Solution-state conformations of natural products from chiroptical spectroscopy: the case of isocorilagin.

Isocorilagin, the α-anomer of the ellagitannin corilagin, has been frequently reported in the literature as a constituent of various plant species. Its identification is based mainly on the smaller value for the coupling constant of its anomeric proton when compared to that of corilagin. A careful investigation of the corilagin structure in both methanol and DMSO solutions using NMR, electronic and vibrational CD, and DFT and MD calculations confirmed that isocorilagin is the result of a solvent-induced conformational transition of corilagin, rather than its diastereoisomer. Corilagin changes from B1,4 and (o)S5 conformations of the ß-glucose core in DMSO-d6 to an inverted (1)C4 conformation in methanol-d4, which accounts for NMR observables attributed to the alleged α-anomer. This misassignment reinforces the risks of relying upon a single technique for structural elucidation and stereochemical analysis of complex natural products, especially those containing saccharide moieties.

Eosinophilic oesophagitis: an enigmatic, emerging disease.

Eosinophilic oesophagitis is a disease that has emerged in recent years. It is often associated with dysphagia and oesophageal food impaction in adults. The disease is characterised by infiltration of eosinophilic granulocytes into the oesophageal mucosa. This infiltrate may be responsible for the subtle peristaltic abnormalities that can be found in these patients. Endoscopic findings are usually absent or nonspecific, although a discrete circular ring pattern of the mucosa may be noticed. Occasionally, overt endoscopic abnormalities (such as exudative changes and shearing of the mucosa) can be found. The presence of at least 15 intraepithelial eosinophilic granulocytes per high-power field in random biopsies from the whole length of the oesophagus is considered to be diagnostic. Gastro-oesophageal reflux needs to be excluded as it may lead to eosinophilic infiltration as well. Adequate diagnosis is relevant for treatment and the prevention of unnecessary further investigations. The disease responds well to the ingestion of fluticasone propionate and its long-term prognosis is generally good. But when fluticasone is discontinued recurrent symptoms are common, and some cases are severe, needing treatment with systemic corticosteroids.

Acetylcholinesterase affinity-based screening assay on Lippia gracilis Schauer extracts.

The use of affinity-based protein assay produced by covalently linking acetylcholinesterase to magnetic beads, followed by chemical characterization of the selective binders using Liquid Chromatography with tandem High-Resolution Mass Spectrometry (LC-HRMS) is herein described for profiling crude aqueous natural product extracts. The fishing assay was first modulated using galanthamine as a reference ligand and then, the assay condition was adjusted for the aqueous leaves extracts obtained from Lippia gracilis Schauer (genotype 201) that was used as the natural combinatory library. From the experiments, a selective binder has been undisclosed with an accurate mass of 449.1131 m/z and identified as eriodictyol 2'-O-glucoside or eriodictyol 3'-O-glucoside. The selectivity of the binding assay was demonstrated, as much as, that erydictiol 7-O-glucoside was not fished, although it was present in the crude aqueous extract. The binding assay platform exhibited high specificity and did not require any sample pretreatment, making it appropriate for profiling binders at natural libraries.

Characterization of the glutathione S-transferase GSTT1 deletion: discrimination of all genotypes by polymerase chain reaction indicates a trimodular genotype-phenotype correlation.

