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Studies on the circumanal skin of a three-week-old puppy have shown that, contrary to the adult dogs, sebaceous glands are absent here (although they are well-developed in other skin sites of the puppy). Hepatoid glands accompany every hair follicle; they differ considerably from hepatoid glands of adult dogs. Hepatoid circumanal glands of the three-week old puppy are represented by two histological varieties of this glandular type which are capable of producing hydrophobic lipids, are rather poor in protein, and do not possess cysts--while in adult dogs they are represented by only one variety incapable of synthesizing lipids, extremely rich in protein, possessing numerous cysts. Discussion concerns histogenesis, distribution, and function of certain hepatoid gland varieties which were first described by the authors in their previous publication.
The nucleotide sequence of RNA-polymerase gene and 3'-terminal untranslated genome region of attenuated foot-and-mouth disease virus (FMDV) strain A(22)645 has been determined. RNA-polymerase gene and predicted amino acid sequences of attenuated FMDV strain A(22)645 were compared with those of the original virulent FMDV strain A(22)550. The examined genome region of strain A(22)645 differed from that of strain A(22)550 by 22 nucleotides and 3 amino acids. Three mutations occurred within nucleotide residues 883 to 1026, which encode an extremely conserved amino acid domain corresponding to RNA polymerization and nucleoside triphosphate binding functions. Mutation in 920n caused substitution of conserved amino acid Asn for Ser in the core motif for nucleotide binding Gxxx-TxxxN(S/T). It is possible that change in functional motif caused the alteration of phenotypic virus and loss of pathogenicity for cattle. This may be finally confirmed only after comparative analysis of complete genomes of the examined virus strains.
This study elucidates some structural and biological features of galactose-binding variants of the cytotoxic proteins ricin and abrin. An isolation procedure is reported for ricin variants from Ricinus communis seeds by using lactamyl-Sepharose affinity matrix, similar to that reported previously for variants of abrin from Abrus precatorius seeds [Hegde, R., Maiti, T. K. & Podder, S. K. (1991) Anal. Biochem. 194, 101-109]. Ricin variants, subfractionated on carboxymethyl-Sepharose CL-6B ion-exchange chromatography, were characterized further by SDS/PAGE, IEF and a binding assay. Based on the immunological cross-reactivity of antibody raised against a single variant of each of ricin and abrin, it was established that all the variants of the corresponding type are immunologically indistinguishable. Analysis of protein titration curves on an immobilized pH gradient indicated that variants of abrin I differ from other abrin variants, mainly in their acidic groups and that variance in ricin is a cause of charge substitution. Detection of subunit variants of proteins by two-dimensional gel electrophoresis showed that there are twice as many subunit variants as there are variants of holoproteins, suggesting that each variant has a set of subunit variants, which, although homologous, are not identical to the subunits of any other variant with respect to pI. Seeds obtained from polymorphic species of R. communis showed no difference in the profile of toxin variants, as analyzed by isoelectric focussing. Toxin variants obtained from red and white varieties of A. precatorius, however, showed some difference in the number of variants as well as in their relative intensities. Furthermore, variants analyzed from several single seeds of A. precatorius red type revealed a controlled distribution of lectin variants in three specific groups, indicating an involvement of at least three genes in the production of Abrus lectins. The complete absence or presence of variants in each group suggested a post-translational differential proteolytic processing, a secondary event in the production of abrin variants.
