Calmodulin inhibitors and 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate do not prevent the inhibitory effect of prostaglandin F2 alpha on cyclic AMP production in isolated rat corpora lutea

In the rat corpus luteum, prostaglandin F2 alpha (PGF2 alpha) rapidly inhibits LH-induced cyclic AMP (cAMP) production when given in vivo or to isolated corpora lutea, but not to broken-cell preparations. The suggestion that increased cytosolic calcium concentration mediates PGF2 alpha action was investigated in corpora lutea of pseudopregnancy induced in immature rats by administration of pregnant mare serum gonadotrophin (15 i.u.). Isolated 10-day-old corpora lutea were incubated for 90 min with LH (5 micrograms/ml), PGF2 alpha (10 mumol/l) and other additions, and cAMP concentration in the tissue was estimated. The putative inhibitor of intracellular calcium release or action, 8-(n,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate (TMB-8; 30 or 150 mumol/l), did not abolish the effect of PGF2 alpha. Similarly ineffective was the combination of TMB-8 (150 mumol/l) and calcium-depleted medium (free ionized calcium concentration, 30 nmol/l). Calmodulin inhibitors of three different chemical structures were then tested. The phenothiazine trifluoperazine, at 300 as well as 30 mumol/l, did not interfere with the inhibitory effect of PGF2 alpha on cAMP, while suppressing (at 300 mumol/l) progesterone secretion in LH-treated tissue. Furthermore, inhibition by PGF2 alpha was not impaired by pimozide, a diphenylbutylpiperidine (25 and 50 mumol/l) nor by N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide (W-7; 15 and 45 mumol/l). In the presence of LH alone, W-7 (45 mumol/l) inhibited and TMB-8 (30 mumol/l augmented cAMP accumulation, indicating that the luteal tissue was effectively exposed to these compounds. Thus, drugs known to inhibit calcium- and calmodulin-dependent processes in a variety of tissues did not abolish the inhibitory action of PGF2 alpha on luteal cAMP production.     

IgA, IgG, IgM, and IgE levels in normal, healthy, non-atopic Israeli children

IgA, IgG, IgM, and IgE levels in healthy, non-atopic, Israeli-born children aged 20 days to 16 years were analyzed and showed similar age-related values and dynamics as those of white populations found in other countries. No significant effect of sex of the individual or ethnic origin of the parents was found on the IgE values at different ages. This may indicate that total IgE levels are strongly influenced by environmental factors. Establishing tolerance limits at 97.5, 95, 75, 25 and 5th percentiles and the geometric mean provides the practitioner with more complete reference values. The use of multivariate control charts with tolerance limits from normal IgA, IgG, IgM, and IgE levels is described and is offered as an additional tool for the diagnosis of an allergic individual.     

Effect of leukocyte hydrolases on bacteria

The bactericidal and bacteriolytic effects of lysolecithin (LL) and egg-white lysozyme (LYZ) on Staph. aureus and group A streptococci and the solubilization of phospholipids from the bacterial membranes by these agents was studied. Low concentrations of lysolecithin (1--10 microgrames/ml) are highly bactericidal for Steph. aureus and group A streptococci, but induce neither bacteriolysis nor solubilization of a substantial amount of membrane phospholipids. On the other hand, while LL at greater than 50 micrograms/ml causes substantial lipid release, a combination of LL and LYZ is absolutely needed to solubilize lipids from streptococci. This combination is, however, not bacteriolytic for this microrganism. The solubilization of lipids from staphylococci by LL is much faster than that induced in streptococci by LL + LYZ. The solubilization of the bulk of membrane lipids from staphylococci can also be achieved by Triton X-100 and by sodium lauryl sulfate and from group A streptococci by Triton X-100 plus LYZ. A variety of other detergents (e.g., Cetavlon, sodium taurocholate, cetyl pyrdinium chloride) have no lipid-releasing properties even in the presence of LYZ. The release of lipids by LYZ (in the presence of LL) from group A streptococci is related to its enzymatic activity, on a still unknown substrate, but not to its cationic nature as this muramidase cannot be replaced by a variety of cation substances (histone, polylysin, leukocyte cationic proteins, polymyxin B, and spermidine). The release of lipids from staphylococci by LL is not inhibited by a variety of anionic and cationic polyelectrocytes (heparin, liquoid, chondroitin sulfate, DNA histone, and polylysine) which markedly inhibit the release of lipids from group A streptococci by LL and LYZ. Streptococci that had been cultivated in the presence of subinhibitory concentrations of penicillin G lose their membrane phospholipids to a larger extent and by much smaller concentrations of LL and LYZ, as compared to controls, suggesting that the interference with the synthesis of the peptidoglycan increases the accessibility of the cell membrane to the lipid-releasing agents. The mechanism by which LL collaborates with LYZ in lipid release is still not known. The possible role of bacterial lipids and lyso compounds in the control of bacterial survival in inflammatory sites is briefly discussed.     

