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Sixteen cobalt(III) complexes incorporating one of the investigated 4N donor tripodal amines in the presence or absence of differently substituted hydroxamates have been synthesized and the effect of the nature of the N-donor, size of the chelates formed and the effect of the type of the substituent(s) at the hydroxamate moiety on the redox properties of the complexes have been studied. The crystal and molecular structures of the new complexes, [Co(uns-penp)(H 2 O)Cl]Cl 2 ·H 2 O ( 4 ), [Co(tren)(phebha)](ClO 4 ) 2 ( 11 ), [Co(tpa)(bha)](ClO 4 ) 2 ·C 2 H 5 OH·H 2 O ( 15 ) and [Co(tpa)(phebha)](ClO 4 ) 2 ( 16 ) have also been determined by single crystal X-ray diffraction method. Cyclic voltammetric (CV) results indicated the irreversible reduction of Co(III) in all the investigated complexes. Out of the four studied tripodal amines, abap was found to decrease the Co(III/II) reduction potential far below the region of bioreductants. Decreasing of two of the chains by one CH 2 in tren compared to abap resulted in less negative reduction potential of the corresponding complex. Further positive shift was observed by introducing two (uns-penp), and especially three (tpa) π-back-bonding pyridyl rings into the chains of tetramines. In agreement with literature results, the 3+ oxidation state of the central cobalt ion was found to be extremely stabilized in the ternary complexes containing the doubly deprotonated benzohydroximate, but the metal ion is significantly more reducible in the ternary complexes with mono-deprotonated benzohydroxamate/derivative ligands. Measurable effect was not found on the redox potential via introduction of chloro or nitro substituents in para position into the phenyl moiety of bha − (Cl-bha − and NO 2 -bha − ). Significant positive shift (ca. 200 mV) was obtained, however, when R N = H was replaced by a phenyl ring in phebha − therefore complexes with this latter ligand can be likely candidates for the in vitro releasing of hydroxamates with proven biological activity.

Complexation of deferasirox (4-[3,5-bis(2-hydroxyphenyl)-1,2,4-triazol-1-yl]benzoic acid, H3LB) and its decarboxy derivative (H2LA) with Ru(III), Os(IV), Co(III) and Ga(III) was studied. These novel complexes with likely multitargeted capabilities are expected to show anticancer potential after biotransformation via both of the metal ion and the liberated ligand. Formation of 1:2 octahedral complexes with a (O,N,O) tridentate coordination of the ligands was found yielding K3[Ru(LB)2], K[Co(LA)2], K3[Co(LB)2] and (guanidinium)Na2[Ga(LB)2], respectively. The molecular structure obtained by single crystal X-ray diffraction also confirmed the octahedral geometry for K[Co(LA)2]∙2PriOH. Unprecedented formation of K2[OsVILB(O)2(OH)] having a tridentate (O,N,O) coordinating ligand, two oxo groups and a hydroxide ion in the coordination sphere of the metal ion with + 6 oxidation state was also found. Simple synthesis and solid state structure of the novel disulfonated derivatives of H3LB, and H2LA, (H5LD and H4LC, respectively, with excellent water solubility) is also presented. Interaction of Ga(III) with H4LD− and H3LC− resulted in the formation of 1:1 and 1:2 complexes with different protonation degree in aqueous solution among them [Ga(LD)2]7− and [Ga(LC)2]5− being the major species under physiologically relevant conditions.

A novel ambidentate type hydroxamic acid derivative (phenhaH, 1) containing an (O,O) and a 1,10-phenantroline (phen) based (N,N) donoratom set together with its heterobimetallic complexes incorporating an octahedral [Co(4 N)]3+ (4 N = tris(2-aminoethyl)amine (tren) or tris(2-methylpyridyl)amine (tpa)) and a half-sandwich type [(η6-p-cym)Ru]2+ entity have been synthesized and characterized using various analytical techniques. Reaction of [Co(4 N)Cl]Cl2 with 1 proved the exclusive (O,O) coordination of the ligand to the [Co(4 N)]3+ core to yield [Co(tpa)(phenha)](ClO4)2, (2). Subsequent treatment of 1 with [Ru(η6-p-cym)Cl2]2 and [Co(4 N)Cl]Cl2 in a one-pot reaction resulted in the formation of [(η6-p-cym)Ru(Cl)(phenha)Co(tren)]Cl(PF6)2 (3) and [(η6-p-cym)Ru(Cl)(phenha)Co(tpa)](PF6)3 (4) in which the organometallic Ru core is coordinated by the phen part while the Co entity by the hydroxamate part of 1. Cyclic voltammetry revealed that 4 could be reduced at a less negative potential and exhibits a reversible Co(III)/Co(II) redox process compared to 3 due to the π-back bonding interaction between the Co(III) centre and the pyridyl-N donors of tpa in 4. Complexes 2–4 were tested for their in vitro cytotoxicity using human-derived cancer cell lines (HeLa, MCF-7, HCT116 and MDA-MB-231) and showed moderate anti-proliferative activity in the double digit micromolar concentration range, 4 being the most active. Complex 4 displayed better activity against MDA-MB-231 cells than cisplatin.