BACKGROUND: Osteogenesis imperfecta (OI) is a clinical and genetic disorder characterised by bone fragility, growth deficiency and skeletal deformity. Ninety per cent of OI cases are attributable to autosomal dominant variants in the COL1A1 and COL1A2 genes. METHODS: Candidate variants were identified and verified through trio whole-exome sequencing (trio-WES), copy number variation sequencing (CNV-seq) and Sanger sequencing. Minigene splicing assays were performed in HeLa and HEK293T cells with pcDNA3.1 and pcMINI-C vectors to investigate the function of the candidate variants. A systematic review of COL1A1 splicing variants and the corresponding genotype-phenotype spectrum was performed. RESULTS: Trio-WES revealed a novel heterozygous variant in the C-terminal region of the COL1A1 gene: NM_000088.4:c.3423+5G>A. Sanger sequencing confirmed the variant in both the proband (II-2) and her foetus (III-1) who were clinically suspected of having OI. The c.3423+5G>A variant causes complete skipping of Exon 46, as demonstrated by a minigene splicing assay. We retrieved 419 COL1A1 splicing variants from PubMed, excluded 15 without phenotypic data and 2 linked to Ehlers-Danlos syndrome and stratified the remaining 402 variants into three types on the basis of splice site location: (1) Variants at canonical splicing sites (77.8%, 313/402) mostly cause mild phenotypes, whereas a minority may be severe. (2) Intron variants in other locations, such as splice region variants (17.9%, 72/402), usually cause mild clinical phenotypes, and deep intronic splice variants (0.4%, 2/402) that may result in severe phenotypes. (3) Other variants (3.7%, 15/402), such as exon variants or fragment loss, are extremely rare. We also preliminarily discuss the mechanisms underlying phenotypic variability and the characteristics of C-terminal variants. CONCLUSIONS: This intron variant in COL1A1 was classified as likely pathogenic and was confirmed to disrupt COL1A1 expression. The summary analysis results also revealed a correlation among splicing variants, C-terminal region variants and disease, suggesting that variant location provides a useful framework for prognosis prediction.
OBJECTIVE: We have assessed the expression levels of messenger RNA for estrogen receptor-alpha and splice variants lacking exon 5 or exon 7 that presumably exert dominant positive (splice variants lacking exon 5) and negative (splice variants lacking exon 7) effects, respectively, on estrogen responses in the human endometrium. STUDY DESIGN: This was a prospective study that was conducted at an academic community-based hospital. The patients, aged 18 to 40 years, underwent hysterectomy for benign gynecologic causes. Eighty-one endometrial specimens (46 proliferative, 35 secretory) were analyzed with the use of reverse transcription-polymerase chain reaction assay for the messenger RNA levels of estrogen receptor-alpha, and splice variants lacking exon 5 and exon 7. RESULTS: Wild-type estrogen receptor-alpha and splice variants splice variants lacking exon 5 and lacking exon 7 messenger RNAs were detected in all endometrial specimens throughout the menstrual cycle. In addition, a double-splice estrogen receptor-alpha messenger RNA variant (splice variants lacking exon 5 and exon 7) was detected at constant low levels of expression. Semiquantitative analysis showed higher levels of estrogen receptor-alpha messenger RNA in the early and mid proliferative endometrial phases than in late proliferative and secretory endometrium ( P <.05). The splice variant lacking exon 7 messenger RNA expression level was about 10-fold higher than the splice variant lacking exon 5 messenger RNA relative to wild-type estrogen receptor-alpha messenger RNA ( P <.001). The expression of splice variants lacking exon 5 compared with wild-type estrogen receptor-alpha messenger RNA is relatively constant throughout endometrial development. In contrast, an examination of the ratio of the levels of splice variants lacking exon 7 to wild-type estrogen receptor-alpha messenger RNA indicated a small, but significantly higher, splice variant lacking exon 7 level in the mid secretory phase (postovulatory days 5-8) than the mid proliferative and early secretory phases ( P <.05). CONCLUSION: We found no evidence of differential coexpression of the positive dominant estrogen receptor variant, splice variants lacking exon 5, with wild-type estrogen receptor-alpha. We did find that the dominant negative splice variant lacking exon 7 was slightly increased relative to wild-type estrogen receptor-alpha in the postovulatory phase. Future investigation is required to suggest the biologic significance of the observed increased relative expression of the splice variants lacking exon 7 in secretory endometrium and to determine the function of splice variants lacking exon 5 and splice variants lacking exon 7.
