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The tungsten formylmethanofuran dehydrogenase from Methanobacterium thermoautotrophicum contains sequence motifs characteristic for enzymes containing molybdopterin dinucleotide.

Formylmethanofuran dehydrogenases are molybdenum or tungsten iron-sulfur proteins containing a pterin dinucleotide cofactor. We report here on the primary structures of the four subunits FwdABCD of the tungsten enzyme from Methanobacterium thermoautotrophicum which were determined by cloning and sequencing the encoding genes fwdABCD. FwdB was found to contain sequence motifs characteristic for molybdopterin-dinucleotide-containing enzymes indicating that this subunit harbors the active site. FwdA, FwdC and FwdD showed no significant sequence similarity to proteins in the data bases. Northern blot analysis revealed that the four fwd genes form a transcription unit together with three additional genes designated fwdE, fwdF and fwdG. A 17.8-kDa protein and an 8.6-kDa protein, both containing two [4Fe-4S] cluster binding motifs, were deduced from fwdE and fwdG. The open reading frame fwdF encodes a 38.6-kDa protein containing eight binding motifs for [4Fe-4S] clusters suggesting the gene product to be a novel polyferredoxin. All seven fwd genes were expressed in Escherichia coli yielding proteins of the expected size. The fwd operon was found to be located in a region of the M. thermoautotrophicum genome encoding molybdenum enzymes and proteins involved in molybdopterin biosynthesis.

Aldehyde Oxidoreductases↗

Kinetics of phosphatidylinositol-3-phosphate acquisition differ between IgG bead-containing phagosomes and Mycobacterium tuberculosis-containing phagosomes.

A key aspect of Mycobacterium tuberculosis pathogenesis is the ability of the bacteria to survive within the host macrophage. A phagosome containing an IgG-coated bead matures into a lysosomal compartment as evidenced by a decrease in pH and an increased acquisition of hydrolytic enzymes. In contrast, when M. tuberculosis is phagocytosed, the maturation of the bacteria-containing phagosome is arrested, and the bacterium resides within a vacuole that retains characteristics of early endosomal compartments. M. tuberculosis-containing phagosomes are delayed in the recruitment of the early endosome autoantigen EEA1. Acquisition of EEA1 is dependent on the presence of phosphatidylinositol-3-phosphate (PI-3-P) generated by the kinase Vps34. We tested the hypothesis that delayed recruitment of EEA1 was due to altered kinetics of PI-3-P accumulation at the phagosomal membrane. Biochemical analysis of the phosphatidylinositol phosphates on M. tuberculosis-containing phagosomes revealed that PI-3-P acquisition was markedly retarded and reduced in comparison to IgG bead-containing phagosomes. Given the role these lipids play in the regulation of phagosome maturation these findings have implications with respect to the mechanisms behind the arrest of phagosome maturation.

Animals↗

Novel non-mecA-containing staphylococcal chromosomal cassette composite island containing pbp4 and tagF genes in a commensal staphylococcal species: a possible reservoir for antibiotic resistance islands in Staphylococcus aureus.

Among methicillin-resistant Staphylococcus aureus isolates, a staphylococcal chromosomal cassette containing the mecA gene (SCCmec) is integrated into the chromosome at a unique site. SCCmec also contains unique ccrAB recombinase genes mediating its integration and excision from the genome and is flanked by characteristic left and right direct- and inverted-repeat sequences. A few non-mecA-containing SCC elements that have the other molecular features described above have recently been described. The origin of these cassettes is not clear. We have identified two new members of the SCC family integrated within orfX in Staphylococcus epidermidis strain ATCC 12228, neither of which carries mecA. One is a 57-kb element flanked by a unique 28-bp SCC direct repeat. It was called the SCC composite island (SCC-CI) because it carries a 19-kb SCC element (SCCpbp4) nested within it. SCCpbp4 contains pbp4 and tagF genes, as well as one pair of ccrAB genes (allotype 2) flanked by classical SCC-specific terminal repeats. External to SCCpbp4, SCC-CI contains a second pair of ccrAB genes (allotype 4), three IS431 elements, and genes mediating resistance to heavy metals. Genes mediating restriction-modification that may facilitate horizontal transfer are also present within SCC-CI, both within and outside SCCpbp4. Several novel arrangements of the SCC direct and inverted repeats were identified. Several long stretches of homology with other SCCs were found within and outside SCCpbp4. In view of the fact that SCC-CI was found in a commensal species, it may represent a reservoir for sequences involved in genetic shuffling between staphylococci and may contribute to the diversity found in SCC elements.