Glutathione S-transferase theta enzyme activity involved in the metabolism of toxic compounds is absent in approximately 20% of Caucasians due to a homozygous deletion of GSTT1 (*0/0). Because the exact manner of the GSTT1 deletion was unknown, current genotyping of GSTT1 was limited to detect the presence versus complete absence of the gene by a GSTT1-specific polymerase chain reaction (PCR). Thus, heterozygous (*A/0) and homozygous (*A/A) samples could not be discriminated. We have characterized the boundaries of the deletion of the human glutathione S-transferase theta (GSTT1) gene: PCR mapping and sequencing revealed a 54251 bp fragment including GSTT1 to be deleted from chromosome 22, most likely by a homologous recombination event between two highly homologous sequence stretches that flank GSTT1. Based on the knowledge of the GSTT1*0 region, a PCR assay was devised for unambiguous discrimination of homozygously deleted (*0/0), heterozygously (*A/0) and homozygously GSTT1 carrying (*A/A) individuals. Genotyping of 180 samples of a Caucasian population revealed that the deletion consists of one defined allele, whose distribution in the population fits the Hardy-Weinberg equilibrium with observed 20% *0/0, 46% *A/0 and 34% *A/A individuals. The number of GSTT1*A alleles detected by this procedure correlated highly significant with the enzyme activity in erythrocytes. Genotype-phenotype comparisons demonstrated a codominant type of inheritance by a gene-dose effect: samples with two active alleles expressed a statistically significant higher enzymatic activity compared to those with one null allele (P < 0.0001, ANOVA).

Characterization of monoclonal antibodies generated against bovine and porcine prostacyclin synthase and quantitation of bovine prostacyclin synthase.

Monoclonal antibodies were raised against prostacyclin synthases purified from bovine and porcine aortae, respectively. Two monoclonal antibodies, RS1 and RS2, were purified and characterized. As shown by enzyme activity precipitation and Western blot analysis, in solubilized bovine and porcine aortae microsomes the monoclonal antibodies reacted only with prostacyclin synthase. The monoclonal antibody RS1 cross-reacts with partially purified prostacyclin synthase from human umbilical veins in an ELISA-based assay. None of the antibodies inhibited the enzyme activity. By combination of the monoclonal antibody RS2 with a polyclonal antibody we established an enzyme-linked immunosorbent assay (ELISA) for quantitation of bovine prostacyclin synthase. ELISA data were confirmed by Western blot analysis. Among different bovine tissues, aortae with 1665 +/- 200 ng/mg microsomal protein showed the highest content of PGIS. Significant lower concentrations were observed in tongue, lung, kidney and thymus ranging from 49 +/- 13.4 to 2.7 +/- 0.9 ng/mg protein. The monoclonal antibody RS1 binds to endothelial cells and vascular smooth muscle cells in human liver tissue.

The use of reverse transcription polymerase chain reaction to analyse large numbers of mRNA species from a single cell.

A PCR method is described for determining the expression of multiple heterogeneous mRNAs from single cells. The total mRNA pool of a single selected cell is subjected to reverse transcription and subsequent tailing with poly(dA). This cDNA is preamplified by a sequence non-specific PCR protocol using oligo(dT)-containing primers. The single cell cDNA library obtained permits the analysis of virtually unlimited numbers of mRNA species per cell using sequence-specific PCR. This procedure of multiple mRNA analysis enables phenotyping of any cell for its mRNA composition and could be used to study the cytokine mRNA expression of individual human T cells ex vivo. The method should greatly facilitate the analysis of combinatorial expression of known genes in any cell.

Selective renal vasoconstriction, exaggerated natriuresis and excretion rates of exosomic proteins in essential hypertension.

