Expression of apoptosis related proteins in brains of patients with Alzheimer's disease.

An increasing number of proteins are implicated in apoptosis and several of them have been shown to be altered in Alzheimer's disease (AD) brain. Because of this apoptosis is thought to be the underlying mechanism of neuronal cell loss in AD. To further substantiate this hypothesis we investigated the expression of a recently identified apoptosis related proteins and other apoptosis regulators in frontal cortex and cerebellum of AD by Western blot and enzyme-linked immunsorbent assay technique. Quantitative analysis revealed unaltered levels of Bax and RAIDD (Receptor interacting protein associated ICH-1 (caspase-2)/CED-3 (Caenorhabditis elegans death protease-3)-homologous protein with death domain) in both regions. ZIP (Zipper interacting protein) kinase, Bim/BOD (Bcl-2 interacting mediator of cell death/Bcl-2 related ovarian death gene) and p21 were significantly increased only in AD frontal cortex (P < 0.05, in all cases). Cerebellar Bcl-2 levels were significantly increased in AD (P < 0.01) while in AD frontal cortex, although the levels tended to increase did not reach significance level. The results indicate that apoptosis indeed account for the neuronal loss in AD. However, it does not seem to involve Bax and RAIDD.

Effect of intravenous iron supplementation on erythropoiesis in erythropoietin-treated premature infants.

OBJECTIVE: To test the efficacy and safety of combining intravenous iron in amounts approximating the in utero iron accretion rate and the postnatal iron loss with erythropoietin (EPO) in very low birth weight (VLBW) infants. METHODS: A prospective, controlled, randomized, unmasked trial lasting 21 days was performed in 29 clinically stable VLBW infants <31 weeks' gestation and <1300 g birth weight not treated with red blood cell transfusions during the study period. Mean (+/- standard error of the mean) age at study entry was 23 +/- 2.9 days. After a 3-day run-in baseline period in which all participants received oral supplements of 9 mg/kg/day of iron polymaltose complex (IPC), participants were randomized to receive 18 days of treatment with: 1) oral IPC alone (oral iron group); 2) 300 U of recombinant human EPO (r-HuEPO) kg/day and daily oral IPC (EPO + oral iron group); 3) 2 mg/kg/day of intravenous iron sucrose, r-HuEPO, and oral iron (intravenous iron + EPO group). To assess efficacy of the 3 treatments, serial blood samples were analyzed for hemoglobin (Hb), hematocrit (Hct), reticulocyte count, red blood cell indices and plasma levels of transferrin, transferrin receptor (TfR), ferritin, and iron. Oxidant injury was assessed before and after treatment by plasma and urine levels of malondialdehyde (MDA) and o-tyrosine. RESULTS: At the end of treatment, Hb, Hct, reticulocyte count, and plasma TfR were markedly higher in both of the EPO-treated groups, compared with the oral iron group. At study exit a trend toward increasing Hb and Hct levels and significantly higher reticulocyte counts were observed in the intravenous iron + EPO group, compared with the EPO + oral iron group. During treatment, plasma ferritin levels increased significantly in the intravenous iron + EPO group and decreased significantly in the other 2 groups. By the end of treatment, ferritin levels were significantly higher in the intravenous iron + EPO group compared with the other 2 groups. Although plasma and urine MDA or o-tyrosine did not differ among the 3 groups, plasma MDA was significantly greater in the subgroup of intravenous iron + EPO participants sampled at the end of the 2-hour parenteral iron infusion, compared with values observed immediately before and after parenteral iron-dosing. CONCLUSIONS: In stable VLBW infants receiving EPO treatment, parenteral supplementation with 2 mg/kg/day of iron sucrose results in a small, but significant, augmentation of erythropoiesis beyond that of r-HuEPO and enteral iron alone. However, to reduce the potential adverse effects of parenteral iron/kg/day on increasing plasma ferritin levels and on causing oxidative injury, we suggest that the parenteral iron dose used should be reduced and/or the time of infusion extended to maintain a serum iron concentration below the total iron-binding capacity.

Decreased alpha-endosulfine, an endogenous regulator of ATP-sensitive potassium channels, in brains from adult Down syndrome patients.

