Variations in Sulfhydryl, Disulfide, and Protein Content during Synchronous and Asynchronous Growth of HeLa Cells

The cellular contents of protein-bound and nonprotein sulfhydry (—SH) and disulfide (—SS—) groups were measured in both asynchronous and synchronous HeLa S3 cultures. About 90% of these groups are associated with proteins, the majority in the —SH form. The content of protein-bound groups, and hence the total content of —SH and —SS— groups (28 × 10^-15 moles/cell, or 1.1 × 10^-6 moles/g protein on average), changes in parallel with the protein content (which varies between 2 and 4 × 10^-10 g/cell) as asynchronous populations pass from the lag through the exponential to the stationary phase of growth. The concentration of nonprotein —SH groups, in contrast, increases 10-fold during lag phase and decreases in stationary phase; it follows the protein concentration closely during the exponential phase, at a level of about 2.8 × 10^-15 moles/cell. In synchronous cultures the protein content doubles during the cell cycle, possibly in an exponential fashion. The total —SH and —SS— content also doubles, but the rate of increase appears to fluctuate. The concentrations of the protein-bound groups show 2- to 3-fold fluctuations per unit protein: protein-bound —SH groups and mixed —SS— linkages rise to maxima while protein-bound —SS— groups fall to a minimum at the G₁/S transition, and fluctuations in these groups occur again during G₂. In addition, the protein-bound —SH concentration falls continuously during the S phase. The nonprotein —SH concentration undergoes the largest (relative) fluctuations, dropping from 4 × 10^-15moles/cell in early G₁ to about 0.4 × 10^-15 moles/cell (of standard protein content) at the end of G₁, and then rising to 30 times this value by the end of S.     

STUDIES ON SULFHYDRYL GROUPS DURING CELL DIVISION OF SEA URCHIN EGG : III. —SH Groups of KCl-Soluble Proteins and Their Change during Cleavage

Sea urchin egg proteins extracted with KCl are mostly TCA-soluble and, conversely, those extracted with TCA are KCl-soluble. Both groups are water-insoluble and show fluctuations in—SH content during the division cycle. The fluctuation of the—SH groups of the KCl-soluble protein of the whole egg is due to a —SH—S—S— interchange within the freely reacting groups and not within the sluggish and masked —SH groups of the protein. The —SH content of the KCl-soluble protein of the egg cortex also fluctuates in a similar way.     

Examining Variation in Recombination Levels in the Human Female: A Test of the Production-Line Hypothesis

The most important risk factor for human aneuploidy is increasing maternal age, but the basis of this association remains unknown. Indeed, one of the earliest models of the maternal-age effect—the “production-line model” proposed by Henderson and Edwards in 1968—remains one of the most-cited explanations. The model has two key components: (1) that the first oocytes to enter meiosis are the first ovulated and (2) that the first to enter meiosis have more recombination events (crossovers) than those that enter meiosis later in fetal life. Studies in rodents have demonstrated that the first oocytes to enter meiosis are indeed the first to be ovulated, but the association between the timing of meiotic entry and recombination levels has not been tested. We recently initiated molecular cytogenetic studies of second-trimester human fetal ovaries, allowing us to directly examine the number and distribution of crossover-associated proteins in prophase-stage oocytes. Our observations on over 8,000 oocytes from 191 ovarian samples demonstrate extraordinary variation in recombination within and among individuals but provide no evidence of a difference in recombination levels between oocytes entering meiosis early in fetal life and those entering late in fetal life. Thus, our data provide a direct test of the second tenet of the production-line model and suggest that it does not provide a plausible explanation for the human maternal-age effect, meaning that—45 years after its introduction—we can finally conclude that the production-line model is not the basis for the maternal-age effect on trisomy.     

