alpha 1-Antitrypsin Christchurch, 363 Glu----Lys: mutation at the P'5 position does not affect inhibitory activity

alpha 1-Antitrypsin Christchurch was isolated from the plasma of a Cambodian woman who was heterozygous for this variant and for the normal M protein. Tryptic peptide maps revealed that the inhibitory-site peptide, 359-365 Ser-Ile-Pro-Pro-Glu,Val,Lys, was missing and replaced by two new peptides Ser-Ile-Pro-Pro,Lys and Val-Lys, indicating a mutation of 363 Glu----Lys. There was no obvious clinical condition associated with this new antitrypsin. Competition experiments showed that antitrypsin Christchurch reacted at the same rate as normal antitrypsin in the presence of limiting amounts of trypsin, chymotrypsin, thrombin and neutrophil elastase. Both inhibitors were inactivated by catalytic amounts of papain. This inactivation was due to cleavage at the phenylalanine residue at the P7 position, seven residues towards the N-terminal of the inhibitory site. A one-step ethanol extraction procedure is described for isolating the papain cleavage products.     

The serum sialyltransferase activity in alpha 1-antitrypsin deficiency.

alpha 1-Antitrypsin phenotypes Pi M and Z, purified by the thiol-disulfide exchange procedure, were desialylated by treatment with neuraminidase covalently coupled to Sepharose and used as acceptors of sialic acid in an assay system for serum sialic acid transferase (CMP-N-acetylneuraminate:D-galactosyl-glycoprotein N-acetylneuraminyltransferase, EC 2.4.99.1) activity. Both asialoantitrypsins were equally effective as acceptors in contrast to native Pi Z antitrypsin which did not accept any sialic acid. Serum sialyltransferase activity was determined in 38 adult alpha 1-antitrypsin deficient individuals (Pi Z, MZ, FZ, SZ) with normal liver function and was found to be of the same magnitude as the activity in normal individuals (Pi M). Equal activities were also found in 5 Pi Z patients with cirrhosis of the liver. The results strongly argue against the concept that sialyltransferase deficiency provides the molecular basis for alpha 1-antitrypsin deficiency.     

Alpha 1 antitrypsin deficiency: a study of the relationship between the Pi system and genetic markers.

Twenty-four members (4 generations) of a family with alpha 1 antitrypsin deficiency were studied in an attempt to determine the chromosomal location of the Pi system locus. Three alpha 1 antitrypsin alleles (PiM, PiI, and PiZ) and five phenotypes (MM, MZ, MI, IZ, and ZZ) were detected in family members. The quinacrine fluorescent banding technique was successfully utilized to reveal eight polymorphic chromosomal markers in family members. Eight red cell antigens and HL-A antigens were identified for each family member. No linkage between the Pi system and chromosomal markers, four polymorphic red cell antigens, and HL-A antigens was detected. On the basis of this family study, the Pi locus as defined by alpha 1 antitrypsin deficiency does not appear to be on chromosomes 2, 3, 13, 14, 21, or 22 within measurable distance of the markers used.     

Catabolism of chylomicron remnants in normolipidemic subjects in relation to the apoprotein E phenotype

The role of the various apolipoprotein E isoproteins in the removal of chylomicrons and their remnants from plasma was studied in 16 normolipidemic subjects with various apoE phenotypes: 5 homozygous for apoE-2, 6 heterozygous for apoE-2 (phenotype E3/2), and 5 without apoE-2 (phenotypes E3/3, E4/4, and E4/3). The subjects were given an oral fat load as cream (50 g/m2). Retinyl palmitate was added as a marker for chylomicrons and their remnants. Blood was sampled at regular time intervals for 8 hr. Remnant particles were isolated from the d less than 1.019 g/ml fraction by heparin-Sepharose chromatography (heparin-bound fraction) after removing the large chylomicrons by flotation at 7,8 X 10(5) g-min. All groups showed a rise in triglycerides in serum and in the chylomicron fraction between 3 and 6 hr to about twice the basal value, followed by a decrease to nearly fasting values. In the homozygous E-2 subjects, fasting lipids in the remnant fraction were increased. In all three groups the fat load did not induce a significant rise in the lipids of the remnant fraction. The homozygous E-2 group showed a strong continuing rise in the retinyl palmitate concentration in the chylomicron and remnant fractions up to 8 hr, whereas in the other groups its maximum was already reached at 5 hr at a much lower level. At 8 hr similar retinyl palmitate concentrations were found in both fractions in the heterozygous E-2 subjects compared to the non-E-2 subjects. These results indicate a delayed removal of chylomicrons and chylomicron remnants in normolipidemic homozygous E-2, but not in heterozygous E-2 subjects.     

