Cerium-Based Demonstration of Phosphatas Activity in Plastic-Embedded Sections: A Comparison with Conventional Methods

Cerium-based methods have been used for the demonstration of seve phatases at alkaline, acid and neutral pH in low temperature acetone-fixed, plastic-e sections. At alkaline pH calcium is used as capturing agent and the precipitated phosphate converted to cerium phosphate. At neutral and acid pH cerium is used d capturing agent. Cerium phosphate is subsequently visualized using the H₂C₂DAB n: comparison has been made with conventional calcium-cobalt and lead methods. It; that calcium-cobalt methods are more susceptible to improvement than lead methods     

Computerized microscopic analysis of prostatic fine needle aspirates. Comparison with breast aspirates

Computerized image analysis was employed to analyze fine needle aspiration smears of the prostate and breast using both high-resolution images of individual cells and medium-resolution images of scenes and clusters (contextual analysis). A linear discriminant analysis was used to demonstrate the computer's ability to discriminate between benign and malignant categories for both types of tissue. Correct classification as benign or malignant using contextual analysis was achieved in 22 of 26 prostatic aspirates and in 15 of 18 breast aspirates, as determined by comparison with histology. The addition of high-resolution single-cell analysis resulted in correct classification of 24 of 26 prostatic aspirates and all breast aspirates. For virtually all features, the distinction between benign and malignant was more subtle for prostatic than for breast tissue. The data indicate that contextual analysis may be less effective as an adjunct to high-resolution single-cell microscopy of prostatic specimens than it is for breast specimens.     

Tellurium-Induced Neuropathy: Metabolic Alterations Associated with Demyelination and Remyelination in Rat Sciatic Nerve

Rats fed a diet containing 1.25% elemental tellurium initiated on postnatal day 20 undergo a transient neuropathy characterized by synchronous demyelination of peripheral nerves. In sciatic nerve, the extent of demyelination was maximal after 5 days of tellurium exposure; there was a loss of 25% of the myelin, as assayed by concentration of myelin-specific P0 protein. Tellurium-induced alterations in the metabolic capacity of Schwann cells were examined by measuring the synthesis of myelin lipids in vitro in isolated sciatic nerve segments. Exposure to tellurium resulted in an early marked decrease of approximately 50% in overall incorporation of [14C]acetate into lipids, with a preferential depression in synthesis of cerebrosides, cholesterol, and ethanolamine plasmalogens (components enriched in myelin). Most dramatically, within 1 day of initiation of tellurium exposure, there was a profound increase in [14C]acetate-derived radioactivity in squalene; 23% of incorporated label was in this intermediate of cholesterol biosynthesis, compared to less than 0.5% in controls. In association with the remyelinating phase seen after 5 days of tellurium exposure, synthesis of myelin components gradually returned to normal levels. After 30 days, metabolic and morphologic alterations were no longer apparent. We suggest that the sequence of metabolic events in sciatic nerve following tellurium treatment initially involves inhibition of the conversion of squalene to 2,3-epoxysqualene, and that this block in the cholesterol biosynthesis pathway results, either directly or indirectly, in the inhibition of the synthesis of myelin components and breakdown of myelin.     

Amino acid sequences of ovomucoid third domain from 25 additional species of birds

Ovomucoids were isolated from 25 avian species other than the 101 studied in Laskowskiet al. (1987,Biochemistry 26, 202–221). These were subjected to limited proteolysis with an appropriate enzyme, and connecting peptide extended ovomucoid third domains were isolated and sequenced to the end in a protein sequencer. Of the 25 new sequences, 13 duplicate ones were already known, and 12 are unique. Probably the most striking findings are a Pro¹⁴ Ser¹⁴ replacement in weka, an Ala¹⁴Thr¹⁵ replacement in Bulwer's pheasant, the discovery of two additional amino acid residues Ile¹⁸ and Gly¹⁸ at the P₁ reactive site position in Kalij pheasant and tawny frogmouth, respectively, and the first finding of a negative (Glu³⁴) rather than positive (Lys³⁴ or Arg³⁴) amino acid residue at the NH₂ terminus of the helix in caracara ovomucoid third domain. These results complete the determination of all the sequences of ovomucoid third domains in the four species genusGallus, in the five species genusSyrmaticus, and in the two species generaAix andPavo.     

Association between XV2c/CS7/KM19/D9 haplotypes and the ΔF508 mutation A study of 57 Belgian families

Summary Using Southern blotting and the polymerase chain reaction, the prevalence of the haplotypes for XV2c, CS7, KM19 and D9 on CF and on normal chromosomes could be determined in 35 Belgian families. A set of primers complementary to the DNA sequence of the CF gene around the ΔF508 deletion was used to amplify this particular segment of the gene. In a total of 57 families, deletion screening showed that 69 out of 116 CF chromosomes (59.5%) carried the ΔF508 deletion. Both the ΔF508 deletion and another mutation(s) showed strong association with the haplotype 1-2-2-2.     

Activation and expansion of cytotoxic T lymphocytes from tumor-draining lymph nodes

Summary Large numbers of cytotoxic T lymphocytes (CTL) could be generated from tumor-draining lymph nodes (DLN) from mice bearing PHS-5 tumor by culturing at low density with autologous tumor cell stimulators and 20 U/ml recombinant interleukin-2 (IL-2). Outgrowth of metastatic tumor cells in culture was prevented by use of this hypoxanthine/aminopterin/thymidine-sensitive mutant of P815, PHS-5. After 9 days in culture, lymphoid cells demonstrated specific cytotoxicity against autologous tumor target cells. Lymph node cells could be expanded continuously in culture with repeated tumor stimulation with up to 7500-fold increase in cell number by 6 weeks; although CTL could be activated from tumor-bearing host spleen cells in short-term culture, they showed no significant growth in long-term cultures. Phenotypically, DLN cells were a mixture of CD8⁺ and CD4⁺ cells immediately after harvest but after 2 weeks in culture they were predominantly CD8⁺ CD4^–. CTL could be generated from tumor-bearing mice 10–14 days after i.d. tumor inoculation into the abdominal wall, but the immune response declined both in spleen and DLN by 21 days. Much greater CTL activity could be generated from axillary DLN that contained metastases than from non-draining popliteal nodes that were free of metastatic tumor cells. Some CTL activity could be generated from DLN with the addition of IL-2 alone but was further increased by the addition of more tumor cells as stimulators. When adoptively transferred to a host with 3-day P815 liver metastases, lymphocytes from DLN activated in vitro were able to reduce or eliminate metastases with very little or no IL-2 administered concomitantly. As few as 10⁶ cells were therapeutically effective, and in vivo efficacy was tumor-specific, since L5178Y liver metastases were not affected.This work was supported in part by grants CA42443, CA48075 and T32-CA09210 from the National Cancer Institute, Department of Health and Human ServicesRecipient of the Canadian Cancer Society McEachern Fellowship.     

Integration of an unstable plasmid into the chromosome of Lactobacillus plantarum

Abstract A 2.3 kb Eco RI restriction fragment of Lactobacillus plantarum genomic DNA was cloned into pSA3 to generate pJRI. Both pSA3 and pJR1 were transformed into L. plantarum . Growth of the transformants in the absence of the selection pressure, erythromycin (both plasmids confer Em^r), led to a stable sub-population of EM^r bacteria in which pSA3 and pJR1 had integrated into the L. plantarum genome. Amplification of the chromosomally located plasmids was observed when the organism was grown in the presence of erythromycin. The integrated plasmid sequences were stably maintained in the absence of selection pressure, and did not affect the growth rate of the lactic acid bacterium in rich media.