Conformation of the Hinge Region and Various Fragments of Human IgG3

A study of the circular dichroic (CD) spectra of various fragments of human IgG3, including the isolated hinge region, Fh, has shown that the hinge region has a high degree of an unusual secondary structure, unique within immunoglobulin material recorded to date. This structure appears to be rigid and aperiodic throughout the hinge region and is compatible with a repeated amino acid sequence. The conformation of the isolated Fh fragment is the same as that of the bound hinge region; also, there is no substantial conformational interaction between the hinge region and the Fab or Fc fragments of human Igtg3. a comparison of the CD spectra of Fc and pFc fragments isolated from an IgG1 and an IgG3 myeloma protein has shown that subclass differences of amino acid sequence do not substantially alter the conformation of these fragments.     

Isolation of a Fragment, Fh, Corresponding to the Hinge Region of Human IgG3

An Fh fragment corresponding to the hinge region of IgG₃ has been isolated by J single α-chymotrypsin treatment of whole IgG₃ Various combinations of two enzymes have also been used, but with somewhat lower yield and less purity The isolated Fh fragment has features similar to those of the bound hinge region in the intact IgG₃ and its F(ab ' )₂ and Fch fragments. The Fh fragment has a char acteristically high content of cystine and proline and seems to lack aromatic amino acids     

Analysis of Dystrophin Gene Deletions Indicates that the Hinge III Region of the Protein Correlates with Disease Severity

We have investigated the frequency of deletions in the dystrophin gene in 108 unrelated Duchenne and Becker muscular dystrophy (DMD/BMD) patients from southern Italy (DMD, n. 47; BMD, n. 61) and identified 89 deletions. The de novo mutation rate (about 30%), and the preferentially maternal origin of deletional mutations, analysed in families in which the maternal grandparents were available or their haplotypes could be unequivocally reconstructed, are in agreement with data reported for other populations. The correlation between BMD phenotype and type of deletion suggests that, in the distal rod domain region, the deletion size may not be as crucial as the particular combination of missing exons. In fact, we provide immunohistochemical and clinical evidence that in‐frame deletion of the hinge III region in the distal rod domain results in a milder phenotype as compared with shorter deletions that do not include the hinge III region. Our data obtained in BMD patients, by confirming inferences arising from minigene transfection experiments in mdx mice, represent an important contribution to gene therapy approaches.     

Comparison of Fch and Fc Fragments of IgG3: Evidence of an Intra-Chain S-S Bridge in the Hinge of γ3 Chains

The Fc and Fch fragments of human IgG₃, appearing late and early during papain digestion, respectively, were antigenically and structurally compared. Only the Fch fragment reacted with an anti-Fh (hinge region-specific) antiserum; thus the main portion of the hinge region is split off during the formation of Fc fragments The Fch fragment has a higher content of inter-chain S-S bridges than the Fc fragments. The Fc fragments are probably a mixture of three related fragments. The Fch fragment appears to be more homogeneous, although there was hydrolysis at the C-terminal site of the hinge region in some of the Fch chains. Evidence is presented that the γ³ chain bridge has an extra intra-heavy-chain disulphidc bridge at the C-terminal end of the hinge region.     

Robertsonian (15q;15q) translocation in a child with Angelman syndrome: Evidence of uniparental disomy

A balanced Robertsonian translocation 45,XY,t(15q15q) was detected in a patient with mental retardation, microcephaly, and hypertonia. Deletion of the 15q11q13 region was unlikely based on fluorescence in situ hybridization studies that revealed hybridization of appropriate DNA probes to both arms of the Robertsonian chromosome. Inheritance of alleles from 13 highly polymorphic DNA markers on chromosome 15 showed paternal uniparental isodisomy. The clinical, cytogenetic, and molecular results are consistent with a diagnosis of Angelman syndrome. © 1996 Wiley-Liss, Inc.     

The Cγ3 Homology Region in Human IgG Subclasses and Allotypes

Amino acid composition, end-group analysis, and antigenic analysis have been performed on isolated pFc' fragments (Cγ3 homology region) from the major allotypic variants of human IgG. A comparison of the amino acid composition of Gm(a+x+) and Gm(a+x−) pFc' fragments showed less serine and more glycine in the Gm(a+x+) proteins. Two IgG2 pFc' fragments were shown to resemble IgG1 Gm(f) pFc' fragments in their amino acid compositions, except that there was more threonine and less alanine in the IgG2 fragments. This was also observed in two IgG3 Gm(g) proteins and may be the basis for the expression of a non b antigen. IgG3 fragments of both Gm(g) and Gm(b) allotypes showed six amino acid differences from the IgG1 subclass. There was more arginine, serine, and isoleucine and less histidine, lysine, and valine in both allotypes. In addition, analysis of a single Gm(b) pFc' fragment showed differences from the Gm(g) proteins; there was more phenylalanine, alanine, and proline and less tyrosine, threonine, and, possibly, aspartic acid.     