Amino Acid Sequence↗

Macrophage activation by an ornithine-containing lipid or a serine-containing lipid.

alpha-N-(3-Acyloxyacyl)-ornithine (or -serine) is the structure of lipoamino acids obtained by us previously from some gram-negative bacteria (Y. Kawai and I. Yano, Eur. J. Biochem. 136:531-538, 1983; Y. Kawai, I. Yano, and K. Kaneda, Eur. J. Biochem. 171:73-80, 1988; Y. Kawai, I. Yano, K. Kaneda, and E. Yabuuchi, Eur. J. Biochem. 175:633-641, 1988). The 3-acyloxyacylamide structure is present in both the lipoamino acids and lipid A of lipopolysaccharide (endotoxin). The efficacy of lipoamino acids (an ornithine-containing lipid and a serine-containing lipid) in activating C3H/HeSlc mouse peritoneal exudate macrophages was compared with that of bacterial lipopolysaccharide, because the two types of substances were expected to exhibit similar biological activities and physiological functions on the basis of their structural similarities. Actually, the lipoamino acids, as well as lipopolysaccharide, strongly activated the macrophages to generate the immunoregulatory substances prostaglandin E2 and interleukin-1, but their effect on the induction of L929 cell cytolytic factor (a possible tumor necrosis factor), another immunoregulatory substance, was weaker than that of lipopolysaccharide. The effect of lipoamino acids on the cytotoxicity of macrophages for EL-4 leukemia cells was very weak. However, all of these activities, as far as tested, were strongly enhanced by synergistic action with gamma interferon. Only the serine-containing lipid killed both C3H/HeSlc and C3H/HeJ macrophages to almost the same degree as endotoxin killed C3H/HeSlc macrophages. On the other hand, lethal toxicity for mice was not found with either the ornithine-containing lipid or the serine-containing lipid, even when 7 mg of compound was injected into a mouse. These studies suggest that the lipoamino acids are nontoxic characteristic immunoactivators.

Animals↗

NITROGEN-CONTAINING AND CARBOHYDRATE-CONTAINING ANTIGEN FROM ACTINOMYCES BOVIS.

Pirtle, E. C. (National Animal Disease Laboratory, Ames, Iowa), P. A. Rebers, and W. W. Weigel. Nitrogen-containing and carbohydrate-containing antigen from Actinomyces bovis. J. Bacteriol. 89:880-888. 1965.-Water-soluble, heat-stable antigens have been isolated from the supernatant fluids of broth cultures of Actinomyces bovis ATCC 10048 after 8 days of growth in a broth medium composed of Casamino Acids, yeast extract, Tween 80 (polyoxyethylene sorbitan monooleate), sodium thioglycolate, dextrose, and sodium chloride. The antigens were precipitated from the culture supernatant fluid with alcohol and purified by fractional precipitation with alcohol in the presence of calcium or zinc and by chromatography on diethylaminoethyl Sephadex. Two serologically active substances which differed in chemical composition and size were characterized. The larger one contained 71% hexose and 2.8% nitrogen, and the smaller one contained 45% hexose and 5.4% nitrogen. Heterogeneity of these fractions could not be demonstrated by electrophoresis in free solution at pH 7.8 or 2, by ultracentrifugal analysis, by double diffusion in agar, or by immunoelectrophoresis. Despite their differences in chemical composition and size, they appeared identical in activity as antigens in complement fixation and in double-diffusion tests in agar. Each was found to contain mannose as its chief component after hydrolysis and paper chromatography with three solvent systems. A small percentage of the total nitrogen may be attributed to the presence of amino sugars but the remainder is as yet unidentified.

