A COMPARISON BETWEEN FLUOROMETRIC AND MASS FRAGMENTOGRAPHIC DETERMINATIONS OF HOMOVANILLIC ACID AND 5-HYDROXYINDOLEACETIC ACID IN HUMAN CEREBROSPINAL FLUID

HVA and 5-HIAA in human cerebrospinal fluid were quantitatively determined by both fluorometry and mass fragmentography. The homovanillic acid values obtained by fluorometry were significantly lower than those obtained by mass fragmentography (P < 0.05). The correlation coefficient between values for HVA obtained by the two methods was high, r= 0.90. For 5-HIAA the concentrations obtained by the two methods were not significantly different while the correlation coefficient was lower, r= 0.55.     

Alpha-1-antitrypsin deficiency and juvenile liver disease Ultrastructural observations compared with light microscopy and routine liver tests

Sixteen children (aged between 1 month and 20 years) with alpha-1-antitrypsin deficiency (PiZ) were investigated by liver biopsy on one or more occasions. Eight patients had suffered from neonatal cholestasis, and two of them were investigated during the cholestatic period as well. The clinical status and liver function tests were compared with the light and electron microscopical findings. According to the light microscopical analyses at the latest investigation, the cholestatic and noncholestatic patients were classified as healthy, fibrotic or cirrhotic cases. All livers displayed periodic acid-Schiff positive, diastase-resistant globules in some but not all periportally located hepatocytes. By electron microscopy accumulation of retained secretory material was found in all PiZ patients. This accumulation was most conspicuous in the smooth endoplasmic reticulum of hepatocytes. alpha-1-antitrypsin deficiency seems to affect some, but not all hepatocytes. In the affected cells disappearance or hypotrophy of the Golgi complex could be observed. The intracellular transport of very low density lipoproteins (VLDL) was apparently not affected. The migration block in alpha-1-antitrypsin deficiency seems to occur before transportation to the Golgi complex. The extent of the involvement was not strictly age-dependent. There was no ultrastructural evidence of subclinical cholestasis as a possible triggering factor in the development of cirrhosis.     

Competitive Advantage Provided by Bacterial Motility in the Formation of Nodules by Rhizobium meliloti

The effect of motility on the competitive success of Rhizobium meliloti in nodule production was investigated. A motile strain formed more nodules than expected when mixed at various unfavorable ratios with either flagellated or nonflagellated nonmotile derivatives. We conclude that motility confers a selective advantage on rhizobia when competing with nonmotile strains.     

Occurrence and characterization of "avium-like" Mycobacteria isolated from animals in Sweden

431 cultures of "avium-like" mycobacteria (ALM) were isolated from wild and domestic animals during 1974--76 at the National Veterinary Institute, Stockholm. Of these, 50 isolates from pigs were studied by growth-chromogenicity, pathogenicity, and biochemical tests. Furthermore, thin-layer chromathography was performed, and on some isolates serotyping. All 50 isolates belonged to Runyon's group III and were pathogenic for chicken; none was capable of splitting oleic acid from Tween 80. 47 gave tellurite reduction within a period of three days; one was arylsulphatase-positive after three days and a further four after 14 days. The biological and biochemical tests permit assignation of the 50 isolates to the M. avium-intracellulare complex. The lipid patterns of the isolates examined were analysed by thin-layer chromatography. Thirty-five of the isolates showed a lipid pattern similar to that of A 2 of the fowl reference strain; three belonged to lipid type A 1 and four to A 3. Eight could not be typed. Of 22 isolates, 14 could be assigned to M. avium serotypes.     

T cell stimulation remodels the latently HIV-1 infected cell population by differential activation of proviral chromatin

The reservoir of latently HIV-1 infected cells is heterogeneous. To achieve an HIV-1 cure, the reservoir of activatable proviruses must be eliminated while permanently silenced proviruses may be tolerated. We have developed a method to assess the proviral nuclear microenvironment in single cells. In latently HIV-1 infected cells, a zinc finger protein tethered to the HIV-1 promoter produced a fluorescent signal as a protein of interest came in its proximity, such as the viral transactivator Tat when recruited to the nascent RNA. Tat is essential for viral replication. In these cells we assessed the proviral activation and chromatin composition. By linking Tat recruitment to proviral activity, we dissected the mechanisms of HIV-1 latency reversal and the consequences of HIV-1 production. A pulse of promoter-associated Tat was identified that contrasted to the continuous production of viral proteins. As expected, promoter H3K4me3 led to substantial expression of the provirus following T cell stimulation. However, the activation-induced cell cycle arrest and death led to a surviving cell fraction with proviruses encapsulated in repressive chromatin. Further, this cellular model was used to reveal mechanisms of action of small molecules. In a proof-of-concept study we determined the effect of modifying enhancer chromatin on HIV-1 latency reversal. Only proviruses resembling active enhancers, associated with H3K4me1 and H3K27ac and subsequentially recognized by BRD4, efficiently recruited Tat upon cell stimulation. Tat-independent HIV-1 latency reversal of unknown significance still occurred. We present a method for single cell assessment of the microenvironment of the latent HIV-1 proviruses, used here to reveal how T cell stimulation modulates the proviral activity and how the subsequent fate of the infected cell depends on the chromatin context.     

Molecular phylogenetics and generic assessment in the tribe Morindeae (Rubiaceae–Rubioideae): How to circumscribe Morinda L. to be monophyletic?