Weight change adjusted equations for assessing resting metabolic rate in overweight and obese adults

BackgroundAlthough over one hundred equations have been developed to predict the energy expenditure of individuals, none are sensitive to weight change in assessment of resting metabolic rate (RMR) before and after weight loss.ObjectiveTo formulate adjusted equations for overweight and obese individuals and to compare their accuracy with existing prediction RMR equations before and after weight loss.Subjects/materialsThis is historical prospective study. Participants included 39 overweight and obese men and women before and after losing 10–20% from baseline weight on a diet and physical activity regimen for at least three months. Pre and post weight loss measured RMR results were compared to estimated RMR using several existing prediction equations: Harris and Benedict, Ravussin and Bogardus, and Mifflin et al. To improve the accuracy of these prediction equations, we suggest new equations adjusted for weight loss, based on measured RMR and evaluated their accuracy.ResultsPre and post weight loss data indicated: significant fat reduction in both genders; reduction in free-fat mass only in men, and a significant decrease in measured RMR only in women. Our suggested equations were the most accurate and closest to measured RMR in both genders, in comparison to the Harris and Benedict, Ravussin and Bogardus, and Mifflin et al equation results. Estimated RMR using the latter equations was significantly lower than measured RMR in both genders at pre and post weight loss (P < 0.01).ConclusionsThis study highlights the need for adjusting RMR equations before and after weight loss in overweight and obese individuals. Further research is needed to validate our suggested equations.     

Specific prognostic factors for secondary pancreatic infection in severe acute pancreatitis

BACKGROUND/AIMS: The aim of the present study was to investigate whether there are specific prognostic factors to predict the development of secondary pancreatic infection (SPI) in severe acute pancreatitis in order to perform a computed tomography-fine needle aspiration with bacteriological sampling at the right moment and confirm the diagnosis. METHODS: Twenty-five clinical and laboratory parameters were determined sequentially in 150 patients with severe acute pancreatitis (SAP) and univariate, and multivariate regression analyses were done looking for correlation with the development of SPI. RESULTS: Only APACHE II score and C-reactive protein levels were related to the development of SPI in the multivariate analysis. A regression equation was designed using these two parameters, and empiric cut-off points defined the subgroup of patients at high risk of developing secondary pancreatic infection. CONCLUSION: The results showed that it is possible to predict SPI during SAP allowing bacteriological confirmation and early treatment of this severe condition.     

Drusen measurement from fundus photographs using computer image analysis

Drusen are yellowish deposits at the level of the retinal pigment epithelium and are frequently associated with age-related maculopathy (ARM). Drusen often change in size and number over time and may be followed by atrophic or exudative macular degeneration. A quantitative method to measure the development of drusen is needed for controlled studies of the natural history, prognosis, and treatment of ARM. An objective method is described using computer image analysis of fundus photographs for the detection and measurement of drusen. This technique enables us to measure both the area of drusen in the macula and the changes in the drusen pattern over time. Evaluation of repeated photographs showed reproducibility of 6.1%, whereas the reproducibility of processing photographic duplicates was 2.3%. Digitization with a high-quality linear array solid state camera did not change reproducibility significantly.     