Individuals carrying melanocortin 1 receptor gene variants have an increased risk for the development of cutaneous melanoma. Melanocortin 1 receptor gene variants are also associated with other risk factors for melanoma such as fair skin and red hair. We evaluated the relationship of melanocortin 1 receptor gene variants, fair skin, red hair and the development of melanoma in 123 patients with cutaneous melanoma and 385 control subjects. To analyze the association between melanocortin 1 receptor gene variants and skin type or hair color we also made use of 453 patients with nonmelanoma skin cancer. We analyzed the coding sequence of the melanocortin 1 receptor gene region by single-stranded conformation polymorphism analysis, followed by DNA sequence analysis. Risk of melanoma dependent on the various melanocortin 1 receptor variant alleles was estimated by exposure odds ratios. The analyses of all different melanocortin 1 receptor gene variants combined, showed that the presence of melanocortin 1 receptor gene variants amounted to a higher melanoma risk, which, in stratified analyses, was independent of skin type and hair color. The odds ratios after adjusting for skin type were 3.6 (95% CI 1.7-7.2) for two variants and 2.7 (95% CI 1.5-5.1) for one variant, respectively. Compound heterozygotes and homozygotes for the Val60Leu, Val92Met, Arg142His, Arg151Cys, Arg160Trp, Arg163Gln, and His260Pro variants had odds ratios of about 4 to develop melanoma, whereas heterozygotes for these variants had half the risk. The presence of the melanocortin 1 receptor gene variant Asp84Glu appeared to impose the highest risk for cutaneous melanoma with odds ratios of 16.1 (95% CI 2.3-139.0) and 8.1 (95% CI 1.2-55.9) in compound heterozygotes and heterozygotes, respectively. The broad confidence intervals, when the different variants were analyzed separately, however, do not allow drawing definite conclusions about the magnitude of these risks. Of the more frequently occurring melanocortin 1 receptor variant alleles the Asp84Glu, Arg142His, Arg151Cys, Arg160Trp, His260Pro, and Asp294His variants were strongly associated with both fair skin and red hair. The Val60Leu, Val92Met, and Arg163Gln variant alleles, however, were only weakly or not associated with fair skin type and/or red hair, which further illustrates the finding that skin type, hair color, and melanoma are independent outcomes of the presence of melanocortin 1 receptor gene variants. We conclude that numerous melanocortin 1 receptor variants predispose to cutaneous melanoma and that possibly the Asp84Glu variant confers the highest risk. This predisposition is largely independent of skin type and hair color.
BACKGROUND: Inherited variants in the LDL (low-density lipoprotein) receptor (LDLR) gene are the most common cause of familial hypercholesterolemia, significantly increasing coronary artery disease risk. Early identification of pathogenic LDLR variants enables prompt lipid-lowering therapy and cascade testing of at-risk relatives; however, most LDLR variants observed in the population have uncertain or absent clinical classifications, leaving many patients without actionable information. METHODS: We developed the first activity-normalized prime editing screening pipeline to measure the impact of 5184 LDLR coding variants on LDL-cholesterol (LDL-C) uptake. Each prime editing guide RNA is paired with a genotypic outcome reporter to correct for variable editing efficiency, overcoming a key limitation of previous pooled genome editing screens. A statistical framework further improves variant effect estimates by jointly analyzing all missense variants at each amino acid position. RESULTS: We show that prime editing of the reporter construct correlates with endogenous variant installation frequency, validating the activity normalization approach. The resulting scores capture a continuous spectrum of functional effects, robustly separate pathogenic versus benign ClinVar variants, and show concordance with LDL-C levels in UK Biobank participants. We calibrate functional evidence strengths to the ACMG/AMP variant interpretation framework, enabling integration into a clinical variant classification workflow. By combining functional, computational, population, and contextual evidence, 322 of 434 LDLR variants currently classified as variants of uncertain significance, conflicting, or absent from ClinVar appear to meet evidence thresholds for reclassification and can be prioritized for expert review, substantially expanding the pool of actionable variant classifications. The screen also reveals a cluster of gain-of-function variants in LDLR class A repeat 5, at least some of which enhance LDL-C uptake through increased apolipoprotein B interaction, with implications for therapeutic genome editing. Last, prime editing uniquely detects splice-altering coding variants missed by cDNA-based screens and pathogenicity predictors, revealing an advantage of endogenous variant installation. CONCLUSIONS: Altogether, activity-normalized prime editing provides a scalable framework for LDLR variant classification that substantially expands the proportion of variants with evidence for genetic diagnosis and reveals novel biology with therapeutic relevance.
