In-vivo effects of ivermection onRhipicephalus appendiculatus: the influence of tick feeding patterns and drug pharmacokinetics

Sublethal effects seen amongstRhipicephalus appendiculatus feeding on ivermectin-treated rabbits were diverse and dependent both on drug dose, pharmacokinetics and tick feeding patterns: changes in drug formulation, the time of infestation relative to treatment, and the tick instar used, profoundly influenced acaricidal activity. Death was a sequel to paralysis only if tick feeding was interrupted for sufficient time to produce irreversible dehydration. Concurrent pharmacokinetic investigations revealed that, for the larvae ofR. appendiculatus, the mean critical lethal dose of ivermectin imbibed over a 5-day engorgement period was 3500 g/kg. This quantity of ivermectin was achieved in the blood-meals of larvae feeding on rabbits treated subcutaneously with a single dose of Ivomec injection (MSD)^*800 g/kg, provided infestation took place within 24 h of treatment. At lower drug doses, or if larval infestations were delayed for>24 h post-treatment, the quantity of circulating ivermectin (and thus imbibed by the tick larvae) fell below 3500 g/kg and an increasing percentage of larvae successfully engorged and detached. More than 90% of such larvae moulted to the nymphal stage. Nymphae and larvae exhibited similar susceptibility to ivermectin on treated rabbits which could be explained by similar feeding patterns. However, adult female and male ticks were markedly less susceptible and interpretation of ivermectin-induced effects was more complex.     

Vitamin K1 reduction in human liver. Location of the coumarin-drug-insensitive enzyme.

The protein kinase C inhibitor staurosporine influenced in different ways the functions of human neutrophils. Staurosporine prevented the enhanced protein phosphorylation in phorbol ester- and N-formylmethyionyl-leucylphenylalanine (fMLP)-stimulated cells, and was a powerful inhibitor of the respiratory burst induced by phorbol myristate acetate [IC50 (concentration causing 50% inhibition) 17 nM] and the chemotactic peptides fMLP and C5a (IC50 24 nM). It did not alter, however, the superoxide production by cell-free preparations of NADPH oxidase. Staurosporine had no effect on agonist-dependent changes in cytosolic free Ca2+ and exocytosis of specific and azurophil granules, and showed only a slight inhibition of the release of vitamin B12-binding protein induced by phorbol myristate acetate (decreased by 40% at 200 nM). On the other hand, staurosporine also exhibited neutrophil-activating properties: it induced the release of gelatinase (from secretory vesicles) and vitamin-B12-binding protein (from specific granules). These effects were protracted, concentration-dependent, insensitive to Ca2+ depletion, and strongly enhanced by cytochalasin B. Staurosporine, however, did not induce the release of beta-glucuronidase or elastase (from azurophil granules). Except for the sensitivity to cytochalasin B, these properties suggest a similarity between the exocytosis-inducing actions of staurosporine and PMA. The results obtained with staurosporine provide further evidence that different signal-transduction processes are involved in neutrophil activation, and suggest that protein phosphorylation is required for the induction of the respiratory burst, but not for exocytosis.     

A human melanoma-derived cell line (IGR39) with a very high number of vasoactive-intestinal-peptide (VIP) receptors. 2. Effect of VIP on cAMP production and on cell-surface VIP-binding sites

Vasoactive intestinal peptide (VIP) stimulated in a dose-dependent manner the accumulation of cAMP in human melanoma-derived cell line IGR39. The maximal effect (about 100 times the basal level) was observed with 10 nM VIP. Half-maximum cAMP production was obtained at 0.78 nM VIP. VIP-related peptides were also potent in stimulating the cAMP production in IGR39 cells. The order of potency was VIP much greater than peptide histidine-methioninamide greater than human growth-hormone-releasing factor(1-44) greater than secretin greater than glucagon. Using the same conditions, IGR37 cells, a metastasic counterpart of IGR39 cells, displayed a weak stimulation of cAMP production. After exposure of IGR39 cells to 10 nM VIP, the cAMP response to a new stimulation by VIP was strongly reduced. This desensitization of IGR39 cells to VIP was rapid (t1/2 less than 2 min) and homologous. Preincubation of IGR39 cells in the presence of native VIP induced disappearance of the VIP-binding sites at the cell surface. This phenomenon was dependent on time and VIP concentration. Maximum effect (loss of 80% of binding capacity) was obtained after exposure of the cells at 37 degrees C with a VIP concentration of 1 microM. The t1/2 of maximum disappearance was less than 2 min and the concentration of VIP giving half-maximum decrease in binding of mono[125I]iodinated VIP (125I-VIP) was 8 nM. This phenomenon was also reversible since 85% of the VIP-binding capacity could be restored in less than 1 h by incubating IGR39 cells in a VIP-free medium. The IGR39 cell line should be a useful model for further study of the structure and function of the human VIP receptor.     

A cytogenetic investigation of the effects of cryopreservation on human sperm.