Acetates↗

Substance P-containing neurons innervating LHRH-containing neurons in the septo-preoptic area of rats.

Neuroanatomical attempts have been made to determine the synapses between luteinizing hormone-releasing hormone (LHRH)-containing neurons and substance P (SP)-containing neurons in the hypothalamus of female rats. Wheat germ agglutinin was injected into the septo-preoptic area (SPA) and found to be incorporated into certain SP-containing neurons within the arcuate nucleus and the ventrolateral portion of the anterior hypothalamus. Hence, we used a preembedding double immuno-staining technique in demonstrating LHRH and SP neurons in the SPA. In light-microscopic preparations LHRH was labeled with 3,3'-diaminobenzidine tetrahydrochloride (DAB) as chromogen while SP was labeled with silver-gold particles; brown LHRH cells appeared to be surrounded by black silver-gold dots. In electron-microscopic preparations, the labelings for LHRH and SP were made reversely; SP was localized with DAB chromogen, and SP-containing axonal terminals appeared to make synaptic contacts on silver-gold-labeled LHRH cell bodies and dendritic processes. The terminals contained numerous small clear vesicles and some large dense-cored vesicles, and the synaptic membrane specialization appeared to be symmetric and asymmetric. These findings indicate that certain SP neurons existing in the arcuate nucleus and the ventrolateral portion of the anterior hypothalamus may project fibers to make synaptic contact with LHRH neurons in the SPA in the rat.

Animals↗

Effect of chemotherapy and reinfection on IgE-containing and IgG-containing circulating immune complexes, serum IgE and IgE antibodies in patients chronically infected with Schistosoma mansoni and Schistosoma haematobium.

The effect of chemotherapy and reinfection on circulating immune complexes (CIC), serum IgE, and parasite-specific IgE antibodies was studied in patients concomitantly infected with Schistosoma mansoni and Schistosoma haematobium. Before chemotherapy IgE- and IgG-containing CIC were present in high concentrations in all patients and IgE antibodies to S. mansoni and to S. haematobium in almost 80% of the cases. Serum IgE was excessively high (median 4,900 ku/1), but decreased to half of its initial value 2 months after chemotherapy. When reinfection with S. haematobium occurred onwards from month 4, the serum IgE reincreased and reached almost the pretreatment level at month 16. Effective chemotherapy was also followed by a rapid decrease of IgE-containing CIC and a slower, but significant, decrease of IgG-containing CIC. However, in contrast to IgE and IgE antibodies, the reinfection did not lead to a reincrease of CIC. It was interesting to note that the concentration of IgE as well as that of IgE-containing CIC was significantly correlated to the intensity of the infection. These investigations demonstrated that high levels of IgE- and IgG-containing CIC are present in chronic human schistosomiasis, but that the concentrations of CIC fall to normal values, when the parasites are eliminated by chemotherapy.

Antigen-Antibody Complex↗

Cholinergic septal afferent terminals preferentially contact neuropeptide Y-containing interneurons compared to parvalbumin-containing interneurons in the rat dentate gyrus.