Solution equilibrium study on Pb(II) complexes of 3-hydroxy-2-methyl-4-pyrone (maltol, maltH) and its two derivatives, 3-hydroxy-2-methyl-4 H -pyran-4-thione (thiomaltol, thiomalH) and 3-hydroxy-1,2-dimethyl-4-pyridinone (dhpH) have been performed by using pH-potentiometry, 1 H NMR, ESI MS methods. Out of the studied ligands, the (S,O)-chelating thiomalt was found to form the most stable mono- and bis-chelated type species, [PbL] + and [PbL 2 ], but limited water solubility hindered the examination on this system above pH 7. The Pb(II)-binding capabilities of malt and especially dhp are still very good and the higher extent of electron delocalization in dhp compared to malt results not only in the increased stability of the 5-membered (O,O) dhp chelate, but also the possibility of some involvement of the 6s 2 lone electron pair of Pb(II) in the bonding. Furthermore, dhp, compared to malt, is not just a more effective Pb(II) chelator, but also shows better selectivity toward Pb(II) against Zn(II).

One of the biggest scandals of the recent history of medicine is the conflict of views between the gerontological establishment and the American Academy of Anti-Aging Medicine (A4M). The style used in that discussion was really rough and unusual. On the one hand, according to some representatives of the American Medical Associations (AMA), the use of human growth hormone (hGH) for anti-aging medical interventions is illegal, criminal, and requires persecution. On the other hand, A4M is of the opinion that all this is "...filled with incorrect, misplaced references and studies, and multiple basic scientific errors, in an apparent attempt to damage the anti-aging medical profession...". It is evident that in the frame of a short article is impossible to treat all the relevant aspects of this complicated story. Nevertheless, this Editorial attempts to point out the main results obtained so far, together with the most important issues of theoretical feasibility of the hGH replacement therapy (hGHRT). The comprehensive explanation of the aging process called "membrane hypothesis of aging" (MHA) offers a solid basis for the interpretation of the observed beneficial effects of the hGH through its practically ubiquitous membrane receptors, and the species specificity of this peptide hormone. The specific activation of these receptors stimulates the membrane transport functions, rehydrates the intracellular colloids, allowing to speed up the protein synthesis and turnover, and activates a great number of cellular functions, all observed so far. The facts known about the adult growth hormone deficiency (AGHD) syndrome, and the beneficial effects of hGHRT in all aspects of this pathology suggest that aging may generally be considered as an AGHD syndrome. If this concept is accepted by most of the gerontologists, we can resolve practically all problems involved in the above outlined controversies. All this requires an independent, open-minded approach to the problem, and pushes us to a better understanding of the results of theoretical aging research. This approach may open a new, realistic way to the development of efficient anti-aging medical interventions.

Oxyradicals are generally considered harmful byproducts of oxidative metabolism, causing molecular damage in living systems. They are implicated in various processes such as mutagenesis, aging, and series of pathological events. Although all this may be justified, evidence is accumulating that it is an oversimplified view of the real situation. We can assume nowadays that the living state of cells and organisms implicitly requires the production of oxyradicals. This idea is supported by experimental facts and arguments as follows. (1) Complete inhibition of the oxyradical production by KCN (or by any block of respiration) kills the living organisms much before the energy reserves would be exhausted. (2) Construction of the supramolecular organization of the cells (especially of their membranous compounds) requires the cross-linking effect of oxyradicals, particularly that of OH* radicals. (3) Blast type cells produce much fewer oxyradicals than do differentiated ones, and interventions increasing the production of OH* radicals induce differentiation of various lines of leukemic (HL-60 and K562) and normal (fibroblasts, chondroblasts, etc.) cells, while SOD expression increases greatly. (4) It is reasonable to assume that the continuous flux of OH* radicals is prerequisite to maintenance of constant electron delocalization on the proteins, which is a semiconductive phenomenon suggested in 1941 by Szent-Györgyi, but it has never been proven experimentally. It is theoretically possible to describe the function of the synapses as that of a single p-n-p transistor, assuming that the free radical flux maintains electron movements on the subsynaptic structures, while the actual membrane potential is governing the electron flux. This theoretical approach may open completely new possibilities for our understanding of the normal functions of living organisms, such as basic memory mechanisms in brain cells, their aging processes, and therapeutic approaches to many degenerative disorders, such as various types of dementia.

This chapter is intended to outline the main results of a research trend realized by the author during the last 45 years, focused on the main role played by the cell membrane in the aging process. It is a very wide field; therefore, the reader cannot expect in this limited space a detailed description, but will be given a wide, interdisciplinary insight into the main facts and theories regarding cellular aging. The central idea described here is the concept called the membrane hypothesis of aging (MHA). The history, the chemical roots, physicochemical facts, biophysical processes, as well as the obligatory biochemical consequences are all touched in by indicating the most important sources of detailed knowledge for those who are more interested in the basic biology of the aging process. This chapter covers also the available anti-aging interventions on the cell membrane by means of the centrophenoxine treatment based on the MHA. It also briefly interprets the possibilities of a just developing anti-aging method by using the recombinant human growth hormone, essential basis of which is the species specificity, and the general presence of receptors of this hormone in the plasma membrane of all types of cells.

These experiments tested the differentiation properties of the PC-12 cell line under conditions of in vitro generation of OH free radicals by Fenton reaction. This involves the simultaneous addition of the following reactants: ADP–Fe 2+ -complex (0.1 mM for iron) and H 2 O 2 (0.025 mM), final concentrations. Superoxide dismutase activity, the increase of which is considered as a marker of differentiation, catalase and glutathione peroxidase enzyme activities were investigated, which all displayed significant increases after single and repeated interventions with hydroxyl free radicals, while the cell number remained nearly at the starting-value. It is known that the differentiation takes place when the cell number has reached a plateau. These data, therefore, suggest that hydroxyl free radicals can induce in vitro cell differentiation, and that they play a more complex role in cell physiology than simply causing oxidative damages. It is interesting that the cells can maintain high levels of these enzyme activities for a relatively long time (2 or 4 days) after a very short flux of hydroxyl free radicals.