A high-performance frontal analysis-capillary electrophoresis (HPFA-CE) method was applied to investigate the effect of pH on the drug binding properties of genetic variants of human alpha1-acid glycoprotein (AGP), A variant and a mixture of F1S variants. The unbound concentrations of a model basic drug, disopyramide (DP), in A variant solutions and in F1S variant solutions were measured by HPFA-CE to evaluate binding constants at pH 4.0, 5.0, 6.0 and 7.4. The binding between DP and A variant was gradually weakened by acidification of background buffer (from pH 7.4 to 4.0), while the binding between DP and FIS variants decreased at first (from pH 7.4 to 6.0), and then gained (from pH 6.0 to 4.0). Consequently, DP was more strongly bound to A variant than to FIS variants at pH 7.4, while at pH 4.0 DP was more strongly bound to F1S variants. At any pH (S)-DP was bound more strongly than (R)-DP, and the enantioselectivity of A variant was significantly higher than that of F1S variants. Electrophoretic mobilities of the AGP genetic variants decreased along with a decrease in pH. Fluorescent emission of these genetic variants indicated a distinct conformational change between pH 5.0 and 4.0. However, there was no significant difference in the electrophoretic mobility and the fluorescent emission spectrum between these variants at any pH. On the other hand, circular dichroism analyses revealed that beta-sheet content in FIS variants diminished as pH decreased, while that in A variant increased. These results suggest that the conformational change induced by acidification of background buffer differs between these genetic variants, and this causes the difference in DP bindability.
BACKGROUND: Several estrogen receptor (ER) variant messenger RNAs (mRNAs) have been identified previously in human breast cancer biopsy samples and cell lines. The relative levels of certain ER variant mRNAs have been observed to increase with breast tumor progression. In vitro assays of the function of polypeptides encoded by some of these variant mRNAs have led to speculation that ER variants may be involved in the progression from hormone dependence to independence in breast cancer. PURPOSE: We set out to establish if ER variant mRNAs are present in normal human breast tissues and, if so, to compare levels of these variants between normal and neoplastic human breast tissues. METHODS: Four human breast tissue samples from reduction mammoplasties and five samples from tissue adjacent to breast tumors were analyzed. The tissue samples were confirmed to be normal (i.e., not malignant) by histopathologic analysis. RNA was extracted immediately from adjacent frozen sections. Human breast tumor specimens originally resected from 19 patients were acquired from a tumor bank and processed in the same way as the normal tissue samples. The RNAs were then reverse transcribed and subsequently amplified with the use of the polymerase chain reaction (PCR). PCR primer sets were designed to detect several different exon-deleted ER variants and a truncated ER variant (i.e., clone 4). A semiquantitative PCR-based method was used to determine the relative expression of exon 5- and exon 7-deleted variants to wild-type ER mRNAs in the nine normal tissues and in 19 ER-positive breast tumor tissues. The Mann-Whitney rank sum test (two-sided) was used to determine P values. RESULTS: ER variant mRNAs corresponding to the clone 4 ER truncated variant and to variants deleted in either exon 2, exon 3, exons 2-3, exon 5, or exon 7 were detected in all normal samples. The results were confirmed by restriction enzyme analyses and sequencing of the PCR products. The expression of exon 5-deleted ER variant relative to the wild-type ER mRNA was significantly lower (P< .001) in normal tissue than in tumor tissue. A similar trend was noted for expression of the exon 7-deleted ER variant mRNA; however, the difference did not achieve statistical significance (P= .476). CONCLUSION: Several ER variant mRNAs are present in normal human breast tissue, but the level of expression of some of these variants may be lower in normal tissue than in tumor tissue. IMPLICATION: These data suggest that the mechanisms generating ER variant mRNAs exist in normal breast tissue and may be deregulated in breast cancer tissues. Further investigation of the role of variant ER expression in development and progression of human breast cancer appears warranted.