Alpha-endosulfine has the ability to block ATP-sensitive potassium (K(ATP)) channels and stimulate insulin release in beta cells like sulfonylurea. Alpha-endosulfine is expressed in a wide range of tissue, including brain and endocrine tissues. Although K(ATP) channels are also present in brain and its regulators have been reported to be involved in the release of neurotransmitters such as acetylcholine that plays an important role in cognitive function, the neurobiological role of alpha-endosulfine has not been studied yet. We examined the expression levels of alpha-endosulfine protein in frontal cortex and cerebellum from patients with Down syndrome (DS) showing Alzheimer's disease (AD) pathology using Western blotting. In frontal cortex, alpha-endosulfine was detected in all of 10 controls, but only 1 (from female) out of 8 DS with weak density. In cerebellum, alpha-endosulfine was also detected in all of 9 controls, but only 1 (from male) out of 6 DS with weak density. The considerably decreased alpha-endosulfine could result in the continuous opening of K(ATP) channels and the subsequent decrease of neurotransmitters release associated with cognition. This study is of significance providing evidence for a biological role of alpha-endosulfine in brain and alpha-endosulfine protein could be a pharmacological target for therapeutic intervention.

Documenting the diet in ancient human populations through stable isotope analysis of hair.

Fundamental to the understanding of human history is the ability to make interpretations based on artefacts and other remains which are used to gather information about an ancient population. Sequestered in the organic matrices of these remains can be information, for example, concerning incidence of disease, genetic defects and diet. Stable isotopic compositions, especially those made on isolates of collagen from bones, have been used to help suggest principal dietary components. A significant problem in the use of collagen is its long-term stability, and the possibility of isotopic alteration during early diagenesis, or through contaminating condensation reactions. In this study, we suggest that a commonly overlooked material, human hair, may represent an ideal material to be used in addressing human diets of ancient civilizations. Through the analysis of the amino-acid composition of modern hair, as well as samples that were subjected to radiation (thus simulating ageing of the hair) and hair from humans that is up to 5200 years old, we have observed little in the way of chemical change. The principal amino acids observed in all of these samples are essentially identical in relative abundances and content. Dominating the compositions are serine, glutamic acid, threonine, glycine and leucine, respectively accounting for approximately 15%, 17%, 10%, 8% and 8% of the total hydrolysable amino acids. Even minor components (for example, alanine, valine, isoleucine) show similar constancy between the samples of different ages. This constancy clearly indicates minimal alteration of the amino-acid composition of the hair. Further, it would indicate that hair is well preserved and is amenable to isotopic analysis as a tool for distinguishing sources of nutrition. Based on this observation, we have isotopically characterized modern individuals for whom the diet has been documented. Both stable nitrogen and carbon isotope compositions were assessed, and together provide an indication of trophic status, and principal type (C3 or C4) of vegetation consumed. True vegans have nitrogen isotope compositions of about 7/1000 whereas humans consuming larger amounts of meat, eggs, or milk are more enriched in the heavy nitrogen isotope. We have also analysed large cross-sections of modern humans from North America and Europe to provide an indication of the variability seen in a population (the supermarket diet). There is a wide diversity in both carbon and nitrogen isotope values based at least partially on the levels of seafood, corn-fed beef and grains in the diets. Following analysis of the ancient hair, we have observed similar trends in certain ancient populations. For example, the Coptics of Egypt (1000 BP) and Chinchorro of Chile (5000-800 BP) have diets of similar diversity to those observed in the modern group but were isotopically influenced by local nutritional sources. In other ancient hair (Egyptian Late Middle Kingdom mummies, ca. 4000 BP), we have observed a much more uniform isotopic signature, indicating a more constant diet. We have also recognized a primary vegetarian component in the diet of the Neolithic Ice Man of the Oetztaler Alps (5200 BP). In certain cases, it appears that sulphur isotopes may help to further constrain dietary interpretations, owing to the good preservation and sulphur content of hair. It appears that analysis of the often-overlooked hair in archaeological sites may represent a significant new approach for understanding ancient human communities.

Evidence that taurine modulates osmoregulation by modification of osmolarity sensor protein ENVZ--expression.

