Assembly of tubulin from cultured cells and comparison with the neurotubulin model

Spontaneous microtubule assembly can be obtained in extracts from a variety of cultured cell lines by including glycerol in the assembly buffer. An analysis of the effects of cultured cell extracts on brain tubulin (neurotubulin) assembly has shown that the extracts contain initiation inhibitors whose effects are diminished by glycerol. By using glycerol during the assembly step, cultured cell tubulin can be purified by assembly-dissassembly procedures. The amount of glycerol necessary for significant spontaneous assembly varies from 1–6 M among the different cell lines and is dependent upon their content of inhibitor. Comparison of the assembly products obtained from NA, C₆ and CHO cells at increasing glycerol concentrations shows that glycerol enhances the purification of tubulin and a polypeptide of molecular weight 49,000 daltons in all three systems. These preparations contain a number of other polypeptides, including a group with gel electrophoretic mobilities characteristic of tau-factor, but lack the high molecular weight microtubule-associated proteins (MAPs) which are present in neurotubulin preparations. Phosphocellulose chromatography of NA tubulin removes several proteins from the tubulin and results in a loss of polymerizability. Among three proteins which are completely removed from the inactive tubulin, the most prominent is the 49K protein. This observation and the co-purification of the 49K protein with tubulin through two assembly-disassembly cycles suggest that it is a true MAP. The difference in MAP proteins between brain and tissue culture cells is parallelled by an absence of ring structures in NA tubulin preparations. NA tubulin, however, does form rings when brain MAPs are added. The early steps of NA tubulin assembly differ from those of neurotubulin; no rings are involved, and the first assembly intermediates are straight protofilament bundles. The differences between MAPs from cultured cells and brain and the absence of ring formation in NA tubulin preparations suggest that the assembly model based on neurotubulin is not completely general. A comparison of extracts from CHO cells grown with and without dibutyryl cAMP revealed no differences between the behavior of these extracts in spontaneous tubulin assembly or in mixture experiments with brain tubulin.     

Opportunities for natural selection on DNA and protein at the Adh locus in Drosophila melanogaster

A study was made of environmental and genetic factors affecting the quantity and disposition of the alcohol dehydrogenase (ADH) protein in Drosophila melanogaster. It was found that the amount of enzyme per fly is greatly influenced by the environmental conditions in which it develops. A critical factor is the concentration of yeast in the medium. A high concentration of yeast can double the quantity of ADH. The yeast appears to act through the provision of protein, and the protein to act through the provision of threonine, which is already known to induce ADH in fungi. Various genetic factors affect the quantity of enzyme. Males have more ADH than females. Files homozygous for the Fast allele have more ADH than those homozygous for the slow allele, and the difference is greater in females than in males. One particular line (ve), homozygous for Slow, has approximately half the normal quantity of enzyme, and the quantity segregates with the electrophoretic allele. Lines differ in the relative amounts of ADH in the gut (including Malpighian tubules) and the fat body. In general it seems that slow lines have relatively more enzyme in the fat body. In a cross between ve and a line homozygous to Fast, the difference in tissue distribution segregated with the electrophoretic allele. It is argued, but not demonstrated, that the differences in quantity and tissue distribution are due to nucleotide substitutions in noncoding regions close to, or within, the structural gene. It seems likely that the observed environmental and genetic differences in the quantity and disposition of ADH will influence the relative selective values of the electrophoretic genotypes.     

Contraction of isolated airway smooth muscle by synthetic leukotrienes C4 and D4

The pharmacology of leukotrienes (LT) C4 and D4 in isolated airway smooth muscle was investigated. In rat trachea, neither LTC4 or D4 elicited a response. In contrast, LTC4 was a potent contractile agonist in guinea-pig trachea, bronchus and parenchymal lung strip. Similar effects were obtained with LTD4 in trachea and parenchyma. In trachea and bronchus, the concentration-response curve to LTC4 was biphasic: indomethacin converted the biphasic response curve to a simple sigmoidal shape and enhanced the maximum contractile response. The SRS-A antagonist FPL 55712 antagonized the effect of LTD4 in both trachea and parenchyma. As regards LTC4-induced contraction of trachea and bronchus, FPL 55712, depending on concentration, either antagonized, or antagonized and enhanced the maximum contractile response. The enhancement of the maximum contractile response by FPL 55712 was not apparent when indomethacin was present. FPL 55712 failed to antagonize the effect of LTC4 in parenchyma.     