The paper of Kitani cited in the title has raised an apparent contradiction regarding the validity of certain aspects of the membrane hypothesis of aging (MHA). He collected data showing that a number of detoxifying liver enzyme activities, although decline with age in male Fischer 344 rats, remain at an unchanged level in females of the same strain. He concluded that the main assumption of the MHA, according to which intracellular enzyme activities generally decline with age, cannot be maintained, and invoked me (p. 312) 'ellipsis to provide in the future ample (and convincing) evidence' in this respect. The present paper answers this criticism by showing that the apparent contradiction mentioned above is based on a misunderstanding on behalf of Kitani. Namely, MHA speaks about the general, density-dependent decline of the catalytic rate constant of any enzyme (k(cat)), i.e., activity per mole of enzyme, being the true specific activity of the enzymes. This parameter inevitably decreases at the increased physical density of the intracellular colloids during aging. This statement derives from the molecular enzyme kinetic models, and has extensively been proven experimentally, too. On the other hand, Kitani speaks about enzyme activities per mg total protein content of certain tissue extracts, which is a very illdefined parameter, since the concentration of the measured enzyme remains unknown. Therefore, this latter parameter is irrelevant from the point of view of MHA in any aspect.

Summary The theoretical basis of adipogenesis has always been a matter of debate. One concept suggests that all types of adipocytes are derived from undifferentiated connective tissue cells, whereas another concept suggests that adipocytes develop from specialized cells only that are able to accumulate fat. Many conflicting data have been published with respect to the transition of fibroblasts into preadipocytes. For example, this transition has been declared as impossible for dermal and perimysial fibroblasts. The present study analysed spontaneous accumulation of fat in various types of fibroblasts from different origin (retroocular, skin, NIH/3T3, and L929). It was found that intense Oil Red O-positive triglyceride-containing droplets accumulated in practically all types of fibroblasts provided that the cells were cultured on glass surface. When the cells were cultured on plastic surfaces, lipid staining was inhibited in a variable manner: inhibition was virtually complete in skin fibroblasts, whereas in other types of fibroblasts, inhibition was only partial. It is concluded that all types of fibroblasts can accumulate fat spontaneously, and thus can be considered as preadipocytes. Therefore, interpretations of data obtained with cultures of fibroblasts with respect to adipogenesis have to be reconsidered.

The lateral diffusion coefficients of lipids (D(l)) and proteins (D(p)) were measured in hepatocyte plasma membranes in freshly prepared liver smears of Brown Norway (BN/BiRijHsd) rats by means of the fluorescence recovery after photobleaching (FRAP) method. D(l) was measured after staining with N-4-nitrobenzo-2-oxa-1,3-diazolyl phosphatidylethanolamine (NBD-PE) in 14 males in four age groups (4.3-30.4 months), and 12 females (5.0-31.0 months). D(p) was measured after the development of peroxide-induced autofluorescence (PIAF) in 16 males in four age groups (4.7-30.8 months) and 15 females in four age groups (4.0-28.1 months). Highly significant negative, linear age-correlations of D(l) (R=-0.972 in males, and -0.840 in females, respectively) were found, the average data being 1.39x10(-9) cm(2)/s in the young male rats, and only 9.26x10(-10) cm(2)/s in the oldest ones, whereas in females, these two extremes were 1.44x10(-9) and 8.97x10(-10) cm(2)/s, respectively. Protein lateral mobility also declined linearly with age; in the male rats it decreased from 2.7x10(-10) to 1.8.x10(-10) cm(2)/s (R=-0.903), while in the females it decreased from 2.64x10(-10) to 2.21x10(-10) cm(2)/s (R=-0.955) between the youngest and oldest age groups. There were also some characteristic differences in the fractional recoveries (FR%), too. These trends are comparable with those of F344 rats studied previously, supporting the conclusion that the changes in the lateral mobility of plasma membrane proteins and lipids represent important biomarkers of cellular aging also in the BN/BiRijHsd rats.

This chapter is intended to outline the main results of a research trend realized by the author during the last 45 years, focused on the main role played by the cell membrane in the aging process. It is a very wide field; therefore, the reader cannot expect in this limited space a detailed description, but will be given a wide, interdisciplinary insight into the main facts and theories regarding cellular aging. The central idea described here is the concept called the membrane hypothesis of aging (MHA). The history, the chemical roots, physicochemical facts, biophysical processes, as well as the obligatory biochemical consequences are all touched in by indicating the most important sources of detailed knowledge for those who are more interested in the basic biology of the aging process. This chapter covers also the available anti-aging interventions on the cell membrane by means of the centrophenoxine treatment based on the MHA. It also briefly interprets the possibilities of a just developing anti-aging method by using the recombinant human growth hormone, essential basis of which is the species specificity, and the general presence of receptors of this hormone in the plasma membrane of all types of cells.

The lateral diffusion constants of lipids (D1) and proteins (Dp) in the plasma membranes of hepatocytes were measured in liver smears of male Fischer 344 rats, by means of the fluorescence recovery after photobleaching (FRAP) method, after ethinyl-estradiol (EE) treatment of 1 or 5 days. The main finding was that EE treatment results in significant decreases in both the D1- and the Dp-values. These observations are interpreted in terms of the role of membrane physicochemistry in functions of the hepatocytes.

