Carrier-free 8-azidoadenosine 5'-[gamma-32P]triphosphate

We found 8-azidoadenosine 5'-diphosphate to be a phosphoryl acceptor in the enzymatic conversion of 1,3-diphosphoglyceric acid to 3-phosphoglycerate. This has allowed us to synthesize in a single-step procedure carrier-free 8-azidoadenosine 5'-[gamma-32P]triphosphate, requiring no further purification of the end product. The synthesized 8-azidoadenosine 5'-[gamma-32P]triphosphate has been characterized and shown to meet all the criteria for a specific photoreactive ATP analogue.     

The leader peptides from bacteriorhodopsin and halorhodopsin are potential membrane-spanning amphipathic helices

We show that the N-terminal leader peptides from the bacterial membrane proteins bacteriorhodopsin and halorhodopsin can be expected to form amphipathic alpha-helics with a highly hydrophobic nonpolar face and a narrow, negatively charged polar face. This finding is discussed in terms of a model for the integration of these proteins into the bacterial membrane.     

Characterization of a heterogeneous camel milk whey non-casein protein

A milk protein, occurring in the whey fraction, has been characterized from camel milk. Determination of the primary structure reveals the existence of two related types of chain with residue differences in at least the N-terminal region. A fragment representing an N-terminal part of the protein was also recovered (heterogeneous at the same positions). The absence of cysteine residues in the protein shows that no disulphide bridges are present. The pattern of fragments and a parent protein resembles that for casein and its fragments, showing that fragments and a multiplicity of forms may be typical for different milk proteins.     

Size selection of latex beads by blackfly larvae (Diptera: Simuliidae) in the laboratory

In laboratory experiments, blackfly larvae collected from a lake outlet, a woodland and a meadow stream were tested for size selection of latex beads of < Ito > 100 μm diameters. 3 suspensions of varying proportions for each size category were supplied to these blackfly larvae in common experiments. Comparisons between the size frequency distributions of particles supplied and the particle compositions in the larval guts showed intra- and interspecific differences and were quantified by calculating Jacobs' electivity index. In all species selection of larger particles increased with the larger larval instars. Although there was a positive selectivity of small particles in some cases, the ingested proportion of large particles increases volumes and biomasses of gut content and may be more important for larval growth than small particles.     

Synthesis and processing of alpha-galactosidase A in human fibroblasts. Evidence for different mutations in Fabry disease

The synthesis and processing of the human lysosomal enzyme alpha-galactosidase A was examined in normal and Fabry fibroblasts. In normal cells, alpha-galactosidase A was synthesized as an Mr = 50,500 precursor, which contained phosphate groups in oligosaccharide chains cleavable by endoglucosaminidase H. The precursor was processed via ill-defined intermediates to a mature Mr 46,000 form. Processing was complete within 3-7 days after synthesis. In the presence of NH4Cl and in I-cell fibroblasts, the majority of newly synthesized alpha-galactosidase A was secreted as an Mr = 52,000 form. For comparison, the processing and stability of alpha-galactosidase A were examined in fibroblasts from five unrelated patients with Fabry disease, which is caused by deficient alpha-galactosidase A activity. In one cell line, synthesis of immunologically cross-reacting polypeptides was not detectable. In another, the synthesis, processing, and stability of alpha-galactosidase A was indistinguishable from that in normal fibroblasts. In a third Fabry cell line, the mutation retarded the maturation of alpha-galactosidase A. Finally, in two cell lines, alpha-galactosidase A polypeptides were synthesized that were rapidly degraded following delivery to lysosomes. These results clearly indicate that Fabry disease comprises a heterogeneous group of mutations affecting synthesis, processing, and stability of alpha-galactosidase A.     

Lysosomal enzyme precursors in coated vesicles derived from the exocytic and endocytic pathways

The molecular forms of two lysosomal enzymes, cathepsin C and cathepsin D, have been examined in lysosomes and coated vesicles (CVs) of rat liver. In addition, the relative proportion of these lysosomal enzymes residing in functionally distinct CV subpopulations was quantitated. CVs contained newly synthesized precursor forms of the enzymes in contrast to lysosomes where only the mature forms were detected. Exocytic and endocytic CV subpopulations were prepared by two completely different protocols. One procedure, a density shift method, uses cholinesterase to alter the density of CVs derived from exocytic or endocytic pathways. The other relies on electrophoretic heterogeneity to accomplish the CV subfractionation. Subpopulations of CVs prepared by either procedure showed similar results, when examined for their relative proportion of cathepsin C and cathepsin D precursors. Within the starting CV preparation, exocytic CVs contained approximately 80-90% of the total steady-state levels of these enzymes while the level in the endocytic population was approximately 10-13%. The implications of these findings are discussed with regard to lysosome trafficking.     