Enhancement of Complement Activation and Cytolysis of Human IgG3 by Deletion of Hinge Exons

The capacity to induce complement-mediated cell lysis is greatly enhanced by truncating the hinge of IgG3 through exon deletions. This was shown by establishing five new cell lines which secreted chimeric IgG3 molecules with specificity for the hapten 4-hydroxy-3-nitrophenacetyl (NP) and having 47,45,32,15, and 0 amino acid hinge regions (the wild-type IgG3 has 62 amino acids in the hinge). Efficient complement activation and complement-mediated cell lysis did not depend on a long total hinge or on a long 'upper' hinge (the stretch from the beginning of the hinge to the first inter-heavy chain S-S bond). On the contrary, the mutant having a 15 amino acid hinge element was up to 10 times more efficient in complement lysis than the wild type. Thus the complement-activation potential appeared to be down-regulated in the wild type. On the other hand, the mutant lacking the hinge altogether did not activate complement or induce complement-mediated cytolysis. These findings have to be taken into account when antibodies are designed for human therapy.     

Feasible Stability Region in the Frontal Plane During Human Gait

The inability to adequately control the motion of the center of mass (COM) in the frontal plane may result in a loss of balance causing a sideways fall during human gait. The primary purposes of this study were (1) to derive the feasible stability region (FSR) in the mediolateral direction, and (2) to compare the FSR with the COM motion state taken from 193 trials among 39 young subjects at liftoff during walking at different speeds. The lower boundary of the FSR was derived, at a given initial COM location, as the minimum rightward COM velocity, at liftoff of the left foot, required to bring the COM into the base of support (BOS), i.e., the right (stance) foot, as the COM velocity diminishes. The upper boundary was derived as the maximum rightward COM velocity, beyond which the left foot must land to the right of the right foot (BOS) in order to prevent a fall. We established a 2-link human model and employed dynamic optimization to estimate these threshold values for velocity. For a range of initial COM positions, simulated annealing algorithm was used to search for the threshold velocity values. Our study quantified the extent to which mediolateral balance can still be maintained without resorting to a crossover step (the left foot lands to the right of the BOS) for balance recovery. The derived FSR is in good agreement with our gait experimental results.     

Morphological Integration of the Orbital Region in a Human Ontogenetic Sample

Most studies on craniofacial morphology have focused on adult individuals, but patterns of variation are the outcome of genetic and epigenetic variables that interact throughout ontogeny. Among cranial regions, the orbits exhibit morphological variation and occupy an intermediate position between neurocranial and facial structures. The main objective of this work was to analyze postnatal ontogenetic variation and covariation in the morphology of the orbital region in a cross‐sectional series of humans from 0 to 31 years old. Landmarks and semilandmarks were digitized on the orbital rim, as well as in neighboring neural and facial structures. Data were analyzed using geometric morphometrics. Results indicated that orbital size increases during the first years of postnatal life, while the shape of the orbital aperture does not change significantly with age. In general, the pattern and magnitude of shape covariation do not vary markedly during postnatal life although some subtle shifts were documented. Additionally, the shape of the orbital aperture is more related to the anterior neurocranium than to zygomatic structures, even when the allometry is adjusted. Although we expected some influence from postnatal craniofacial growth and from some functional factors, such as mastication, on the development of the orbits, this assumption was not completely supported by our results. As a whole, our findings are in line with the prediction of an early influence of the eyes and extraocular tissues on orbital morphology, and could be interpreted in relation to processes promoting early neural development that coordinately affects orbital traits and the neurocranial skeleton. Anat Rec, 299:70–80, 2016. © 2015 Wiley Periodicals, Inc.     

Difference in methylation patterns within the D15S9 region of chromosome 15qll–13 in first cousins with Angelman syndrome and Prader–Willi syndrome

Abnormalities of chromosome region 15qll–13 are associated with Angelman syndrome (AS) and Prader–Willi syndrome (PWS). Differences between the methylation patterns of the region of chromosome 15qll–13 which hybridizes to the highly conserved DNA, DN34, in normal individuals and in patients with AS and PWS have been described. We report on a family in which first cousins are affected by AS and PWS as a result of a familial paracentric inversion of 15qll–ql3. The results of the studies on this family demonstrate the differences in the methylation patterns in the 2 conditions and the phenomenon of genomic imprinting, whereby genetic information is expressed differently dependent on the parent of origin. © 1993 Wiley-Liss, Inc.     

Coding Region Polymorphisms of Human T-Cell Receptor Vβ6.9 and Vβ21.4

Two new TCRV beta coding region polymorphisms were identified: V beta 6.9a/b and V beta 21.4a/b. In both cases, a single nucleotide difference gives rise to an amino acid exchange. Genomic typing by the PCR/sequence-specific oligonucleotide probing technique was performed to study a possible contribution of these two new polymorphisms in susceptibility to autoimmune diseases. However, there was no association with insulin-dependent diabetes mellitus, rheumatoid arthritis, juvenile rheumatoid arthritis, multiple sclerosis, myasthenia gravis or coeliac disease. On the other hand, significant differences were found between Caucasoid and Oriental populations in frequencies of the V beta 6.9 and V beta 21.4 alleles.     