Studies on Sulfhydryl Groups during Cell Division of Sea Urchin Egg : V. Change in contractility of the thread model in relation to cell division

The contractility of the thread model prepared from the KCl-soluble proteins of the egg and in vivo factors for the contraction are investigated in Hemicentrotus, Anthocidaris, and Pseudocentrotus eggs. The contractility of the thread model induced by metal ions or cystine changes during development in the characteristic pattern of high at the metaphase and low at the monaster and the interkinetic stages. The change in contractility is paralleled by the change in the —SH content of the protein. The water-soluble fraction of the eggs has activity in causing contraction of the thread model. This activity changes during development in the same way as the contractility itself. The contraction of the thread induced by the water-soluble fractions is accompanied by a decrease in the —SH content of the thread. The activity of the water-soluble fraction in inducing the contraction is proportional to its ability to decrease the number of —SH groups. On boiling, the activity is largely destroyed. The activity is due to two components, one being non-dialyzable and the other dialyzable. Separately each component has little effect, but when mixed, the activity of the original sample is completely restored.     

Sulfhydryl groups and the structure of hemoglobin

1. Addition of 2 moles of mersalyl, mercuric chloride, p-chloromercuribenzoate (PCMB), or methyl mercury hydroxide per mole of hemoglobin greatly reduces heme-heme interactions (n), yet these substances have quite different effects on the oxygen affinity (-log p₅₀). Mersalyl and mercuric chloride at this concentration each increase the oxygen affinity, while PCMB and methyl mercury have little or no effect on the oxygen affinity. These effects are primarily associated with the binding of —SH groups, and are largely reversed on the addition of glutathione. —SH groups do not appear to be responsible for the Bohr effect. 2. Evidence is presented for the belief that the two hemes of each half-molecule of horse hemoglobin are situated on either side of a cluster of—SH groups. 3. The mechanism of interaction between the hemes is discussed. It is concluded that the reorganization of the protein architecture which accompanies oxygenation plays a central role in this interaction, in agreement with the views of Pauling and Wyman.     

Studies on Sulfhydryl Groups during Cell Division of Sea Urchin Egg : IV. Contractile properties of the thread model of KCl-soluble protein from the sea urchin egg

A KCl-soluble protein fraction can be extracted from the water-insoluble residue of the homogenate of sea urchin eggs which contains three major components separable by ultracentrifugation. When the extract is stirred in acetone or distilled water, a fibrous precipitate appears which has a strong birefringence positive in the direction of the long axis. When the fraction is squirted through a slender tubing, it precipitates in the form of a thread. The thread model contracts vigorously under the action of di-, tri-, and tetravalent metal ions; the contraction can be reversed by EDTA. The contraction and the elongation of the thread model can be repeated many times. The thread model contracts also in the presence of dehydroascorbic acid, cystine, oxidized glutathione, or other oxidizing agents, and this contraction is reversed by reducing agents such as cysteine or ascorbic acid. When —SH groups of the thread model are blocked by various —SH reagents, the contraction by metal ions is inhibited to some extent. As mechanisms of the contraction, electrostatic forces between metal ions and negative charges of the thread model are essential for metal ion-induced contraction and oxidation of —SH groups of the thread model for the contraction by oxidizing reagents.     

STUDIES ON CELL ADHESION : III. Adhesion of Baby Hamster Kidney Cells

Normal and transformed baby hamster kidney (BHK) cells attach to Falcon polystyrene with the same first order rate constant. The longer the cells are attached to the bottles, the more difficult they are to remove. Sulfhydryl (—SH) binding reagents inhibit both the attachment of BHK cells and the increase in adhesive strength of attached cells. Attached BHK cells bind fewer molecules of [1-¹⁴C]N-ethylamleimide (an —SH binding reagent) than do suspended cells. Incubation of cells with high concentrations of trypsin results in a reversible loss of cell adhesiveness. The recovery of adhesiveness of trypsin-treated cells is inhibited by cycloheximide.     