AIM: In essential hypertension (EH), the regulation of renal sodium excretion is aberrant. We hypothesized that in mild EH, (i) abnormal dynamics of plasma renin concentration (PRC) and atrial natriuretic peptide (ANP) are responsible for the exaggerated natriuresis, and (ii) exosomic protein patterns reflect the renal tubular abnormality involved in the dysregulation of sodium excretion. METHODS: After 2-week drug washout and 4-day diet, systemic and renal hemodynamics, cardio-renal hormones, glomerular filtration and renal excretion were studied in male patients during saline loading (SL). Excretion rates of exosome-related urinary proteins including apical membrane transporters were determined by proteomics-based methods. RESULTS: In patients, baseline renal vascular conductance was reduced (-44%, P < 0.001), but non-renal vascular conductances were normal while PRC was reduced and ANP elevated (both P < 0.01). SL induced exaggerated natriuresis and reduced PRC (P < 0.01), at normal suppression rate. SL increased arterial pressure in patients (+11 mmHg, P < 0.001), but not in controls; however, during time control, patients showed identical increases (+10 mmHg, P < 0.005) apparently dissociating arterial pressure from natriuresis. At baseline, excretion rates of 438 proteins ranged from 0.07 to 49.8 pmol (mmol creatinine)(-1); 12 proteins were found in all subjects, and 21 proteins were found in two or more patients, but not in controls. In patients, the excretion rate of retinoic acid-induced gene 2 protein was reduced, and excretion rates of other proteins showed increased variances compatible with pathophysiological and clinical applicability. CONCLUSION: Essential hypertension patients exhibit selective renal vasoconstriction and individually varying excretion rates of several exosome-related proteins. Hormonal changes, rather than arterial pressure, seem to cause exaggeration of natriuresis.

Type B lactic acidosis in solid malignancies.

BACKGROUND: Type B lactic acidosis is thought to be a rare complication of malignancy. It was first described in patients with acute leukaemia by Field et al. in 1963. Since then, it has been observed more often, in particular in haematological malignancies and rarely in solid tumours. METHODS: Previously reported cases of lactic acidosis in solid malignancy are reviewed. In addition, we report a case of type B lactic acidosis in a woman with metastatic breast cancer. Afterwards, we speculate on the elusive pathophysiology of this oncological emergency. RESULTS: 14 cases of lactic acidosis due to solid malignancies, without prior chemotherapy, were identified. The cases were published from the year 1978 to 2006. DISCUSSION: Several theories concerning the mechanism for type B lactic acidosis in solid malignancy have been postulated. During the last decade, more and more evidence supports the role of overproduction of lactic acid due to ischaemia in the neoplastic tissue bed and with cancer cells having an aberrant energy production.

Nude mice are nonpermissive towards the anti-dextran response of congenic cells.

Nude mice bearing allotype Ighb on a BALB/c genetic background (= CB nu/nu) are nonresponders to alpha (1----3)dextran (Dex), in contrast to BALB/c or BALB/c nu/nu. Although CB nu/nu mice accept transplants of congenic BALB/c, or BALB nu/nu lymphocytes, as shown by the expression of donor allotype Igha, they are not permissive for a primary anti-Dex response by the grafted cells. BALB/c or BALB nu/nu cells, however, give a strong anti-Dex response when grafted onto irradiated CB nu/nu or CB 23 (Ighb) euthymic mice. A thymus-independent, radiation-sensitive suppressor cell population is postulated, which specifically hinders the anti-Dex response, and which is exhibited by strains bearing that portion of chromosome 12 which codes for CH allotype Ighb, not containing the germ-line anti-Dex V/D genes. The suppressive action of Ighb lymphocytes could be demonstrated directly in staggered co-transfer experiments.

Idiotype suppression by maternal influence.

In BALB/c mice, antibodies to the alpha-(1-3) glucosidic linkage of some dextrans (Dex) carry the idiotype of the BALB/c myeloma protein J558. Both specific antibody and idiotype are inherited in a dominant fashion, linked to the immunoglobulin (Ig) (heavy chain) allotype Igla of BALB/c mice (Eur. J. Immunol. 1975. 5: 775). In F1 hybrid mice from the parent strains SJL and BALB/c, we were able to suppress the expression of anti-Dex antibodies by immunizing prospective SJL mothers to the J558 idiotype. The state of suppression in the progeny was ascertained by immunization with Dex, and tests for the following were carried out: (a) antibodies specific for Dex; (b) inhibition of such antibodies (if present) by antiidiotypic serum to protein J558; (c) presence of the J558 idiotype; and (d) concentration of lambda1 chains (which are associated with the 558 idiotype) in the serum. SJL mothers, once immunized, conferred suppression upon several successive litters, spanning a period of 4-5 months. Suppression in F1 progeny animals lasted for 16 weeks or more. Spleen cells from suppressed F1 mice which had neither been treated with Dex nor with J558 protein, were able to confer suppression to further F1 newborn mice.