Single-dose metyrapone test at 06.00 h: an accurate method for assessment of pituitary-adrenal reserve

One dose of metyrapone (1.5g) administered at 06.00 h, with subsequent measurement of 11-deoxycortisol and 17-hydroxycorticosteroid (17-OHCS) levels in plasma at 12.00 and 14.00 h, allowed accurate assessment of the pituitary-adrenal reserve. Normal response was defined as achieving a serum 17-OHCS level of more than 10.0 micrograms/100 ml and a 11-deoxycortisol level of more than 6.0 micrograms/100 ml at either 12.00 or 14.00 h. These criteria are based on a group of 18 persons with normal pituitary-adrenal axis, and 86 additional cases responded in this normal range. In this group of 104 subjects, 11-deoxycortisol levels rose to 9.2 +/- 3.5 micrograms/100 ml at noon and 17-OHCS levels to 15.4 +/- 4.7 micrograms/100 ml at 14.00 h. Post-metyrapone 17-OHCS levels were significantly higher than normal cortisol levels at these times (P less than 0.001) and than those observed at 08.00 h on the day of the test, demonstrating stimulation of adrenal corticoid production in addition to blockade of cortisol production by metyrapone. Thirty-one patients found to suffer from secondary adrenal failure showed impaired response. All these patients had limited pituitary-adrenal reserve, either proven by other pituitary-adrenal tests or implicated by severe pituitary disease.     

Effect of prolactin and prostaglandins on the stimulation of prolactin binding sites in the male rat liver

Induction of prolactin hepatic receptors in the male rat by exogenous prolactin and the possible involvement of prostaglandins in this process were studied. When ovine prolactin 500 microgram/kg was administered sc in saline containing 10% PVP (polyvinylpyrrolidone), twice daily for 6.5 days and the rats killed 48 h after the last injection, the specific binding of 125I-labelled ovine prolactin to prolactin hepatic receptors was raised 26-fold, while administration of prolactin in saline only caused a 7-fold increase. A well correlated log dose-response relationship was demonstrated between 12.5 and 500 microgram of prolactin in saline-PVP, with lowest dose causing an 18-fold increase in binding. A shorter 4.5 day treatment of prolactin in saline-PVP caused only a small 3-fold increase in prolactin binding. Scatchard analysis showed that these increases resulted from increases in receptor concentration. The effect of prolactin on the induction of the hepatic receptors could not be mimicked by PGE1, PGE2 or PGF2 alpha, nor could PGF2 alpha synergize with a short treatment with prolactin. Further, indomethacin caused no significant effect on this action of prolactin. It seems that prolactin does not induce its own receptors in the rat liver by stimulation of prostaglandins in this tissue.     

Synonymous Mutations in RNASEH2A Create Cryptic Splice Sites Impairing RNase H2 Enzyme Function in Aicardi–Goutières Syndrome

Aicardi–Goutières syndrome is an inflammatory disorder resulting from mutations in TREX1, RNASEH2A/2B/2C, SAMHD1, or ADAR1. Here, we provide molecular, biochemical, and cellular evidence for the pathogenicity of two synonymous variants in RNASEH2A. Firstly, the c.69G>A (p.Val23Val) mutation causes the formation of a splice donor site within exon 1, resulting in an out of frame deletion at the end of exon 1, leading to reduced RNase H2 protein levels. The second mutation, c.75C>T (p.Arg25Arg), also introduces a splice donor site within exon 1, and the internal deletion of 18 amino acids. The truncated protein still forms a heterotrimeric RNase H2 complex, but lacks catalytic activity. However, as a likely result of leaky splicing, a small amount of full‐length active protein is apparently produced in an individual homozygous for this mutation. Recognition of the disease causing status of these variants allows for diagnostic testing in relevant families.