Comparison of three methods for determining aflatoxins in melon seeds

The suitability of 3 methods for determining aflatoxins in melon seeds was examined. The first 2 are the Contaminants Branch (CB) method and the Best Foods (BF) method, both official methods for determining aflatoxins in peanuts and peanut products. The third method, the modified CB method-Rapid Modification of the Cottonseed (CB-RCS-Mod) method, devised in this work, was derived by combining steps from the CB method and the Rapid Modification of the Cottonseed method. The CB method was superior to the other 2 methods for quantitation of aflatoxins. It gave better recoveries and cleaner extracts that exhibit less fluorescent interference for thin-layer chromatography (TLC) than the BF method. Also, its solvent efficiency was better than that of the CB-RCS-Mod method. With the CB method, recoveries from spiked samples were 85.0% for aflatoxin B1 and 90.0% for aflatoxin B2. Recoveries of G aflatoxins were more variable, averaging 90.0% for aflatoxin G1 and 72.5% for aflatoxin G2. Total aflatoxin recovery was 86.5% for the CB method. At a low aflatoxin contamination level (8 micrograms B1/kg sample), aflatoxin B1 was detectable by the CB method but not by the BF method. Detection of aflatoxins in BF method sample extracts by TLC was not improved by the use of chloroform-acetone-water (88 + 12 + 1), benzene-ethanol-water, or ether-methanol-water (96 + 3 + 1) in place of the standard chloroform-acetone (88 + 12) developer. Use of ether-methanol-water (96 + 3 + 1) for detecting aflatoxins by TLC in the CB method extracts increased interference compared with the standard chloroform-acetone (88 + 12) developer.     

Components of a calmodulin-dependent protein kinase cascade. Molecular cloning, functional characterization and cellular localization of Ca2+/calmodulin-dependent protein kinase kinase beta

Ca2+/calmodulin-dependent protein kinases I and IV (CaMKI and CaMKIV, respectively) require phosphorylation on an equivalent single Thr in the activation loop of subdomain VIII for maximal activity. Two distinct CaMKI/IV kinases, CaMKKalpha and CaMKKbeta, were purified from rat brain and partially sequenced (Edelman, A. M., Mitchelhill, K., Selbert, M. A., Anderson, K. A., Hook, S. S., Stapleton, D., Goldstein, E. G., Means, A. R., and Kemp, B. E. (1996) J. Biol. Chem. 271, 10806-10810). We report here the cloning and sequencing of cDNAs for human and rat CaMKKbeta, tissue and regional brain localization of CaMKKbeta protein, and mRNA and functional characterization of recombinant CaMKKbeta in vitro and in Jurkat T cells. The sequences of human and rat CaMKKbeta demonstrate 65% identity and 80% similarity with CaMKKalpha and 30-40% identity with CaMKI and CaMKIV themselves. CaMKKbeta is broadly distributed among rat tissues with highest levels in CaMKIV-expressing tissues such as brain, thymus, spleen, and testis. In brain, CaMKKbeta tracks more closely with CaMKIV than does CaMKKalpha. Bacterially expressed CaMKKbeta undergoes intramolecular autophosphorylation, is regulated by Ca2+/CaM, and phosphorylates CaMKI and CaMKIV on Thr177 and Thr200, respectively. CaMKKbeta activates both CaMKI and CaMKIV when coexpressed in Jurkat T cells as judged by phosphorylated cAMP response element-binding protein-dependent reporter gene expression. CaMKKbeta activity is enhanced by elevation of intracellular Ca2+, although substantial activity is observed at the resting Ca2+ concentration. The strict Ca2+ requirement of CaMKIV-dependent phosphorylation of cAMP response element-binding protein, is therefore controlled at the level of CaMKIV rather than CaMKK.     

Fetal cardiac measurements derived by transvaginal and transabdominal cross-sectional echocardiography from 14 weeks of gestation to term

OBJECTIVE: Most of the routine ultrasound screening in our institution consists of early transvaginal examinations at 14-17 weeks. Complete fetal echocardiography is performed in every case. However, normal values for most fetal cardiac structures at this stage of gestation are not available. Our aim was to construct normal ranges for fetal cardiac structures, derived from cross-sectional echocardiography, at 14-40 weeks of gestation. DESIGN: A prospective study was performed. The study group consisted of 637 pregnant women referred for a routine sonographic examination. Women with abnormal prenatal or postnatal outcome were not included in the study. Transvaginal examinations were used for 14-17 weeks of gestation. More advanced pregnancies were examined transabdominally. RESULTS: We constructed normal ranges for the left and right end-diastolic transverse ventricular diameters (n = 637), left/right ventricular ratio (n = 637), aortic root diameter (n = 637), pulmonary artery diameter (n = 637), aortic/pulmonary ratio (n = 490), left and right transverse atrial diameters (n = 201) and left/right atrial ratio (n = 201). CONCLUSIONS: The results provide the examiner with normal ranges for fetal cardiac structures for the early transvaginal examination. The continuity of all curves from 14 to 40 weeks of gestation allows follow-up of any specific fetus to term.     