A novel substitution at the translation initiator codon (ATG-->ATC) of the lipoprotein lipase gene is mainly responsible for lipoprotein lipase deficiency in a patient with severe hypertriglyceridemia and recurrent pancreatitis

A patient with severe hypertriglyceridemia and recurrent pancreatitis was found to have significantly decreased lipoprotein lipase (LPL) activity and normal apolipoprotein C-II concentration in post-heparin plasma. DNA analysis of the LPL gene revealed two mutations, one of which was a novel homozygous G-->C substitution, resulting in the conversion of a translation initiation codon methionine to isoleucine (LPL-1). The second was the previously reported heterozygous substitution of glutamic acid at residue 242 with lysine (LPL-242). In vitro expression of both mutations separately or in combination demonstrated that LPL-1 had approximately 3% protein mass and 2% activity, whereas LPL-242 had undetectable activity but normal mass. The combined mutation LPL-1-242 exhibited similar changes as for LPL-1, with markedly reduced mass, and for LPL-242, with undetectable activity. These results suggest that the homozygous initiator codon mutation rather than the heterozygous LPL-242 alteration was mainly responsible for the patient phenotypes.     

Partial uncoupling of the mitochondrial membrane by a heterozygous null mutation in the gene encoding the gamma- or delta-subunit of the yeast mitochondrial ATPase

Prior genetic studies indicated that the yeast mitochondrial ATP synthase can be assembled into enzyme complexes devoid of the gamma-, delta-, or epsilon-subunits (Lai-Zhang, J., Xiao, Y., and Mueller, D. M. (1999) EMBO J. 18, 58-64). These subunit-deficient complexes were postulated to uncouple the mitochondrial membrane thereby causing negative cellular phenotypes. This study provides biochemical and additional genetic data that support this hypothesis. The genetic data indicate that in a diploid cell, a heterozygous deletion mutation in the gene encoding the gamma- or delta-subunit of the ATPase is semidominant negative due to a decrease in the gene number from 2 to 1. However, the heterozygous atp2Delta mutation is epistatic to the heterozygous mutation in the gene encoding gamma or delta, suggesting that the semidominant negative effect is because of a gain of activity in the cells. Biochemical studies using mitochondria isolated from the yeast strains that are heterozygous for a mutation in gamma or delta indicate that the mitochondria are partially uncoupled. These results support the hypothesis that the negative phenotypes are caused by the formation of a gamma- or delta-less ATP synthase complex that is uncoupled.     

On the robertsonian polymorphism found in the Japanese raccoon dog (Nyctereutes procyonoides viverrinus)

Karyotypes of 39 Japanese raccoon dogs (NPV) which appeared in the literature and of 7 previously unreported specimens were examined. Thirty four individuals showed the standard karyotype 2K = 26M + 10A + (M)X + (A)Y + Bs (2n = 38 + Bs), where Bs are supernumerary chromosomes. The remaining 11 individuals had 2K = 25M + 12A + XY + Bs (2n = 39 + Bs) and one was 2K = 23M + 16A + XY + Bs (2n = 41 + Bs). The G- and C-banding analyses of both somatic and germ cells revealed that these karyotypes with odd numbers are heterozygous (M/A) for a single Robertsonian rearrangement of chromosomes 2, 5, 6, 8, or 11, and one is M/A heterozygous for three autosomes: 5, 6, and 11.     

