The biochemistry and medical significance of the flavonoids.

(First of Two Parts) COLLAGEN is the major macromolecule of most connective tissues, and it is widely believed that many human diseases, particularly chronic diseases, will be found to involve this protein either directly or indirectly. Since collagen biosynthesis was last reviewed in the Journal in 1972,1 much new information has been gained about the relatively complex pathway by which it is synthesized. We should like to consider this new information and some of its consequences in terms of the following questions: What is the structural information in the collagen molecule, and how does it determine the structure of the . . .

Biosynthesis of collagen and its disorders

The Biochemistry of glycoproteins and proteoglycans

In our view, gene therapy may ameliorate some human genetic diseases in the future. For this reason, we believe that research directed at the development of techniques for gene therapy should continue. For the foreseeable future, however, we oppose any further attempts at gene therapy in human patients because (i) our understanding of such basic processes as gene regulation and genetic recombination in human cells is inadequate; (ii) our understanding of the details of the relation between the molecular defect and the disease state is rudimentary for essentially all genetic diseases; and (iii) we have no information on the short-range and long-term side effects of gene therapy. We therefore propose that a sustained effort be made to formulate a complete set of ethicoscientific criteria to guide the development and clinical application of gene therapy techniques. Such an endeavor could go a long way toward ensuring that gene therapy is used in humans only in those instances where it will prove beneficial, and toward preventing its misuse through premature application. Two recent papers have provided new demonstrations of directed genetic modification of mammalian cells. Munyon et al. (44) restored the ability to synthesize the enzyme thymidine kinase to thymidine kinase-deficient mouse cells by infection with ultraviolet-irradiated herpes simplex virus. In their experiments the DNA from herpes simplex virus, which contains a gene coding for thymidine kinase, may have formed a hereditable association with the mouse cells. Merril et al. (45) reported that treatment of fibroblasts from patients with galactosemia with exogenous DNA caused increased activity of a missing enzyme, alpha-D-galactose-l-phosphate uridyltransferase. They also provided some evidence that the change persisted after subculturing the treated cells. If this latter report can be confirmed, the feasibility of directed genetic modification of human cells would be clearly demonstrated, considerably enhancing the technical prospects for gene therapy.

https://science.sciencemag.org/content/sci/175/4025/949.full.pdf

Gene Therapy for Human Genetic Disease

Antioxidant activity and biologic properties of a procyanidin-rich extract from pine (Pinus maritima) bark, pycnogenol.

The effects of the administration of normal human plasma to patients affected by mucopolysaccharidoses I and II (Hurler's and Hunter's syndromes) have been evaluated. The infusion was followed by a decreased urinary excretion of relatively large molecular weight glycosaminoglycans and by an increased excretion of their products of degradation. Among the latter, products of the degradation of dermatan sulfate and heparan sulfate could be demonstrated. 

The results indicate that normal human plasma may contain those “factors” that are involved in the normal degradation of dermatan sulfate and heparan sulfate, that are missing in the diseased states.

Induced Degradation of Glycosaminoglycans in Hurler's and Hunter's Syndromes by Plasma Infusion

A method is proposed for the measurement of glycosaminoglycans (GAG) on 5-10 ml of plasma. It is based on the adsorption of GAG on small ECTEOLA columns followed by measurement of the hexuronic acid in the NaCl eluates. Routine use of the method has indicated the presence of a GAG fraction that adsorbs readily on ECTEOLA ("free" GAG) and of another that adsorbs on it only after treatment with papain ("bound" GAG). "Free" and "bound" GAG have been measured in normal adults, normal children, and children affected by mucopolysaccharidosis type I; the results obtained are in good agreement with those previously reported in the literature.Various analyses performed on purified "free" and "bound" GAG have confirmed that chondroitin-4-sulfate is the main glycosaminoglycan of normal human plasma where it occurs both free and bound to protein and at various levels of sulfation. The presence of small amounts of heparan sulfate and keratan sulfate has also been demonstrated. Metabolic experiments performed in rabbits have indicated that plasma GAG derive from peripheral tissues and increase sharply after papain injection. In young animals the "free" GAG have a faster turnover than the "bound," possibly a reflection of active processes of remodeling and calcification. The synthesis of the "free" and "bound" GAG, as measured with (85)S-sulfate incorporation, seems to proceed at the same rate, and the hypothesis has been advanced that as a result of the action of tissue proteases, part of the "bound" GAG may be transformed into "free" GAG, the latter being immediately extruded from the tissues into the circulatory system.