The lateral diffusion constant of lipids (D 1 ) in hepatocyte plasma membranes was measured in liver smears by means of the fluorescence recovery after photobleaching (FRAP) method, applying the label, N -4-nitrobenzo-2-oxa-1,3-diazolyl phosphatidylethanolamine (NBD-PE). Nineteen ad libitum fed, male Fischer-344 rats in four age groups (2.1–29.8 months of age) were studied. A highly significant negative linear age-correlation of D 1 (cc = 0.958) was found. D 1 values were 1.39 x 10 −9 cm2/s in the young rats, and only 6.77 x 10 −10 cm2/s in the oldest rats. Lipid lateral mobility is changing in parallel with that of proteins, having been measured previously also with the FRAP method by the authors. Fractional recovery values (FR%) of the lipids were lower than those of proteins even in the young ages, but also decreased linearly with age, therefore, the parameter, D, x FR decreased even steeper with age than D 1 itself. D 1 was also measured in a group of six male Fischer 344 rats having been kept on dietary restriction (DR) since their age of 1 month until 30 months of age (applying the every-other-day (EOD) feeding). DR caused an increase of D 1 , compared with the age-matched ad libitum fed animals: the mean was 9.24 x 10 −10 cm 2 /s. FR% and D I , x FR again increased considerably under DR. The results are interpreted in terms of the increased protein and lipid turnover under DR.

Oxyradicals have been considered as harmful byproducts causing molecular damage during aging. However, evidence is accumulating to show that the actual situation is more complex: the living state implicitly involves the production of oxyradicals. (1) Blast type cells produce much less oxyradicals than the differentiated ones, and an increased production of OH' radicals induces differentiation of various lines of leukemia cells; meanwhile, their superoxide dismutase expression increases to a very high extent. (2) The supramolecular organization of the cells is developed by means of “useful” crosslinking effects OH' radicals. (3) Repiratory inhibition of oxyradical production (KCN-intoxication, suffocation, etc.) would kill living organisms prior to the exhaustion of energy reserves. It is assumed that the continuous flux of OH' radicals is a prerequisite for a constant electron delocalization on the proteins, which is a semiconduction of p-type, proposed already in 1941 by Albert Szent-Gyorgyi, and refuted on a ″theoretical″ basis. It has become clear by now that the carbon-based semiconduction is possible because diamond transistors are known to exist. The recently developed atomic force microscopy offers some real possibilities for experimental testing of this assumption. This concept may lead us to new horizons in interpretation of living functions, such as the basic memory mechanisms in brain cells and their impairment during aging.

The enzyme activities of trypsin (using an artificial substrate, Nalpha-benzoyl-L-arginine-ethylester = BAEE), xanthine oxidase (XOD) and superoxide dismutase (SOD) were measured in the absence and presence of various concentrations of the following inert, water-soluble polymer viscogens: polyvinylpyrrolidone (PVP-40), polyethyleneglycol (PEG-6000) and bovine serum albumin (BSA). Enzyme activities measured in the absence of viscogens were taken as 100%. In the presence of the viscogens, enzyme activities decreased considerably as follows: (i) Trypsin: to 2 or 12% in reaction mixtures containing 64 mg/ml PVP-40 or 481 mg/ml PEG-6000, respectively. (ii) XOD: to 29.3% in a reaction mixture containing 116 mg/ml PVP-40, to 68.9% in a medium containing 266 mg/ml PEG-6000, and 38.1% in the presence of 138 mg/ml BSA. (iii) SOD: to 40.0, 19.9 and 16.6% in the same media as listed for XOD, respectively. The observations are consistent with the predictions of the molecular enzyme kinetic model (MEKM), and are also of importance for the membrane hypothesis of aging, since the latter explains the loss of cell functions by an age-dependent increase of intracellular density which may cause serious enzyme inhibitions.

Catalase-mediated oxygen evolution from H2O2 was measured with a Clark electrode in a closed, buffered aqueous medium in a properly designed experimental model. Addition of various molecules (bovine serum albumin, egg albumin, dextrane, starch and PEG-8000) in increasing concentrations up to greater than 20% by volume decreased the rate of oxygen evolution in a linear fashion, although there were some quantitative differences between the effects of various components. The rotational correlation time of the spin label TMPN in BSA and egg albumin increased considerably in both protein solutions indicating a very significant increase of the microviscosity of these media. The results are interpreted in terms of the molecular enzyme kinetic model (MEKM) predicting that apart from microviscosity, the average mass distribution and the distance of the nearest two lattice points formed by components of the medium are also inversely proportional to both the forward and backward rate constants of the formation of the enzyme-substrate complex. These observations, on one hand, lend further experimental support to the MEKM and on the other one, are consistent with the membrane hypothesis of aging which explains the loss of cell functions by an age-dependent increase of intracellular density.

Experiments have been performed using oxidized (ID-O) and reduced (ID-H) idebenone in various spin trapping systems. The following results were obtained. (1) ID-O does not have any detectable scavenger effect on the OH• free radicals when they are generated by means of Fenton reaction and trapped using DMPO, PBN or 4-POBN. (2) ID-H represents a serious competition for all spin traps used in capturing OH• free radicals. The mechanism of this competition is, however, not a direct reaction, but it is based on the rather quick autoxidation of the dissolved ID-H generating O 2 • radicals. They interact with the OH• radicals (or with the OH-spin adducts) by means of electron donation to them. This statement has been proven by showing (i) that O2• radicals generated during the autoxidation of ID-H can directly be trapped on DMPO; (ii) the effect of ID-H on the OH• free radicals is abolished if SOD is added to the system; (iii) the O 2 • radicals generated by ID-H autoxidation reduce directly the OH-spin adducts on various kinds of nitroxide type spin traps (e.g. TEMPO, TMPN). (3) The rate of autoxidation of ID-H in solution is about an order of magnitude faster than that of ubiquinone. Similar differences could be observed in the rate of reduction of the oxidized forms of both compounds by Na-borohydride. The results fully explain the in vitro protective effect of ID-H against lipid peroxidation of artificial membranes.