Relationship between the bovine major histocompatibility complex (BoLA), erythrocyte markers and susceptibility to mastitis in Icelandic cattle

Summary. Milk and blood samples were obtained from three Icelandic dairy herds. The herds were monitored regularly for mastitis incidence. Cell counts, adenosine triphosphate (ATP) and antitrypsin levels of the milk samples were recorded. In addition, red cell and BoLA typing were performed on the blood. Although cell counts and ATP levels showed significant associations with mastitis, antitrypsin levels did not. Red blood cell antigens N'₂ and S₁ and the lymphocyte antigen detected by the monoclonal antibody M7 were associated with low cell counts, whilst BoLA w6 and w6.1 were associated with high cell counts. BoLA w6.2 and w11 showed significant association with high antitrypsin levels. Only ED116 showed a significant association with mastitis.     

Alpha 1 antitrypsin M1: a new common genetically determined variant.

A new common variant (M1) of alpha 1 antitrypsin was detected by isoelectric focusing of serum in a pH gradient of 3.5-5.0 in polyacrylamide gels. The variant can be clearly distinguished from the common M type only when alpha 1 antitrypsin M is present in the same serum. It cannot be recognized on starch gel electrophoresis. The gene frequency in a population sample of United States whites was .09.     

A turbidimetric method for measuring the activity of trypsin and its inhibition

Microscopic poly(styrene-divinylbenzene) beads coated with a monomolecular film of fibrinogen agglutinate when stirred in the presence of thrombin, a consequence of interbead fibrin formation. Trypsin, by digesting bead-bound fibrin, dissociates bead aggregates at a rate proportional to the amount of enzyme activity. The agglutination of beads and the dissociation of bead aggregates can be monitored turbidimetrically using a platelet aggregometer or other photometric device equipped with a stirred cell. We have exploited the behavior of aggregates of fibrin-coated beads to develop a rapid, sensitive, and accurate method for measuring the activity of trypsin and its inhibition, in aqueous media, including serum. The new method yields serum antitrypsin activity levels that correlate well with immunological levels of alpha1-antitrypsin and, thus, may prove useful for assessing antitrypsin activity in clinical specimens.     

Haploinsufficiency of cyfip1 produces fragile x-like phenotypes in mice

Background

Copy number variation (CNV) at the 15q11.2 region, which includes a gene that codes for CYFIP1 (cytoplasmic FMR1 interacting protein 1), has been implicated in autism, intellectual disability and additional neuropsychiatric phenotypes. In the current study we studied the function of Cyfip1 in synaptic physiology and behavior, using mice with a disruption of the Cyfip1 gene.

Methodology/Principal Findings

We observed that in Cyfip1 heterozygous mice metabotropic glutamate receptor (mGluR)-dependent long-term depression (LTD) induced by paired-pulse low frequency stimulation (PP-LFS) was significantly increased in comparison to wildtype mice. In addition, mGluR-LTD was not affected in the presence of protein synthesis inhibitor in the Cyfip1 heterozygous mice, while the same treatment inhibited LTD in wildtype littermate controls. mGluR-agonist (RS)-3,5-dihydroxyphenylglycine (DHPG)-induced LTD was also significantly increased in hippocampal slices from Cyfip1 heterozygous mice and again showed independence from protein synthesis only in the heterozygous animals. Furthermore, we observed that the mammalian Target of Rapamycin (mTOR) inhibitor rapamycin was only effective at reducing mGluR-LTD in wildtype animals. Behaviorally, Cyfip1 heterozygous mice showed enhanced extinction of inhibitory avoidance. Application of both mGluR5 and mGluR1 antagonist to slices from Cyfip1 heterozygous mice reversed the increase in DHPG-induced LTD in these mice.

Conclusions/Significance

These results demonstrate that haploinsufficiency of Cyfip1 mimics key aspects of the phenotype of Fmr1 knockout mice and are consistent with the hypothesis that these effects are mediated by interaction of Cyfip1 and Fmrp in regulating activity-dependent translation. The data provide support for a model where CYFIP1 haploinsufficiency in patients results in intermediate phenotypes increasing risk for neuropsychiatric disorders.     

A study of rabbit plasma progressive antithrombinic activity during acute phase inflammatory reaction and in experimental nephrotic syndrome.

