Association of butyrylcholinesterase K variant with cholinesterase-positive neuritic plaques in the temporal cortex in late-onset Alzheimer's disease

In confirmed late-onset (>65 years) Alzheimer's disease, we found a greater load, both of overall neuritic plaques and of cholinesterase-positive neuritic plaques, in the temporal cortex of carriers of the butyrylcholinesterase K variant (BCHE-K) aged <80 years than of all other patients. The differences were most striking in the case of cholinesterase-positive neuritic plaques. Among BCHE-K carriers, densities of such plaques were over six times higher in patients <80 years at death than in those >80 years (P=0.01). Furthermore, in subjects <80 years, BCHE-K carriers had nearly six-fold greater densities of these plaques than non-carriers (P=0.009). We consider three potential explanations for these findings: that the K variant binds more readily to plaque constituents, that it promotes fibril formation or that it induces aberrant neurite growth.     

Cytokine biosynthesis by tumor-infiltrating T lymphocytes from human non-small-cell lung carcinoma

The purpose of this work was to assess the capacity of tumor-infiltrating leukocytes (TIL) from human non-small-cell lung carcinoma (NSCLC) specimens to synthesize type-1 and type-2 cytokines. Methods: TIL were isolated from tumors following digestion with collagenase/DNase and further enriched by ficoll-hypaque gradient centrifugation. Membrane phenotypes and intracellular cytokine protein expression of TIL were assessed by flow cytometry. Results: The majority of TIL expressed the CD3 antigen with a CD4:CD8 ratio of approximately 2:1. Other leukocytes such as macrophages (CD14), B lymphocytes (CD20), and natural killer (NK) cells (CD56) were also found to infiltrate the tumors, but in significantly lower numbers. Owing to the limited recovery of non-CD3⁺ leukocytes, our analysis of cytokine biosynthesis has focused on T lymphocytes. In the absence of activation, a small percentage of CD3⁺ TIL synthesized cytokines ( <4%). Following activation with anti-CD3+interleukin-2 (IL-2), CD3⁺ TIL synthesized predominantly a type-1 cytokine profile; however, the type-2 cytokines, IL-6 and IL-10, were also detected in a small percentage of infiltrating cells. Following activation with phorbol 12-myristate 13-acetate + ionomycin, CD3⁺ TIL also expressed more type-1 than type-2 cytokines and in significantly greater numbers of cells. The CD3⁺CD8⁺ component of the TIL synthesized only type-1 cytokines, whereas the CD3⁺CD4⁺ component synthesized both type-1 and type-2 cytokines. Conclusion: These results show that the majority of the TIL isolated from NSCLC specimens are T lymphocytes with the capacity to synthesize type-1 cytokines. Received: 24 March 1999 / Accepted: 9 September 1999     

Bacillus thuringiensisδ-Endotoxin Proteins Show a Correlation in Toxicity and Short Circuit Current Inhibition Against Helicoverpa zea

Pesticidal activity of Bacillus thuringiensisδ-endotoxins, Cry1Aa, Cry1Ab, Cry1Ac, and Cry2A, was determined by using the force-feeding bioassay method to 4^th instar larvae of Helicoverpa zea. H. zea was susceptible to Bt toxins in the order Cry1Ac > Cry1Ab > Cry1Aa > Cry2A with 63.60, 89.04, 159.65, and 375.78 ng/larvae respectively. The abilities of selected Bacillus thuringiensis toxins to inhibit short circuit current (I_SC) in midgut epithelia of H. zea were also investigated by voltage clamp assay. The voltage-clamp studies were conducted on isolated midguts, measuring the inhibition of short circuit current (I_SC) by activated toxin. A Cry1Aa toxin dilution of 33.3 and 500 ng/ml resulted in inhibition of I_SC of −2.29 μA/min (lag time 15 min) and −4.48 μA/min (lag time, 2 min) respectively. The Cry1Ab dilution of 25 ng/ml inhibited I_SC to −1.39 μA/min, a lag time of 14 min, and 333.3 ng/ml dilution resulted in decay of I_SC−2.49 μA/min, lag time 1 min respectively. The Cry1Ac lower dilution 16.7 ng/ml inhibited I_SC to −1.39 μA/min, lag time 4 min, and a high dilution 333.3 ng/ml decay I_SC to −2.44 μA/min, lag time 1 min. The inhibition of I_SC (−1.10 μA/min, lag time 25) at lower dilution (33.3 ng/ml) and high dilution (500 ng/ml), decay (−2.38 μA/min, lag time 5 min), showed a correlation between toxin concentration and inhibitory response with Cry2A toxin. The lag time decreased with increasing concentration of toxin applied, which is additional evidence of dose response besides direct correlation of toxicity assays and I_SC. Received: 7 April 2000 / Accepted: 2 May 2000     