SULFHYDRYL AND DISULFIDE GROUPS OF PROTEINS : IV. SULFHYDRYL GROUPS OF THE PROTEINS OF MUSCLE

1. In the denatured proteins of skeletal muscle, the ratio of SH to S-S groups is higher than in the mixed denatured proteins of other tissues, with a single exception—the proteins of the crystalline lens. 2. The number of active SH groups in the proteins of minced muscle or in any of the protein fractions of muscle is only a fraction of the number found after the proteins have been treated with a denaturing agent. 3. The SH groups of the native proteins of muscle are activated by a rise in pH. 4. The relation between pH and number of active SH groups in the proteins of minced muscle and in the various protein fractions of muscle shows that little, if any, denatured protein is present in minced muscle.     

Protein Degradation, Meiosis and Sporulation in Proteinase-Deficient Mutants of SACCHAROMYCES CEREVISIAE

During the process of sporulation, a/α diploids degrade about 50% of their vegetative proteins. This degradation is not sporulation specific, for asporogenous diploids of a/a mating type degrade their vegetative proteins in a fashion similar to that of their a/α counterparts. Diploids lacking carboxypeptidase Y activity, prc1/prc1, show about 80% of wild-type levels of protein degradation, but are unimpaired in the production of normal asci. Diploids lacking proteinase B activity, prb1/prb1, show about 50% of wild-type levels of protein degradation. The effect on degradation of the proteinase B deficiency is epistatic to the degradation deficit attributable to the carboxypeptidase Y deficiency. The prb1 homozygotes undergo meiosis and produce spores, but the asci and, possibly, the spores are abnormal. Diploids homozygous for the pleiotropic pep4–3 mutation show only 30% of the wild-type levels of degradation when exposed to a sporulation regimen, and do not undergo meiosis or sporulation. Neither proteinase B nor carboxypeptidase Y is necessary for germination of spores.——Approximately half of the colonies arising from a/a or α/α diploids exposed to the sporulation regiment that express an initially heterozygous drug-resistance marker (can1) appear to arise from mating-type switches followed by meiosis and sporulation.     

DETERMINATION OF CERTAIN AMINO ACIDS IN CHYMOTRYPSINOGEN,AND ITS MOLECULAR WEIGHT

1. A preparation of chymotrypsinogen, obtained from Dr. M. Kunitz, was analyzed for sulfur, the sulfur amino acids, tyrosine, and tryptophane. 2. The protein sulfur of chymotrypsinogen was accounted for as methionine, cysteine, and cystine. 3. A method is presented for calculating the minimum molecular weight of a protein from the distribution of the sulfur amino acids. In the case of chymotrypsinogen, the calculated minimum molecular weight was found to be the actual molecular weight. 4. The molecular weight of chymotrypsinogen is 36,700 by amino acid analysis as compared to 36,000 by osmotic pressure measurements of Kunitz and Northrop. Chymotrypsinogen contains per mol 17 atoms of sulfur, 3 residues of methionine, 4 of cysteine, 10 of half-cystine (i.e. 5 S—S linkages), 6 of tyrosine, and 10 of tryptophane. 5. The tryptophane content of chymotrypsinogen (5.51 per cent) is the highest of any protein so far on record. 6. Chymotrypsinogen contains no reactive SH groups, although it yields cysteine on hydrolysis. This may be due either to preformed but unreactive SH groups or to S—X groups. The term S—X group is used to denote the substitution of the sulfhydryl hydrogen by a constituent X; hydrolysis yields SH groups: S—X + HOH = SH + X—OH.     