The effect of Hydron burn wound dressing on burned rat and rabbit ear wound healing.

The effect of Hydron burn wound dressing was evaluated using a standard Walker burned rat model (N = 156) and a rabbit ear wound model (N = 34). In both studies Hydron was administered only once in one group and changed twice weekly in another. Animals were predesignated as controls and for pathologic examination. Gross observations, including photographic documentation, and the pathologic analyses revealed no gross purulence or sepsis. No deaths occurred in the rabbit study. Of the 156 animals in the rat study only two died; a culture of the wounds revealed no sepsis. Statistical analyses revealed that in the rat study the once only application was significantly better at 7 days postburn than the twice weekly treatment and control groups. No significant differences among the treatment and control groups used in the rabbit study were revealed. The application of Hydron burn wound dressing did not adversely affect the wound healing.

Demonstration that milk cells invade the suckling neonatal mouse.

Mouse milk cells were stained with rhodamine or fluorescein isothiocyanate and fed to young suckling mice. By visual examination of serial sections and by flow cytofluorometry, we were able to demonstrate directly the presence of these cells in peripheral tissues. It was estimated that at least 0.1% of the fed cells might infiltrate the young mouse, which is initially immunologically defenseless. This is in accordance with evidence from many sources for activity of maternally-derived lymphoid cells in young rodents.

Structural comparison of the external glycoproteins of human and simian immunodeficiency virus.

The structural variability of the external glycoproteins of primate immunodeficiency viruses, has, so far, been investigated exclusively by sequence comparison of the respective proviral genomes. We have examined the structural relationship amount the external glycoproteins from three specific human immunodeficiency viruses (HIF-1, HIV-2), three specific simian immunodeficiency viruses from macaques (SIVmac) and three specific SIV from African green monkeys (SIVagm) by peptide mapping. Differences among glycoproteins were most pronounced between HIV-1 and SIVmac, as well as HIV-2. Two specific glycoproteins from independent SIVagm isolates were closely related to HIV-1, whereas the glycoprotein from a third SIVagm isolate was more similar to those of SIVmac and HIV-2. Our analysis reflects the classification of primate immunodeficiency viruses into three groups, the HIV-2 and SIVmac viruses, the green monkey isolates and HIV-1.

p100: a novel proliferation-associated nuclear protein specifically restricted to cell cycle phases S, G2, and M.

By immunization with nuclear lysates of L428 cells, we raised a monoclonal mouse antibody, Ki-S2 (IgG1). In Western blots, this antibody recognizes a nuclear antigen with an apparent molecular mass of 100 kD, termed p100. Protein sequencing of p100 showed that this is a hitherto unknown protein. Immunohistochemical examination of cryostat and paraffin sections of nearly all human tissue types and neoplasms showed that p100 was exclusively expressed in the nuclei of a fraction of proliferating cells. Cell sorting and fluorescence-activated cell sorting analysis of stimulated peripheral blood mononuclear cells showed that p100 was exclusively expressed in proliferating cells from the transition G1/S until the end of cytokinesis. During mitosis, this protein is strictly associated with the spindle pole and with the mitotic spindle, whereas during S and G2, p100 is diffusely distributed throughout the cell nucleus. Immediately after completion of cytokinesis, p100 was rapidly degraded. In L428 cells, p100 is phosphorylated at least during mitosis. It has a turnover time of about 1 hour. Studies on routinely processed paraffin sections of specimens of malignant lymphoma, benign and malignant nevocellular tumors, and breast cancer showed that in all cases less than 40% of the Ki-67-positive growth fraction expressed p100. Thus, p100 might prove to be a more reliable measure of cellular proliferation and one that is more closely correlated to cancer prognosis, beyond its general biologic relevance as a cell cycle protein.