The name idebenone (CV-2619) was given to a synthetic compound the chemical structure of which is 6-(10-hydroxydecyl)-2,3-dimethoxy-5-methyl-1,4-benzoquinone (commercial name in Japan: Avan). Although it is insoluble in water, suspensions of it can be applied and are absorbed relatively well from the intestine. Its acute toxicity is very low, and is well tolerated also in subacute and chronic treatments. It displayed no immunogenic or mutagenic side effects in the models studied so far. Pharmacological studies of idebenone revealed that (i) it acts as a free radical scavenger and protects the mitochondrial membrane against lipid peroxidation; (ii) it prevents platelet aggregation; (iii) it improves vascular disorders due to strokes or experimental cerebral ischemia; (iv) it recovers the cholinergic and monoaminergic changes in the ischemic brain; (v) it improves overall brain function (including memory, also) in various animal models. Pharmacokinetic studies have confirmed that it reaches the brain cells even after per os administration. The main conclusion from the experimental data was that idebenone may be of beneficial effect for the neurological disorders related to cerebral ischemia even in humans: the results obtained so far in human clinical trials confirmed the validity of this expectation for cases of human cerebrovascular disease.

Fritz Verzar, founder and one of the most important operator of experimental gerontology, was born 120 years ago at Budapest (Hungary) and passed away in 1979 at Basel (Switzerland) at the age of 93. This short paper, as well as the following one in this issue by Robert [Robert, L., 2006. Fritz Verzar was born 120 years ago: his contribution to experimental gerontology through the collagen research as assessed after half a century. Arch. Gerontol. Geriatr. 43, 13-43], intends to commemorate his human and scientific merits, which remained valid even after long decades after his death. The author of this paper was the last Hungarian pupil of Verzar; therefore, a personal touch of this commemoration could not be avoided and was not even wanted. Verzar was an exceptionally clever and realistic scientist and at the same time, a warm-hearted and honest man, who is remembered with deep respect and love.

As was shown in a recent review by this author (Ann. N.Y. Acad. Sci., 928: 187-199, 2001), oxyradicals cannot be considered only as harmful by-products of the oxidative metabolism, but living cells and organisms implicitly require their production. This idea is supported by numerous facts and arguments, the most important of which is that the complete inhibition of the oxyradical production by KCN (or by any block of respiration) kills the living organisms long before the energy reserves would be exhausted. This new theoretical approach not only helps our understanding of the normal functions of the living organisms, such as the basic memory mechanisms in the brain cells, but also helps in identifying the site-specific, radical-induced damaging mechanisms that represent the undesirable side effects of oxygen free radicals. First of all, these effects make the cell plasma membrane vulnerable and cause a series of intracellular functional disorders, as described by the membrane hypothesis of aging (MHA). The logical way for any antiaging intervention therefore should be to increase the available number of loosely bound electrons inside the plasma membrane that are easily accessible for OH(*) free radical scavenging. The present review summarizes the available knowledge regarding the theory of the use of membrane-related antiaging pharmaca, like centrophenoxine (CPH), tested in both animal experiments and human clinical trials. A modified, developed version of CPH coded as BCE-001 is also reported.

The membrane hypothesis of aging (MHA) explains the biological waste product (lipofuscin) formation as a disbalance between the rates of protein synthesis and damage, as well as of elimination of the damaged components. Although, this concept has not been refuted on the basis of any experimental evidence, it has neither been widely accepted. During the last decade the general interest has turned toward the molecular genetics so intensely, that research aimed at clarifying cell biological mechanisms became so to say hibernated. Nowadays it is being recognized more and more that after the complete description of the human genetic code, attention has to be dedicated again to the cellular mechanisms explaining the function of the gene products (proteins). In this context, our experimental findings described during the recent years may become again the subject of interest. We have shown that the in vivo inhibition of the lysosomal thiol-proteinase functions by sublethal doses of leupeptin in young, adult and old mice results in a considerable increase (about 30%) of the immobile fraction of membrane proteins in hepatocyte plasma membrane, meanwhile the lateral diffusion constant of the still mobile membrane proteins increased. These observations were interpreted as signs of a general slowing down of protein turnover in the plasma membrane, just by inhibiting the elimination mechanisms in the lysosomes. This paper will discuss the theoretical conclusions and significance of these findings for the biological waste product formation, as a basic cell biological function.

The lateral diffusion coefficients of proteins (D(p)) were measured in hepatocyte plasma membrane in freshly prepared liver smears by means of the fluorescence recovery after photobleaching (FRAP) method. D(p) was measured after development of peroxide-induced autofluorescence (PIAF) in a total of 115 senescence accelerated mice (SAM), distributed in three strains, at least five age-groups in each, as follows: (i) SAMR1TA (25 males and 22 females), medium life span (MLS) in months, under specific pathogen free (SPF) conditions, MLS(spf) 20.1 and 20.0, respectively, while under conventional conditions, MLS(Deltapf)=18.9 in average for both sexes; (ii) SAMP6/Ta (18 males and 17 females), MLS(spf)=17.1 and 15.3, respectively, and MLS(cc)=8.1 for both sexes; (iii) SAMP6/Ta (17 males and 16 females), MLS(spf)=15.6 and 14.7, respectively, and MLS(cc)=10.0 for both sexes. A highly significant negative linear age-correlation of D(p) (R=0.975 or higher) was found in each strain, being roughly proportional with the MLS(cc) values. Since the studied mice kept under SPF conditions survived longer, than under conventional conditions, the actual age-dependent decay rates of D(p) values did not differ significantly in two pairs of comparisons (female R1/P6 and female R1/P8), whereas they did in all other possible pairs, including also the normal C57BL/6 mice. The main conclusion can be drawn that the D(p) of hepatocyte membranes is a good biomarker of aging and survival also in SAM, as in all other inbred and outbred rodents, studied so far.

