Certification of vitamins and carotenoids in SRM 3280 multivitamin/multielement tablets.

A new multivitamin/multielement dietary supplement Standard Reference Material (SRM) has been issued by the National Institute of Standards and Technology (NIST), with certified and reference concentration values for 13 vitamins, 24 elements, and 2 carotenoids. The constituents have been measured by multiple analytical methods with data contributed by NIST and by collaborating laboratories. This effort included the first use of isotope dilution mass spectrometry for value assignment of both fat-soluble vitamins (FSVs) and water-soluble vitamins (WSVs). Excellent agreement was obtained among the methods, with relative expanded uncertainties for the certified concentration values typically ranging from <2% to 15% for vitamins.

Total selenium and selenomethionine in pharmaceutical yeast tablets: assessment of the state of the art of measurement capabilities through international intercomparison CCQM-P86.

Results of an international intercomparison study (CCQM-P86) to assess the analytical capabilities of national metrology institutes (NMIs) and selected expert laboratories worldwide to accurately quantitate the mass fraction of selenomethionine (SeMet) and total Se in pharmaceutical tablets of selenised-yeast supplements (produced by Pharma Nord, Denmark) are presented. The study, jointly coordinated by LGC Ltd., UK, and the Institute for National Measurement Standards, National Research Council of Canada (NRCC), was conducted under the auspices of the Comité Consultatif pour la Quantité de Matière (CCQM) Inorganic Analysis Working Group and involved 15 laboratories (from 12 countries), of which ten were NMIs. Apart from a protocol for determination of moisture content and the provision of the certified reference material (CRM) SELM-1 to be used as the quality control sample, no sample preparation/extraction method was prescribed. A variety of approaches was thus used, including single-step and multiple-step enzymatic hydrolysis, enzymatic probe sonication and hydrolysis with methanesulfonic acid for SeMet, as well as microwave-assisted acid digestion and enzymatic probe sonication for total Se. For total Se, detection techniques included inductively coupled plasma (ICP) mass spectrometry (MS) with external calibration, standard additions or isotope dilution MS (IDMS), inductively coupled plasma optical emission spectrometry , flame atomic absorption spectrometry and instrumental neutron activation analysis. For determination of SeMet in the tablets, five NMIs and three academic/institute laboratories (of a total of five) relied upon measurements using IDMS. For species-specific IDMS measurements, an isotopically enriched standard of SeMet (76Se-enriched SeMet) was made available. A novel aspect of this study relies on the approach used to distinguish any errors which arise during analysis of a SeMet calibration solution from those which occur during analysis of the matrix. To help those participants undertaking SeMet analysis to do this, a blind sample in the form of a standard solution of natural abundance SeMet in 0.1 M HCl (with an expected value of 956 mg kg(-1) SeMet) was provided. Both high-performance liquid chromatography (HPLC)-ICP-MS or gas chromatography (GC)-ICP-MS and GC-MS techniques were used for quantitation of SeMet. Several advances in analytical methods for determination of SeMet were identified, including the combined use of double IDMS with HPLC-ICP-MS following extraction with methanesulfonic acid and simplified two-step enzymatic hydrolysis with protease/lipase/driselase followed by HPLC-ICP-IDMS, both using a species-specific IDMS approach. Overall, satisfactory agreement amongst participants was achieved; results averaged 337.6 mg kg(-1) (n = 13, with a standard deviation of 9.7 mg kg(-1)) and 561.5 mg kg(-1) (n = 11, with a standard deviation of 44.3 mg kg(-1)) with median values of 337.6 and 575.0 mg kg(-1) for total Se and SeMet, respectively. Recovery of SeMet from SELM-1 averaged 95.0% (n = 9). The ability of NMIs and expert laboratories worldwide to deliver accurate results for total Se and SeMet in such materials (selensied-yeast tablets containing approximately 300 mg kg(-1) Se) with 10% expanded uncertainty was demonstrated. The problems addressed in achieving accurate quantitation of SeMet in this product are representative of those encountered with a wide range of organometallic species in a number of common matrices.

Growth and metastasis of human breast cancers in athymic nude mice.

