Studies on a genetically determined albumin dimer

An electrophoretic variant of human albumin has been characterized as a dimer involving both disulfide and noncovalent bonds. The variant protein was isolated by starch block electrophoresis and gel filtration and extensively investigated by gel electrophoresis and immunoelectrophoresis under a variety of conditions. No significant differences were found between normal albumin and the propositus's albumin in sulfhydryl reactivity or content or in the tryptic fingerprint.Contribution No. 140 from the Blood Research Laboratory, American National Red Cross.     

A simple in vitro culture system for tracheal cartilage development

Semi-circular tracheal cartilage is a critical determinant of maintaining architectural integrity of the respiratory airway. The current effort to understand the morphogenesis of tracheal cartilage is challenged by the lack of appropriate model systems. Here we report an in vitro tracheal cartilage system using embryonic tracheal–lung explants to recapitulate in vivo tracheal cartilage developmental processes. With modifications of a current lung culture protocol, we report a consistent in vitro technique of culturing tracheal cartilage from primitive mouse embryonic foregut for the first time. This tracheal culture system not only induces the formation of tracheal cartilage from the mouse embryonic foregut but also allows for the proper patterning of the developed tracheal cartilage. Furthermore, we show that this culture technique can be applied to culturing other types of cartilage in vertebrae, limbs, and ribs. We believe that this novel application of our in vitro culture system will facilitate the manipulation of cartilage development under various conditions and thus enabling us to advance our current limited knowledge on cartilage biology and development.     

Human apolipoprotein E isoprotein subclasses are genetically determined.

In a recent communication, we showed that human very low density lipoprotein (VLDL) apolipoprotein E (Apo E) from different individuals appears upon two-dimensional gel electrophoretic analysis in either one of two complex patterns. These have been designated class alpha and class beta. Mixing of VLDL from different subjects revealed that not all alpha or beta apo E patterns were the same. In this manner, we identified three subclasses of class alpha (alpha II, alpha III, and alpha IV) and three subclasses of class beta (beta II, beta III, and beta IV). We report here the results of family studies that reveal that the subclasses (alpha II, alph III, and alpha IV and beta II, beta III, and beta IV) of apo E are determined at a single genetic locus with three common alleles, epsilon II, epsilon III, and epsilon IV. The class beta phenotypes (beta II, beta III, and beta IV) represent homozygosity for two identical apo E alleles (epsilon). In contrast, class alpha phenotypes (alpha II, alpha III, and alpha IV) represent heterozygosity for two different apo E alleles. The apo E subclasses and their corresponding genotypes are as follows: beta II = epsilon II/epsilon II; beta III = epsilon III; beta IV = epsilon IV/epsilon IV; alpha II = epsilon II/epsilon III; alpha III = epsilon III/epsilon IV; and alpha IV = epsilon II/epsilon IV. To estimate the frequencies of the apo E alleles in the general population, apo E subclasses were then investigated in 61 unrelated volunteers and the results were: beta II = 1 (2%), beta III = 30 (49%), alpha II = 9 (15%, alpha III = 13 (31%), and alpha IV = 2 (3%). Utilizing the frequencies of these phenotypes, the gene frequencies were calculated to be epsilon II = 11%, epsilon III = 72%, and epsilon IV = 17%. In addition, apo E subclasses were studied in a clinic for individuals with plasma lipid disorders and the apo E subclass beta IV was found to be associated with type III hyperlipoproteinemia. There was no association of any apo E subclass with type II, type IV, or type VI hyperlipoproteinemia or plasma HDL cholesterol levels. This study explains the genetic basis for the common variation in a human plasma protein, apo E. Since the apo E subclass beta IV is associated with type III hyperlipoproteinemia, a disease characterized by xanthomatosis and premature atherosclerosis, understanding the genetic basis of the apo E subclasses should provide insight into the genetics of type III hyperlipoproteinemia.     

Mitochondrial involvement in genetically determined transition metal toxicity I. Iron toxicity

Iron that is not specifically chaperoned through its essential functional pathways is damaging to biological systems, in major part by catalyzing the production of reactive oxygen species. Iron serves in several essential roles in the mitochondrion, as an essential cofactor for certain enzymes of electron transport, and through its involvement in the assembly of iron-sulfur clusters and iron-porphyrin (heme) complexes, both processes occurring in the mitochondrion. Therefore, there are mechanisms that deliver iron specifically to mitochondria, although these are not well understood. Under normal circumstances the mitochondrion has levels of stored iron that are higher than other organelles, though lower than in cytosol, while in some disorders of iron metabolism, mitochondrial iron levels exceed those in the cytosol. Under these circumstances of excess iron, protective mechanisms are overwhelmed and mitochondrial damage ensues. This may take the form of acute oxidative stress with structural damage and functional impairment, but also may result in long-term damage to the mitochondrial genome. This review discusses the evidence that mitochondria do indeed accumulate iron in several genetic disorders, and are a direct target for iron toxicity when it is present in excess. We then consider two classes of genetic disorders involving iron and the mitochondrion. The first include defects in genes directly regulating mitochondrial iron metabolism that lead to Friedreich's ataxia and the various sideroblastic anemias, with excessive mitochondrial iron accumulation. Under the second class, we discuss various primary hemochromatoses that lead to direct mitochondrial damage, with reference to mutations in genes encoding HFE, hepcidin, hemojuvelin, transferrin receptor-2, ferroportin, transferrin, and ceruloplasmin.     