Genetic control of malate dehydrogenase isozymes in maize

At least six nuclear loci are responsible for the genetic control of malate dehydrogenase (L-malate: NAD oxidoreductase; EC 1.1.1.37; MDH) in coleoptiles of maize. Three independently segregating loci (Mdh1, Mdh2, Mdh3) govern the production of MDH isozymes resistant to inactivation by ascorbic acid and found largely or solely in the mitochondria. A rare recessive allele found at a fourth nuclear locus (mmm) causes increased electrophoretic mobility of the MDH isozymes governed by the Mdh1, Mdh2 and Mdh3 loci.—Two loci (Mdh4, Mdh5) govern MDH isozymes that are selectively inactivated by homogenization in an ascorbic acid solution and that appear to be nonmitochondrial (soluble). Mdh4 and Mdh5 segregate independently of each other and independently of Mdh1, Mdh2 and Mdh3. However, there is close linkage between the migration modifier and Mdh4.——Multiple alleles have been found for all of the Mdh loci except the migration modifier, and electrophoretically "null" or near "null" alleles (as expressed in standardized sections of maize coleoptile) have been found for all loci except Mdh4. Duplicate inheritance commonly occurs for Mdh1 and Mdh2 and also for Mdh4 and Mdh5.——Inter- and intragenic heterodimers are formed between sub-units specified by the three loci governing the mitochondrial MDH isozymes. The same is true of the alleles and nonalleles at the two loci governing the soluble variants. No such heterodimers are formed by interactions between mitochondrial and soluble MDH isozymes.     

The Effect of Framing and Normative Messages in Building Support for Climate Policies

Deep cuts in greenhouse gas emissions are required to mitigate climate change. However, there is low willingness amongst the public to prioritise climate policies for reducing emissions. Here we show that the extent to which Australians are prepared to reduce their country''s CO₂ emissions is greater when the costs to future national income are framed as a “foregone-gain”—incomes rise in the future but not by as much as in the absence of emission cuts—rather than as a “loss”—incomes decrease relative to the baseline expected future levels (Studies 1 & 2). The provision of a normative message identifying Australia as one of the world''s largest CO₂ emitters did not increase the amount by which individuals were prepared to reduce emissions (Study 1), whereas a normative message revealing the emission policy preferences of other Australians did (Study 2). The results suggest that framing the costs of reducing emissions as a smaller increase in future income and communicating normative information about others'' emission policy preferences are effective methods for leveraging public support for emission cuts.     

Starch metabolism in the leaf sheaths and culm of rice

The levels of starch and dextrin, free sugars, soluble protein, and enzymes involved in starch metabolism—α-amylase, β-amylase, phosphorylase, Q-enzyme, R-enzyme, and ADP-glucose starch synthetases—were assayed in the leaf sheaths and culm of the rice plant (Oryza sativa L., variety IR8) during growth.     

Chromosome pairing in maize

This report summarizes our observations at pachytene on opposite-arms intercrosses between stocks of interchanges that involve chromosomes 1 and 5 in maize.—Pairing does not begin at the centromeres in these intercrosses.—We propose a model which assumes different probability values along each chromosome arm for the initial or primary site of pairing. Observations on the frequencies of the different types of configurations at pachytene were used to estimate probability values which satisfactorily fit the data.—There is a relatively low probability (of the order of.1 to.3) for the initial pairing to be in a short terminal segment (about.1 of the arm length). Initial pairing in the one or two short segments adjacent to the tip segment is much higher. Initial pairing is much lower in segments successively closer to the middles of the chromosome arms, and then zero or nearly zero in the proximal half of the arm. This means that the initial pairing may fail occasionally even in a relatively long interchanged segment and produce a T-shaped (3-armed) configuration.—After the initial pairing has occurred, the average probability that a secondary site of pairing is adjacent to the centromere in a segment.3 to.4 the length of an arm is low (.13, ranging from.02 to.29).—We can predict that in an intercross in which both breakpoints in both parental interchanges are far out on the chromosomes, "pairs" will be formed with nonhomologous ends (homologous differential segments paired). In these pairing could have begun at any point in the interstitial segments, but not likely in segments close to the centromeres.—Multiple secondary sites which vary in time or in order of pairing will explain the variation in position of the cross-shaped pachytene configuration in interchange heterozygotes.—The observed configuration in any one cell is the result of a particular combination of pairing events at the various sites. This is a very different concept of pairing from previous interpretations which described it as a result of zipper-like action, and the variation in position of the pachytene cross-configuration as the result of "shifts" in position.—Our cytogenetic results and their interpretation are in close agreement with reports on chromosome ultrastructure and molecular events in the early stages of meiosis, i.e. the attachment of chromosome ends to the nuclear membrane, the manner in which synaptonemal complexes develop, and the regions of DNA whose replication is delayed until zygonema.     