To evaluate critically the merit of utilizing a wound model for growing human tumors, a series of increasingly difficult human tumor types were tested for growth at sites of trauma in athymic nude mice. In vitro tumor lines as well as fresh tumors from the breast, colon, rectum, lung, and a metastasis from an unknown primary were intraperitoneally injected into mice subjected to intra-abdominal organ injury. Successful xenografts were obtained from nine of 10 cell lines and 14 of 24 fresh tumors. The latter included five of six (83%) colon cancers, one lung tumor, metastatic tumor of unknown primary, three of four (75%) metastatic breast cancers and four of six (67%) estrogen receptor (ER)-negative breast primary tumors. Six ER-positive breast tumors tested failed to grow in mice without estrogen supplementation. Xenografts from two breast, two colon and the lung cancers formed spontaneous metastases and all xenografts tested were able to yield serial transplants in the surgical wound model. Histologically, all xenografts and their metastases were identical to their respective donor tumors. Transplantability in mice without exogenous estrogen supplementation was linked to the absence of estrogen and progesterone receptors in breast tumors. Transplantability of the cell lines was associated with the expression of cell surface receptors for fibronectin and hyaluronic acid. Receptors for other extracellular matrix components, namely, laminin, vitronectin, collagen, fibrinogen or von Willebrand factor were not associated with transplantability. These results demonstrate that a large proportion of human tumors, including the breast tumors, can be successfully xenografted into athymic mice by providing them with a healing wound environment, and that such xenografts grown at ectopic sites exhibit metastatic ability.

The role of fibronectin in tumor implantation at surgical sites.

Fibronectins are a family of glycoproteins with modular functional domains. They mediate cell-cell and cell-matrix interactions which are important in embryogenesis, wound healing, metastasis and other processes. We present data on the influence of fibronectin on wound implantation of a murine mammary carcinoma line, TA3Ha. Fibronectin used in these studies was derived from bovine plasma, human serum, human foreskin fibroblasts, and mouse embryo cultures. TA3Ha cells rarely form tumors in the liver of syngeneic mice when injected intravenously but after hepatic wedge resection, 45% (107/240) of the mice develop tumors in the hepatic wound. Wound implantation is markedly reduced when the cells are pre-exposed to 200 micrograms/ml bovine plasma fibronectin (13%, P = 0.007), human serum fibronectin (0%, P = 0.02), human cellular fibronectin (0%, P = 0.02), or mouse cellular fibronectin (0%, P = 0.04). Lung colonization is also reduced by these fibronectins. These effects are not due to a cytotoxic action of fibronectin, since intraperitoneally injected fibronectin-treated cells form ascites tumor as effectively as do control untreated cells. Local application of a solution containing 0.25 mg/ml mouse cellular fibronectin to the hepatic wound reduces the frequency of tumor implantation from 45% to 5% (1/21, P = 0.001). No tumor implantation inhibition is seen when only suspending medium or albumin in suspending medium is used. The mechanism by which topical application of fibronectin reduces hepatic wound implantation of tumor cells is unclear, but this finding raises an exciting possibility of preventing local recurrence of cancer.

No evidence for histamine-mediated morphine analgesia.

A role for brain histamine in the acute action of morphine was studied in mice. In contrast to earlier reports, whole brain histamine levels were not changed 30 min after morphine (1-56 mg/kg). Levels of the brain histamine metabolite, tele-methylhistamine were also unchanged. Morphine (1.8-10 mg/kg) caused a dose-dependent antinociceptive response that was unaffected by histamine H1-antagonists (i.p.), H2-antagonists (i.p. or i.vent.), or metoprine (i.p.), an inhibitor of histamine metabolism. alpha-Fluoromethylhistidine, the inhibitor of brain histamine synthesis, unexpectedly potentiated the response to low doses of morphine. These results find no evidence for a role of brain histamine in opiate analgesia.

Substituted salicylanilides as inhibitors of two-component regulatory systems in bacteria.

A new class of inhibitors of the two-component regulatory systems (TCS) of bacteria was discovered based on the salicylanilide screening hits, closantel (1) and tetrachlorosalicylanilide (9). A systematic SAR study versus a model TCS, KinA/Spo0F, demonstrated the importance of electron-attracting substituents in the salicyloyl ring and hydrophobic groups in the anilide moiety for optimal activity. In addition, derivatives 8 and 16, containing the 2, 3-dihydroxybenzanilide structural motif, were potent inhibitors of the autophosphorylation of the KinA kinase, with IC50s of 2.8 and 6. 3 µM, respectively. Compound 8 also inhibited the TCS mediating vancomycin resistance (VanS/VanR) in a genetically engineered Enterococcus faecalis cell line at concentrations subinhibitory for growth. Closantel (1), tetrachlorosalicylanilide (9), and several related derivatives (2, 7, 10, 11, 20) had antibacterial activity against the drug-resistant organisms, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VREF).

Neurotoxic effects of colchicine: differential susceptibility of CNS neuronal populations.