The effects of cryopreservation on the frequency and type of chromosome abnormalities in human sperm have been investigated for the first time. With a technique which enables direct visualization of human sperm chromosomes following in vitro penetration of hamster oocytes, sperm samples from 13 normal men were examined before and after being frozen in liquid nitrogen. The overall abnormality frequencies of 17.8% for fresh semen and 13.4% for previously frozen semen were not significantly different (chi 2(1) df = 3.04, p = 0.08). When specific abnormality types were analyzed, only the category of hypohaploidy was significantly different (chi 2(1) df = 6.75, p = 0.009) before (7.5%) and after (3.4%) freezing. Hypohaploidy was significantly higher than hyperhaploidy both prefreeze and postfreeze, and chromosome loss was random. Because the observed excess of hypohaploid cells may be attributable to technical artifact, the aneuploidy levels were estimated by doubling the number of hyperhaploid cells. Neither the adjusted numerical abnormality frequencies (1% prefreeze vs. 0% postfreeze) nor the overall abnormality frequencies (11.8% prefreeze vs. 10.4% postfreeze) were significantly different. The types and distributions of karyotypically abnormal sperm complements (numerical, structural, or combined) observed before and after freezing were not different. Interdonor variability in sperm chromosome abnormality frequencies and a possible donor-dependent response to cryopreservation were suggested by the data. The sex ratios were not affected by cryopreservation and did not differ significantly from the theoretical 50%. It is concluded that cryopreservation does not affect the type or frequencies of chromosome abnormalities or alter the sex ratio in human sperm.     

Chromobindin A, a Ca2+- and ATP-dependent chromaffin granule-binding protein, is found in a variety of tissues and in yeast

Chromobindin A is a ring shaped, multisubunit protein which exhibits Ca2+ and ATP-dependent binding to chromaffin granule membranes. Here we report biochemical and immunochemical evidence for the presence of chromobindin A in a surprisingly broad range of tissues: The protein is abundant in bovine skeletal muscle, pancreas, adrenal cortex and in brain gray and white matter; it is present in low quantities in the parotid, intestine and spleen and is undetectable in lung. Interestingly, chromobindin A was also detected in extracts of yeast. Electron micrographs of the yeast protein reveal a morphology virtually identical to the mammalian protein. These results suggest that chromobindin A is an important protein in a wide variety of cell types and that it has been highly conserved through evolution.     

Identification of a 42-kilodalton phosphotyrosyl protein as a serine(threonine) protein kinase by renaturation.

We have surveyed fibroblast lysates for protein kinases that might be involved in mitogenesis. The assay we have used exploits the ability of blotted, sodium dodecyl sulfate-denatured proteins to regain enzymatic activity after guanidine treatment. About 20 electrophoretically distinct protein kinases could be detected by this method in lysates from NIH 3T3 cells. One of the kinases, a 42-kilodalton serine(threonine) kinase (PK42), was found to possess two- to fourfold-higher in vitro activity when isolated from serum-stimulated cells than when isolated from serum-starved cells. This kinase comigrated on sodium dodecyl sulfate-gels with a protein (p42) whose phosphotyrosine content increased in response to serum stimulation. The time courses of p42 tyrosine phosphorylation and PK42 activation were similar, reaching maximal levels within 10 min and returning to basal levels within 5 h. Both p42 tyrosine phosphorylation and PK42 activation were stimulated by low concentrations of phorbol esters, and the responses of p42 and PK42 to TPA were abolished by chronic 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. Chronic TPA treatment had less effect on serum-induced p42 tyrosine phosphorylation and PK42 activation. PK42 and p42 bound to DEAE-cellulose, and both eluted at a salt concentration of 250 mM. Thus, PK42 and p42 comigrate and cochromatograph, and the kinase activity of PK42 correlates with the tyrosine phosphorylation of p42. These findings suggest that PK42 and p42 are related or identical, that PK42 is activated by tyrosine phosphorylation, and that this tyrosine phosphorylation can be regulated by protein kinase C.     

Acetyl-l-carnitine as a precursor of acetylcholine

Synthesis of [³H]acetylcholine from [³H]acetyl-l-carnitine was demonstrated in vitro by coupling the enzyme systems choline acetyltransferase and carnitine acetyltransferase. Likewise, both [³H] and [¹⁴C] labeled acetylcholine were produced when [³H]acetyl-l-carnitine andd-[U-¹⁴C] glucose were incubated with synaptosomal membrane preparations from rat brain. Transfer of the acetyl moiety from acetyl-l-carnitine to acetylcholine was dependent on concentration of acetyl-l-carnitine and required the presence of coenzyme A, which is normally produced as an inhibitory product of choline acetyltransferase. These results provide further evidence for a role of mitochondrial carnitine acetyltransferase in facilitating transfer of acetyl groups across mitochondrial membranes, thus regulating the availability in the cytoplasm of acetyl-CoA, a substrate of choline acetyltransferase. They are also consistent with a possible utility of acetyl-l-carnitine in the treatment of age-related cholinergic deficits.     

Indole-3-acetic acid content and glutamine synthetase activity in the pericarp, and peroxidase activity and isoenzymes in the meso- and exocarp of growing peach fruits

The indole-3-acetic acid (IAA) content in peach pericarp (Prunus persica L. Batsch cv. Merry) was highest at early stage I of development (200 ng/g fresh wt), decreased to the lowest level during stage II, and rose again at stage III to 60–70 ng/g fresh wt. High activity of glutamine synthetase was found in the pericarp during stage I. The soluble peroxidase activity was highest in the meso- and exocarp at stage II, and isoenzymatic changes in this fraction corresponded to the transition from cationic isoenzymes, predominant at stage I, to anionic isoenzymes at stage III. The ionically bound peroxidase activity in these tissues was highest at stage I. The three developmental stages showed marked differences in auxin content and enzyme activities; for peroxidases these changes reflect a developmental expression pattern for the isoenzymes.