A meiotic uv-sensitive mutant that causes deletion of duplications in neurospora

The meiotic-3 (mei-3) mutant of Neurospora crassa has several effects: (1) When homozygous, it almost completely blocks meiosis and ascospore formation, (2) it is sensitive to UV, (3) its growth is inhibited by histidine and, (4) it increases the instability of nontandem duplications. This was shown for duplications produced by five different rearrangements and was demonstrated by two different criteria. The effects on meiosis and duplication instability are expressed strongly at 25°; the effects on sensitivity to UV and to histidine are expressed strongly at 38.5° but only slightly at 25°. Nevertheless, all four effects were shown to be due to a single gene. mei-3 is not allelic with previously reported UV-sensitive mutants.—Two other results were obtained that are not necessarily due to mei-3: (1) A cross involving mei-3 produced a new unlinked meiotic mutant, mei-4, which is not sensitive to UV or histidine, and (2) a burst of several new mutants occurred in a different mei-3 stock, including a partial revertant of mei-3.—mei-3 has previously been shown to cause frequent complete loss of a terminal duplicate segment, beginning exactly at the original rearrangement breakpoint. Possible mechanisms are discussed by which a UV-sensitive mutant could cause such precise deletions.     

Exocytosis Precedes and Predicts the Increase in Growth in Oscillating Pollen Tubes

We examined exocytosis during oscillatory growth in lily (Lilium formosanum and Lilium longiflorum) and tobacco (Nicotiana tabacum) pollen tubes using three markers: (1) changes in cell wall thickness by Nomarski differential interference contrast (DIC), (2) changes in apical cell wall fluorescence in cells stained with propidium iodide (PI), and (3) changes in apical wall fluorescence in cells expressing tobacco pectin methyl esterase fused to green fluorescent protein (PME-GFP). Using PI fluorescence, we quantified oscillatory changes in the amount of wall material from both lily and tobacco pollen tubes. Measurement of wall thickness by DIC was only possible with lily due to limitations of microscope resolution. PME-GFP, a direct marker for exocytosis, only provides information in tobacco because its expression in lily causes growth inhibition and cell death. We show that exocytosis in pollen tubes oscillates and leads the increase in growth rate; the mean phase difference between exocytosis and growth is –98° ± 3° in lily and –124° ± 4° in tobacco. Statistical analyses reveal that the anticipatory increase in wall material predicts, to a high degree, the rate and extent of the subsequent growth surge. Exocytosis emerges as a prime candidate for the initiation and regulation of oscillatory pollen tube growth.     

Oocyte Polarization Is Coupled to the Chromosomal Bouquet,a Conserved Polarized Nuclear Configuration in Meiosis