The working hypothesis assuming that oxygen free radicals cannot be considered only as harmful by-products of the oxidative metabolism has been experimentally tested. Human fibroblasts were grown in culture from the following five types of tissues: (1) normal orbital fat; (2) orbital fat of patients with endocrine ophtalmopathy (EOP); (3) normal orbital muscle; (4) orbital muscle of EOP patients; (5) skin. These fibroblasts (second to 12th passages) were treated for 2x72 h with the Fenton reactants: ADP-Fe(2+)-complex (0.1 mM for iron) and H(2)O(2) (0.055 mM), final concentrations. This treatment caused a slowing down of the cell proliferation, induced various morphological signs of differentiation, and significantly increased (40-150%) the total superoxide dismutase (SOD) and catalase (CAT) activities of the fibroblasts. Authors suggest that the increased expression of these enzymes may play a general role in the cell differentiation mechanisms, meaning that the generation of oxygen free radicals is an essential, useful factor even during the early phases of development, and may not be taken only as a harmful process during aging.

In order to study the effect of interferon alpha on the levels of acute phase complement proteins in vivo, serum concentrations of C9 and C1-inhibitor (C1-INH) were measured in patients with chronic hepatitis C before and 3 months after the beginning of interferon alpha2b therapy. Serum levels of the activation product of terminal complement pathway, C5b-9, HCV RNA and IL-6 were also determined. IFN alpha treatment significantly (P

The lateral diffusion coefficient (Dp) of the Con-A receptor protein was measured in the sarcolemma of the quadriceps femoris (QF) muscle of male and female C57BL/6JNia mice in four age groups between 2 and 26 months. Freshly prepared, ex vivo taken muscle strips were stained with Con-A-FL conjugate for 10 min, and fluorescence recovery after photobleaching (FRAP) measurements were carried out on 20-30 cells per animal, at 37 degrees C. Using this technique, Dp, and the fractional recovery (mobile fraction = FR%) of these proteins can be measured. In the youngest male and female age groups, Dp values of 5.72E-10 and 5.43E-10 cm2/s, and FR% values of 43.3 and 36.3%, were found, respectively. Dp displayed a characteristic, significant, negative, linear correlation with age in both sexes. The slope of the linear regression line calculated per month of age was 1.06E-11 and 0.96E-11 cm2/s for males and females, respectively; both of them differ from zero highly significantly. FR% values tended to increase slightly with age, yet the estimated average Dp = D(FR), calculated for the total Con-A receptor pool, maintained its significant, negative, linear age-correlation. The physiological significance of these changes needs to be clarified in the future.

ADP-Fe2+ (or ATP-Fe2+) complex and H2O2, components of the Fenton reaction, were added to K562 cells, then cultured for 96 h. Ara-C-induced differentiation served as a basis for comparison. Cell numbers, viability, benzidine staining, thymidine incorporation, and cell-cycle distribution by means of flow cytometry were determined. The Fenton reagents reduced the growth rate and thymidine incorporation of leukemic cells in a dose-dependent manner as regards the added H2O2 (from 0.01 to 1.0 mM), accompanied by an accumulation of hemoglobin in them. Differentiation of the cells was accompanied by considerable changes in total SOD and catalase activities. Ara-C caused an increase of SOD to 366%, and of catalase to 235%, while the complete Fenton reaction resulted in SOD increase to 705% and catalase decrease to 38% of the untreated control cultures. These shifts in enzyme inductions suggest the existence of a higher H2O2 flux in the differentiating cells. The results are consistent with the assumption that products of the Fenton reaction, among them OH. radicals deriving from H2O2 by heterolysis, may play a causal role in cell differentiation, whereas an overproduction of these radicals causes aging or death of the cells.

The OH free radical formation can be increased in the brain by intralumbar iron injections into the cerebrospinal fluid (CSF) through the Fenton reaction. Ferrous ammonium sulphate was injected to male CFY rats of 3 months of age. The animals survived a single dose of 4 mumoles, and this treatment was repeated daily for 3 or 6 days. The total iron contents of the large brain and the cerebellum were measured by atomic absorption spectroscopy; 3 days of iron administration resulted in significant increases of iron contents only in the cerebellum, whereas after 6 days, both parts of the brain showed considerably higher iron contents. Electron microscopic point-counting morphometric analysis revealed a 65% increase of volume density of the lipofuscin in the large cells of parietal cortex after 6 days of iron-treatment. The Purkinje cells displayed a particularly frequent occurrence of an "apoptosis"-like structural alteration under the effect of iron. This kind of experimental approach is complicated by the facts that (i) the increase of OH radical flux above a certain level is lethal, and (ii) the young animals have a very active elimination mechanism of the waste products. Nevertheless, the enhancement of OH radical yield by an increased availability of iron for Fenton reaction resulted in an accumulation of lipofuscin even in young rats.