Heavy metal composition of polysomal fractions following cadmium challenge

Endogeneous levels of zinc and copper were found to be 1.2±0.1×10⁻² and 0.3±0.1×10⁻² μg/A260 unit, respectively, in polysomal fractions from control animals; cadmium, however, was undetectable. In experimental animals (injected with cadmium) zinc, copper, and cadmium were found in polysomal fractions isolated by two different methods. One hour after a cadmium injection there was a rise in both the zinc and copper content of the polysomal fractions, which then declined steadily to below control levels by 16 h. Neither zinc nor cadmium were dialyzable from these fractions by a TRIS buffer; however, addition of 0.01M EDTA to the buffer resulted in removal of 75% of the zinc and all of the detectable cadmium. The addition of cadmium (CdCl₂) to control supernatants (adjusted to the cadmium concentration present in supernatants 6 h after in vivo exposure) resulted in metal binding to polysomal fractions in levels comparable to those observed after in vivo exposures to the metal. When cadmium was added in the form of cadmium thionein, a smaller fraction of the metal was isolated with the polysomal fraction. Cadmium bound to polysomal fractions in vivo (24 h after exposure) was sensitive to release by protease digestion, but insensitive to release by ribonuclease digestion.     

Bioaccumulation of marine pollutants.

Bioaccumulation of pollutants can occur from sea water, from suspended particles, from sediments and through food chains. The rate at which accumulation occurs in an organism depends not only on the availability of the pollutant but also on a whole range of biological, chemical and environmental factors. The ultimate level which is reached is governed by the ability of the organism to excrete the pollutant or, alternatively, store it. This latter course often leads to the attainment of very high concentrations and sometimes no equilibrium level is ever reached. Two particular topics which are considered are the biological amplification of pollutants along food chains and the development of tolerance which sometimes occurs.     

Antibody-coated bacteria in the urine of preschool and school-aged girls with asymptomatic bacteriuria.

Urine samples from 3564 girls aged 2 to 13 years were screened for evidence of infection. Cultures were positive (bacteria count, more than 10(5)/ml) in 61 (1.7%) by the dipslide method and in 55 (1.5%) by standard culture techniques. In 13 (23.6%) of the 55, antibody-coated bacteria (ACB) were detected in the urine. The clinical, bacteriologic, radiologic and urinalysis findings in children with ACB were no different from those in children in whom the bacteria were not coated. Direct examination of uncentrifuged urine under high power revealed one or more bacteria per two high-power fields in 96% of infected urine samples and in only 7% of noninfected samples. Five or more leukocytes per high-power field in centrifuged urine were detected in 36.7% of infected urine samples but not in noninfected samples. The ACB test did not differentiate between asymptomatic bacteriuria with parenchymal scarring or vesicoureteral reflux or both and asymptomatic bacteriuria without these abnormalities.     

Effect of increased gas density on pulmonary gas exchange in man.

Pulmonary gas exchange was measured in seven resting supine subjects breathing air or a dense gas mixture containing 21% O2 in sulfur hexafluoride (SF6). The mean value of the alveolar-arterial oxygen difference (AaDO2) decreased from 12.4 on air to 7.0 on SF6 (P less than 0.01), and increased again to 13.4 when air breathing resumed (P less than 0.01). No differences occurred between gas mixtures for O2 consumption, respiratory quotient, minute ventilation, breathing frequency, heart rate, or blood pressure, and the improved oxygen transfer could not be attributed to changes in cardiac output or mixed venous oxygen content in the one subject in which they were measured. These results are best explained by an altered distribution of ventilation during dense gas breathing, so that the ventilation-perfusion ratio (VA/Q) variance was reduced. Of several considered mechanisms, we favor one in which SF6 promotes cardiogenic gas mixing between peripheral parallel units having different alveolar gas concentrations. This mechanism allows for observed increases in arterial carbon dioxide tension and dead space-to-tidal volume ratio during dense gas breathing, and suggests that intraregional VA/Q variance accounts for at least one-half of the resting AaDO2 in healthy supine young men.     

Mucociliary tracheal transport rates in man.

A new method for measuring mucociliary tracheal transport rates (MTTR's) is described. An aqueous aerosol containing albumin microspheres labeled with 99mTc was inhaled in such a manner that it was deposited in local concentrations in the large airways. These boli of microspheres were transported up the trachea and their MTTR's measured using a gamma camera. MTTR's were measured in 42 healthy nonsmoking adults (32 men and 10 women, mean age 28 yr). The mean MTTR's appeared to be log normally distributed with a geometric mean of 3.6 mm/min and a coefficient of variation of 75%. The MTTR's of men and women were similar. Each individual's short-term coefficient of variation was 25%. Twenty-two repeat studies 1 wk to 15 mo apart showed the variation within individuals was less than between individuals. The parasympatholytic drug, atropine (0.6 mg iv) decreased MTTR's for at least 3 h. Inhalation of the sympathomimetic drug, Th1165a increased MTTR's. Chronic and acute smoking did not appreciably change the MTTR'S.