Septal cholinergic neurons may affect hippocampal memory encoding and retrieval by differentially targeting parvalbumin (PARV)-containing basket cells and neuropeptide Y (NPY) interneurons. Thus, the cellular associations of cholinergic efferents, identified by the low-affinity, p75 neurotrophin receptor (p75(NTR)), with interneurons containing either PARV or NPY in the hilus of the rat dentate gyrus were examined in single sections using dual labeling immunoelectron microscopy. Most profiles immunoreactive (IR) for PARV and NPY were perikaryal and dendritic and found within the infragranular and central hilar regions, respectively, whereas most profiles with p75(NTR)-labeling were unmyelinated axons and axon terminals. Although PARV-labeled profiles were more numerous, p75(NTR)-labeled axons and terminals contacted few PARV-IR profiles compared to NPY-labeled profiles (2% of 561 for PARV vs 12% of 433 for NPY). Moreover, structures targeted by p75(NTR)-IR axon terminals varied depending on the presence of PARV or NPY immunoreactivity. p75(NTR)-IR terminals primarily contacted PARV-IR dendrites (87%) compared to somata (13%); however, they contacted more NPY-IR somata (57%) than dendrites (43%). p75(NTR)-labeled terminals formed exclusively symmetric (inhibitory-type) synapses with PARV-IR somata and dendrites; however, they formed mostly symmetric but also asymmetric (excitatory-type) synapses with NPY-IR somata and dendrites. These results suggest that septal cholinergic efferents in the dentate gyrus: (1) preferentially innervate NPY-containing interneurons compared to PARV-containing basket cells; and (2) may provide a more powerful (i.e., somatic contacts), yet functionally diverse (i.e., asymmetric and symmetric synapses), modulation of NPY-containing interneurons. Moreover, they provide evidence that neurochemical subsets of hippocampal interneurons can be distinguished by afferent input.

Afferent Pathways↗

Abnormal concentrations of CII, CIII, and E apolipoproteins among apolipoprotein B-containing, B-free, and A-I-containing lipoprotein particles in hemodialysis patients.

Serum concentrations of total cholesterol, triglycerides, and apolipoproteins (apo) A-I, B, CII, CIII, and E in 36 hemodialysis patients and nine anephric patients were compared with the concentrations in 34 normolipidemic subjects. The dialysis patients displayed a moderate hypertriglyceridemia (1.94 +/- 0.12 vs 1.09 +/- 0.11 mmol/L in controls, mean +/- SEM; P less than 0.001), apo CIII concentrations were also increased (130.2 +/- 2.1 vs 108.4 +/- 0.7 mg/L; P less than 0.001), whereas apo CII (34.5 +/- 0.5 vs 36 +/- 0.5 mg/L; P less than 0.05), apo E (22.7 +/- 0.3 vs 27.9 +/- 0.2 mg/L; P less than 0.001), and apo A-I (1.18 +/- 0.05 vs 1.31 +/- 0.04 g/L; P less than 0.05) were decreased. Concentrations of serum apo B were normal (0.86 +/- 0.03 vs 0.97 +/- 0.07 g/L). In the hemodialysis patients, apo CIII concentrations were increased in apo B-containing lipoproteins (30.1 +/- 0.5 vs 25.0 +/- 0.1 mg/L; P less than 0.001), whereas CII and E were decreased below control values (14.4 +/- 0.2 vs 16.8 +/- 0.1, and 8.2 +/- 0.2 vs 11.4 +/- 0.1 mg/L, respectively; P less than 0.001 each). By calculation, non-B-containing lipoproteins in the hemodialysis group had increased concentrations of apo CIII (100.1 +/- 2.1 vs 83.3 +/- 0.7 mg/L; P less than 0.001) and decreased amounts of apo E (14.5 +/- 0.4 vs 16.4 +/- 0.3 mg/L; P less than 0.001); apo CII content was unchanged (20.1 +/- 0.5 vs 19.3 +/- 0.5 mg/L). Results for apo CII, CIII, and E among apo A-I-containing lipoproteins in both normolipidemic and hemodialysis groups were similar to those in non-B-containing lipoproteins. Finally, the sole significant (P less than 0.01) difference between the anephric and hemodialysis groups was the lower apo E concentrations in the former group. Accumulation of triglyceride-rich lipoproteins in hemodialysis patients may thus be related to the enrichment of apo CIII in apo B-containing lipoproteins and to a marked decrease in the apo CII and E contents.

Adult↗

Copper- and zinc-containing superoxide dismutase, manganese-containing superoxide dismutase, catalase, and glutathione peroxidase in normal and neoplastic human cell lines and normal human tissues.

