Intracellular Zn2+ increases contribute to the progression of excitotoxic Ca2+ increases in apical dendrites of CA1 pyramidal neurons.

Sustained intracellular Ca(2+) elevation is a well-established contributor to neuronal injury following excessive activation of N-methyl-d-aspartic acid (NMDA)-type glutamate receptors. Zn(2+) can also be involved in excitotoxic degeneration, but the relative contributions of these two cations to the initiation and progression of excitotoxic injury is not yet known. We previously concluded that extended NMDA exposure led to sustained Ca(2+) increases that originated in apical dendrites of CA1 neurons and then propagated slowly throughout neurons and caused rapid necrotic injury. However the fluorescent indicator used in those studies (Fura-6F) may also respond to Zn(2+), and in the present work we examine possible contributions of Zn(2+) to indicator signals and to the progression of degenerative signaling along murine CA1 dendrites. Selective chelation of Zn(2+) with N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) significantly delayed, but did not prevent the development and progression of sustained high-level Fura-6F signals from dendrites to somata. Rapid indicator loss during the Ca(2+) overload response, which corresponds to rapid neuronal injury, was also not prevented by TPEN. The relationship between cytosolic Zn(2+) and Ca(2+) levels was assessed in single CA1 neurons co-loaded with Fura-6F and the Zn(2+)-selective indicator FluoZin-3. NMDA exposure resulted in significant initial increases in FluoZin-3 increases that were prevented by TPEN, but not by extracellular Zn(2+) chelation with Ca-EDTA. Consistent with this result, Ca-EDTA did not delay the progression of Fura-6F signals during NMDA. Removal of extracellular Ca(2+) reduced, but did not prevent FluoZin-3 increases. These results suggest that sustained Ca(2+) increases indeed underlie Fura-6F signals that slowly propagate throughout neurons, and that Ca(2+) (rather than Zn(2+)) increases are ultimately responsible for neuronal injury during NMDA. However, mobilization of Zn(2+) from endogenous sources leads to significant neuronal Zn(2+) increases, that in turn contribute to mechanisms of initiation and progression of progressive Ca(2+) deregulation.

Fatty acid composition of the milk lipids of Nepalese women: correlation between fatty acid composition of serum phospholipids and melting point.

Milk was collected from 36 Nepalese women, 15 to 32 years of age, in order to investigate relationships between the proportions of intermediate chain-length (C10-C14) fatty acids and critical n-3 and n-6 polyunsaturated fatty acids in the milk lipids they were producing. Serum was also obtained from these lactating women and the fatty acid composition of their serum phospholipid fraction was determined and compared with that of the corresponding milk lipid fraction. Compared to women in technologically advanced parts of the world, the serum phospholipids of the Nepalese women contained nutritionally adequate proportions of linoleic acid (LA) (16.8%), alpha-linolenic acid (ALA) (0.53%), arachidonic acid (AA) (5.69%), and docosahexaenoic acid (DHA) (1.42%). However, although the milk lipids contained adequate proportions of ALA (1.81%), AA (0.43%), and DHA (0.23%), the lipids contained low to moderate percentages of LA (mean, 9.05%). Positive correlations were observed between the proportions of AA (P=0.001, r=0.50) and ALA (P=0.03, r=0.36) in the serum phospholipids and milk lipids of the women. As the proportion of C10-Cl4 fatty acids in the milk lipids increased from 10% to 40%, there was preferential retention of three critical n-3 and n-6 fatty acids (ALA, AA, and DHA) at the expense of two relatively abundant nonessential fatty acids, namely stearic acid and oleic acid. In addition, using fatty acid melting point data and the mol fraction of the 9 most abundant fatty acids in the milk, we estimated the mean melting point (MMP) of the milk lipids of the Nepalese women. The MMPs ranged from 29.3 to 40.5 degrees C (median, 35.5 degrees C). These results indicate that: 1) the levels of AA and ALA in the blood of lactating mothers influence the levels of these fatty acids in the milk they produce; 2) when the mammary gland produces a milk that is rich in C10-Cl4 fatty acids, it somehow regulates triglyceride synthesis in such a way as to ensure that the milk will provide the exclusively breast-fed infant with the amounts of the critical n-3 and n-6 fatty acids it requires for normal growth and development; and 3) the melting point of the milk lipid fraction is determined mainly by the mol % of the intermediate chain-length (C10-C14) fatty acids, oleic acid, linoleic acid, and alpha-linolenic acid.