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The glycosaminoglycans of human plasma

Two maternal cousins affected by the X–linked form of Ehlers–Danlos syndrome have been observed. Both had congenital heart disease, “floppy valve syndrome”, hernias, short stature, stretchable skin and moderate joint hyper–mobility. Both excreted normal amounts of urinary glycosaminoglycans, almost entirely represented by dermatan sulfate, whose degradation appeared to be inadequate. They also excreted large amounts of hydroxylysine glycosides and L–valyl–proline, considered to be products of degradation of collagen and elastin, respectively. Cultured skin fibroblasts of the propositus synthesized excessively soluble collagen and had a low lysyl oxidase activity. These findings suggest that the increased degradation of structural proteins may be secondary to the defective cross–linking processes caused by the enzymic defect. Addition of (+) catechin, a flavonoid, to the propositus's cultured fibroblasts decreased the abnormal solubility of their collagen.

Lysyl oxidase deficiency in Ehlers-Danlos syndrome type V.

[1-3H]Galactitol-6-sulfate, N- [1-3H]acetylgalactosaminitol-6-sulfate, N-[1-3H]acetylglucosaminitol-6-sulfate, N-acetylglucosamine-6-sulfate, and 6-sulfated tetrasaccharides from chondroitin-6-sulfate have been used for the measurement of 6-sulfatase activity of extracts of normal skin fibroblasts and of fibroblasts cultured from patients with genetic mucopolysaccharidoses. With these substrates, extracts of fibroblasts derived from Morquio patients lack or have greatly reduced activities for galactitol-6-sulfate, N-acetylgalactosaminitol-6-sulfate, and 6-sulfated tetrasaccharides but have normal activity for N-acetylglucosamine-6-sulfate and its alditol; those derived from a patient with a newly discovered mucopolysaccharidosis have greatly reduced activity for N-acetylglucosamine-6-sulfate and its alditol but normal activity for galactitol-6-sulfate, N-acetylgalactosaminitol-6-sulfate, and the 6-sulfated tetrasaccharides. These findings demonstrate the existence of two different hexosamine-6-sulfate sulfatases, specific for the glucose or galactose configuration of their substrates. Their respective deficiencies, causing inability to degrade keratan sulfate and heparan sulfate in one case and keratan sulfate and chondroitin-6-sulfate in the other, are responsible for different clinical phenotypes.

Deficiencies of glucosamine-6-sulfate or galactosamine-6-sulfate sulfatases are responsible for different mucopolysaccharidoses.

Treatment of radioactively labeled guinea-pig skin soluble collagen or calf skin collagen with the flavonoid (+)-catechin makes the collagen resistant to the action of mammalian collagenase but not to the action of bacterial collagenase Complete resistance to the action of the mammalian enzyme may be achieved by incubating 06 mg of collagen (dry weight) with 01 mM (+)-catechin, followed by dialysis to remove the unbound flavonoid Since incubation of the mammalian enzyme with (+)-catechin does not inhibit its activity, it is postulated that (+)-catechin binds tightly to collagen and modifies its structure sufficiently to make it resistant to enzyme degradation

Patricia V. Donnelly

Papers

Induced Degradation of Glycosaminoglycans in Hurler's and Hunter's Syndromes by Plasma Infusion

The glycosaminoglycans of human plasma

Lysyl oxidase deficiency in Ehlers-Danlos syndrome type V.

Deficiencies of glucosamine-6-sulfate or galactosamine-6-sulfate sulfatases are responsible for different mucopolysaccharidoses.

Collagen treated with (+)-catechin becomes resistant to the action of mammalian collagenase.