Structural and functional properties of full-length and truncated human proapolipoprotein AI expressed in escherichia coli

Utilizing the Escherichia coli/pGex vector expression system incorporating a thrombin cleavage site, full-length (residues -6-243) and truncated forms of proapolipoprotein AI (proapoAI), terminating at amino acid residues 222, 210, 150, and 135, were purified to levels of at least 5 mg/L, after thrombin cleavage. Assessed by circular dichroism, the helical contents of L-alpha-dimyristoylphosphatidylcholine-associated forms of human plasma-derived apolipoprotein AI (apoAI) and recombinant proapoAI were comparable, being 69% and 65%, respectively. Circular dichroism measurements of the lipid-associated complexes of the truncated forms showed that between the sequence of residues 150-222 no additional helicity was gained until the carboxyl-terminal sequence was present in the molecule, indicating that the carboxyl terminus of the protein is required for the formation of helix within this central region. While tryptophan residues were more than 86% accessible, as assessed by iodide quenching, in the two truncated forms, proapoAI-6-135 and proapoAI-6-150, for both free and complexed protein, this figure fell to about 50% for full-length recombinant proapoAI, further indicating the influence of the carboxyl terminus on the structure of the whole protein. While cross-linking human plasma apoAI in solution with dithiobis-(succinimidyl propionate) revealed high molecular weight oligomers by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, recombinant proapoAI did not strongly form complexes larger than trimers. None of the truncated proapoAI molecules formed oligomers larger than trimers. The shortest form, proapoAI-6-135, only dimerized. Initial results from lecithin:cholesterol acyltransferase activation (apoAI peptide concentration 0.2 microM) indicated that truncation of the 21 carboxy-terminal amino acids resulted in a drop of approximately 53% in activation and 33 residues a drop of 67% relative to the full-length protein. Overall these results indicate the important influence of the carboxyl terminus on the structure of apoAI.     

Strain difference in the induction of T-cell activation-associated, interferon gamma-dependent hepatic injury in mice

A single intravenous injection of concanavalin A (Con A) induces T-cell activation-associated inflammatory injury selectively in the liver. This study investigated the strain difference in the development of Con A-induced hepatic injury. Normal C57BL/6 and BALB/c spleen cells produced comparable levels of T-cell-derived lymphokines (interferon gamma [IFN-gamma], tumor necrosis factor alpha [TNF-alpha], and interleukin-2 [IL-2]) following in vitro stimulation with Con A. A single intravenous injection of Con A to C57BL/6 mice induced the plasma levels of TNF-alpha and IL-2 comparable with or slightly higher than those observed in BALB/c mice, whereas the same treatment resulted in an apparently lower level of IFN-gamma production in C57BL/6 mice. RNA from livers of Con A-treated C57BL/6 mice exhibited lower levels of IFN-gamma mRNA than RNA of BALB/c livers. Unexpectedly, a dramatic difference in the severity of hepatic injury was observed between C57BL/6 and BALB/c. Namely, the peak alanine transaminase (ALT) level was more than 15,000 U/L and inducible as early as 8 hours after injection of 0.2 mg Con A per mouse in the C57BL/6 strain, whereas the peak was approximately 3,000 U/L and induced as late as 24 hours after Con A injection in the BALB/c strain. The increase in plasma ALT levels was limited to less than 10% by injection of anti-IFN-gamma monoclonal antibody (mAb) in both strains. The C57BL/6 strain inducing lower levels of IFN-gamma exhibited higher IFN-gamma responsiveness as exemplified by the intrahepatic expression of an IFN-gamma-inducible gene, an inducible type of nitric oxide (NO) synthase (iNOS). These results indicate that, while IFN-gamma produced in vivo by activated T cells induces hepatic injury, there exists a striking strain difference in the induction of IFN-gamma-dependent hepatic injury.     

Idiopathic intracranial hypertension in female homozygous twins

The authors report on female homozygous twins with idiopathic intracranial hypertension. At the age of 12 years, both twins simultaneously developed visual disturbances with photophobia. At the age of 19 years, an ophthalmological examination disclosed papilloedema in both their eyes. At the age of 22 years, a lumbar puncture showed raised CSF pressure over (200 mm H2O) in both twins. Their neurological and radiological examinations were extremely similar; both of them had severely impaired visual acuity and impaired visual field, bilateral optic nerve atrophy, intracranial hypertension, an enlarged and partial empty sella turcica, digital markings of the calvalium, and an enlarged frontal subarachnoid space. This is the first case report describing idiopathic intracranial hypertension occurring in homozygous twins.     

Diagnosing At-Speed Scan BIST Circuits Using a Low Speed and Low Memory Tester

Numerous solutions have been proposed to reduce test data volume and test application time during manufacturing testing of digital devices. However, time to market challenge also requires a very efficient debug phase. Error identification in the test responses can become impractically slow in the debug phase due to large debug data, slow tester speed, and limited memory of the tester. In this paper, we investigate the problems and solutions related to using a relatively slow and limited memory tester to observe the at-speed behavior of fast circuits. Our method can identify all errors in at-speed scan BIST environment without any aliasing and using only little extra overhead by way of a multiplexer and masking circuit for diagnosis. Our solution takes into account the relatively slower speed of the tester and the reload time of the expected data to the tester memory due to limited tester memory while reducing the test/debug cost. Experimental results show that the test application time by our method can be reduced by a factor of 10 with very little hardware overhead to achieve such advantage.     