Does analysis of the antigenic specificities of anti-neutrophil cytoplasmic antibodies contribute to their clinical significance in the inflammatory bowel diseases?

BACKGROUND: The clinical relevance of anti-neutrophil cytoplasmic antibodies (ANCA) in inflammatory bowel diseases is unclear. Definition of their antigenic specificities may improve their diagnostic significance. METHODS: We studied the target antigens of ANCA in 96 patients with ulcerative colitis (UC) and 112 patients with Crohn disease (CD) by indirect immunofluorescence, antigen-specific enzyme-linked immunosorbent assays, and immunodetection on Western blot. We related the presence of antibodies of defined specificity to clinical symptoms. RESULTS: By indirect immunofluorescence, ANCA were present in 58% of UC patients and in 21% of CD patients. Major antigens were catalase, alpha-enolase, and lactoferrin. In UC, ANCA titers correlated with disease activity. In CD, both ANCA, by indirect immunofluorescence, and antibodies to lactoferrin were associated with colonic localization of the disease. Neither ANCA, by indirect immunofluorescence, nor antibodies of defined specificity were associated with duration of disease, use of medication, or a history of bowel resection. CONCLUSIONS: ANCA are useful as markers for UC and colonic localization in CD. Definition of the antigenic specificities of ANCA in inflammatory bowel disease does not significantly contribute to their clinical significance.

Inactivation of glutathione reductase by 4-hydroxynonenal and other endogenous aldehydes.

4-Hydroxynonenal, a product of oxidative degradation of unsaturated lipids, is an endogenous reactive alpha,beta-unsaturated aldehyde with numerous biological activities. 4-Hydroxynonenal rapidly inactivated glutathione reductase in an NADPH-dependent reaction. Inactivation appears to involve the initial formation of an enzyme-inactivator complex, K(D) = 0.5 microM, followed by the inactivation reaction, k = 1.3 x 10(-2) min(-1). alpha,beta-Unsaturated aldehydes such as acrolein, crotonaldehyde, and cinnamaldehyde also inactivated glutathione reductase, although rates varied widely. Inactivation of glutathione reductase by alpha,beta-unsaturated aldehydes was followed by slower NADPH-independent reactions that led to formation of nonfluorescent cross-linked products, accompanied by loss of lysine and histidine residues. Other reactive endogenous aldehydes such as methylglyoxal, 3-deoxyglucosone, and xylosone inactivated glutathione reductase by an NADPH-independent mechanism, with methylglyoxal being the most reactive. However, 2-oxoaldehydes were much less effective than 4-hydroxynonenal. Inactivation of glutathione reductase by these 2-oxoaldehydes was followed by slower reactions that led to the formation of fluorescent cross-linked products over a period of several weeks. These changes were accompanied by loss of arginine residues. Thus, the sequence of events is different for inactivation and modification of glutathione reductase by alpha,beta-unsaturated aldehydes compared with 2-oxoaldehydes with respect to kinetics, NADPH requirements, fluorescence changes, and loss of amino acid residues. The ability of 4-hydroxynonenal at low concentrations to inactivate glutathione reductase, a central antioxidant enzyme, suggests that oxidative degradation of unsaturated lipids may initiate a positive feedback loop that enhances the potential for oxidative damage.

Synthesis and anti-HIV activity of 1,1'-dideoxygossypol and related compounds.