Copper- and zinc-containing superoxide dismutase, manganese-containing superoxide dismutase, catalase, and glutathione peroxidase form the primary enzymic defense against toxic oxygen reduction metabolites. Such metabolites have been implicated in the damage brought about by ionizing radiation, as well as in the effects of several cytostatic compounds. These enzymes were analyzed in 31 different human normal diploid and neoplastic cell lines and for comparison in 15 normal human tissues. The copper- and zinc-containing superoxide dismutase appeared to be slightly lower in malignant cell lines in general as compared to normal tissues. The content of manganese superoxide dismutase was more variable than the content of the copper- and zinc-containing enzyme. Contrary to what has been suggested before, this enzyme did not appear to be generally lower in malignant cells compared to normal cells. One cell line, of mesothelioma origin (P27), was extremely abundant in manganese-containing superoxide dismutase; the concentration was almost an order of magnitude larger than in the richest normal tissue. Catalase was very variable both among the normal tissues and among the malignant cells, whereas glutathione peroxidase was more evenly distributed. In neither case was a general difference between normal cells and tissues and malignant cells apparent. The myocardial damage brought about by doxorubicin has been linked to toxic oxygen metabolites; particularly, an effect on the glutathione system has been noted. The heart is one of the tissues which have a low concentration of enzymes which protect against hydroperoxides. However, the deviation from other tissues is probably not large enough to provide a full explanation for the high doxorubicin susceptibility. In the present survey, no obvious relationship between generally assumed resistance to ionizing radiation or to radical-producing drugs and cellular content of any of the enzymes could be demonstrated.

Catalase↗

Elevation of the metastable limits and absence of container surface nucleation for calcium oxalate crystallization in a urothelial-lined system as compared to glass containers.

We studied the metastable limits of calcium oxalate supersaturation in glass tubes and in urothelial-lined rat bladders from a solvent system consisting of 0.9 per cent saline. The metastable limit was significantly elevated at 30 min in the urothelial-lined system as compared to the glass-surfaced containers. Heterogeneous nucleation on the container surfaces consistently occurred with the saline solvent in glass tubes as the metastable limit was exceeded. Intraluminal or free-floating crystals (? homogenous nucleation) without container-adherent crystals occurred with the saline solvent in urothelial-lined containers. This study reveals important properties of crystallization in urothelium and emphasizes the potential errors in extrapolating from crystallization studies in vitro in glass containers to crystallization phenomena in biological systems.

Animals↗

Synthesis and hybridization properties of oligonucleotides containing polyamines at the C-2 position of purines: a pre-synthetic approach for the incorporation of spermine into oligodeoxynucleotides containing 2-(4,9,13-triazatridecyl)-2'-deoxyguanosine.

We have developed a synthesis of spermine-containing oligonucleotides (ODN-sper) which allows incorporation of multiple polyamine residues. This approach was based on the pertrifluoroacetylated 5'DMT-dGsper phosphoramidite synthon. Its coupling yield with resin-bound ODN decreased dramatically when close to the 3'-end. Optimization of the coupling conditions allowed 22-mer ODNs containing up to six spermine residues to be synthesized. Several ODNs of different sequences with 1-4 pendent spermines could be purified and their hybridization properties were evaluated. Duplex melting temperatures increased linearly with the number of polyamine residues (deltaTm/sper = 3.0 +/- 0.2 degrees C in 100mM NaCl). This compares very favorably with values reported for duplexes of similar initial stability containing other cation-substituted bases. Moreover, the stability increase was neither sequence nor position-dependent, and even contiguous spermine residues did not cross-talk. Extrapolation based on these findings leads to the conclusion that a duplex formed with a 22-mer oligonucleotide containing seven spermine residues would be as stable as genomic DNA, which highlights its potential for DNA strand invasion.

Aza Compounds↗

Artificial extracellular matrix proteins contain heparin-binding and RGD-containing domains to improve osteoblast-like cell attachment and growth.