The onset of the respiratory burst in human neutrophils. Real-time studies of H2O2 formation reveal a rapid agonist-induced transduction process

A real-time study of the initiation of the respiratory burst in human neutrophils was made. The cells were stimulated with fMet-Leu-Phe (fMLP) C5a, platelet-activating factor, leukotriene B4, phorbol myristate acetate (PMA), or ionomycin, and H2O2 production was determined by chemiluminescence. Identical average onset times (2.4 s) and closely comparable values for the apparent first-order rate constant (kapp) for the induction of NADPH-oxidase activity (0.21-0.29 s-1) were obtained following stimulation with fMLP, C5a, platelet-activating factor, or leukotriene B4, suggesting that different agonists act through a common transduction sequence. Much longer onset times and lower kapp values were obtained upon stimulation with PMA or ionomycin. Pretreatment with PMA consistently shortened the onset time of the neutrophil's responses to agonists by about 1 s. When H2O2 production was initiated with PMA, a subsequent stimulation with the agonist fMLP elicited an immediate response (onset time less than 0.2 s) which preceded further changes in fura-2-detected [Ca2+]i. The results are consistent with a mechanism in which agonist signals appear to be transduced by two sequences acting in concert--a rate-limiting one liberating Ca2+ and diacylglycerol and turning on the Ca2+/phospholipid-dependent enzyme protein kinase C, and an essentially instantaneous one which does not appear to require further changes in cytosolic Ca2+.     

An estimation of drag in front crawl swimming

Propulsive arm forces of twelve elite male swimmers during a front crawl swimming-like activity were measured. The swimmers pushed off against grips which are attached to a 23 m tube at 0.8 m under the water surface. The tube was fixed to a force transducer. Since at constant speed, mean propulsive force equals mean drag force this method also provides the mean active drag on a moving swimmer. The mean propulsive force at a speed of v = 1.48 m s-1 appeared to be 53.2 +/- 5.8 N which is two to three times smaller than what is reported by other authors for active drag but which is in agreement with values reported for passive drag on a (towed) swimmer who is not moving. Discrepancies with indirect active drag measurements are discussed.     

Epidermal growth factor receptor expression during morphological differentiation of pheochromocytoma cells, induced by nerve growth factor or dibutyryl cyclic AMP

Rat pheochromocytoma cells (clone PC12) possess functional surface receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF). PC12 cells respond to NGF as well as to dibutyryl cyclic AMP (dbcAMP) by arrest of cell proliferation and initiation of morphological differentiation, while EGF acts as a mitogen. Exposure of PC12 cells to NGF for several days resulted in a complete loss of rapid EGF responses, such as membrane ruffling and activation of active K+ transport. EGF binding studies revealed that this loss of EGF responses was due to an almost complete reduction of the number of EGF binding sites. In contrast, exposure of PC12 cells to dbcAMP for 2 days did not affect the rapid EGF responses, despite the morphological differentiation. Moreover, EGF binding studies demonstrated a twofold increase in the number of high-affinity binding sites and a small increase in the number of low-affinity sites. In addition, exposure of the cells to dbcAMP caused a twofold increase of EGF-receptor phosphotyrosine kinase activity. These results indicate that neither EGF-binding or the presence of EGF receptors nor the rapid EGF responses are sufficient for persistent proliferation, on one hand, or sufficient to avoid morphological differentiation, on the other.     

An immunoenzyme triple-staining method using both polyclonal and monoclonal antibodies from the same species. Application of combined direct, indirect, and avidin-biotin complex (ABC) technique

For immunohistological analysis, simultaneous detection of multiple cellular epitopes, as compared to single staining of serial sections, is sometimes needed. Therefore, immunoenzyme triple-staining protocols were tested with polyclonal and monoclonal antibodies on tissue sections and cytospin preparations. Various immunoconjugates were used in different combinations of methods, of which not all proved to be suitable. Of the tested protocols, one yielded superior results for both monoclonal and polyclonal antibodies, with optimal preservation of their original avidity. The method consists of a combination of indirect, direct, and avidin-biotin complex technique. The three antigens can be distinguished clearly and selectively by the reaction products of the enzyme activities of beta-galactosidase (green), alkaline phosphatase (blue), and horseradish peroxidase (red).