Although the involvement of taurine in osmoregulation is well-documented and widely accepted, no detailed mechanism for this function has been reported so far. We used subtractive hybridization to study mRNA steady state levels of genes up- or downregulated by taurine. Rats were fed taurine 100 mg/kg body weight per day for a period of three days and hearts (total ventricular tissue) of experimental animals and controls were pooled and used for mRNA extraction. mRNAs from two groups were used for subtractive hybridization. Clones of the subtractive library were sequenced and the obtained sequences were identified by gen bank assignment. Two clones were found to contain sequences which could be assigned to the osmolarity sensor protein envZ, showing homologies of 61 and 65%. EnvZ is an inner membrane protein in bacteria, important for osmosensing and required for porine gene regulation. It undergoes autophosphorylation and subsequently phosphorylates OmpR, which in turn binds to the porine (outer membrane protein) promoters to regulate the expression of OmpF and OmpC, major outer membrane porines. This is the first report of an osmosensing mechanism in the mammalian system, which was described in bacteria only. Furthermore, we are assigning a tentative role for taurine in the osmoregulatory process by modifying the expression of the osmoregulatory sensor protein ENVZ.

Serotonin (5-HT) in brains of adult patients with Down syndrome.

Down syndrome (DS) is a genetic disease with developmental brain abnormalities resulting in early mental retardation and precocious, age dependent Alzheimer-type neurodegeneration. Furthermore, non-cognitive symptoms may be a cardinal feature of functional decline in adults with DS. As the serotonergic system plays a well known role in integrating emotion, cognition and motor function, serotonin (5-HT) and its main metabolite, 5-hydroxyindol-3-acetic acid (5-HIAA) were investigated in post-mortem tissue samples from temporal cortex, thalamus, caudate nucleus, occipital cortex and cerebellum of adult patients with DS, Alzheimer's disease (AD) and controls by use of high performance liquid chromatography (HPLC). In DS, 5-HT was found to be age-dependent significantly decreased in caudate nucleus by 60% (DS: mean +/- SD 58.6 +/- 28.2 vs. Co: 151.7 +/- 58.4 pmol/g wet tissue weight) and in temporal cortex by about 40% (196.8 +/- 108.5 vs. 352.5 +/- 183.0 pmol/g), insignificantly reduced in the thalamus, comparable to controls in cerebellum, whereas occipital cortex showed increased levels (204.5 +/- 138.0 vs. 82.1 +/- 39.1 pmol/g). In all regions of DS samples, alterations of 5-HT were paralleled by levels of 5-HIAA, reaching significance compared to controls in thalamus and caudate nucleus. In AD, 5-HT was insignificantly reduced in temporal cortex and thalamus, unchanged in cerebellum, but significantly elevated in caudate nucleus (414.3 +/- 273.7 vs. 151.7 +/- 58.4 pmol/g) and occipital cortex (146.5 +/- 76.1 vs. 82.1 +/- 39.1 pmol/g). The results of this study confirm and extend putatively specific 5-HT dysfunction in basal ganglia (caudate nucleus) of adult DS, which is not present in AD. These findings may be relevant to the pathogenesis and treatment of cognitive and non-cognitive (behavioral) features in DS.

Expression of DNA excision-repair-cross-complementing proteins p80 and p89 in brain of patients with Down Syndrome and Alzheimer's disease.

Although deficient DNA repair was proposed for neurodegenerative disorders including Down syndrome (DS), repair proteins for nucleotide excision repair have not been studied in brain yet. As one of the hypotheses for the pathogenesis of brain damage in DS and Alzheimer's disease (AD), is oxidative stress, and cells of patients with DS were shown to be more susceptible to ionizing irradiation. We decided to study expression of excision repair-cross-complementing (ERCC) gene products, proteins 80 and 89, representatives of repair genes known to be involved in the repair of different types of DNA damage. ERCC2-protein 80 kDa and ERCC3-protein p89 were determined in five individual brain regions of controls, aged DS and AD patients. Although different in the individual regions, DNA repair proteins were consistently higher in temporal and frontal lobes of patients with DS and higher in all brain regions of patients with AD. Our results are the first to describe DNA repair gene protein patterns in human brain regions providing the basis for further studies in this area. We showed that DNA repair genes ERCC2 and ERCC3 (excision-repair-cross-complementing) for nucleotide excision repair were increased at the protein level with the possible biological meaning that this increase may be compatible with and indicate ongoing (oxidative?) DNA damage.

Hydroxyl radical generation in oxygen-treated infants.

