Differential regulation of the Kar3p kinesin-related protein by two associated proteins, Cik1p and Vik1p

The mechanisms by which kinesin-related proteins interact with other proteins to carry out specific cellular processes is poorly understood. The kinesin-related protein, Kar3p, has been implicated in many microtubule functions in yeast. Some of these functions require interaction with the Cik1 protein (Page, B.D., L.L. Satterwhite, M.D. Rose, and M. Snyder. 1994. J. Cell Biol. 124:507-519). We have identified a Saccharomyces cerevisiae gene, named VIK1, encoding a protein with sequence and structural similarity to Cik1p. The Vik1 protein is detected in vegetatively growing cells but not in mating pheromone-treated cells. Vik1p physically associates with Kar3p in a complex separate from that of the Kar3p-Cik1p complex. Vik1p localizes to the spindle-pole body region in a Kar3p-dependent manner. Reciprocally, concentration of Kar3p at the spindle poles during vegetative growth requires the presence of Vik1p, but not Cik1p. Phenotypic analysis suggests that Cik1p and Vik1p are involved in different Kar3p functions. Disruption of VIK1 causes increased resistance to the microtubule depolymerizing drug benomyl and partially suppresses growth defects of cik1Delta mutants. The vik1Delta and kar3Delta mutations, but not cik1Delta, partially suppresses the temperature-sensitive growth defect of strains lacking the function of two other yeast kinesin-related proteins, Cin8p and Kip1p. Our results indicate that Kar3p forms functionally distinct complexes with Cik1p and Vik1p to participate in different microtubule-mediated events within the same cell.     

Compartmentalization of low molecular mass GTP-binding proteins among neutrophil secretory granules

Neutrophils contain several distinct classes of secretory granules that may sequentially fuse with the phagosome after the ingestion of particulates, or that may be differentially exocytosed after cellular activation with soluble stimuli. The exocytosis of neutrophil secretory granules has been shown to be GTP-dependent at a step distal to activation of the transductional G proteins. Inasmuch as ras-related low molecular mass GTP-binding proteins have been shown to play regulatory roles in vesicle sorting in the secretory pathway in yeast, the differential mobilization of neutrophil granules might be regulated by distinct GTP-binding proteins. We therefore explored the distribution and identity of low molecular mass GTP-binding proteins in neutrophil secretory granules and other subcellular fractions. After lysis by nitrogen cavitation, four highly resolved fractions were harvested from discontinuous Percoll gradients: a microsomal fraction enriched for plasma membranes, specific granules, primary granules, and cytosol. At least seven bands of distinct Mr were detected by probing protein blots with [32P]GTP. Microsomes contained a prominent GTP-binding band at 26 kDa and weaker ones at 24 and 22.5 kDa; specific granules contained bands at 26, 24, 22, and 20 kDa; primary granules showed bands at 24 and 23 kDa; cytosol showed strong bands at 23.5 and 19 kDa and a weak band at 26 kDa. Antiserum against ADP-ribosylation factor reacted strongly with the 19-kDa band in cytosol but with none of the membrane fractions. None of these proteins was recognized by antibodies against ras or against Sec4p. Botulinum exoenzyme C3 labeled bands of molecular mass 20 and 21 kDa in cytosol and microsomes that have distinct mobilities from all the blotted [32P]GTP-binding proteins. The highly compartmentalized subcellular distribution of the blotted [32P]GTP-binding proteins in neutrophils is consistent with a regulatory role in the differential mobilization of granule compartments during cellular activation.     

Selective A1-adenosine receptor antagonists identified using yeast Saccharomyces cerevisiae functional assays.

Evaluation of a biased "library" of pyrrolo[2,3-d]pyrimidines using yeast-based functional assays expressing human A1- and A2a-adenosine receptors, led to the A1 selective antagonist 4b. A direct correlation between yeast functional activity and binding data was established. Practical compounds with polar residues at C-4 of the pyrrolopyrimidine system required H-bond donor functionality for high potency.     

Midlatency respiratory-related somatosensory activity and perception of oral pressure pulses in normal humans.

A direct relationship exists within subjects between midlatency features (<100 ms poststimulus) of respiratory-related evoked potentials and the perceived magnitude of applied oral pressure pulse stimuli. We evaluated perception in 18 normal subjects using cross-modality matching of applied pressure pulses via grip force and estimated mechanoafferent activity in these subjects by computing the global field power (GFP) from respiratory-related evoked potentials recorded over the right side of the scalp. We compared across subjects 1) the predicted magnitude production for a standard pressure pulse and 2) the slope (beta) and 3) the intercept (INT) of the Stevens power law to the summed GFP over 20-100 ms poststimulus. Both the magnitude production for a standard pressure pulse and the beta showed an inverse relationship with the summed GFP over 20-100 ms poststimulus, although there was no relationship between INT and the summed GFP. This may partially reflect characteristics of the mechanosensors and surely includes aspects of cognitive judgment, because we found and corrected for a high correlation between, respectively, beta (and INT) for pressure pulses and beta (and INT) for estimation of line lengths, a nonrespiratory modality. The relatively shallow, even inverse GFP-to-perception relationship suggests that, despite marked differences in the magnitude of afferent traffic, normal subjects seem to perceive things similarly.     

Kynurenine metabolism in rats: some hormonal factors affecting enzyme activities.

Six male subjects (19–23 years old) underwent a 7-day control period with respect to diet, temperature (22C), and sleep (7.5 hrs), followed by a 2-day exposure to 15C and a 2-day recovery period (22C). Urine collections were made every 8 hours commencing at 2300 hours; MHPG and VMA were assayed using gas-liquid chromatography. During the control period a diurnal rhythmicity was demonstrated for MHPG and VMA with maxima at 0700–1500 hours. The mean excretory rates for MHPG and VMA were 0.71 ± 0.04 μg and 2.6 ± 0.2 μg per milligram creatinine (± S.E.), respectively. Cold exposure abolished the rhythms for MHPG and VMA and caused an 18% increase in MHPG excretion. In contrast, VMA excretion was not altered. Significant correlations were obtained with MHPG excretion and both urinary cortisol and rectal temperature. The data suggest that MHPG excretion may be indicative of changes in norephinephrine metabolism in the central nervous system, although alterations in peripheral degradative pathways cannot be ruled out. Careful interpretation of changes in MHPG excretion in clinical studies is emphasized due to the relative ease of altering MHPG metabolism.     

Aerobic Gram-Positive and Gram-Negative Bacteria Exhibit Differential Sensitivity to and Transformation of 2,4,6-Trinitrotoluene (TNT)

A systematic evaluation of the ability of different bacterial genera to transform 2,4,6-trinitrotoluene (TNT), and grow in its presence, was conducted. Aerobic Gram-negative organisms degraded TNT and evidenced net consumption of reduced metabolites when cultured in molasses medium. Some Gram-negative isolates transformed all the initial TNT to undetectable metabolites, with no adsorption of TNT or metabolites to cells. Growth and TNT transformation capacity of Gram-positive bacteria both exhibited 50% reductions in the presence of approximately 10 μg TNT ml⁻¹. Most non-sporeforming Gram-positive organisms incubated in molasses media amended with 80 μg TNT ml⁻¹ became unculturable, whereas all strains tested remained culturable when incubated in mineral media amended with 98 μg TNT ml⁻¹, indicating that TNT sensitivity is linked to metabolic activity. These results indicate that the microbial ecology of soil may be severely impacted by TNT contamination. Received: 2 December 1996 / Accepted: 3 February 1997