Photodimerization of Coumarin‐Derived Pentacyclo[9.5.1.13,9.15,15.17,13]octasiloxane to Fabricate a Three‐Dimensional Organic–Inorganic Hybrid Material

An organic–inorganic hybrid material was prepared from a pentacyclo[9.5.1.1^3,9.1^5,15.1^7,13]octasiloxane bearing eight coumarin groups. The coumarin groups were photodimerized by UV‐light irradiation to connect the monomer units of the octasiloxane. Using a concentrated solution, the intermolecular photodimerization of these coumarin groups leaded to the cross‐linking of the octasiloxane, fabricating an organic–inorganic hybrid material with a three‐dimensional nanostructure. Whereas in dilute solution the intramolecular photodimerization was preferred to the intermolecular reaction, resulting in no formation of polymeric material.     

Induction of metallothionein synthesis by bismuth in cultured vascular endothelial cells

The accuracy and reliability of the TMJ Scale were originally determined in cross-validation studies on large, research-based patient samples. It had been assumed that the demographic characteristics and test responses of these research-based samples would be representative of the clinical population in which the TMJ Scale would ultimately find use. The present study on more than 10,000 patients that were evaluated for temporomandibular disorders in clinical practice demonstrates that the test scores, demographic variables, and the patterns of symptom severity that characterize the original TMJ Scale research sample accurately represent the general temporomandibular disorder patient population in which the TMJ Scale is now being used. The results suggest a high degree of confidence in the clinical efficacy of this assessment tool. The overall symptom severity of temporomandibular disorders was found to be normally distributed in the patient population. Women with temporomandibular disorders report a higher level of severity of all physical and psychological symptoms than men. This may explain the high female-to-male ratio in patients seeking treatment. However, a higher percentage of male temporomandibular disorder patients has clinically significant psychological and stress-related problems than do women. The severity and prevalence of symptoms associated with joint dysfunction and range of motion limitation are lower in older age groups, and the overall symptom severity of temporomandibular disorders is not higher in older age groups. However, the severity and prevalence of symptoms associated with joint dysfunction are greater in groups in which temporomandibular disorders have existed for longer durations, although pain levels do not follow this trend. There is also an association between time duration of the temporomandibular disorder and the severity of psychological problems and chronicity. Patients with chronic problems are symptomatically more impaired than those with acute problems.     

Differential interleukin 12 responsiveness for interferon gamma production in advanced stages of cancer patients correlates with performance status

Interleukin 12 (IL-12) has been shown to exhibit potent antitumor activity in murine tumor models through various mechanisms including the capacity to stimulate IFN-gamma production by T cells and natural killer cells. The aim of the present study was to examine the efficacy of IL-12 in inducing IFN-gamma secretion in cancer patients. A comparison was made between healthy individuals who served as controls and cancer patients for IFN-gamma production induced after the stimulation of whole blood samples with 1000 pg/ml IL-12. Samples from all healthy individuals showed positive IL-12 responsiveness. Approximately half of the samples from patients displayed levels of IFN-gamma production comparable to those observed for controls, whereas the rest of the samples exhibited almost-null responses. The incidences for reduced capacity of IFN-gamma production and null IL-12 responsiveness in cancer patients at all cancer stages or at a given advanced stage (stage IV) increased along with performance status. However, these correlated with neither the number of lymphocytes contained in the blood samples nor the tumor types. When peripheral blood mononuclear cells were isolated from patient blood samples showing null/marginal responses, and their responsiveness was examined, 7 of 13 samples exhibited positive responses. Whereas enhanced tumor necrosis factor alpha production was also observed in some patients after IL-12 stimulation, the elevation of tumor necrosis factor alpha was induced only in blood samples that showed IL-12-stimulated IFN-gamma production. These observations indicate that a remarkable difference exists in IL-12 reactivity among cancer patients, and that differential IL-12 responsiveness depends largely on performance status.     

Entamoeba dispar: cultivation with sterilized Crithidia fasciculata

Four isolates of Entamoeba dispar identified by their hexokinase and phosphoglucomutase isoenzyme profile and by their failure to react with Entamoeba histolytica-specific monoclonal antibody (4G6) could be grown in either Diamond's BI-S-33 medium, newly developed BCSI-S (Biosate cysteine starch iron-serum) medium, or casein-free YI-S medium in the presence of Crithidia fasciculata (ReF-1:PRR) sterilized by heating 56 degrees C for 30 min and subsequent incubation with 1% hydrogen peroxide for 24 hours at 4 degrees C. After the cultures were maintained for over 50 passages, the amebae were identified as E. dispar by isoenzyme analysis, polymerase chain reaction with E. histolytica- and E. dispar-specific primers, i.e. p11 plus p12 and p13 plus p14, respectively, and by negative reactivity with monoclonal antibody 4G6. The flagellates added to the culture were judged to be metabolically inactive based on the results of nuclear magnetic resonance spectroscopy, electron microscopy, and polarographic analysis. All of these findings suggest that E. dispar can grow in vitro with metabolically inactive C. fasciculata as a culture associate.     

Uncertainty evaluation of an in vivo near-field exposure setup for testing biological effects of cellular phones

In designing an in vivo near-field exposure setup for testing biological effects of cellular phones, one generally uses a small still animal because a plastic holder is used to restrain it. One also takes no account of the exposure box with radio wave absorbers as well as the plastic holder. In this paper, for the in vivo exposure setup developed in the National Institute of Information and Communications Technology (NICT), which was used for testing the promoting effect of 1.439- and 1.95-GHz digital cellular phones on rat brain carcinogenesis, we investigated the effects of the above-mentioned factors on the dosimetry design using the finite-difference time-domain (FDTD) method in conjunction with an anatomical rat model. As a result, we found that the specific absorption rate (SAR) averaged in the brain was 18% higher at maximum than the previously designed level due to the existence of the exposure box and the plastic holder and that the variation due to the rotation of the rat's head inside the plastic holder was within 10%. The backward movement of the rat along the plastic holder was more serious, which yielded a decrease of nearly 20% for the average SAR in the brain.