Colchicine injected into the dentate gyrus of the hippocampus in adult rats preferentially destroys dentate granule cells. In the present study, we examine the light- and electron-microscopic correlates of the degeneration and evaluate whether the selectivity is preserved across the range of doses between 0.18 and 25 micrograms. Colchicine in a similar dose range was also injected into the cerebellum, olfactory bulb, striatum and cerebral cortex to examine local and regional differences in susceptibility to colchicine. The morphological changes accompanying degeneration in the dentate gyrus include fragmentation of the granule cell layer, appearance of small dark staining bodies in the cell layer, massive microglial invasion and profound disruption of granule cell axons and dendrites. Electron-microscopic observations suggest that the small dark bodies are probably condensed nuclei. The preferential vulnerability of dentate granule cells following intrahippocampal injection was observed at all doses. At doses between 0.18 and 2.5 micrograms there was little evidence of damage to neurons other than dentate granule cells. At the highest dose tested (25 micrograms) some pyramidal cells of regio superior near the injection site were destroyed, while granule cell destruction extended several mm from the injection site. Injection of 0.5-25 micrograms into the cerebellum resulted in the destruction of both granule cells and Purkinje cells, while cells which appeared to be neurons in the molecular layer were less affected. Following injection of 0.5 microgram into the olfactory bulb, granule cells were extensively destroyed and there appeared to be some loss of mitral cells and an overall shrinkage of the injected bulb. Neuronal destruction in the striatum was observed with colchicine injections ranging from 2.5 to 25 micrograms, but at a given dose, the destruction was less extensive than for any other region tested except cerebral cortex. A possible application of this method and the implications of these results for other investigators using colchicine in the brain are discussed.

Post-source decay in the analysis of polystyrene by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Various secondary series are observed in matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectra of polystyrene. The number and positions of the series depend on the choice of matrix and added cation. For a given treatment, series observed in linear mode are not necessarily observed in reflectron mode, and vice versa. Post-source decay analysis was used to determine that the secondary series arise at least in part from formation and decay of adducts of polystyrene with matrix species. There is some treatment-to-treatment variation, but adduct formation and decay were observed for all tested treatments. The multiplicity of secondary series makes it unclear whether post-source decay occurs for the main series (polystyrene + cation)+ ions under the conditions normally used for polystyrene analysis. Such ions do undergo post-source decay at laser fluences greater than normally used. Although only polystyrene was investigated in this work, other polymers may also produce adduct and decay series in MALDI analysis. Their presence can mask the presence of minor components in a sample, but at least as observed here, do not have a strong influence on molecular mass determinations.

Inhibitors of bacterial two-component signalling systems.

Bacterial two-component regulatory systems (TCS) play a pivotal role in the process of infection. These signal transduction systems enable bacterial pathogens to mount an adaptive response and cope with diverse environmental stresses, including nutrient deprivation, antibiotic onslaught and phagocytosis. Interest in these systems as novel bacterial targets has been rekindled by the recent discovery of several essential systems in important Gram-positive and Gram-negative pathogens. Several series of TCS inhibitors derived from broad screening approaches have been reported in the literature, however, most appear to suffer from poor selectivity, excessive protein binding and/or limited bioavailability. Consequently, pharmaceutical chemists have turned to alternate strategies, such as the design of substrate-based inhibitors, the generation of combinatorial libraries and the isolation of natural products, to identify inhibitors with more desirable properties. Recent structural studies of the histidine protein kinase and response regulator proteins that constitute TCS may provide a foundation for a structure-based design approach to TCS inhibitors.

Effectiveness of fluoroquinolones against gram-positive bacteria.

Fluoroquinolones are broad-spectrum and therapeutically effective antibacterial agents that have retained high activity against methicillin-susceptible Staphylococcus aureus (MSSA) and streptococci. Primary targets of these agents in Gram-positive bacteria are DNA topoisomerase and DNA gyrase. High-level resistance is associated with at least two mutations in either target, or combinations of at least two mutations in multiple targets, particularly affecting the ParC subunit of topoisomerase IV and the GyrA subunit of DNA gyrase. This resistance may be enhanced when combined with mutations that increase fluoroquinolone efflux. Data from fluoroquinolone-resistant clinical isolates suggest that as yet unidentified mutations may also be involved in clinical resistance.

Modulation of alloreactivity in transplant recipients by phenotypic manipulation of donor endothelium.