Through the recombinant DNA technology, it is possible to create artificial extracellular matrix (aECM) proteins with domains chosen to modulate cellular behaviors. In this study, we constructed three aECM proteins containing both heparin-binding and RGD-containing domains (387RGDS, Tri-FN10, and TNC-FN3) produced in Escherichia coli. Promotion of MG-63 cell attachment and growth in two-dimensional (2D) cultures (tissue culture plate, polycaprolactone membrane) and 3D cultures (Cytodex 1 and Plastic Plus microcarriers) were demonstrated on these three aECM protein-coated surfaces. These three aECM proteins improved MG-63 cell attachment and growth in the order TNC-FN3 > Tri-FN10 > 387RGDS in both 2D and 3D cultures. This study is the first report of the construction of aECM proteins that contain both heparin-binding and RGD-containing domains used in osteoblast tissue engineering applications.

Amino Acid Sequence↗

A missense mutation in the RING finger motif of PEX2 protein disturbs the import of peroxisome targeting signal 1 (PTS1)-containing protein but not the PTS2-containing protein.

SK24 and PT54 mutant cells, which are peroxisome-deficient Chinese hamster ovary (CHO) cells isolated using peroxisomal forms of green fluorescent protein (GFP), were found to be defective in the PEX2 gene. The nucleotide sequences of PEX2 cDNA from the mutant cells were determined to identify mutation sites in the mutant cells. The mutation in SK24 cells changed cysteine to tyrosine at amino acid position 258, which is a component of the RING finger (C(3)HC(4)) motif in the carboxyl terminus of the protein. PT54 cells contained a nonsense mutation in the codon for glutamine at position 101, resulting in premature termination. The immunocytochemical analyses revealed distinct phenotypes between mutant cells defective in the PEX2 gene. Both mutant cells exhibited cytosolic mislocalizations on catalase and urate oxidase containing PTS1. On the other hand, on 3-ketoacyl-CoA thiolase containing PTS2, PT54 cells exhibited cytosolic mislocalization, but SK24 cells exhibited peroxisomal localization. When wild-type or mutant-type PEX2 cDNA was transfected into both mutant cells, the stable transformants restored the phenotype in accordance with the transfected cDNA. These observations indicate that an amino acid substitution, cysteine-258 to tyrosine, in the RING finger motif of PEX2 protein, whose function is required for peroxisomal localizations of both PTS1- and PTS2-containing proteins, results in a complete defect in the PTS1 pathway but not in the PTS2 pathway.

Amino Acid Sequence↗

Synaptic regulation of paraventricular arginine vasopressin-containing neurons by neuropeptide Y-containing monoaminergic neurons in rats. Electron-microscopic triple labeling.

Synaptic regulation of arginine vasopressin (AVP)-containing neurons by neuropeptide Y (NPY)-containing monoaminergic neurons was demonstrated in the paraventricular nucleus of the rat hypothalamus. NPY and AVP were immunolabeled in the pre- and the post-embedding procedures, respectively, and monoaminergic fibers were marked by incorporating 5-hydroxydopamine (5-OHDA), a false neurotransmitter. The immunoreaction for NPY was expressed by diaminobenzidine (DAB) chromogen, and that for AVP by gold particles. The DAB chromogen was localized on the surface of the membrane structures, such as vesicles or mitochondria, and on the core of large cored vesicles. Gold particles were located on the core of the secretory granules within the AVP cell bodies and processes. The incorporated 5-OHDA was found as dense cores within small or large vesicular structures. From these data, three types of nerve terminals were discernible: NPY-containing monoaminergic, NPY-containing non-aminergic, and monoaminergic fibers. The AVP cell bodies appeared to have synaptic junctions formed by these nerve terminals as well as by the unlabeled nerve terminals which have small clear vesicles and large cored vesicles. These different types of nerve terminals were frequently observed in a closely apposed position on the same AVP cell bodies. The functional relationships of these three types of neuronal terminals are discussed.