OBJECTIVE: Because the hydroxyl radical is capable of oxidizing phenylalanine to O-tyrosine, we sought to determine whether increased levels of O-tyrosine are found in urine of infants treated with supplemental oxygen. METHODS: A total of 39 consecutively admitted neonates to an intensive care unit were included. Twenty-seven received supplemental oxygen therapy for respiratory disease, and 12 did not. Urinary O-tyrosine levels were determined on two or more occasions using high-performance liquid chromatography with results expressed as a percentage of the urinary phenylalanine concentration. Using simple and stepwise multiple linear regression analyses, urinary O-tyrosine was examined for associations with relevant clinical conditions and laboratory measurements. RESULTS: Infants supplemented with oxygen showed significantly higher mean +/- SEM urinary O-tyrosine levels (0.40% +/- 0.028) compared with those remaining in room air (0.18% +/- 0.012). Mean daily FIO2 was the clinical and laboratory variable most highly correlated with urinary O-tyrosine (r = 0.66). In the stepwise regression, significant associations were also found for renal fractional sodium excretion and Apgar score at 5 minutes. CONCLUSIONS: Hydroxylation at the O position of phenylalanine, a specific direct marker for the hydroxyl radical attack, was strongly associated with oxygen treatment in neonates. This finding increases our understanding of the pathogenesis of oxygen injury and suggests a basis for developing therapeutic approaches.

Evidence against increased glycoxidation in patients with Alzheimer's disease.

Neuropathological findings of Alzheimer's disease (AD) are intracellular (neurofibrillary tangles) and extracellular (senile plaques) filamentous protein aggregates. Non-enzymatic glycation has been proposed as a primary factor in this pathogenesis, leading to increased insolubility of tau protein and beta-amyloid. The aim of our study was to test the hypothesis that increased glycoxidation, i.e. increased levels of oxidized products from non-enzymatic glycation could be found in brains of patients with AD and of aged Down syndrome (DS) subjects with abundant AD-like neuropathological lesions. Frontal cortex specimens were assayed for pentosidine (Pent) and N-epsilon-carboxymethyl-lysine (CML) by reversed phase high performance liquid chromatographical methods. Pent and CML levels in AD (n = 10; Pent, 35.5 +/- 4.84 mumol/g wet-weight tissue; CML, 135.2 +/- 5.0 mumol/g wet-weight tissue) were comparable to DS (n = 9; Pent, 36.4 +/- 3.21; CML, 133.5 +/- 4.7) and controls (n = 10; Pent, 35.2 +/- 3.55; CML, 136.9 +/- 3.3). We conclude that the results are not compatible with the concept of increased glycoxidation in AD compared to normal aging.

Kynurenic acid and kynurenine aminotransferase in heart.

Kynurenic acid (KYNA) is a tryptophan metabolite and represents the only known endogenous compound acting as an antagonist to excitatory amino acid receptors in the mammalian CNS. Blocking of these receptors in CNS by KYNA affects cardiac function. As it is not known whether human heart is able to synthesize this neuromodulatory amino acid, we investigated the biosynthesizing enzyme of kynurenine aminotransferase (KAT) in the human heart and compared the activity with that of the human brain. The activities of heart and brain KATs were assayed by the conversion of L-kynurenine (L-KYN) to KYNA and quantitated by HPLC with fluorescence detection. Using either pyruvate or 2-oxoglutarate as cosubstrates, heart KAT was found to have a shallow pH optimum between 8 and 9. Highest heart KAT activity was seen in the presence of 2-oxoglutarate, followed by pyruvate. 2-oxoadipate, and 2-oxoisocaproate. Kinetic analyses, performed at pH 8.5, and using various concentrations of L-KYN (from 0.125 to 22.8 mM) in the presence of 2-oxoglutarate (1 and 5 mM) or pyruvate (5 mM) revealed apparent K(m) values in the millimolar range, for L-KYN 1.5, 27, and 20 mM, respectively. Heart KAT activities were compared with those in human brain KAT I and KAT II showing different pH optima 7.4 and 9.6, respectively. In contrast to brain KAT I, heart KAT activity was not inhibited by an excess of 2 mM L-tryptophan, L-glutamine, or L-phenylalanine at pH 9.6, as well as at pH 8 or 7.4. Our study demonstrates that human heart is capable of synthesizing KYNA from low concentrations of L-KYN selectively. A shallow pH optimum of KAT activity, i.e. between 8.0 and 9.0, pronounced 2-oxoacid specificity, and a lack of sensitivity to inhibition by L-glutamine, L-phenylalanine, and L-tryptophan indicate that the heart KAT system displays enzymatic characteristics different from those of human brain KAT I or KAT II. Fluctuation of L-KYN and 2-oxoacid levels may markedly influence the KYNA synthesis and subsequent KYNA effect on cardiac activity. KYNA synthesis in the human heart suggests a neurophysiologic role. Our studies from the basis for purification and further characterization of KAT protein in human heart as well as for physiologic studies.

