Band 6, Heft 2

A simple synthesis in solution, of the series of six consecutive t-butyl N-(4-propargylaminobenzoyl)-yoligo( L-glutamate)s (8-13) containing from two to seven L-glutamic acid residues (Scheme 1) is described. a-t-Butyl N-(4-propargylaminobenzoyl)-L-glutamate (1), the versatile synthon is coupled with one of the t-butyl y-oligo(L-glutamate)s (2-7) by means of 2-chloro-l-methylpyridinium tosylate to give the title compounds in 91-95% yield subsequent to edulcoration of a post-reaction mixture and crystallization. These compounds were characterized by melting points, elemental analyses and HPLC and are 96.7-100.0% pure according to the last method (Table I).

The V /K kinetic parameters for glyceryl-ether monooxygenase with 3-1 '-n-alkoxypropane-I,2-diols having alkyl sidechains varying from Cll to C22 (lb-k, 2a and b, and 3d) were determined. Maximum activity was found with the CI6 ethers, dropping almost to zero with ethers that have sidechains shorter than Cl2 and longer than C19. The thioether analogues with C16 and CI8 sidechains were poorer substrates than the respective oxygen ethers by one order of magnitude. The kinetic parameters for 2-1 '-n-hexadecyloxy- (6a) and 2-1 '-n-octadecyloxy- (6d) ethanol revealed that they were also better substrates than the respective thio-ethers by one order of magnitude and with these compounds those with C16 sidechains were better substrates than the C 18 analogues. L2-bis-3-1 '-n-Hexadecyloxypropan-3-01 (3c) and 1,3-bis-3- 1'-n-hexadecyloxypropan-3-01 and 2- 01 (4a) were inactive but 3-1'-n-hexadecyloxy- 2-methoxypropan-I-ol (3b) and 3-1'-n-hexadecyloxy-l-methoxypropan-2-01 (4b) which have one long fatty chain were viable substrates because they possessed a free hydroxy group, but the ethers (6b, c and e), (7c and d) and (5) which do not have a free hydroxy group were inactive. These data, taken with those reported previously, have allowed us to postulate a model for the active site of the alkyl-ether substrates of the monooxygenase and to define the contours of the hydrophobic and hydrophilic pockets.

6-Pyruvoyl tetrahydropterin synthase is involved in the synthesis of pteridine eye pigments in Drosophila. The purple gene which was known to be one of the target loci of the suppressor mutation su(sj2 has been identified to encode the enzyme, and its cDNA has been cloned recently. The cDNA encoding the 19.3 kDa subunit of the 6-pyruvoyl tetrahydropterin synthase was expressed as fusion proteins in E. coli. The recombinant protein was shown to be active and purified from the E. coli crude extract by metal-chelation chromatography. The fused metal-chelating oilgopeptide was removed by thrombin for further characterization. Apparent Km for the substrate dihydroneopterin triphosphate was determined to be 590 IlM, which was slightly higher than the value of the native enzyme. The isoelectric point of 6.4 was also different from the known value of 4.3 determined by the native enzyme. Heat stability and the stimulatory effect of reducing agents were similar to the native enzyme. The modification of cysteine residues in the recombinant enzyme, one of which is known to be conserved in human and rat enzymes, by iodoacetamide inhibited its activity by up to 80%.

Besides elevated levels of lipids and lipoproteins, ievels of the physiological amino acid homocysteine as well as whole blood folates are now known to play a role in atherogenesis. The role of folates in atherosclerosis is rather peculiar in that it helps to catalyse the remethylation of homocysteine (a proatherogenic agent) to methionine thus preventing the accumulation of homocysteine in plasma and hence its antiatherogenic role. We estimated whole blood folate levels of 69 normotensive, 34 diabetic and 94 hypertensive individuals and have attempted to correlate these values with lipid and non-lipid parameters. Whole blood levels of simple, conjugated and total folates were significantly higher among the diabetics as well as the hypertensives when compared to the normotensives. In all the study groups, levels of conjugated folates correlated significantly to those of total folates even after adjustments for age and body mass index were introduced. Among the normotensive individuals the significance of the inverse correlation between simple folates and total cholesterol/high density lipoprotein cholesterol ratio was lost after adjusting for age and body mass index. Levels of total and conjugated folates were positively correlated to plasma levels of thromboxane B2 among the diabetics. In the same group the positive correlation between total folates and thromboxane B2 and that between total folates and the eicosanoid ratio remained significant even after adjustments for age and body mass index were introduced. None of the other metabolic variables correlated with whole blood levels of simple folates in any of the study groups.