3,3'-Diaminobenzidine↗

ELISA quantitation of apolipoproteins in plasma lipoprotein fractions: ApoE in ApoB-containing lipoproteins (Lp B:E) and ApoB in ApoE-containing lipoproteins (Lp E:B).

Growing clinical evidence suggests that metabolic behavior and atherogenic potential vary within lipoprotein subclasses that can be defined by apolipoprotein variation. Variant constituency of apolipoproteins B and E (apoB and apoE) may be particularly important because of the central roles of these apolipoproteins in the endogenous lipid delivery cascade. ApoB is the sole protein of low-density lipoprotein (LDL), and like LDL cholesterol, the plasma apoB level has been positively correlated with risk for atherosclerotic disease. ApoE is a major functional lipoprotein in the triglyceride-rich lipoproteins, and may be crucial in the conversion of very low density lipoprotein (VLDL) to LDL. Based on work by others that enabled the quantititation of apoB-containing particles by content of up to two other types of apolipoprotein, we have developed a method for determining the amount of apoE in apoB-containing lipoproteins (Lp B:E) and the amount of apoB in apoE-containing lipoproteins (Lp E:B). From the Lp B:E and Lp E:B concentrations, the molar ratio of apoE to apoB in lipoproteins containing apoB and/or apoE in plasma can be determined. The methodology is fast, specific, and sensitive and should prove extremely useful in further categorizing lipoproteins and characterizing their behavior. In applying this method to clinical groupings of normo- and hyperlipidemia, we found that the plasma triglyceride level correlated with the apoE and Lp B:E concentrations in plasma, while the total cholesterol level correlated with the apoB and Lp E:B levels.

Antibody Specificity↗

Stability of sufentanil citrate in a portable pump reservoir, a glass container and a polyethylene container.

The stability of sufentanil citrate (100 ml, 5 micrograms/ml) in an admixture with sodium chloride 0.9% injection was investigated when filled in a portable pump reservoir with PVC wall, a glass container and a polyethylene container, at 32 degrees C, 4 degrees C and -20 degrees C for up to 21 days. No change in colour was visually observed in any of the samples during the 21-day storage period. A slight precipitation was noticed in three out of nine portable pump reservoirs, one at each storage temperature. There was a slight rise in pH at each storage temperature in all samples. There was approximately 13% loss of sufentanil citrate in the portable pump reservoirs stored at 32 degrees C during 2 days and 60% loss after 21 days, due to absorption of sufentanil citrate in the reservoir wall. No loss of sufentanil citrate could be detected in the portable pump reservoirs when stored at -20 degrees C and 4 degrees C. However, a serious inhomogeneity of the sufentanil citrate solution occurred after thawing at room temperature in the portable pump reservoirs which had been kept at -20 degrees C. The homogeneity could be restored by shaking for approximately 10 min. There was no change in the sufentanil citrate concentrations in the glass containers and polyethylene containers stored at the three temperatures. The portable pump reservoirs stored at 32 degrees C also showed a significant loss of vehicle due to evaporation (1.0 +/- 0.1 ml a week). This could not be detected in any of the other samples.

Absorption↗

Serotonin containing and histamine containing cells in human stomach. Identification of these cells by fluorescent microscope and electron microscope.

Paraformaldehyde (PF) treatment of epithelial cells of human stomach revealed intense yellow fluorescent granules within the cells. These granules were identified as serotonin on microspectrofluorometry. Serotonin containing cells were also identified as enterochromaffin cells (EC cells) under electron microscope and by the histochemical analysis. O-phthalaldehyde treatment (OPT) of epithelial cells in human stomach demonstrated whitish blue intracellular granules under fluorescence microscope. The cells containing these granules were identified as histamine containing cells on microspectrofluorometry, because of accordance in excitation and emission spectra with authentic histamine. Histamine-containing cells were considered to be identical to the mast cells, because of metachromatic property on toluidine blue staining and of close structural similarities under electron microscope.

Epithelial Cells↗