1. The development of the progressive antithrombinic activity in rabbit plasma as a function of the level of the two alpha-macroglobulins in two experimental pathologies: acute phase inflammatory reaction and nephrotic syndrome, were studied. 2. In the first case, the antithrombinic activity is a result of the increased biosynthesis of plasmatic antithrombins: antithrombin III, alpha 1 antitrypsin and alpha macroglobulins. 3. In the nephrotic syndrome, this activity follows the increase in the alpha M levels, the other antithrombins having been massively eliminated from the circulation in the urine.     

Probe the function of histone lysine 36 methylation using histone H3 lysine 36 to methionine mutant transgene in mammalian cells

Chondroblastoma is a cartilaginous tumor that typically arises under 25 y of age (80%). Recent studies have identified a somatic and heterozygous mutation at the H3F3B gene in over 90% chondroblastoma cases, leading to a lysine 36 to methionine replacement (H3.3K36M). In human cells, H3F3B gene is one of 2 genes that encode identical H3.3 proteins. It is not known how H3.3K36M mutant proteins promote tumorigenesis. We and others have shown that, the levels of H3K36 di- and tri-methylation (H3K36me2/me3) are reduced dramatically in chondroblastomas and chondrocytes bearing the H3.3K36M mutation. Mechanistically, H3.3K36M mutant proteins inhibit enzymatic activity of some, but not all H3K36 methyltransferases. Chondrocytes harboring the same H3F3B mutation exhibited the cancer cell associated phenotypes. Here, we discuss the potential effects of H3.3K36M mutation on epigenomes including H3K36 and H3K27 methylation and cellular phenotypes. We suggest that H3.3K36M mutant proteins alter epigenomes of specific progenitor cells, which in turn lead to cellular transformation and tumorigenesis.     

The M2 muscarinic receptor mediates in vitro bladder contractions from patients with neurogenic bladder dysfunction

Bladder muscle specimens from seven patients with neurogenic bladder dysfunction were analyzed to determine whether the muscarinic receptor subtype mediating contraction shifts from M(3) to the M(2) subtype as found in the denervated, hypertrophied rat bladder. Seven bladder specimens were analyzed from six female and one male patients. Six of the patients had traumatic cervical spinal cord injuries (C(4)-C(7)), and the other patient had an L(1) congenital myelomeningocele. This was compared with results from bladder specimens obtained from eight organ transplant donors. The affinities of three subtype-selective muscarinic receptor antagonists for inhibition of carbachol-induced contractions were determined. The affinity of the M(3) selective antagonists darifenacin or p-fluoro-hexahydrosiladifenadol (p-F-HHSiD) was determined in six of the seven spinal injury patient specimens. The affinity was consistent with M(2)-mediated contractions in four of these six specimens, intermediate between M(2) and M(3) in one specimen, and within the M(3) range in one specimen. The other specimen, tested only with the M(2) selective antagonist methoctramine, showed an M(3) affinity. In the organ donors, the affinity of p-F-HHSiD was within the M(2) range for six of seven specimens, whereas the affinity of darifenacin was within the M(3) range for five of six and intermediate between M(2) and M(3) for the other specimen tested. The affinity of methoctramine in both organ donor specimens tested was within the M(3) range. Whereas normal detrusor contractions are mediated by the M(3) receptor subtype, in patients with neurogenic bladder dysfunction as well as certain organ transplant donors, contractions can be mediated by the M(2) muscarinic receptor subtype.     

Jmjd3 is essential for the epigenetic modulation of microglia phenotypes in the immune pathogenesis of Parkinson's disease

Classical activation (M1 phenotype) and alternative activation (M2 phenotype) are the two polars of microglial activation states that can produce either detrimental or beneficial effects in the central nervous system (CNS). Harnessing the beneficial properties of microglia cells by modulating their polarization states provides great potential for the treatment of Parkinson''s disease (PD). However, the epigenetic mechanism that regulates microglia polarization remains elusive. Here, we reported that histone H3K27me3 demethylase Jumonji domain containing 3 (Jmjd3) was essential for M2 microglia polarization. Suppression of Jmjd3 in N9 microglia inhibited M2 polarization and simultaneously exaggerated M1 microglial inflammatory responses, which led to extensive neuron death in vitro. We also observed that the suppression of Jmjd3 in the substantia nigra (SN) in vivo dramatically caused microglial overactivation and exacerbated dopamine (DA) neuron death in 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-intoxicated mouse model of PD. Moreover, we showed that the Jmjd3 level was lower in the midbrain of aged mice, which was accompanied by an elevated level of H3K27me3 and an increased ratio of M1 to M2 markers, suggesting that aging is an important factor in switching the microglia phenotypes. Overall, our studies indicate that Jmjd3 is able to enhance the polarization of M2 microglia by modifying histone H3K27me3, and therefore it has a pivotal role in the switch of microglia phenotypes that may contribute to the immune pathogenesis of PD.     