Incorporation of fatty acids into phospholipids has been investigated using samples of rat live tissue homogenate, Krebs-Ringer-phosphate buffer (pH = 7A) containing 0.3% albumin, farry acid mixture and glycerol. The addition of sepiapterin, 7,8-dihydrobiopterin and 5.6.7.8-tetrahydrobiopterin (5 and 30 pmol g wet weight) to incubation medium induced a decrease of saturated (stearic acid) and an increase of polyunsaturated (arachidonic acid) fatty acids incorporation into phospholipids. Cholesterol content decreased, but phospholiplid content did not change in samples containing sepiapterin, 7,8-dihydrobiopterin and 5,6,7,8-tetrahydrobiopterin. No changes of fatty acid incorporation into phospholipids as well as of the content of cholesterol and phospholipids were observed in samples after the addition of xanthopterin (4 and 20 nmol/g wet weight) to incubation medium for phospholipid biosynthesis in vitro. The observations made by incubation with 7,8-dihydrobiopterin, 5,6,7,8-tetrahydrobiopterin and sepiapterin where in opposite to those made earlier employing neopterin and using the same incubation procedure.

Despite its low incidence septic arthritis remains a serious problem in orthopaedic practice. General or local immunosuppression predisposes for septic arthritis. Thus we see an increasing incidence in elderly patients, in patients suffering from rheumatic diseases and after alloplastic arthroplasty of the hip and of the knee. In septic arthritis the first line of host defense mechanisms is overwhelmed and bacterial multiplication proceeds within the joint. The pathogens found are in 50% to 75% of cases Staphylococci and in 15% to 20% Streptococci. Histologically we initially find an edema of the synovial membrane and a dramatic polymorph-nuclear-cell infiltration. After developing all signs of inflammation, a persistent purulent effusion accumulates and cartilage and bone destruction ensues as a result of proteolytic enzyme activity released from the bacteria and the polymorph nuclear leukocytes. Modern therapy consists of early surgical intervention (synovectomy, debridement) combined with local and systemic antibiotic treatment. The early intervention is one of the main factors for successful management. Thus the interest of early detection of septic arthritis is obvious. The production of neopterin can be related to the activity of the specific antibacterial cell mediated immune response. In the current, study weexamined neopterin levels in 12 patients with septic arthrits and in 8 patients with an infected prosthesis. The results were related to a new inflammation marker (PMN-elastase) and to 4 other 'classical' inflammation markers (ESR, leukocytes, fibrinogen and C-reactive protein). All markers showed a high sensitivity in the detection of septic arthritis. 10 patients with septic arthritis and 5 patients with an infected prosthesis showed pathological neopterin levels > 10 nmol/l.

Behçet’s Disease (BD) is a multisystem inflammatory disorder characterized by recurrent oral and genital ulcerations with uveitis. Clinical features are the only diagnostic and follow up parameters and there is no unique laboratory test to indicate both diagnosis and disease activity. We studied urinary neopterin levels by using high performance liquid chromatography in 21 consecutive patients with Behçet’s Disease in clinically active (n = 13) and inactive (n = 8) phase of the disease and in 14 healthy controls to investigate the correlation between this immunologic parameter and the disease status. In comparison to inactive phase, significantly higher urinary neopterin levels were found in active period (p<O.05). Mean urinary neopterin levels were 210.83 ± 157.19 vs 138.85 ± 56.99 I-imol neopterin/mol creatinine in active and inactive t1hase respectively (p<O.05). There was no significant difference between inactive phase and control values. In only 2 patients, both active and inactive phase urine neopterin levels could be measured in the same individual and increased neopterin levels were also determined in the active phase of disease. In conclusion, urinary neopterin levels were found to be elevated in active period and can be considered as an active phase marker in BD. Larger study groups with own controls of BD patients and other vasculitic disease controls are required before the final judgement.