Similar deficits of central histaminergic system in patients with Down syndrome and Alzheimer disease.

In order to study whether Alzheimer-like neuropathological changes involve the central histaminergic system we measured the concentration of histamine, its precursor histidine as well as the activity of histidine decarboxylase (HDC) and histamine-N-methyl-transferase (HMT) in frontal cortex of aging Down syndrome (DS) patients, Alzheimer patients and control individuals. The study populations were also investigated for choline acetyltransferase (ChAT) activity, since reduced ChAT activity is an established biochemical hallmark in DS and Alzheimer disease (AD). HDC and ChAT activity were reduced in brains of both DS and Alzheimer patients versus control patients. Additionally, we observed a significant decrease of histamine levels in the DS group. Histamine levels in AD brains tended to be decreased. Histidine concentrations and HMT activities were comparable between the three groups. Thus, our results for the first time show histaminergic deficits in brains of patients with DS resembling the neurochemical pattern in AD. Neuropathological changes may be responsible for similar neurochemical alterations of the histaminergic system in both dementing disorders.

Excitatory amino acids and monoamines in parahippocampal gyrus and frontal cortical pole of adults with Down syndrome.

Aspartate (ASP), glutamate (GLU), noradrenaline (NA), dopamine (DA) and its acidic metabolites DOPAC and HVA, serotonin (5-HT) and its metabolite 5-HIAA were simultaneously investigated in post-mortem tissue samples from right parahippocampal gyrus (temporal cortex) and frontal cortical pole (frontal cortex) of adults with Down syndrome (DS), and of neurologically healthy controls by use of high performance liquid chromatography (HPLC). In parahippocampal gyrus, ASP, GLU, NA, DOPAC and 5-HT levels were significantly decreased in patients with DS, compared to levels found in control subjects (approximately 50%). No significant changes were observed in frontal pole. ASP and GLU levels were significantly lower in parahippocampal gyrus than in frontal pole of DS, a regional distribution that could not be observed in control subjects. In conclusion, the results of this study suggest that the temporal cortex would be more affected than the frontal cortex in adult patients with DS, a finding in line with reports showing a marked hypometabolism and extensive cell loss in temporal cortex of DS, and with those showing that parahippocampal gyrus abnormality may correlate with the extent of mental retardation affecting this type of patients.

The effect of estradiol and ovariectomy on tyrosine hydroxylase, tyrosine aminotransferase and phenylalanine hydroxylase.

Although the regulatory activity of steroid hormones on amino acid metabolism has been described, no information is published on the effect of ovariectomy. We studied the influence of ovariectomy in Wistar rats determining the amino acids such as phenylalanine and tyrosine in liver, kidney and urine. 32 animals were used in the study, 12 animals were sham operated, 9 animals were ovariectomized and 11 rats were ovariectomized and supplemented with estradiol. No quantitative changes were detected comparing liver and kidney phenylalanine and tyrosine between the groups. The phenylalanine/tyrosine ratio in liver and urine of ovariectomized rats showed a significant decrease of the quotient, which was restored by estradiol replacement, whereas the ratio was unaffected in the kidney. Tyrosine hydroxylase was in liver (0.04 +/- 0.04 U/min), kidney (0.4 +/- 0.04 U/min) in the sham operated rats; liver (0.06 +/- 0.02 U/min), kidney (0.3 +/- 0.2 U/min) in ovariectomized and liver (0.07 +/- 0.055 U/min), kidney (0.4 +/- 0.1 U/min) in the ovariectomized with estradiol replacement. Tyrosine aminotransferase was in liver (0.004 +/- 0.002 U/h), kidney (0.01 +/- 0.002 U/h) in the sham operated panel; liver (0.005 +/- 0.001 U/h), kidney (0.01 +/- 0.004 U/h) in the ovariectomized and liver (0.005 +/- 0.002 U/h) and kidney (0.012 +/- 0.007 U/h) in the ovariectomized with estradiol supplementation. Phenylalanine hydroxylase was in liver (0.12 +/- 0.07 U/min), kidney (0.5 +/- 0.3 U/min) in the sham operated group; liver (0.19 + 0.16 U/min), kidney (0.5 +/- 0.2 U/min) in the ovariectomized and liver (0.15 +/- 0.09 U/min), kidney (0.4 +/- 0.2 U/min) in the ovariectomized rats with estradiol replacement. No significant difference in the enzyme activities between the groups was found. The impairment of the phenylalanine/tyrosine ratio in liver could be assigned to an endocrine control of amino acid uptake and/or transport, in the urine by different pools, proteolytic and synthetic enzyme activites, filtration/reabsorptioneffects.

