrocyte binding ligand-1 (EBL-1), erythrocyte binding antigen-140 (EBA-140) recognize erythrocyte surface sialoglycoproteins - glycophorins A, B, C, respectively. EBA-140 merozoite ligand hijacks glycophorin C (GPC), a minor erythrocyte sialoglycoprotein, to invade the erythrocyte through an alternative invasion pathway. A homolog of P. falciparum EBA-140 protein was identified in P. reichenowi. The amino acid sequences of both EBA-140 ligands are very similar, especially in the conservative erythrocyte binding region (Region II). It has been suggested that evolutionary changes in the sequence of EBL proteins may be associated with Plasmodium host restriction. In this study we obtained, for the first time, the recombinant P. reichenowi EBA-140 ligand Region II using baculovirus expression vector system. We show that the ape EBA-140 Region II is host specific and binds to chimpanzee erythrocytes in the dose and sialic acid dependent manner. Further identification of the erythrocyte receptor for this ape ligand is of great interests, since it may reveal the molecular basis of host restriction of both P. reichenowi and its deadliest human counterpart , P. falciparum.
Uveal melanoma is the most common primary intraocular malignancy in adults. In advanced cases, the prognosis is very poor. Thus far, no effective methods of pharmacotherapy of this cancer have been ...found. The aim of the study was to evaluate the expression of PARP-1, the best-known member of the family of poly(ADP-ribose) polymerases, in uveal melanoma and its associations with clinicopathological parameters, overall survival, and disease-free survival.
The study included 91 patients who underwent enucleation due to uveal melanoma. PARP-1 expression was assessed by immunohistochemistry.
High PARP-1 expression was associated with more frequent chromosome 3 loss, higher histopathological grade, bigger tumor size, and absence of intrascleral extension. High PARP-1 expression was associated with shorter overall survival time and disease-free survival time.
The above findings indicate that high expression of PARP-1 can be considered as an unfavorable prognostic factor in uveal melanoma.
Carcinogenesis in various tissues is accompanied by alterations in protein and lipid glycosylation, such as changes in the expressions of Lewis histo-blood group antigens. Neoplastic transformation ...is often followed by changes in expression of one or more of these oligosaccharides in a pattern that is typical for the tissue. These alterations correlate with changes in the expressions of specific glycosyltransferases. Overexpression of Lewis antigens in some types of cancer might be a significant prognostic factor. Upregulation of Lewis(y), Lewis(b), and other alpha1,2-fucosylated oligosaccharides is linked to an increased tendency to metastasis and resistance to treatment. For example, invasive breast cancer of poor prognosis reveals high expression levels of Lewis(y) and Lewis(b). Lewis(x) is also overexpressed in breast cancer cells. Overexpression of Lewis(a) also occurs in precancerous states of stomach tissue. Upregulation of sialyl Lewis(a) correlates with the metastatic potential of colon and pancreatic malignancies and inversely with survival rate of patients. A similar relation was observed between sialyl Lewis(x) overexpression and malignancies of the lungs, prostate, urinary bladder, stomach, breast, kidney, and liver. Some of the Lewis antigens are ligands of specific receptors and adhesion molecules. For example, Lewis(x) is recognized by scavenger receptor C-type lectin (SRCL), which belongs to the group of innate immunity receptors. Sialyl Lewis(a) and sialyl Lewis(x) specifically interact with E- and P-selectins, which are key molecules in leukocyte rolling process. Ongoing research shows an increasing number of potential therapeutic applications related to the upregulation of Lewis antigens.
Human Gb3/CD77 synthase (α1,4-galactosyltransferase) is the only known glycosyltransferase that changes acceptor specificity because of a point mutation. The enzyme, encoded by
A4GALT
locus, is ...responsible for biosynthesis of Gal(α1–4)Gal moiety in Gb3 (CD77, P
k
antigen) and P1 glycosphingolipids. We showed before that a single nucleotide substitution c.631C > G in the open reading frame of
A4GALT
, resulting in replacement of glutamine with glutamic acid at position 211 (substitution p. Q211E), broadens the enzyme acceptor specificity, so it can not only attach galactose to another galactose but also to
N
-acetylgalactosamine. The latter reaction leads to synthesis of NOR antigens, which are glycosphingolipids with terminal Gal(α1–4)GalNAc sequence, never before described in mammals. Because of the apparent importance of position 211 for enzyme activity, we stably transfected the 2102Ep cells with vectors encoding Gb3/CD77 synthase with glutamine substituted by aspartic acid or asparagine, and evaluated the cells by quantitative flow cytometry, high-performance thin-layer chromatography and real-time PCR. We found that cells transfected with vectors encoding Gb3/CD77 synthase with substitutions p. Q211D or p. Q211N did not express P
k
, P1 and NOR antigens, suggesting complete loss of enzymatic activity. Thus, amino acid residue at position 211 of Gb3/CD77 synthase is critical for specificity and activity of the enzyme involved in formation of P
k
, P1 and NOR antigens. Altogether, this approach affords a new insight into the mechanism of action of the human Gb3/CD77 synthase.
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