Determination of deuterium-labeled tryptophan in proteins by means of high-performance liquid chromatography and thermospray mass spectrometry

In order to develop direct methods for determining the extent of metabolic incorporation of isotopically labeled amino acids into a protein, the determination of deuterated tryptophan in [2H5]tryptophan-bacteriorhodopsin was investigated. The isotopically modified protein was subjected to alkaline hydrolysis. After phenyl isothiocyanate derivatization of the hydrolysate, the mixture was separated by reversed-phase liquid chromatography. Field desorption mass spectrometry and thermospray mass spectrometry were investigated for their ability to determine the ratio between [2H5]tryptophan and total tryptophan in the collected fractions. In order to check the procedure a set of known tryptophan/[2H5]tryptophan mixtures were passed through the same derivatization, HPLC separation, and lyophilization procedure as used for the biological samples.     

Fibronectin initiates fibrin production by rabbit corneal endothelial cells in vitro

Confluent rabbit corneal endothelial cells incubated in the absence of serum do not produce fibrinogen. When exogenous fibronectin is added to these cultures, fibrinogen production is observed. Fibronectin concentrations stimulate fibrinogen synthesis by endothelial cells in a dose-response fashion. This direct interaction of fibronectin and fibrinogen may be important in both wound healing processes and pathological states.     

Analysis of the binding of p-chlorophenyl-methoxybenzyl-ketoxime (CPMB-oxime) to mitochondrial cytochrome c reductase.

Cytochrome c reductase is inhibited by p-chlorophenyl-methoxybenzyl-ketoxime (CPMB-oxime). CPMB-oxime induces a red-shift of the reduced spectrum of cytochrome b. The inhibitor blocks the oxidation of ubihydroquinone at the QP center of this enzyme in a non-competitive way. The binding stoichiometry equals one inhibitor molecule per Qp center. The apparent Kd in a red-shift assay was 6.9 +/- 0.6 microM. All binding characteristics analysed in this study were very similar to those of the E-beta-methoxyacrylate inhibitors, although the chemical structure is different from these inhibitors. This result is interpreted as a support for the inhibitory mechanism based on the model of a 'catalytic switch' proposed recently for the E-beta-methoxyacrylate inhibitors (MOA-inhibitors (Brandt and von Jagow, Eur. J. Biochem.     

METABOLIC CHANGES IN THE BRAINS OF MICE FROZEN IN LIQUID NITROGEN

Abstract— Autolytic changes in the mouse brain, occurring during immersion of the animal in liquid nitrogen, were evaluated by measuring the tissue concentrations of glucose, lactate, pyruvate, α-oxoglutarate, phosphocreatine, creatine, ATP, ADP and AMP. The values thus obtained were compared with those obtained in paralysed mice under nitrous oxide anaesthesia, the brains of which were frozen in such a way that arterial blood pressure and oxygénation were upheld during the freezing. Immersion of unanaesthetized mice in liquid nitrogen gave rise to significant alterations in phosphocreatine, creatine, lactate, lactate/pyruvate ratio, ADP and AMP. A comparison with values obtained in paralysed and anaesthetized mice that were frozen by immersion in liquid nitrogen showed that the metabolic changes observed in the unanaesthetized animals could not be caused by an anaesthetic effect on the metabolic pattern. It is concluded that autolysis in the mouse brain occurs during immersion of the animal in a coolant, mainly because arterial hypoxia develops before the tissue is frozen. A comparison with previous results on rat cerebral cortex indicates that mice offer no advantage for studies of cerebral metabolites in unanaesthetized animals. In both species, accurate analyses of labile cerebral metabolites require that the brain is frozen in a way that prevents arterial hypoxia during the fixation of the tissue.     

Cyclic nucleotide alterations in mixed leukocyte reaction: a preliminary report

Endogenous cyclic adenosine and guanosine monophosphate (cAMP, cGMP) levels were studied in human peripheral blood lymphocytes during mixed leukocyte reactions (MLR). cAMP level was consistently elevated in one-way MLR, with good correlation to 3H-thymidine uptake in these reactions. In contrast, cGMP level was practically unchanged. Irradiation of reacting cell populations resulted in inhibition of cyclic nucleotide phosphodiesterase (PDE) activity. These results suggest that metabolic alterations in cAMP may be associated with immune reactions of cellular recognition.     