Polyamines in frontal cortex of patients with Down syndrome and Alzheimer disease.

Polyamines may play an important role in brain development, mature brain function and also in neurodegenerative conditions. We investigated polyamine levels in frontal cortex of human post-mortem brain samples of elderly patients with Down syndrome (DS), Alzheimer disease (AD) and normal controls by means of chromatographic separation after dansylation. Spermidine and spermine concentrations were markedly decreased in DS and AD. Polyamine levels were neither related to age and post-mortem interval nor to choline acetyltransferase activity, as indicator of neuronal loss. Our results support the idea that besides other neurotransmitter systems, endogenous polyamine levels are altered in dementing illnesses such as Alzheimer disease and Down syndrome.

Increased kynurenic acid levels and decreased brain kynurenine aminotransferase I in patients with Down syndrome.

Excitatory amino acid (EAA) receptors are central to brain physiology and play important roles in learning and memory processes. Kynurenic acid (KYNA), a metabolite of tryptophan in the brain blocks all three classical ionotropic EAA receptors and also serves as an antagonist at the glycine site associated with the N-methyl-D-aspartate receptor (NMDA) complex. We measured the endogenous levels of KYNA and activities of KYNA synthesizing enzymes kynurenine aminotransferase I (KAT I) and kynurenine aminotransferase II (KAT II) in the frontal and temporal cortex of elderly Down syndrome (DS) patients (aged 46-69 years). Compared with control specimens (0.21 +/- 0.06 pmol/mg tissue), the measurement of KYNA content revealed a significant 3-fold increase in frontal cortex of DS patients (0.67 +/- 0.13 pmol/mg tissue; p < or = 0.01). In temporal cortex KYNA levels were increased by 151% (p < or = 0.05) of control (0.41 +/- 0.09 pmol/mg tissue) Using crude cell free homogenate KAT's activities were determined in the presence of the 1 mM 2-oxoacid as a co-substrate at their pH optima of 10.0 for KAT I and 7.4 for KAT II. KATs activities in the presence of 1 mM pyruvate were 2.79 +/- 0.52 and 4.55 +/- 1.98 pmol/mg protein/h for KAT I and 0.98 +/- 0.07 and 1.09 +/- 0.14 pmol/mg protein/h for KAT II in frontal cortex and temporal cortex, respectively. When compared with the brain samples of controls the activity of KAT I was reduced in frontal cortex (9.8 +/- 2.4%; p < or = 0.01) and temporal cortex (25.8 +/- 6.4 %) of DS patients, while KAT II levels were within the normal range. Measurement of the neuronal, cholinergic marker choline acetyltransferase (ChAT) in the frontal cortex, revealed a significant reduction (36.6 +/- 4.3% of control; p < or = 0.01) in DS. Our data demonstrate the involvement of KYNA-metabolism in the cellular mechanisms underlying altered cognitive function in patients with DS. Although the localisation of both, KAT I and KAT II is not stated yet the reduction of KAT I may suggest impairment of KYNA metabolism in neuronal and/or nonneuronal compartments.

Low-dose dietary L-arginine increases plasma interleukin 1 alpha but not interleukin 1 beta in patients with diabetes mellitus.