Recently we characterized three distinct GnRH receptors in the bullfrog (bfGnRHR-1, bfGnRHR-2, and bfGnRHR-3). In the present study, we further investigated the expression and function of splice variants, generated from the primary bfGnRHR-3 transcript by exon skipping (splice variant 1), intron retention (splice variants 2 and 3), and/or transcriptional slippage (splice variant 4), apart from the constitutively spliced form (wild-type). Cellular expression and function of the splice variants were examined using a transient expression system. Immunoblot analysis revealed that the wild-type receptor and all splice variant proteins were expressed in transfected HeLa cells with no significant differences in expression levels. These splice variants showed a very low binding affinity to ligand and did not induce signal transduction in response to GnRH treatment. Interestingly, cotransfection of the wild-type with splice variants 2--4, but not with splice variant 1, significantly inhibited wild-type receptor-mediated signaling. Subcellular localization analysis of green fluorescent protein-tagged wild-type and splice variant proteins revealed that the wild-type receptor protein was mainly localized in the cell membrane, whereas the splice variant 1 protein was exclusively detected in the cytoplasm. The splice variant 2--4 proteins, however, were found in both the cell membrane and cytoplasm. The inhibition of wild-type receptor signaling by splice variants 2--4 and the subcellular localization of splice variants 2-4 suggest a possible physical interaction of splice variants 2--4 with the wild-type receptor protein. In addition, the ratio of mRNA levels of the wild-type to splice variants 2--4 significantly varied from hibernation (wild-type < splice variants 2--4) to the prebreeding season (wild-type > splice variants 2--4). Collectively, these results suggest that alternative splicing of the bfGnRHR-3 primary transcript plays a role in fine-tuning GnRH receptor function in amphibians.
OBJECTIVE: alpha1-acid glycoprotein (AAG) has three main genetic variants, F1, S, and A variants. There are few reports on the correlation between AAG variants and binding activity of drug enantiomers. We studied the differences between the binding characteristics of enantiomers of disopyramide (DP), which is a basic drug. The aim of this study was to elucidate the cause of the differences between the binding characteristics of DP enantiomers. METHODS: The variants in human AAG were separated by hydroxyapatite chromatography. Binding of DP enantiomers to AAG variants was studied by the ultrafiltration method. The characteristics of the binding of DP enantiomers to total variants and each variant were examined by Scatchard analysis within a range of concentrations from 0.5 to 50.0 microg/ml. RESULTS: The binding capacity of S-DP was significantly higher than that of R-DP in variant 3, although the binding capacities of DP enantiomers were almost the same in variant 2. On the other hand, the binding capacities for both S-DP and R-DP in variant 3 were significantly higher than those in variant 2. Furthermore, there was an almost 2.4-fold difference in the dissociation constant (Kd) between S-DP and R-DP in variant 3, although no significant difference was observed in the number of binding sites (N). In variant 2 no significant differences between DP enantiomers were observed in either the dissociation constant or number of binding sites per molecule of AAG. On the other hand, significant differences between variants 2 and 3 in the dissociation constant for both S-DP and R-DP were observed. The differences in dissociation constant between variants 2 and 3 were 4.0-fold in S-DP and 1.7-fold in R-DP. CONCLUSION: The difference between the binding capacities of S-DP and R-DP is due to differences in the association of DP to variants 3-6, and the role of the variants 1 and 2 in the binding of drugs to AAG is minor.
