CDK1 Inactivation Regulates Anaphase Spindle Dynamics and Cytokinesis In Vivo

Through association with CDK1, cyclin B accumulation and destruction govern the G₂/M/G₁ transitions in eukaryotic cells. To identify CDK1 inactivation-dependent events during late mitosis, we expressed a nondestructible form of cyclin B (cyclin BΔ90) by microinjecting its mRNA into prometaphase normal rat kidney cells. The injection inhibited chromosome decondensation and nuclear envelope formation. Chromosome disjunction occurred normally, but anaphase-like movement persisted until the chromosomes reached the cell periphery, whereupon they often somersaulted and returned to the cell center. Injection of rhodamine-tubulin showed that this movement occurred in the absence of a central anaphase spindle. In 82% of cells cytokinesis was inhibited; the remainder split themselves into two parts in a process reminiscent of Dictyostelium cytofission. In all cells injected, F-actin and myosin II were diffusely localized with no detectable organization at the equator. Our results suggest that a primary effect of CDK1 inactivation is on spindle dynamics that regulate chromosome movement and cytokinesis. Prolonged CDK1 activity may prevent cytokinesis through inhibiting midzone microtubule formation, the behavior of proteins such as TD60, or through the phosphorylation of myosin II regulatory light chain.     

Spectroscopic characterization of quinone-site mutants of the bacterial photosynthetic reaction center

Site-specific mutations in the quinone binding sites of the photosynthetic reaction center (RC) protein complexes of Rhodobacter (R.) capsulatus caused pronounced effects on sequential electron transfer. Conserved residues that break the twofold symmetry in this region of the RC – M246Ala and M247Ala in the QA binding pocket, and L212Glu and L213Asp in the QB binding pocket – were targeted. We constructed a QB-site mutant, L212Glu-L213Asp Ala-Ala, and a QA-site mutant, M246Ala–M247Ala Glu-Asp, to partially balance the differences in charge distribution normally found between the two quinone binding sites. In addition, two photocompetent revertants were isolated from the photosynthetically-incompetent M246Glu-M247Asp mutant: M246Ala–M247Asp and M246Gly–M247Asp. Sequential electron transfer was investigated by continuous light excitation and time-resolved electron paramagnetic resonance (EPR), and time-resolved optical techniques. Several lines of EPR evidence suggested that the forward electron transfer rate to QA, kQ, was slowed in those strains containing altered QA sites. The slower rates of secondary electron transfer were confirmed by time-resolved optical results with the M246Glu-M247Asp mutations in the QA site resulting in a dramatically lowered secondary electron transfer efficiency [kQ < (2 ns)-1] in comparison with either the native R. capsulatus RC or the QB site mutant [kQ (200 ps)-1]. Secondary electron transfer in the two revertants was intermediate between that of the native RC and the QA mutant. The P+ QA- PQA charge recombination rates were also changed in the strains that carried altered QA sites. We show that local mutations in the QA site, presumably through local electrostatic changes, significantly alter binding and electron transfer properties of QA.     

Co-Occurrence of Preference Functions and Acceptance Thresholds in Female Choice: Mate Discrimination in the Lesser Wax Moth

Basic economic models adapted from foraging theory predict that decisions in mate choice may be determined either by ‘best‐of‐n’ preference functions or by sequential rules incorporating acceptance thresholds. However, in some species, more complex determinations that incorporate versions of both protocols are found. To understand the functions of co‐occurring protocols, we studied mating decisions in the lesser wax moth, Achroia grisella (Lepidoptera: Pyralidae), an acoustic species in which females prefer males, the advertisement songs of which are delivered at relatively high ‘pulse‐pair’ rates. In addition to this preference, A. grisella females avoid mating with a male, the song of which does not exceed a minimum pulse‐pair rate, and they hold to this criterion even when no other singing males are present and regardless of song amplitude. Thus, mating decisions are not simply based on acoustic power (pulse‐pair rate × amplitude). We recorded male songs and female responses in an A. grisella population and found that male pulse‐pair rates showed a median of 87/s and ranged from 50 to 115/s, while female acceptance thresholds for male song showed a median of 60/s and ranged from 30 to 105/s. The distributions of thresholds were approximately normal and were not significantly skewed toward the right. Male song rates declined slightly with age, but female thresholds remained stable over the adult lifespan. Both the male and female traits showed significant repeatability within individuals. Whereas phylogenetic inference indicates that hearing in pyralid moths originated as a means of avoiding predation by insectivorous bats, the specific distribution of female acceptance thresholds suggests that currently this protocol does not primarily function to preclude inappropriate, and potentially lethal, responses to bat echolocations: pulse rates in the searching‐phase echolocations used by either aerial‐hawking or substrate‐gleaning bats mostly range from 10 to 20/s, and the lack of positive skew in the distribution of thresholds indicates an absence of directional selection from the left. Rather, we infer that thresholds augment preference functions in A. grisella by precluding mating with males which are markedly inferior in a critical song character. In general, co‐occurring protocols may be important where population density fluctuates markedly, as preference functions may be ineffective in preventing mating with inferior males when density is low.     