1,1'-Dideoxygossypol (DDG), 1,1'-dideoxygossylic acid (DDGA), 8-deoxyhemigossypol (DHG), and 8-deoxyhemigossylic acid (DHGA) were synthesized and tested for their ability to inhibit the replication of HIV in vitro. The EC50 for DDGA was < 1 microM, and its threshold cytotoxicity was approximately 20 microM. DDG was less effective than DDGA against HIV and showed considerable toxicity at 5 microM. DHGA was ineffective against HIV and had very low cytotoxicity. DHG showed some anti-HIV activity, but the threshold cytotoxicity was 5 microM. The dissociation constants for the binding of the four compounds to human serum albumin were determined by fluorescence quenching titrations, and all four were found to have much lower affinities for albumin than the parent compound gossypol.

Substrate specificity of human aldose reductase: identification of 4-hydroxynonenal as an endogenous substrate.

Aldose reductase, which catalyzes the reduction of glucose to sorbitol as part of the polyol pathway, has been implicated in the development of diabetic complications and is a prime target for drug development. However, aldose reductase exhibits broad specificity for both hydrophilic and hydrophobic aldehydes, which suggests that aldose reductase may also be a detoxification enzyme. Several series of structurally related aldehydes were compared as substrates in order to deduce the structural features that result in low Michaelis constants. Aldehydes that contain an aromatic ring are generally excellent substrates, consistent with crystallographic data which suggest that aldose reductase possesses a large hydrophobic substrate binding site. However, there is little discrimination among different aromatic aldehydes. In addition, small hydrophilic aldehydes exhibit low Km values if the alpha-carbon is oxidized. Analysis of the binding of NADPH by fluorescence quenching techniques indicates that aldose reductase exhibits higher affinity for NADPH than NADP, suggesting that this enzyme is normally primed for reductive metabolism. Thus aldose reductase appears to have evolved to catalyze the reduction of a very broad range of aldehydes. Structural features of substrates that bind to aldose reductase with low Km values were used to identify potential endogenous substrates. 4-Hydroxynonenal, a reactive alpha-beta unsaturated aldehyde produced during oxidative stress, is an excellent substrate (Km = 22 microM, kcat/Km = 4.6 x 10(6) M-1 min-1). Reductive metabolism of endogenous aldehydes in addition to glucose, catalyzed by aldose reductase, may play an important role in the development of diabetic complications.

Healthy individuals possess circulating antibodies against their indigenous faecal microflora as well as against allogenous faecal microflora: an immunomorphometrical study.

Healthy persons were shown to possess circulating antibodies of both IgA, IgG and IgM isotype directed against the bacteria of their faecal microflora, assessed by immunomorphometry. After removal, by absorption, of the fraction of antibodies directed against the autochthonous faecal bacteria or cross-reacting with allogenous faecal bacteria, there were still antibodies left directed against allogenous faecal bacteria of both the IgA, IgG and IgM isotype. However, relatively more antibodies of the IgA isotype appeared to be directed against allogenous bacteria than against indigenous faecal bacteria. Persons who reacted with specific antibodies to many bacteria of their own flora also tended to react specifically to bacteria in the allogenous microflora of the other volunteers. The patterns of antibodies directed to faecal bacteria of different morphologies (morphotypes) were unique for each individual.

Left coronary artery originating from the pulmonary artery correction and total myocardial blood flow measurements.

In a boy aged 12 years correction of an anomalous coronary artery system was performed by suture of the left coronary artery origin at the pulmonary artery trunk and a vein by-pass to the proximal LAD. Peroperative blood flow gives values for myocardial perfusion before and after by-pass with relation to patency of the vein graft. Before correction pure coronary blood flow was 540 ml/min, shunt flow 540 ml/min; in total 1,080 ml/min through the right coronary artery. After correction the vein by-pass conducted 310 ml/min and the right coronary artery 180 ml/min. At follow-up 2 months later coronary angiography showed a normal right coronary artery without collateral connections to the left coronary artery system which was well perfused from the by-pass. Left heart hypertrophy was reduced from 568 ml/m2. A weak systolic murmur at the apex could be a slight unimportant mitral insufficiency.