Presacral phlegmons and meningitis as the complications of a foreign body in the rectosigmoid

A 24-year-old patient presented with severe back pain in the lumbar and sacral regions and septic temperatures up to 40 degrees C. Lasègue's sign was bilaterally positive. An enhanced cell count was seen in the CSF (1877 cells/microliters, erythrocytes 1024/microliters). Total protein concentration was 4440 mg/dl. The patient also suffered from granulocytic pleocytosis. X-ray as well as scintigrams revealed inflammation of the presacral soft parts involving the os sacrum and bone marrow. Treatment with doxycycline (200 mg/d), fosfomycin (5 g/d), netilmicin (400 mg/d) and ofloxacin (200 mg/d) succeeded in curing the meningitis, whereas the presacral phlegmon persisted. Endoscopic examination showed a rod-shaped foreign body in the rectum that had penetrated deeply into the mucosa and required deformation and removal by laser. The plastic rod of 10 cm length was probably a liquorice stick. The patient denied introducing it per anum; he suspected having swallowed it while drunk. The inflammation subsided rapidly after removal of the foreign body.     

Cytomegalovirus-specific IFNgamma and IL-4 are produced by antigen expanded human blood lymphocytes from seropositive volunteers

The cytokine responses to cytomegalovirus (CMV) antigen in seropositive and seronegative individuals were measured using a combination of antigenic expansion and intracellular staining. Intracellular IFNgamma and IL-4 were produced in a dose-dependent manner by T cells in response to CMV only in the seropositive population. The potential for individual cells to produce both Th1 and Th2 cytokines simultaneously was clear, as IL-4 was most often produced in those cells with the highest IFNgamma production. The cytokine-specific nature of this response was demonstrated by fluorescence microscopy, which showed granular cytoplasmic staining, and at the mRNA level by ribonuclease protection assays. These methods expand our ability to evaluate the immune response to CMV, and can now be correlated to a number of clinical conditions.     

A nonamer peptide derived from Listeria monocytogenes metalloprotease is presented to cytolytic T lymphocytes

Listeria monocytogenes is an intracellular bacterium that secretes proteins into the cytosol of infected macrophages. Major histocompatibility complex (MHC) class I molecules bind peptides that are generated by the degradation of bacterial proteins and present them to cytolytic T lymphocytes (CTL). In this study we have investigated CTL responses in L. monocytogenes-immunized mice to peptides that (i) derive from the L. monocytogenes proteins phosphatidylinositol-specific phospholipase C, lecithinase (most active on phosphatidylcholine), metalloprotease (Mpl), PrfA, and the ORF-A product and (ii) conform to the binding motif of the H2-Kd MHC class I molecule. We identified a nonamer peptide, Mpl 84-92, that is presented to L. monocytogenes-specific CTL by H2-Kd MHC class I molecules. Unlike other motif-conforming peptides derived from the secreted Mpl of L. monocytogenes, Mpl 84-92 is bound with high affinity by H2-Kd. Mpl 84-92 is the fourth L. monocytogenes-derived peptide found to be presented to CTL by the H2-Kd molecule during infection and demonstrates the importance of high-affinity interactions between antigenic peptides and MHC class I molecules for CTL priming.     

A giant follicular cyst of the ovary in a newborn infant

The purpose of this study was to evaluate the accuracy of clinical diagnosis of acute pelvic inflammatory disease (PID). Data were obtained on 176 consecutive women admitted to St. Elizabeth Hospital Medical Center with a clinical diagnosis of PID. All underwent diagnostic laparoscopy. PID was established laparoscopically in 134 (76.1%) of the patients. Statistical tests for significant associations between PID and each of 21 clinical indicators of the disease were conducted using the chi 2 and Mann-Whitney tests. Stepwise logistic regression was performed on those variables whose univariate tests of significant association with PID resulted in P values < 0.20. An optimal set of PID indicators consisted of adnexal tenderness, lower abdominal pain of < one week's duration and an elevated white blood cell count. Use of these indicators resulted in a test with an estimated sensitivity and specificity of 86.6% and 45.7%, respectively. Estimated predictive values for positive and negative test results were 0.84 and 0.52, respectively. These results confirm the fact that laparoscopy is the definitive diagnostic modality in PID.