Oral high-dose arginine supplementation is used for the experimental immunotherapy of tissue trauma and sepsis. Yet the adequate dosage required for immunomodulation has to be established and the toxicity of high-dose arginine has not been fully elucidated. Following a protocol for the treatment of diabetic long-term complications (oral daily doses of 30 mg/kg BW; blind, placebo-controlled prospective study with crossing-over design) we studied plasma levels of interleukins 1 alpha (IL-1 alpha) and 1 beta reflecting immunostimulation. Arginine supplementation in 29 patients with diabetes mellitus prompted a 2-fold increase of IL-1 alpha from baseline levels (P < 0.001) while IL-1 beta was unaffected. Implications for the treated panel of diabetic patients could be a reduction of collagen accumulation by enhanced collagenolysis and clearance of advanced-stage non-enzymatic glycosylation products. Based upon our data, low-dose arginine protocols for further immunotherapeutical studies should be discussed.

The effect of ovariectomy on phenylalanine and tyrosine metabolism.

Although the regulatory activity of steroid hormones on amino acid metabolism has been described, no information is published on the effect of ovariectomy. We studied the influence of ovariectomy in Wistar rats determining the amino acids phenylalanine and tyrosine in liver, kidney, plasma and urine. 32 animals were used in the study, 12 animals were sham operated, 9 animals were ovariectomized and 11 rats were ovariectomized and supplemented with estradiol. No quantitative changes were detected comparing liver and kidney phenylalanine and tyrosine between the groups (sham operated rats liver phenylalanine 2,53nM/mg ± 1,07; liver tyrosine 1.95nM/mg ± 0.92; kidney phenylalanine 2.16nM/mg ± 0.53; kidney tyrosine 1.80nM/mg ± 0.39. Ovariectomized rats showed liver phenylalanine 3.07nM/mg ± 1.14; liver tyrosine 2.63nM/mg ± 1.01; kidney phenylalanine 2.30 nM/mg ± 0.74; kidney tyrosine 1.93nM/mg ± 0.63. Ovariectomized and estradiol supplemented rats presented with liver phenylalanine 2.84nM/mg ± 1.40; liver tyrosine 2.35nM/mg ± 1.28; kidney phenylalanine 1.91nM/mg ± 0.26, kidney tyrosine 1.67nM/mg ± 0.23.). When, however, the phenylalanine/tyrosine ratio in the liver was evaluated, ovariectomized rats showed a significant decrease of the quotient (p = 0.001). The phenylalanine/tyrosine ratio was restored by estradiol replacement. Our findings show that phenylalanine and tyrosine metabolism is under estradiol control. The effect on the metabolic changes could be mediated by enzyme systems as phenylalanine hydroxylase, tyrosine hydroxylase and tyrosine aminotransferase. Our results would be compatible with previous reports on the stimulatory effect of estradiol on these enzymes. The kidney phenylalanine/tyrosine ratio was unaffected by ovariectomy and/or estradiol replacement which can be easily explained by different pools, enzyme activities, filtration/reabsorption effects, etc.The urinary P/T ratio was decreased by ovariectomy and restored by estradiol replacement indicating endocrine control of renal reabsorption and secretion mechanisms.

[5-fluorouracil inhibits bacterial collagenase and the collagenolytic system of a human rhabdomyosarcoma]. / 5-Fluorouracil hemmt bakterielle Kollagenase und das kollagenolytische System eines menschlichen Rhabdomyosarkoms.

Protease inhibitory activity of eight cytostatic drugs on 5 proteases was tested by applying an immunoelectrophoretic system, a rhabdomyosarcoma's collagenolytic activity was investigated and inhibition studies were performed. We found 5-fluorouracil stopping collagenolytic activity of clostridial origin and the enzyme released by the tumor. No other cytostatic drug showed protease inhibition. The collagenolysis of the rhabdomyosarcoma was examined for its origin. Negative inhibition studies with ethylene-diamine-tetraacetate make the leukocyte origin highly improbable so that one can suggest that the hydrolytic enzyme derived from the tumor. Our findings could contribute to characterize the enzyme released by the tumor. We suggest its role as a mediator of invasion and metastasis and on the other hand we detected its relatively specific inhibitor, 5-fluorouracil, which could influence tumorous and normal growth, regeneration and the intermediary protein metabolism.