The precursor of interleukin-1 alpha is phosphorylated at residue serine 90

Mononuclear phagocytes release interleukin-1 (IL-1), a 17-kDa polypeptide with diverse biological activities. IL-1 is synthesized as a precursor (31 kDa) which lacks a signal sequence or hydrophobic domains that could facilitate transmembrane translocation. Possible postsynthetic modifications of IL-1 that might account for its cellular transport were examined. We found that lipopolysaccharide stimulated, but not unstimulated, murine macrophages incorporated 32PO4 into the IL-1 alpha precursor (31 kDa) predominantly at residue serine 90. Released IL-1 alpha (17 kDa) is not phosphorylated in agreement with peptide sequence data that the site of 32P incorporation is in the amino-terminal one-third of the precursor. Approximately 10% of the phosphorylated IL-1 alpha precursor is membrane bound and associated with a fraction enriched in lysosomal vesicles. Together these data suggest mechanisms by which the postsynthetic proteolysis of the IL-1 alpha precursor may be modified and cellular transport of IL-1 alpha is accomplished.     

Aphanizomenon ovalisporum (Forti) in Lake Kinneret, Israel

The filamentous cyanobacterium Aphanizomenon ovalisporum wasobserved for the first time in Lake Kinneret in August 1994and formed a prominent bloom from September through October.Aphanizomenon ovalisporum reappeared in diminished amounts inthe summer and fall of 1995. These events are the first recordof significant quantities of a potentially toxic nitrogen-fixingcyanobacterium in this lake. No definite provenance of inoculumhas been identified, although A.ovalisporum was also observedin a newly reflooded area (Lake Agmon) in the catchment. Unusuallyhigh water temperatures and low wind inputs were observed priorto and during the A.ovalisporum bloom period. These, togetherwith possibly enhanced availability of phosphorus or other growthfactors, may have contributed to the cyanobacterium growth in1994. Phosphorus limi tation, as indicated by high cellularalkaline phosphatase activity, the onset of stormy conditionsand a fall in water temperatures led to the demise of the 1994bloom. Although the A. ovalisporum bloom in 1994 had no seriousdirect impact on water quality, the continued presence of apotentially toxic cyanobacterium in Lake Kinneret, a major nationalwater supply source, is a cause for serious concern.     

A method for flow cytometric cell cycle analysis of normal and psoriatic human epidermis based on a detergent/citric acid technique for suspension of nuclei

A new method is described for flow cytometric cell cycle analysis of normal and psoriatic human epidermis, based on non-enzymatic tissue disaggregation. The epidermis was isolated by treatment with acetic acid and stored by freezing. After thawing, the epidermis was disintegrated into a nuclear suspension by 3 steps: incubation with dithiotreitol, whirling in a buffer (pH 7.4) with the non-ionic detergent Nonidet P40, EGTA, RNase and spermine, and whirling after addition of citric acid to a final concentration of 1% (pH 2.4). The suspension was stained with propidium iodide and filtered before flow cytometry. The yield of suspended nuclei was approximately 70% of the original number of cells in the tissue. The detergent/citric acid method was found to be preferable to an ultrasonication method previously used on human epidermis. All cell cycle and cell maturation stages were represented in the detergent/citric acid suspension, in contrast to the selection of immature G1, S and G2 stages with enzymatic methods. In the analysis of psoriatic epidermis inadequately matured (parakeratotic) cells were present in the suspension and had to be discriminated by gating on light scattering intensity, as they were not susceptible to lysis and did not stain properly. The fraction of S phase nuclei was on average 1.9% in normal and 7.7% in psoriatic epidermis, thus confirming the results of other investigators using enzymes. The presence of mitotic figures in the suspension was demonstrated by flow sorting. In this way the mitotic fraction was estimated to 0.06% in normal and 0.22% in psoriatic epidermis, confirming histological data of other investigators.