Evidence of Steroid Receptors in Human Osteoblast-Like Cells

A nuclear binding assay was used to demonstrate steroid receptors in normal human osteoblast-like cells. Nuclear binding of [³H] estradiol was found in 27 of 30 (90%) cell strains and nuclear binding of [³H] androgen was identified in 21 of 25 (84%) separate osteoblast cell strains. The nuclear binding was saturable and steroid-specific. Estrogen and androgen receptor gene expression was confirmed by RNA blot analysis. These data suggest that circulating sex steroids may act directly on normal human osteoblasts through receptor-mediated mechanisms.     

Ciliated Cells in the Human Gastric Mucosa Evidence of Metaplastic Change

In the gastric mucosa of Japanese patients, ciliated cells were found in association with intestinal metaplasia. The cells occurred frequently in the pyloric mucosa of nearly half of the cases examined but rarely in the cardiac mucosa of total 12 cases, but never adjacent to the chief cells of gastric glands. The ciliated cells were always found in the basal part of cardiac and pyloric glands, but never in the surface or in the foveolar epithelium. Furthermore, ciliated cells containing a few small mucus granules and simultaneously possessing numerous cilia and basal bodies were noted. Ciliated cells in the gastric mucosa have been found mainly in elderly Japanese patients, but were also observed exceptionally in one Chinese, two Swedes and one American. These ciliated cells are not present in the normal human gastric and intestinal mucosa, and therefore a new term, "ciliated metaplasia", is proposed for their occurrence. Acta Pathol Jpn 40: 98–106, 1990.     

Effect of 15-lipoxygenase metabolites on angiogenesis: 15(S)-HPETE is angiostatic and 15(S)-HETE is angiogenic

Objective

15(S)-Hydroxyeicosatetraenoic acid [15(S)-HETE] and 15(S)-hydroperoxyeicosatetraenoic acid [15(S)-HPETE] are the products of arachidonic acid formed in the 15-lipoxygenase pathway. They have opposing effects on the inflammatory process. The present study was designed to examine the role of these metabolites on angiogenesis, which is critically associated with inflammatory conditions.

Methods

Chick chorio-allantoic membrane (CAM), rat aortic rings and human umbilical vein endothelial cells (HUVECs) in culture were used to study the effect of 15(S)-HETE and 15(S)-HPETE on angiogenesis. Biochemical markers of angiogenesis were analysed by ELISA.

Results

15(S)-HETE increased vessel density in chick CAM, induced sprouting in rat aortic rings and increased endothelial cell–cell contact and formation of tubular network-like structures in HUVECs. Furthermore, it up-regulated the expression of CD31, E-selectin and vascular endothelial growth factor (VEGF) in HUVECs, indicating its pro-angiogenic effect. 15(S)-HPETE, on the other hand, decreased vessel density in chick CAM, down-regulated the expression of E-selectin (<35?%), VEGF (<90?%) and CD31 (<50?%) and did not produce sprouting in aortic rings, suggesting an anti-angiogenic property. 15(S)-HETE-mediated up-regulation of CD 31 and VEGF was reversed by treatment with 15(S)-HPETE.

Conclusion

These results indicate the divergent effects of hydroxy and hydroperoxy products of 15-LOX on angiogenesis, highlighting the role of these products in the co-dependence of inflammation and angiogenesis.     

A mitochondrial frameshift-suppressor (+) of the yeast S. cerevisiae maps in the mitochondrial 15S rRNA locus

Summary The first case of a +1 extrageneic frameshift suppressor (MF1), mapping in the yeast mitochondrial 15S rRNA gene is reported. The suppressor was identified by genetic analyses in a leaky mitochondrial oxi1 frameshift mutant and the respective wild-type strain 777-3A of the yeast S. cerevisiae. This is in accordance with the finding that all mitochondrial frameshift mutants isolated from this strain tend to be leaky to a variable degree. MF1 does not suppress known nonsense mutations created by a direct basepair exchange in strain 777-3A. These mutants exhibit a non-leaky phenotype (Weiss-Brummer et al. 1984).     

Evidence for a new microdeletion syndrome in 15q21

We report on the fourth known case with an interstitial deletion in 15q21. In the present case the breakpoints have been determined by GTG-banding, microdissection and the recently developed multicolor banding (MCB) technique as 15q21.1-q21.3. Common features in all four cases are mental retardation, growth retardation, a beak-like nose with hypoplastic alae nasi and a thin upper lip. Additional frequent features are small hands and feet, hypotonia, low hair implantation, low set ears, clinodactyly and obesity. The possibility that a critical region for a new microdeletion-syndrome is situated in 15q21 is discussed.