Mitochondrial involvement in genetically determined transition metal toxicity II. Copper toxicity

Copper, like iron, is an essential transition metal ion in which its redox reactivity, whilst essential for the activity of mitochondrial enzymes, can also be a source of harmful reactive oxygen species if not chelated to biomolecules. Therefore, both metals are sequestered by protein chaperones and moved across membranes by protein transporters with the excess held in storage proteins for future use. In the case of copper, the storage proteins in the mitochondria are a distinct ceruloplasmin and metallothionein (MT). If the cell accumulates too much copper or copper is needed by other cells, then copper can be chaperoned to the trans-Golgi secretory compartment where it is transported into the Golgi by ATP-dependent pumps ATP7A/B. In liver, the copper is then incorporated into ceruloplasmin in vesicles that travel to the plasma membrane and release ceruloplasmin into the plasma. This paper reviews the genetic basis for diseases associated with copper deficit or excess, particularly those attributed to defective ATP7A/B transporters, with special emphasis on pathologies related to a loss of mitochondrial function.     

Distribution of sibship size in families manifesting a genetically determined disorder.

The effect on sibship size distribution of the birth of a child with a genetical defect is considered for several different conditions. Family size continues to be over-dispersed in such cases, rather than showing any sign of reduced variation, though theoretical expectations about the correlation between numbers of normal and affected children are not well-supported by the data.     

Studies on cartilage formation. XXII. Investigations of certain oxidative metabolic processes in regenerating articular cartilage

The distal articular surface of the femur was surgically removed in 57 dogs. Succinate dehydrogenase and cytochrome oxidase activities were assayed on postoperative days 7, 20, 26, 33 and 70 in the regenerating, chondrifying articular surface and in the granulation tissue adhering to the capsule. In the 70-day samples, the cyanide-induced inhibition of oxygen consumption was determined and enzyme histochemical reactions (cytochrome oxidase, monoamine oxidase, xanthine oxidase, peroxidase and "catalase") were performed. The succinate dehydrogenase activity was the highest in the early postoperative stage in both tissues. This was followed by a definite decrease and a subsequent significant increase in activity when chondrification took place. Measurement of cytochrome oxidase activity could not reveal any convincing result, presumably because of the properties of the tissues studied. The oxygen consumption by the chondrifying articular surface at 70 days was inhibited to about 50% by cyanide, and about 90% inhibition was observed in the tissue adhering to the capsule. The cells of the regenerating articular surface possess cytochrome oxidase and a cyanide- (and sodium azide-) resistant oxidase activity. The enzyme activity of the cartilaginous islets exceeded that of their connective tissue environment. The cytochrome oxidase activity increased in the cells during cartilage differentiation. Presumably, some further cyanide-sensitive and cyanide-resistant oxidases are present in chondroblasts and young chondrocytes.     

A rare genetically determined variant of psuedocholinesterase in two German families with high plasma enzyme activity.

Activity of pseudocholinesterase (acylcholine-acyl-hydrolase) elevated up to four times has been detected in sera of members of two German families. The catalytic concentrations of the pseudocholinesterase of the afflicted members of both families (male and female) varied between 4800 U/l and 10 200 U/o (acetylthiocholine iodide substrate). The pseudocholinesterase of the propositi exhibits isoenzyme separation patterns in polyacrylamide electrophoresis as well as in electrofocussing which are different from those of pseudocholinesterase from normal persons. No differences could be seen as regards the Km of substrates or the inhibition by dibucaine, fluoride or succinyldiocholine.     

Early glomerular alterations in genetically determined low nephron number

An association between low nephron number and subsequent development of hypertension in later life has been demonstrated. The underlying pathomechanisms are unknown, but glomerular and postglomerular changes have been discussed. We investigated whether such changes are already present in prehypertensive "glial cell line-derived neurotrophic growth factor" heterozygous mice (GDNF+/-) with lower nephron number. Twenty-six-week-old mice [22 GDNF+/-, 29 C57B6 wild-type control (wt)] were used for in vivo experiments with intra-arterial and tail cuff blood pressure measurements. After perfusion fixation, kidneys were investigated with morphological, morphometric, stereological, and immunohistochemical techniques and TaqMan PCR analysis. As expected at this age, blood pressure was comparable between GDNF+/- and wt. Nephron number per kidney was significantly lower in GDNF+/- than in wt (-32.8%, P < 0.005), and mean glomerular volume was significantly higher (+49.5%, P < 0.001). Renal damage scores, glomerular and tubular proliferation, analysis of intrarenal arteries and peritubular capillaries, expression of relevant tubular transporter proteins, as well as gene expression of profibrotic, proinflammatory, or prohypertensive markers were not significantly different between GDNF+/- and wt. Compensatory glomerular hypertrophy in GDNF+/- was accompanied by higher numbers of endothelial and mesangial cells as well as PCNA-positive glomerular cells, whereas podocyte density was significantly reduced. Further electron microscopic analysis showed marked thickening of glomerular basement membrane. In conclusion, lower nephron number is associated with marked early glomerular structural changes, in particular lower capillary supply, reduced podocyte density, and thickened glomerular basement membrane, that may predispose to glomerular sclerosis.