Two types of antibodies which previously were found to be inversely associated with CD4 + cell counts and which may contribute to the progression of HIV disease were measured in parallel in 55 serum samples of 7 longitudinally tested HIV-infected patients (4 homosexual men, 3 haemophilic men) and in 15 serum samples from 15 patients with advanced AIDS. HIV-infection enhancing antibodies were determined in the presence of near-physiologic human complement concentration using a complement receptor type 2 (CR2) carrying HIV-target cell line. IgG and IgA class autoantibodies directed against human IgG-Fab fragments were measured in specific ELISA assays. In agreement with our previous studies obtained in HIV-seropositive haemophilic patients, significant negative correlations were found between CD4 + cell counts and IgG anti-Fab and IgA anti-Fab antibodies (Spearman correlation coefficient r =−0.587, P r =−0.269, P =0.024, respectively). A significant positive correlation was observed between complement-dependent enhancing antibodies and IgA anti-Fab antibodies ( r =0.408, P =0.003), whereas the correlation with IgG anti-Fab antibodies was only weak ( r =0.288, P =0.034). Serum samples with high titres of complement-dependent enhancing antibodies had almost 3 times higher IgA anti-Fab autoantibody activity than sera with low titres ( P =0.0038). Our findings indicate that the two disease markers in HIV disease, enhancing antibodies and autoantibodies directed against the Fab moiety of IgG, are not identical. However, anti-Fab antibodies may contribute to complement-dependent HIV infection enhancement.

OBJECTIVE To study the association between the progression of HIV disease and HIV neutralization and enhancement measured in the presence of human complement. DESIGN Two studies were performed: (1) longitudinal measurement of the complement-dependent enhancing antibodies in parallel with T-cell subset determination in 55 serum samples from seven HIV-infected patients, and (2) determination of the titres of neutralizing and enhancing antibodies in stored samples of 21 HIV-asymptomatic patients obtained between 1986 and 1987 and follow-up of the patients until October 1992. METHODS HIV-1 [human T-lymphotropic virus (HTLV)IIIB strain, 100 median tissue culture infective dose (TCID50)] was incubated with twofold dilutions of sera in the presence of human complement (final dilution, 1:4) and added to MT-4 cells. HIV growth was monitored daily for 5 days using the reclustering inhibition and p24 immunofluorescence assays. RESULTS A significant negative correlation between the titres of enhancing antibodies and CD4+ cell count was found in longitudinal measurements. In the prospective studies, marked differences were observed between patients with undetectable, low, or high titres of enhancing antibodies in the clinical course of HIV disease: CD4+ cell counts and percentages decreased more rapidly in the high titre group within 3 years. After 5 years, AIDS developed in five out of six patients in the high titre group but only in five out of 15 of the low titre group (P < 0.05). A similar difference was observed between patients with and without neutralizing antibodies. CONCLUSIONS Measurement of HIV neutralization and enhancement in complement-containing serum samples using a complement receptor carrying target may provide data of clinical relevance. Neutralization appears to be associated with a favourable prognosis whereas high titre enhancing antibodies predict rapid progression of HIV disease.

The liver cells of male and female wild mice (Peromyscus leucopus and Mus musculus) have been studied in young, adult, and old age groups. Because P. leucopus displays a life expectancy of 7-8 years, the age groups of this strain were from 5.5 to 68 months. M. musculus survives only to about 2.5 years; therefore, the age groups of this strain ranged from 6 to 27 months. Protein lateral diffusion in hepatocyte membranes was measured by means of fluorescence recovery after photobleaching (FRAP) in liver smears. The peroxide-induced autofluorescence was utilized as a fluorescent label, which in H2O2 treated liver smears is caused by the oxidation of riboflavin bound uniformly to all proteins of the cell membrane. This way the average lateral diffusion constant (D) and the fractional recovery of those proteins can be measured. In both males and females of both strains D displayed a negative linear age correlation. Intraspecies sex differences were small and statistically insignificant. However, interspecies comparisons of males or females resulted in a strongly significant difference: although the intercept points of the regression lines describing D were identical in both strains (3.1 x 10(-10) cm2/sec), the decay of D with aging was about 2.5 times faster in the short-living M. musculus than in the long-living P. leucopus. By the end of life expectancy, both strains reached practically identical values (about 1.9 x 10(-10) cm2/sec). The results lend further support to the membrane hypothesis of aging.

Tumor cells usually contain lower superoxide dismutase (SOD) activity than differentiating cells, suggesting the involvement of oxygen free radicals in cell maturation. The effects of a system known to produce the OH. radicals were tested on HL-60 cells cultured under optimum conditions for 96 hr. Hydroxyl radicals were generated by a Fenton reaction, involving an ADP-Fe2+ (or ATP-Fe2+) complex and H2O2. Changes induced by OH. were compared to the effects of DMSO-induced differentiation of HL-60 cells. Cell numbers, viability, thymidine incorporation, TPA-induced NBT reduction and propidium iodide staining in flow cytometry were determined. The OH. generating system inhibited the growth and thymidine incorporation of leukemic cells in a manner dependent on the dose of added H2O2 (from 0.005 to 0.05 mM). In addition, an increasing proportion of the treated cells displayed signs of cell differentiation. In DMSO-treated cells, SOD and catalase activities increased after 6 days of culturing. The results show that a portion of the OH. free radicals derived from H2O2, produced by the action of SOD, may be a necessary prerequisite for differentiation, whereas an overproduction of OH. causes cell lethality or aging. We suggest that OH. free radicals may have a more complex role in cell physiology than simply causing oxidative damage.