CHANGES IN THE PROTEIN COMPOSITION OF MOUSE BRAIN MYELIN DURING DEVELOPMENT

Abstract— Myelin was isolated from the brains of mice at various ages by a procedure involving a final purification on a continuous CsCl gradient. Myelin protein accumulated throughout development, increasing from 0.25 mg of protein/brain at 8 days of postnatal age to 3.5 mg of protein/brain at 300 days, although the rate of accumulation was greatest at about 21 days of age. Quantitative studies of the protein composition of these samples were carried out, utilizing discontinuous polyacrylamide gel electrophoresis in buffers containing sodium lauryl sulphate. Mouse brain myelin, contained (in order of increasing molecular weight) two basic proteins, an uncharacterized doublet, proteolipid protein, and a group of high molecular weight proteins. There were marked changes in the quantitative distribution of these proteins with increasing postnatal age. The basic protein fraction of total myelin protein increased from about 18 per cent at 8 days to 30 per cent at 300 days of age. Proteolipid protein increased even more dramatically, from 7 to 27 per cent in the same time interval. These chemical studies were correlated with ultrastructural investigations, both of the developing myelin sheath in situ and the isolated myelin obtained from mice of various ages. A hypothesis, relating the observed changes in protein composition of myelin during development to its mode of formation, is developed. Another subcellular fraction, separated from myelin, by virtue of its greater density in a CsCl gradient, was also studied. It was a vesicular, membranous fraction present at a level of 0.35 mg of protein/brain at all ages and was related to myelin in terms of protein composition.     

The nature of the organic nitrogen of soils

Summary Examination of the 6N HC1 hydrolysates from 14 different proteins indicated that a considerable proportion of the total protein nitrogen in the hydrolysates, as determined by the micro-Kjeldahl method, was not accounted for by the NH₄-N and the α amino nitrogen found in the hydrolysates. It seems clear that this hydrolysable unidentified nitrogen (HUN) originates mainly from non-amino nitrogen atoms present in arginine, tryptophan, lysine and proline. These nitrogen atoms do not satisfy the conditions necessary for reaction with ninhydrin. The amounts of each amino acid in a particular protein determine the HUN value which will be obtained for 6N HC1 hydrolysates of that protein. There is good agreement between the HUN values for a wide range of proteins when calculated from the amino acid composition of the protein and when determined experimentally. It is concluded that these findings indicate a considerably higher content of amino acid nitrogen in the organic nitrogen of soils and leaf litter than was previously considered to be the case. It is suggested that the findings support the contention that the organic nitrogen of soils contains leaf protein complexes.     

Nonfunctional Tricarboxylic Acid Cycle and the Mechanism of Glutamate Biosynthesis in Acetobacter suboxydans

Acetobacter suboxydans does not contain an active tricarboxylic acid cycle, yet two pathways have been suggested for glutamate synthesis from acetate catalyzed by cell extracts: a partial tricarboxylic acid cycle following an initial condensation of oxalacetate and acetyl coenzyme A. and the citramalate-mesaconate pathway following an initial condensation of pyruvate and acetyl coenzyme A. To determine which pathway functions in growing cells, acetate-1-(14)C was added to a culture growing in minimal medium. After growth had ceased, cells were recovered and fractionated. Radioactive glutamate was isolated from the cellular protein fraction, and the position of the radioactive label was determined. Decarboxylation of the C5 carbon removed 100% of the radioactivity found in the purified glutamate fraction. These experiments establish that growing cells synthesize glutamate via a partial tricarboxylic acid cycle. Aspartate isolated from these hydrolysates was not radioactive, thus providing further evidence for the lack of a complete tricarboxylic acid cycle. When cell extracts were analyzed, activity of all tricarboxylic acid cycle enzymes, except succinate dehydrogenase, was demonstrated.     

Sexual dimorphism inDryopithecus africanus

Among the numerous specimens presently classified withinDryopithecus africanus only one can be identified as a male of this species. Poor sampling is not the reason for the unequal numbers of male and female specimens. Rather, the males have been classified elsewhere, specifically withinDryopithecus nyanzae and Kenyapithecus africanus. The specimens to be transferred from these two taxa are proved to be males ofD. africanus. The newly transferred males are compared with the females to show the cranial dimorphism of the species.