The source of symmetry breaking in vertebrate oocytes is unknown. Animal—vegetal oocyte polarity is established by the Balbiani body (Bb), a conserved structure found in all animals examined that contains an aggregate of specific mRNAs, proteins, and organelles. The Bb specifies the oocyte vegetal pole, which is key to forming the embryonic body axes as well as the germline in most vertebrates. How Bb formation is regulated and how its asymmetric position is established are unknown. Using quantitative image analysis, we trace oocyte symmetry breaking in zebrafish to a nuclear asymmetry at the onset of meiosis called the chromosomal bouquet. The bouquet is a universal feature of meiosis where all telomeres cluster to one pole on the nuclear envelope, facilitating chromosomal pairing and meiotic recombination. We show that Bb precursor components first localize with the centrosome to the cytoplasm adjacent to the telomere cluster of the bouquet. They then aggregate around the centrosome in a specialized nuclear cleft that we identified, assembling the early Bb. We show that the bouquet nuclear events and the cytoplasmic Bb precursor localization are mechanistically coordinated by microtubules. Thus the animal—vegetal axis of the oocyte is aligned to the nuclear axis of the bouquet. We further show that the symmetry breaking events lay upstream to the only known regulator of Bb formation, the Bucky ball protein. Our findings link two universal features of oogenesis, the Bb and the chromosomal bouquet, to oocyte polarization. We propose that a meiotic—vegetal center couples meiosis and oocyte patterning. Our findings reveal a novel mode of cellular polarization in meiotic cells whereby cellular and nuclear polarity are aligned. We further reveal that in zygotene nests, intercellular cytoplasmic bridges remain between oocytes and that the position of the cytoplasmic bridge coincides with the location of the centrosome meiotic—vegetal organizing center. These results suggest that centrosome positioning is set by the last mitotic oogonial division plane. Thus, oocytes are polarized in two steps: first, mitotic divisions preset the centrosome with no obvious polarization yet, then the meiotic—vegetal center forms at zygotene bouquet stages, when symmetry is, in effect, broken.     

Cytogenetics of chromosome pairing in wheat

Meiotic chromosome pairing in Triticum aestivum is controlled by genetic systems promoting and reducing pairing. The pairing of homoeologous chromosomes is prevented principally by the activity of a single locus (Ph) distally located on the long arm of chromosome 5B. In certain hybrids, supernumerary chromosomes (B chromosomes) from Aegilops species can compensate for the absence of chromosome 5B preventing or reducing homoeologous pairing. Temperature-dependent variants and colchicine sensitivity have been used to show that there are at least two stages in the G1 of meiosis at which the occurrence of meiotic pairing is determined. Wheat may differ from lily in the detailed organization of meiosis.     

Efficient Synthesis and Anti-Tubercular Activity of a Series of Spirocycles: An Exercise in Open Science

Tuberculosis afflicts an estimated 2 billion people worldwide and causes 1.3 million deaths annually. Chemotherapeutic solutions rely on drugs developed many years ago, with only one new therapeutic having been approved in the last 40 years. Given the rise of drug-resistant strains, there is an urgent need for the development of a more robust drug development pipeline. GlaxoSmithKline recently placed the structures and activities of 177 novel anti-tubercular leads in the public domain, as well as the results of ongoing optimisation of some of the series. Since many of the compounds arose from screening campaigns, their provenance was unclear and synthetic routes were in many cases not reported. Here we present the efficient synthesis of several novel analogues of one family of the GSK compounds—termed “Spiros”—using an oxa-Pictet–Spengler reaction. The new compounds are attractive from a medicinal chemistry standpoint and some were potent against the virulent strain, suggesting this class is worthy of further study. The research was carried out using open source methodology, providing the community with full access to all raw experimental data in real time.     

Effect of Mating Structure on Variation in Linkage Disequilibrium

Measurement of linkage disequilibrium involves two sampling processes. First, there is the sampling of gametes in the population to form successive generations, and this generates disequilibrium dependent on the effective population size (N_e) and the mating structure. Second, there is sampling of a finite number (n) of individuals to estimate the population disequilibrium.——Two-locus descent measures are used to describe the mating system and are transformed to disequilibrium moments at the final sampling. Approximate eigenvectors for the transition matrix of descent measures are used to obtain formulae for the variance of the observed disequilibria as a function of N_e, mating structure, n, and linkage or recombination parameter.——The variance of disequilibrium is the same for monoecious populations with or without random selfing and for dioecious populations with random pairing for each progeny. With monogamy, the variance is slightly higher, the proportional difference being greater for unlinked loci.