The oligosaccharide chains of pituitary glycoprotein hormones such as human thyroid-stimulating hormone (hTSH) have been shown to be important in biosynthesis, subunit association, secretion and bioactivity. However, the exact biological significance of these glycosylation variants (isoforms) remains controversial. The aim of this paper is to investigate the role of hTSH glycosylation variants in signal transduction. Human pituitary standard TSH (2nd International Reference Preparation 80/558; IRP-hTSH) was treated with neuraminidase, fractionated by isoelectric focusing (IEF) and affinity chromatography using the lectins concanavalin A (Con A) and lentil. To determine the in vitro bioactivity of these hTSH isoforms, simultaneous measurement of cAMP formation and inositol phosphates release was applied in two different cell systems (CHO cells stably and Cos-7 cells transiently transfected with hTSHR cDNA). Desialylated TSH variants showed a significantly increased ratio of bioactivity to immunoreactivity for cAMP production in CHO-R cells (B/I ratio desialylated variants: 3.54 +/- 0.005; B/I ratio sialylated variants: 2.84 +/- 0.01 P < 0.05). Testing the bioactivity of hTSH glycosylation variants isolated by IEF, we found basic variants to be significantly more active than acidic ones in stimulating the cAMP formation in CHO-R cells (B/I ratio basic variants: 9.92 +/- 0.64; neutral variants: 5.98 +/- 0.07; acidic variants: 2.80 +/- 0.12; P < 0.01). There were no differences in stimulation of IP-release. High-mannose TSH variants (firmly bound to Con A) showed greater potency to stimulate cAMP formation and IP-release in both CHO-R and Cos-7 cells than biantennary TSH variants (weakly bound to Con A). Both core-fucosylated (lentil-bound) and core-unfucosylated (lentil-unbound) TSH variants proved to be strong stimulators of cAMP release in CHO and Cos-7 cells. In CHO-R (Cos-7) cells, 400 microU/ml core-fucosylated TSH stimulated cAMP formation 14(2.6)-fold, core-unfucosylated TSH 7.3(2.3)-fold over control values. In contrast to our findings of cAMP activation by both core-fucosylated and core-unfucosylated TSH variants, release of IPs was stimulated only by, core-fucosylated (lentil-bound) TSH variants and not by TSH variants lacking core-fucose residues (lentil-unbound TSH). This was true for both CHO-R and Cos-7 cells. The lentil-unbound TSH therefore showed an identical differential activation of signal transduction pathways in two different cell systems: strong stimulation of the cAMP-cascade without activation of IPs release (P < 0.05). In conclusion, we showed for the first time for TSH that the two dominant intracellular signal transduction systems (cAMP formation and IPs release) are activated to different degrees by hTSH glycosylation variants.
Plaque purification of the M variant of encephalomyocarditis (EMC) virus resulted in the isolation of two stable variants: one diabetogenic and designated D and the other nondiabetogenic and designated B. When the D variant was inoculated into SJL/J male mice, hypoinsulinemia and hyperglycemia developed in > 90% of the animals. In contrast, none of the mice inoculated with the B variant developed diabetes. Histologic examination of pancreata from mice infected with the D variant revealed insulitis and necrosis of beta cells, whereas islets from mice infected with the B variant showed little, if any, change. When islets were assayed for infectious virus, approximately 10 times more virus was recovered from animals inoculated with the D as compared with the B variant. Moreover, approximately 60% of islet cells from mice infected with the D variant contained viral antigens when stained with fluorescein-labeled anti-EMC virus antibody, whereas < 5% of islet cells from animals infected with the B variant contained viral antigens. Co-infection experiments showed that the induction of diabetes by the D variant was inhibited by the B variant. When the B and D variants were mixed together at B:D ratios of 1, 9, and 99, diabetes developed in 60, 11, and 0% of the mice, respectively. Tissue-culture experiments revealed that the B variant induced considerably more interferon than the D variant, and studies in animals showed that interferon appeared earlier and in greater amounts in the circulation of mice infected with the B as compared with the D variant. These studies suggest that the induction of interferon by the B variant is, at least in part, responsible for the inhibition of diabetes by the D variant.