Most of the species of the family Rubiaceae with flowers arranged in head inflorescences are currently classified in three distantly related tribes, Naucleeae (subfamily Cinchonoideae) and Morindeae and Schradereae (subfamily Rubioideae). Within Morindeae the type genus Morinda is traditionally and currently circumscribed based on its head inflorescences and syncarpous fruits (syncarps). These characters are also present in some members of its allied genera, raising doubts about the monophyly of Morinda. We perform Bayesian phylogenetic analyses using combined nrETS/nrITS/trnT-F data for 67 Morindeae taxa and five outgroups from the closely related tribes Mitchelleae and Gaertnereae to rigorously test the monophyly of Morinda as currently delimited and assess the phylogenetic value of head inflorescences and syncarps in Morinda and Morindeae and to evaluate generic relationships and limits in Morindeae. Our analyses demonstrate that head inflorescences and syncarps in Morinda and Morindeae are evolutionarily labile. Morinda is highly paraphyletic, unless the genera Coelospermum, Gynochthodes, Pogonolobus, and Sarcopygme are also included. Morindeae comprises four well-supported and morphologically distinct major lineages: Appunia clade, Morinda clade (including Sarcopygme and the lectotype M. royoc), Coelospermum clade (containing Pogonolobus and Morinda reticulata), and Gynochthodes–Morinda clade. Four possible alternatives for revising generic boundaries are presented to establish monophyletic units. We favor the recognition of the four major lineages of Morindeae as separate genera, because this classification reflects the occurrence of a considerable morphological diversity in the tribe and the phylogenetic and taxonomic distinctness of its newly delimited genera.     

Quantitative affinity chromatography: increased versatility of the technique for studies of ligand binding

The potential of affinity chromatography for the characterization of strong solute-ligand interactions is explored by studying the NADH-dependent elution of rabbit muscle lactate dehydrogenase from a column of trinitrophenyl-Sepharose in 0.067 M phosphate, pH 7.2. An interesting development is the simplification of the general affinity chromatography theory that emanates from the use of affinity matrices with a high concentration of immobilized reactant groups. The resultant expression allows evaluation of the intrinsic association constant for solute-ligand interactions from a single series of either zonal or frontal affinity chromatographic experiments conducted in the presence of a range of free ligand concentrations. Thus, contrary to previous belief, an affinity matrix designed for solute purification work should prove to be an asset for, rather than an impediment to, the study of solute-ligand interactions by quantitative affinity chromatography.     

Fatty acids increase paracellular absorption of aluminium across Caco-2 cell monolayers

Passive paracellular absorption, regulated by tight junctions (TJs), is the main route for absorption of poorly absorbed hydrophilic substances. Surface active substances, such as fatty acids, may enhance absorption of these substances by affecting the integrity of TJ and increasing the permeability. It has been suggested that aluminium (Al) absorption occurs mainly by the paracellular route. Herein, we investigated if physiologically relevant exposures of fully differentiated Caco-2 cell monolayers to oleic acid and docosahexaenoic acid (DHA), which are fatty acids common in food, increase absorption of Al and the paracellular marker mannitol. In an Al toxicity test, mannitol and Al absorption through Caco-2 cell monolayers were similarly modulated by Al concentrations between 1 and 30 mM, suggesting that absorption of the two compounds occurred via the same pathways. Exposure of Caco-2 cell monolayers to non-toxic concentrations of Al (2 mM) and ¹⁴C-mannitol in fatty acid emulsions (15 and 30 mM oleic acid, 5 and 10 mM DHA) caused a decreased transepithelial electrical resistance (TEER). Concomitantly, fractional absorption of Al and mannitol, expressed as percentage of apical Al and mannitol retrieved at the basolateral side, increased with increasing dose of fatty acids. Transmission electron microscopy was applied to assess the effect of oleic acid on the morphology of TJ. It was shown that oleic acid caused a less structured morphology of TJ in Caco-2 cell monolayers. Taken together our findings indicate that fatty acids common in food increase the paracellular intestinal absorption of Al. These findings may influence future risk assessment of human Al exposure.     

Reproducibility of protein identification of selected cell types in Barrett's esophagus analyzed by combining laser-capture microdissection and mass spectrometry

Barrett's esophagus (BE) is associated with increased risk of esophageal adenocarcinoma (EAC) and characterized by replacement of normal esophageal squamous epithelium by columnar epithelium. These alterations are also reflected in changes in the protein-expression profiles of the cell types involved. To separately investigate the proteomes of selected cell-types we combined laser-capture microdissection (LCM) and liquid chromatography-mass spectrometry (LC-MS). Aims were to determine the sensitivity, specificity, and technical reproducibility of the sampling method, and the biological variability within and between biopsies and patients. Frozen biopsies were cryo-sectioned, samples of around 2000 epithelial or stroma cells microdissected, digested and measured by Orbitrap LC-MS. Proteins were then identified by MS/MS database search and quantified by label-free analysis. An average of 366 protein-groups were identified per sample, and more protein-groups were found in epithelial samples than in stromal samples (442 vs 301, p < 0.0001). Altogether, 1254 distinct protein-groups were found, 289 and 88 of them significantly more often in epithelial and stroma samples, respectively. We assessed five different types of reproducibilities (run-to-run, intrabiopsy, biopsy-to-biopsy, experiment-to-experiment, and patient-to-patient) for protein identification and protein quantification. Reproducibility of protein identification ranged from 78 to 57%, and standard deviation of protein quantification was on patient-to-patient level four times higher than for run-to-run. We conclude that sampling around 2000 cells requires groups of 32 samples to detect significant, over 10-fold differences in protein abundances and thus creates a successful compromise between throughput and quality of results. We therefore believe that this method is suitable for investigating protein-expression profiles during carcinogenesis.