Four groups of male C57BL/6 mice (ages 2, 3.8, 11.8–12.5 and 24 months) were administered leupeptin at 5 mg/100 g body weight/day via intraperitoneally implanted osmotic minipumps. Both untreated and saline-treated mice served as controls. The time and dose-dependence of the effects of leupeptin on the lateral mobility of proteins in hepatocyte surface membranes were analyzed first in mice treated for 2, 5, 8, 11 and 14 days, respectively, by using the fluorescence recovery after photobleaching (FRAP) technique. The age dependence of the response to this treatment was also studied after 14 days of treatment in mice of various ages. The average lateral diffusion constant (D) and the fractional recovery (FR) were measured, and D × FR calculated. Leupeptin treatment at a daily dose of 5 mg/100g for 2 weeks increased plasma glutamic-pyruvlc transaminase levels 2-fold in all age groups. Leupeptin treatment caused a linear increase of D and a decrease of FR with respect to the duration of the treatment in adult mice. In all age groups about 30% of the membrane proteins became immobile after 14 days of treatment, whereas the still mobile fraction displayed a large increase of D. The values of D × FR in the leupeptin-treated groups were slightly higher than those in the untreated mice until the age of 15 months but decreased after this age. The results are interpreted in terms of the known inhibitory effect of leupeptin on cytoplasmic and lysosomal thiol proteases, causing a general slowing down of the protein turnover and, specifically, of proteins in the hepatocyte membrane.

This review summarizes the main points of an interdisciplinary, theoretically established approach to the problem of cell senescence, called membrane hypothesis of aging (MHA). The main knowledge and some new suggestions regarding the damaging and sometimes useful roles of the oxygen free radicals is outlined. The most important experimental results are listed, which harmonize with the MHA. It is emphasized that MHA is not an alternative to the other aging hypotheses but represents a synthesis of most of them. It is pointed out that the new drug design based on the MHA resulted in a useful new molecule which is able to improve the key cell parameters deteriorated by advancing age. The necessity of a closer international cooperation is emphasized, in order to achieve a breakthrough in experimental gerontology within this century.

A mixture of ADP, ferrous ions, and hydrogen peroxide (H2O2) generates hydroxyl radicals (OH) that attack the spin trap DMPO (5,5-dimethyl-pyrollidine-N-oxide) to yield the hydroxyl free radical spin-adduct, degrade deoxyribose and benzoate with the release of thiobarbituric acid-reactive material, and hydroxylate benzoate to give fluorescent products. Inhibition studies, with scavengers of the OH radical, suggest that the behavior of iron-ADP in the reaction is complicated by the formation of ternary complexes with certain scavengers and detector molecules. In addition, iron-ADP reacting with H2O2 appears to release a substantial number of OH radicals free into solution. During the generation of OH radicals the ADP molecule was, as expected, damaged by the iron bound to it. Damage to the iron ligand in this way is not normally monitored in reaction systems that use specific detector molecules for OH radical damage. Under certain reaction conditions the ligand may be the major recipient of OH radical damage thereby leading to the incorrect assumption that the iron ligand is a poor Fenton reactant.

Protein lateral diffusion in hepatocyte membranes was measured in 23 male and 16 female C57BL/6 mice in four groups of age 5 to 32 months by means of fluorescence recovery after photobleaching (FRAP). The peroxide-induced autofluorescence (PIAF) in H2O2 treated liver smears is due to the oxidation of flavins bound uniformly to all proteins of the cell membrane, i.e., an average lateral diffusion constant (D) of those proteins can be measured. D showed a negative linear age-correlation in both sexes: D male = (2.81 - 0.016X)10(-10), and D female = (2.67 - 0.020X)10(-10) cm2/sec, (r greater than 0.94), where X is the age in months. Sex differences are significant (p less than .001) in the intercept. Fractional recovery did not prove to be age-dependent; it was, on average, 91% in males and 100% in females. The findings agree well with the previously obtained data on Fischer 344 and Wistar rats, and strengthen the gerontological relevance of D.

Water-insoluble protein fractions increase in the brain cortical tissue and liver of rats during aging in both sexes. This suggests a possible increase in the cross-linking of proteins which may be due to the formation of, for example, hydroxyl free radicals during several metabolic processes. In vivo application of centrophenoxine causes a reversal of this phenomenon in old rats. In vitro experiments show that the generation of hydroxyl free radicals by chemical systems like homolysis of H2O2 by redox coupling with Fe2+ leads to Fe3+ conversion, results in the cross-linking of bovine serum albumin and the mixed proteins of liver or brain homogenates of young rats. The cross-linked proteins have a very much increased molecular weight, they become mostly insoluble even in 6 M urea. Dimethylaminoethanol, the effective part of the centrophenoxine molecules, is able to diminish the extent of cross-linking, acting most probably as a free-radical scavenger. The results are discussed in terms of the "membrane hypothesis of aging". A molecular basis is proposed for the anti-aging effect of centrophenoxine.

Intracellular water content (IWC) was measured in freeze-fractured biological bulk specimens by means of energy-dispersive X-ray microanalysis. The method is based on the concentration differences of certain elements (potassium and phosphorus) between frozen-hydrated and frozen-dried states of the tissues as applied formerly to sectioned material by others. A new mathematical formula has been derived giving rather precise figures for IWC. No elemental standards are necessary for the measurement: one has to obtain only the peak to background ratios in wet and dry states of the cells. The method is sensitive enough to reveal age-dependent as well as drug-induced changes of IWC in liver and brain cells. The values obtained are quite comparable with the theoretically expected ones. Technical problems of the application of this method are discussed in detail.