[Experimental microbiological research on instrument and denture disinfection with a disinfectant spray for dental practice and the use of patients. 2. Virological studies; the evaluation summary]. / Mikrobiologisch-experimentelle Untersuchungen zur Instrumenten- und Prothesendesinfektion mit Desinfektionsmittel-Sprays für die stomatologische Praxis und zu Händen des Patienten. 2. Mitteilung: Virologische Untersuchungen; zusammenfassende Bewertung.

Carrier experiments using Echo- and Influenza-viruses on denture basis resins and fragments of orthodontic appliances resulted in good and even very good virucidal effects of disinfectant sprays Desident and Fesia-sept. The application of the disinfectant as spray seem to be advantageous, but is not recommendable. The diverse causes of this conclusion are discussed and an alternative way is proposed.

[Experimental microbiological research on instrument and denture disinfection with a disinfectant spray for dental practice and the use of patients. 1. The formulation of the problem and bacteriological studies]. / Mikrobiologisch-experimentelle Untersuchungen zur Instrumenten- und Prothesendesinfektion mit Desinfektionsmittel-Spray für die stomatologische Praxis und zu Händen des Patienten. 1. Mitteilung: Problemstellung, bakteriologische Untersuchungen.

Three disinfectant sprays (Arugeen, Desident and Fesia-sept) proved to be very efficient against pure cultures of aero bacteria species, Cand. albicans, saliva flora and entero- and influenza viruses. The methods included carrier experiments using denture basis resins, fragments of orthodontic appliances and screws (instead of instruments).

[Atrophy of the corpus mucosa of the stomach simulating polyposis]. / Atrophie der Corpusschleimhaut des Magens unter dem Bild einer Polypose.

We report on five cases of mucosal atrophy of the gastric body, under the aspect of a polyposis: These polyps showed intact mucosa with abundant fundic glands and were situated in the diffuse atrophic mucosa of the gastric body. Four of these cases were manifestations of type A gastritis, in one case the lesion surrounded a peptic ulcer. Laboratory findings of serologically analysed cases with type A gastritis showed elevated parietal cell antibodies in all cases, two of three cases showed a high level of gastrin, the values for vitamin B12 showed different levels; no patients revealed antibodies against the intrinsic factor, and no patient had pernicious anemia. The family history revealed three cases with cancer of the stomach on one side of the parents. It is important to biopsy polyps and the flat mucosa separately in order to verify this form of atrophy of the gastric mucosa and to also exclude small polypous tumors of the mucosa. Observation of the number of polyps allows a control of the extent of the atrophy of the mucosa.

Polyclonal B-cell activation by a synthetic analogue of bacterial lipoprotein is functionally different from activation by bacterial lipopolysaccharide.

The reactivity of 38 murine strains to a synthetic analogue of bacterial lipoprotein, tripalmitoyl-pentapeptide (TPP), was tested and compared with the reactivity to lipopolysaccharide (LPS). These strains include common laboratory mice and H-2 recombinant inbred lines, as well as some newly bred lines originating from animals recently captured in different regions of Europe. All animals analysed were reactive to TPP and polyclonally activated to proliferation and immunoglobulin synthesis. Large differences in mitogen reactivities of various H-2 recombinant inbred strains suggest that MHC or closely linked gene products influence the reactivity to the LPS and TPP mitogens. By analysing the frequencies of precursor cells reactive to TPP or LPS and the isotype patterns obtained after stimulation, we demonstrated that both mitogens activate individual B cells in different ways.

High-dose intravenous immunoglobulin for post-transfusion purpura.

Five patients with post-transfusion purpura (four due to Zw(a), one presumably due to HLA antibodies) were treated with intravenous immunoglobulin (IgG) at doses of 0.4 g per kg body weight. IgG therapy was immediately effective as indicated by cessation of bleeding and rise of platelet counts in four out of five cases.