Colony formation by subpopulations of human T lymphocytes. III. Antigenic phenotype and function of colony-forming cells, colony cells, and their expanded progeny

The antigenic phenotype of individual PHA-induced T lymphocyte colonies was studied with a direct immunofluorescence technique using fluorescein-labeled anti-Leu-2a and anti-Leu-3a antibodies. Of the colonies grown from mononuclear peripheral blood cells 85% were Leu-3a+ (inducer/helper phenotype), 12% were Leu-2a+ (suppressor/cytotoxic phenotype), and 3% contained equal numbers of Leu-2a+ and Leu-3a+ cells. Fluorescence-activated cell sorter (FACS) separated T-cell subsets showed that Leu-2a+ cells and Leu-3a+ cells form exclusively Leu-2a+ and Leu-3a+ colonies, respectively. Leu-3a+ cells formed colonies in both the absence and presence of conditioned medium (PHA-CM), whereas colony formation by Leu-2a+ cells was absolutely dependent on PHA-CM. Mixing experiments with FACS-separated T-cell subsets showed that Leu-2a+ cells inhibit colony formation by Leu-3a+ cells in a cell dose-dependent manner both in the presence and absence of PHA-CM. Phenotype analysis of individual colonies from mixing experiments strongly suggested monoclonal proliferation in the present colony assay system. The majority of expanded T-cell colonies showed helper activity in a reverse hemolytic plaque-forming B-cell assay, although to a lesser degree as compared to that of freshly isolated T lymphocytes.     

Brain Tryptophan Hydroxylation in the Portacaval Shunted Rat: A Hypothesis for the Regulation of Serotonin Turnover In Vivo

Regional and whole-brain tryptophan-hydroxylating activity and serotonin turnover were investigated in portacaval shunted (PCS) rats using an in vivo decarboxylase inhibition assay. To saturate tryptophan hydroxylation with amino acid substrate, rats were administered a high dose of tryptophan 1 h prior to analysis of brain tryptophan, 5-hydroxytryptophan, serotonin, and 5-hydroxyindoleacetic acid. The analysis revealed, as expected, higher brain concentrations of tryptophan and 5-hydroxyindoles and increased serotonin synthesis rate in PCS rats as compared with shamoperated controls. Saturating levels of brain tryptophan were achieved in both PCS and sham animals after exogenous tryptophan administration. The tryptophan load resulted in increased brain serotonin turnover in all regions and in whole brain compared with rats that did not receive a tryptophan load. Tryptophan-loaded PCS rats showed increased brain serotonin turnover compared with tryptophan-loaded sham rats. Regionally, this supranormal tryptophan-hydroxylating activity was most pronounced in the mesencephalon-pons followed by the cortex. It is concluded that, at least in the PCS rat, brain tryptophan hydroxylation is an inducible process. Since it is known that brain tissue from PCS rats undergoes a redox shift toward a reduced state and that the essential cofactor tetrahydrobiopterin is active in tryptophan hydroxylation only when present in its reduced form, it is hypothesized that this is the reason for the supranormal tryptophan-hydroxylating activity displayed by the PCS rats. The hypothesis further suggests that alterations in tetrahydrobiopterin availability may serve as a mechanism by which brain tryptophan hydroxylation, and therefore serotonin turnover, can be regulated with high sensitivity in vivo.     

Pharmacological and Biochemical Properties of the γ-Aminobutyric Acid–Benzodiazepine Receptor Protein from Codfish Brain

The gamma-aminobutyric acidA (GABAA) receptor of codfish brain has been purified to homogeneity and contains a single polypeptide band of 56 kDa molecular mass. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE) of codfish GABA receptor photoaffinity-labeled by both [3H]flunitrazepam ([3H]Flu) and [3H]muscimol showed a single radioactive peak with molecular mass of 56 kDa, in contrast to the multiple subunits found in other vertebrate species. The codfish receptor, purified using benzodiazepine (BZ, Ro 7-1986/1) affinity chromatography, contains an apparent single band both by isoelectric focussing and on a silver-stained SDS gel. The receptor density and affinity constants for [3H]muscimol and [3H]Flu binding are comparable to those in mammalian brain, and the specific activity (greater than 1,000 pmol/mg of protein) is comparable to that of preparations purified from those sources. The pharmacological specificity of the codfish GABA-BZ receptor is generally similar to that of mammalian brain, including GABA-BZ coupling. The BZ binding exhibits homogeneous kinetic properties resembling those of the mammalian BZ2 receptor type, and shows strong GABA enhancement of [3H]Flu binding and weaker pentobarbital potentiation. This is consistent with other observations of an earlier phylogenetic, as well as ontogenetic, emergence in mammals of the BZ2 receptor subtype than the BZ1. Codfish GABA receptor is postulated to be a homo-oligomer in which the conformation of GABA and BZ recognition sites is very similar to that in the mammalian hetero-oligomeric GABAA receptor. The codfish receptor appears to be encoded by an ancestral gene and indicates an early development of BZ-GABA coupling.     