Phenotypic manipulation of allograft endothelium to reduce immunogenicity would have a significant impact on transplantation. In this study we have demonstrated that random seeding of a heart allograft with endothelium, of host origin, not only promotes long-term survival, but reduces the requirement for pharmacologic immunosuppression. We propose that this simple technology could easily be extrapolated to the clinical arena where hypothermia and preservation solutions have allowed allografts to remain ex vivo for extended periods.

Infective endocarditis caused by Paecilomyces varioti.

The first reported case of Paecilomyces varioti endocarditis occurring on a porcine heterograft prosthesis is presented and the clinical and pathological features described in the four previously reported cases of prosthetic valvular endocarditis caused by this organism are reviewed. In each case, infection became apparent more than two months after cardiac surgery. Three of the four patients with infected aortic prosthesis developed outflow obstruction secondary to valvular vegetations. All patients died regardless of medical or surgical intervention. Appropriate therapy remains unknown.

On the role of hippocampal connections in the performance of place and cue tasks: comparisons with damage to hippocampus.

Behavioral changes following interruption of the main connections of hippocampus and closely related areas (entorhinal cortex, mammillary bodies, dentate gyrus) were determined and compared with findings of previous research that involved direct damage to hippocampus. By a within-subjects design, rats were trained to run in a radial maze with a procedure that involved two kinds of learning (place and cue) and two memory functions (working and reference memory). Rats with fimbria-fornix and entorhinal cortex lesions were impaired on both the place and the cue task. Specifically, the animals suffered a general impairment in working memory on both tasks but were impaired in reference memory only on the place task. Animals with lesions of the dentate gyrus and mammillary bodies were able to perform the complex place and cue tasks with minimal problems. In previous research it was found that direct damage to hippocampus (including all cell fields, alveus, fimbria) resulted in impaired performance only on the place task (Jarrard, 1983). Taken together, these findings indicate that interruption of hippocampal input/output pathways and/or damaging some closely related structures has a greater effect on the behaviors studied than does direct damage to hippocampus.

Fate of afferents to the dentate gyrus following destruction of granule cells with colchicine.

Granule cells of the dentate gyrus can be selectively destroyed by intrahippocampal injections of colchicine. The present study evaluates the consequences of this selective neuronal destruction on the afferent axon terminals which have been deprived of their normal targets. The area of the neuropil in the dentate gyrus (the molecular layer) was evaluated in sections stained using the Timm's method for heavy metals, which selectively marks the terminal fields of the different afferent systems. The molecular layer was examined electron microscopically to determine the fate of afferent terminals. Anterograde transport of HRP or [3H]proline was used to define the location and extent of afferent terminal fields of the entorhinal and commissural projections to the dentate gyrus in which the granule cells had been destroyed. There was a substantial reduction in the size of the dentate gyrus molecular layer after destruction of granule cells with colchicine. Electron microscopic analyses revealed that there were very few axon terminals or synapses remaining in the shrunken molecular layer. Tract tracing methods revealed that both the entorhinal and commissural pathways were still present in their normal terminal zones in the dentate gyrus, however, the density of the projections was greatly reduced. There was no evidence to suggest the formation of ectopic projections to unusual locations, such as the contralateral dentate gyrus. Pathways passing through the hippocampus appeared to survive the colchicine injections. These results suggest that target destruction in adult animals leads to the disappearance of the afferent axon terminals which normally innervate the cells which die.

Retrograde regulation of neuronal size in the entorhinal cortex: consequences of the destruction of dentate gyrus granule cells with colchicine.

Studies in developing animals have documented that manipulations which increase or decrease the size of a neuron's axon arbor lead to increases or decreases respectively in the size of the neuron's soma. The present study evaluates whether similar dependencies exist in adult animals, by analyzing changes in cell size in the entorhinal cortex after selective destruction of dentate granule cells with colchicine. Neurons in layer II of the entorhinal cortex which had been deprived of their normal targets decreased in size by 32% relative to their contralateral homologs. Neurons in layer III which project to regio superior of the hippocampus were affected to only a slight extent, decreasing in size by 8% relative to their contralateral homologs. Neurons in layer V, which do not project to the hippocampus, were unaffected by colchicine injections into the hippocampus. These results indicate that neurons in adult animals which retract terminal arbors as a consequence of target loss also decrease in size.

Improved detection of metastases to lymph nodes and estrogen receptor determination.