ATP independent proteasomal degradation of NQO1 in BL cell lines

Human NAD(P)H: quinone oxidoreductase 1 (NQO1) catalyzes the obligatory two-electron reduction of quinones. For this peculiar catalytic mechanism, the enzyme is considered an important cytoprotector. The NQO1 gene is expressed in all human tissues, unless a polymorphism due to C609T point mutation is present. This polymorphism produces a null phenotype in the homozygous condition and reduced enzyme activity in the heterozygous one. We previously demonstrated that two cell lines of haematopoietic origin, HL60 and Raji cells, possess the same heterozygous genotype, but different phenotypes; as expected for a heterozygous condition the HL60 cell line showed a low level of enzyme activity, while the Raji cell line appeared as null phenotype. The level of NQO1 mRNA was similar in the two cell lines and the different phenotype was not due to additional mutations or to expression of alternative splicing products. Here we show that in Raji BL cell line with heterozygous genotype the null NQO1 phenotype is due to 20S proteasome degradation of wild type and mutant protein isoforms and is not directly linked to C609T polymorphism. This finding may have important implications in B-cell differentiation, in leukaemia risk evaluation and in chemotherapy based on proteasome inhibitors.     

Features of the milk whey protein partitioning in polyethyleneglycol-sodium citrate aqueous two-phase systems with the goal of isolating human alpha-1 antitrypsin expressed in bovine milk

Partitioning behaviour of the bovine whey proteins (bovine serum albumin, alpha-lactoalbumin and beta-lactoglobulin) and human alpha-1 antitrypsin in aqueous two-phase systems prepared with polyethyleneglycol (molecular masses: 1000, 1450 and 3350)-sodium citrate was analysed at pH 5.2, 6.2 and 8.2. Alpha lactoalbumin concentrated in the polyethyleneglycol rich-phase, while beta-lactoglobulin, bovine serum albumin and alpha-1 antitrypsin showed affinity for the citrate rich-phase. In aqueous two-phase systems of high medium pH and high polyethyleneglycol molecular mass the protein partitioning equilibrium is displaced to the citrate rich-phase. The polyethyleneglycol 1450-pH 5.2 system with a top/bottom phase-volume ratio of 3 showed to have the best capability of recovering the alpha-1 antitrypsin from a mixture prepared with natural milk whey and human alpha-1 antitrypsin. The recovery of this protein in the bottom phase was of 90% and the purity of the obtained product was of 98%. The method appears to be suitable as a starting point to isolate other human proteins expressed in transgenic bovine milk.     

Relationships among growth and different NOR phenotypes in a specific stock of rainbow trout (Oncorhynchus mykiss).

Growth is one of the most important aspects in the genetic improvement of cultured fish species. Consequently, genetic parameters related to this feature and their response to selection have been the focus of most research in this area. Such research indicates that, in general, there is enough additive genetic variance related to growth, justifying the use of selection. Based on the usefulness of cytogenetic and molecular markers in the fish culture, the aim of the present work was to analyze the possible relationships among cytogenetic characteristics, specifically the NOR phenotypes, and the increase in length and weight in specimens of the rainbow trout (Oncorhynchus mykiss), resultant from directed mating between homozygous females and heterozygous males according to their NOR phenotypic patterns. The equations of the relationship between length and weight of the analyzed specimens followed the model Wt = a Lt(b), showing b values higher than 3, determinant of a positive allometric growth. The results showed that the different NOR phenotypes were not related with the growth values for length and weight in any statistical test.