Organocatalysis with endogenous compounds: Towards novel non-enzymatic reactions

The aldol reaction of the endogeneous compounds acetone and methylglyoxal has been studied using organocatalysis in relation to biologically relevant non-enzymatic reactions. Under preparative conditions, 3-hydroxy-2,5-hexadione, known as Henze’s ketol, is formed in high yield and with enantioselectivities up to 88% ee. Furthermore, Henze’s ketol is also formed under simulated physiological conditions at micromolar scale, indicating that this reaction might take place in living organisms.     

Decreased Number of Benzodiazepine Receptors in Frontal Cortex of Rat Brain Following Long-Term Lithium Treatment

Chronic administration of lithium led to a decreased number of benzodiazepine receptors (ca. 20%) in frontal cortex of rat brain, whereas no change was observed in the binding characteristics in the remaining part of the cortex and in the hippocampus and the cerebellum. Long-term lithium treatment did not change the binding of [3H]lysergic acid diethylamide and [3H]quinuclidinyl benzilate to membranes of various brain regions in the rat. We concluded that the effect of lithium on the benzodiazepine receptor is brain region specific and cannot be explained as a consequence of a reduced gamma-aminobutyric acid-ergic stimulation of benzodiazepine receptor, as the change in receptor binding was due to a change in the number of receptors rather than in the affinity constant.     

The differential cardio-respiratory responses to ambient hypoxia and systemic hypoxaemia in the South American lungfish, Lepidosiren paradoxa

Lungfishes (Dipnoi) occupy an evolutionary transition between water and air breathing and possess well-developed lungs and reduced gills. The South American species, Lepidosiren paradoxa, is an obligate air-breather and has the lowest aquatic respiration of the three extant genera. To study the relative importance, location and modality of reflexogenic sites sensitive to oxygen in the generation of cardio-respiratory responses, we measured ventilatory responses to changes in ambient oxygen and to reductions in blood oxygen content. Animals were exposed to aquatic and aerial hypoxia, both separately and in combination. While aerial hypoxia elicited brisk ventilatory responses, aquatic hypoxia had no effect, indicating a primary role for internal rather than branchial receptors. Reducing haematocrit and blood oxygen content by approximately 50% did not affect ventilation during normoxia, showing that the specific modality of the internal oxygen sensitive chemoreceptors is blood PO(2) per se and not oxygen concentration. In light of previous studies, it appears that the heart rate responses and the changes in pulmonary ventilation during oxygen shortage are similar in lungfish and tetrapods. Furthermore, the modality of the oxygen receptors controlling these responses is similar to tetrapods. Because the cardio-respiratory responses and the modality of the oxygen receptors differ from typical water-breathing teleosts, it appears that many of the changes in the mechanisms exerting reflex control over cardio-respiratory functions occurred at an early stage in vertebrate evolution.     

Increased levels of soluble CD40L in African tick bite fever: possible involvement of TLRs in the pathogenic interaction between Rickettsia africae, endothelial cells, and platelets

The pathophysiological hallmark of spotted fever group rickettsioses comprises infection of endothelial cells with subsequent infiltration of inflammatory cells. Based on its ability to promote inflammation and endothelial cell activation, we investigated the role of CD40L in African tick bite fever (ATBF), caused by Rickettsia africae, using different experimental approaches. Several significant findings were revealed. 1) Patients with ATBF (n = 15) had increased serum levels of soluble CD40 ligand (sCD40L), which decreased during follow-up. 2) These enhanced sCD40L levels seem to reflect both direct and indirect (through endothelial cell activation involving CX3CL1-related mechanisms) effects of R. africae on platelets. 3) In combination with sCD40L, R. africae promoted a procoagulant state in endothelial cells by up-regulating tissue factor and down-regulating thrombomodulin expression. 4) Although the R. africae-mediated activation of platelets involved TLR2, the combined procoagulant effects of R. africae and sCD40L on endothelial cells involved TLR4. 5) Doxycycline counteracted the combined procoagulant effects of R. africae and sCD40L on endothelial cells. Our findings suggest an inflammatory interaction between platelets and endothelial cells in ATBF, involving TLR-related mechanisms. This interaction, which includes additive effects between sCD40L and R. africae, may contribute to endothelial inflammation and hypercoagulation in this disorder.     

Proton nuclear magnetic resonance spectroscopy based investigation on propylene glycol toxicosis in a Holstein cow

Background  

It is unknown which metabolites are responsible for propylene glycol (PG)-induced toxicosis, and a better understanding of the underlying mechanisms explaining incidences of abnormal behaviour of dairy cows fed PG is therefore needed.