Volatile Organic Compounds Associated with Microbial Growth in Automobile Air Conditioning Systems

Volatile organic compounds from Penicillium viridicatum and Methylobacterium mesophilicum growing on laboratory media and on component materials of automobile air conditioners were analyzed with gas chromatography and mass spectrometry. P. viridicatum produced compounds such as 4-methyl thiazole, terpenes and alcohols, whereas M. mesophilicum produced dimethyl disulfide, dimethyl trisulfide, and chlorophenol with growth on laboratory media. In comparison with laboratory media, fewer volatiles were detected from colonized foam insulation materials. Biofilms of M. mesophilicum on aluminum evaporator components produced mainly dimethyl disulfide. These biofilms, after inoculation with P. viridicatum, produced offensive smelling alcohols and esters such as 2-methyl propanol, 3-penten-2-ol, and the ethyl ester of butanoic acid. The moisture and substrates innate to the automobile air conditioning systems provided an environment suitable for microbial biofilm development and odor production. Reduction of retained moisture in the air conditioning system coupled with use of less susceptible or antimicrobial substrates are advised for remediation of the noxious odors. Received: 26 February 2000 / Accepted: 2 May 2000     

Microgravity culture reduces apoptosis and increases the differentiation of a human colorectal carcinoma cell line

Our hypothesis is that rotation increases apoptosis in standard tissue culture medium at shear stresses of greater than approximately 0.3 dyn/cm2. Human MIP-101 poorly differentiated colorectal carcinoma cells were cultured for 6 d in complete medium in monolayers, on Teflon-coated nonadherent surfaces (static three-dimensional [3D]) or in rotating 3D cultures either in microgravity in low-earth orbit (3D microg) or in unit gravity on the ground (3D 1g). Apoptosis (determined morphologically), proliferation (by MIB1 staining), and the expression of epidermal growth-factor receptor (EGF-R), TGF-alpha, or TGF-beta were assessed by immunohistochemistry, while the expression of the differentiation marker carcinoembryonic antigen (CEA) was assessed on Western blots. Over the course of 6 d, static 3D cultures displayed the highest rates of proliferation and lowest apoptosis. This was associated with high EGF-R, TGF-alpha, and TGF-beta expression which was greater than that of a monolayer culture. Both rotated 3D lg and 3D microg cultures displayed lower expression of EGF-R, TGF-alpha, or TGF-beta and proliferation than that of monolayer or static 3D cultures. However, rotated 3D microg displayed significantly less apoptosis and greater CEA expression than rotated 3D 1g cultures. When rotated cultures of MIP-101 cells were grown uncler static conditions for another 3 d, proliferation increased and apoptosis decreased. Thus, rotation appears to increase apoptosis and decrease proliferation, whereas static 3D cultures in either unit or microgravity have less apoptosis, and reduced rotation in microgravity increases CEA expression.     