We present a new method for detection of micrometastases to axillary lymph nodes and estrogen receptor determination. Cellular suspensions from primary infiltrating ductal breast carcinoma or level I axillary lymph nodes of patients who underwent mastectomies were obtained, by loosely grinding fresh tumors or lymph nodes through a grid and then transferring the matrix to a slide using cytocentrifugation. Tumor samples were analyzed for estrogen receptor status using an immunocytochemical kit and compared with the dextran-coated charcoal method. Thirty-eight of 48 correlated (20 were estrogen positive, and 18 were estrogen negative). Seven of 46 were estrogen positive while results from the dextran-coated charcoal method were estrogen negative. One of 46 was estrogen negative, while the results from the dextran-coated charcoal method were estrogen positive. Lymph node slide preparations were stained to detect tumor cells using antikeratin monoclonal antibodies. Three of 8 node-negative patients were found to have micrometastases. Four of 15 node-positive patients had additional nodes with tumor. Our method combines the advantages of serial sectioning and immunohistochemical staining.

The importance of DNA flow cytometry in node-negative breast cancer.

DNA flow cytometric analysis and conventional clinical factors were compared with disease outcome in 257 patients with node-negative infiltrating ductal carcinoma who had been treated between 1976 and 1983. Median follow-up was 80 months; none of the patients received adjuvant therapy. The relative prognostic importance of clinical variables, ploidy, and S-phase fraction was analyzed by Cox multivariate analysis. Ploidy was analyzable for 198 tumors and did not predict survival. Nuclear grade predicted disease-free survival for all patients. For 71 patients with diploid tumors, only high S-phase had a statistically significant association with relapse. For 127 patients with aneuploid tumors, tumor diameter predicted both disease-free survival and cancer death; histologic grade was also significant for predicting disease-free survival. In conclusion, flow cytometric determination of ploidy and S-phase fraction can provide valuable predictive information in node-negative breast cancer in addition to conventional variables.

Comparison of the neurotoxic effects of colchicine, the vinca alkaloids, and other microtubule poisons.

Previous studies have revealed that colchicine is selectively toxic to certain neuronal populations in the CNS, particularly granule cells of the dentate gyrus. The present study evaluates whether other microtubule poisons exhibit similar neurotoxic effects. Equimolar solutions of colchicine, colcemid, podophyllotoxin, vinblastine, vincristine and lumicolchine, the non-binding analog of colchicine, were injected into the dentate gyrus. Neurotoxicity was evaluated histologically. As previously reported, colchicine selectively destroyed dentate granule cells with minimal damage to other neurons including hippocampal pyramidal cells. Vincristine was very toxic and was not selective for granule cells. Vinblastine was relatively selective in destroying granule cells, but was not as potent as colchine. Colcemid and podophyllotoxin had minimal toxic effects. Lumicolchine injections caused no more damage than injections of vehicle. This ordering appears to correlate with the reversibility of binding tubulin.

Mast cells in rat thalamus: nuclear localization, sex difference and left-right asymmetry.

Mast cells were positively identified in rat brain by a combination of staining and histochemical procedures. These cells stained positively with toluidine blue and Astrablau at low pH, indicating the presence of a proteoglycan similar to that found in peripheral mast cells. Brain mast cells also fluoresced after o-phthalaldehyde exposure, indicating that they contain histamine. Mast cells varied greatly in number among brains, but their distribution was almost exclusively thalamic; within thalamus, the ventral complex, medial dorsal, lateral, and paraventricular nuclei contained the most mast cells. Mast cell numbers were greater in brains of females than of males, and greater in left than in right hemispheres. These findings suggest that mast cells have a specialized function in thalamus and/or that the vascular environment of the thalamus is particularly conducive to mast cell accumulation.

Normal levels of histamine and tele-methylhistamine in mast cell-deficient mouse brain.

To determine the contribution by mast cells to the brain content of histamine (HA) and its metabolite tele-methylhistamine (t-MH), the number of mast cells, as well as the levels of HA and t-MH were measured in brain regions of mast cell-deficient (W/Wv) and control (+/+) mice. In agreement with earlier studies, mast cells were identified in control mouse brains, whereas W/Wv brains were devoid of mast cells. Contrary to earlier studies, no differences between these strains were found in the HA levels of any brain region, implying that mouse brain mast cells do not contribute significantly to brain HA levels. Brain t-MH levels were also not different between strains, except in hypothalamus, where W/Wv levels were higher; a significantly smaller W/Wv hypothalamus accounted for this difference. It is not certain that such differences are due to the absence of mast cells, since the W/Wv mutant is pleiomorphic, and the biochemical nature of this mutation remains uncertain. However, the absence of mast cells and presence of HA in the W/Wv mouse brain is direct evidence for the existence of non-mast cell HA in the brain. These results also show that mouse brain t-MH levels are predictive of non-mast cell HA in brain.