Effects of High-Potassium-Induced Depolarization on Amino Acid Chemistry of the Dorsal Cochlear Nucleus in Rat Brain Slices

High K⁺ was used to depolarize glia and neurons in order to study the effects on amino acid release from and concentrations within the dorsal cochlear nucleus (DCN) of brain slices. The release of glutamate, -aminobutyrate (GABA) and glycine increased significantly during exposure to 50 mM K⁺, while glutamine and serine release decreased significantly during and/or after exposure, respectively. After 10 min of exposure to 50 mM K⁺, glutamine concentrations increased in all three layers of DCN slices, to more than 5 times the values in unexposed slices. In the presence of a glutamate uptake blocker, L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC), glutamine concentrations in all layers did not increase as much during 50 mM K⁺. Similar but smaller changes occurred for serine. Mean ATP concentrations were lower in 50 mM K⁺-exposed slices compared to control. The results suggest that depolarization, such as during increased neural activity, can greatly affect amino acid metabolism in the cochlear nucleus.     

A cluster of genes for the biosynthesis of spinosyns, novel macrolide insect control agents produced by Saccharopolyspora spinosa

Spinosyns A and D are the active ingredients in a family of insect control agents produced by fermentation of Saccharopolyspora spinosa. Spinosyns are 21–carbon tetracyclic lactones to which are attached two deoxysugars. Most of the genes involved in spinosyn biosynthesis are clustered in an 74 kb region of the S. spinosa genome. This region has been characterized by DNA sequence analysis and by targeted gene disruptions. The spinosyn biosynthetic gene cluster contains five large genes encoding a type I polyketide synthase, and 14 genes involved in modification of the macrolactone, or in the synthesis, modification and attachment of the deoxysugars. Four genes required for rhamnose biosynthesis (two of which are also required for forosamine biosynthesis) are not present in the cluster. A pathway for the biosynthesis of spinosyns is proposed.     

Detection of Cyclospora cayetanensis in Wastewater

Cyclospora cayetanensis causes diarrheal disease worldwide without a confirmed mode of transmission. Wastewater was examined for the presence of this organism. Oocysts were detected microscopically, and their identity was confirmed by molecular techniques. These findings verify that current techniques can isolate Cyclospora oocysts and suggest that fecally contaminated water may act as a vehicle of transmission.     

Analysis of the Influence of Environmental Parameters on Clostridium botulinum Time-to-Toxicity by Using Three Modeling Approaches

This study used the technique of waiting time modeling to analyze the combined effects of temperature, pH, carbohydrate, protein, and lipid on the time-to-toxicity of Clostridium botulinum 56A. Waiting time models can be used whenever the time to the occurrence of some event is the variable of interest. In the case of the time-to-toxicity data, the variable is the time from the beginning of an experiment until a tube is identified as positive. The statistical analysis used the SAS procedure LIFEREG and included determination of the form of the response surface, identification of the error distribution, and simplification of the response surface. We found that increasing the macromolecule concentration decreased the probability of toxin formation. The probability of toxin formation also decreased at lower temperatures and at pHs further from the optimum. The waiting time modeling approach to developing models for botulinal toxin formation compared favorably with other approaches but had one specific advantage. Waiting time models have the inherent advantage that safety concerns regarding predictions are automatically quantified in the analysis by formally identifying a distribution of times-to-toxicity. The use of this time-to-toxicity distribution permits a customizable margin of safety (e.g., one in a million) not possible with other approaches.     

Regulation of Escherichia coli secA by Cellular Protein Secretion Proficiency Requires an Intact Gene X Signal Sequence and an Active Translocon

secA is translationally regulated by the protein secretion proficiency state of the Escherichia coli cell. This regulation was explored by making signal sequence mutations in the gene upstream of secA, gene X, which promotes secA translational coupling. Gene X signal sequence mutants were constitutive for secA expression, while prlA alleles partially restored secA regulation. These results show that interaction of the pre-gene X protein with the translocon is required for proper secA regulation. Furthermore, gene X signal sequence mutations disrupted secA regulation only in the cis configuration. We propose that nascent pre-gene X protein interacts with the translocon during its secretion to constitute the secretion sensor.