Microscopic anatomy of the ovary ofAlouatta caraya

The microscopic structure of theAlouatta caraya ovary is studied in different ages and reproductive stages. The most significant feature seems to be the presence in adult ovaries of abundant glandular interstitial tissue which occupies both the cortex and medulla. It seems to be derived from the theca interna of atretic follicles. Discrete luteinized masses are present in the medulla in all the ovaries observed. Invaginations of the surface epithelium are seen only in infant and juvenile ovaries. The development of cystic follicles seems to be a common pathway of atresia.     

Amino acid transport in pig lymphocytes Enhanced activity of transport system asc following mitogenic stimulation

Changes in neutral amino acid transport activity caused by addition of phytohaemagglutinin-P to quiescent peripheral pig lymphocytes have been evaluated by measurements of ¹⁴C-labelled neutral and analogue amino acids under conditions approaching initial entry rates. Utilizing methylaminoisobutyric acid, the best model substrate of System A, we confirmed our previous report (Borghetti, A.F., Kay, J.E. and Wheeler, K.P. (1979) Biochem. J. 182, 27–32) on the absence of this transport system in quiescent cells and its emergence following stimulation. Furthermore, we demonstrated the presence in quiescent cells of an Na⁺-dependent transport system for neutral amino acids that has been characterized as System ASC by several criteria including intolerance to methylaminoisobutyric acid, strict Na⁺-dependence, the property of transtimulation and specificity for pertinent substrates such as alanine, serine, cysteine and threonine. Analysis of the relationship between influx and substrate concentration revealed that two independent saturable components contribute to entry of alanine in quiescent cells: a low affinity ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= ≈4}\text{mM}$) and a high affinity ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}\text{= ≈0.2}\text{mM}$) component. The high affinity component could be inhibited in a competitive way by serine, cysteine and threonine, but methylaminoisobutyric acid did not change appreciably its constants. The enhanced activity of alanine transport through the ASC system observed in activated cells resulted from a large increase in the capacity ($\text{V}$) of the high affinity component without any substantial change in the apparent affinity constant ($\text{K}{}_{\mathrm{<mtext>m</mtext>}}$).     

Autosomal synaptonemal complexes and sex chromosomes without axes in Triatoma infestans (Reduviidae; Hemiptera)

Meiotic and somatic cells at interphase in Triatoma infestans are characterized by the formation of a large chromocenter, which was assumed to contain the whole of the three large pairs of autosomes and the sex chromosomes. Observations with C-banding techniques show that the chromocenter is formed only by the terminal and subterminal heterochromatic blocks of the three large pairs of autosomes and the sex chromosomes. During pachytene the two largest autosomal pairs loop on themselves and their condensed ends form the chromocenter, together with the single heterochromatic end of the third autosomal pair. The X and Y chromosomes seem to associate with these condensed ends by their affinity for C-heterochromatin. During a very short pachytene stage, bivalents and synaptonemal complexes (SCs) are observed. Pachytene is followed by a very long diffuse stage, during which SCs are disassembled, multiple complexes aggregate on the inner face of the chromocenter and finally all complexes disappear and a dense material is extruded to the cytoplasm through the annuli. The 3-dimensional reconstruction of early pachytene chromocenters show 3 SCs entering and tunnelling the chromocenter, while during mid-pachytene 4 SCs enter this mass and a 5th SC is in a separate small mass. The looping of a whole SC which has both ends in the chromocenter was shown by the reconstructions. These data are interpreted as the progressive looping of the two largest bivalents during pachytene, forming finally the association of 5 bivalent ends corresponding to the 5 C-banding blocks of the large autosomal pairs. No single axis or SC that could be ascribed to the sex chromosomes was found. This agrees with the pachytene microspreads, which show only 10 SCs corresponding to the autosomal bivalents. The X and Y chromosomes are enclosed in the chromocenter, as shown by the unravelling chromocenters at diplotene-diakinesis. Thus the sex chromosomes do not form axial condensations, and this fact may be related to the ability of the X and Y chromosomes to divide equationally at metaphase I. SCsThis paper is dedicated to the memory of the late Professor Francisco A. Saez     

The synaptic behaviour of the X and Y chromosomes in the marsupial Monodelphis dimidiata

The pairing behaviour of the X and Y chromosomes of Monodelphis dimidiata was studied with light and electron microscopy. Pairing of the sex chromosomes is delayed with respect to autosome synapsis. Both the X and the minute Y chromosome show an axis attached by its two ends to the nuclear envelope. Synapsis of the sex chromosomes occurs by the joining of the chromatin sheaths that surround the axes and by a small, three-layered structure close to the nuclear envelope. The X and Y chromosomes remain joined to each other during the diffuse stage and diplotene-diakinesis but they do not show a synaptonemal complex. During the diffuse stage a dense plate is formed at the boundary between the X-Y body and the nuclear envelope. During early metaphase a folded sheet is attached to the periphery of the X-Y body. This sheet is formed by a piece of the nuclear envelope carrying the dense plate and it shows transverse fibrils and a central element similar to synaptonemal-complex remains. No evidence of a non-chiasmate segregation mechanism was observed. Polarization of the axial ends of the sex chromosomes is observed after X-Y synapsis. These important departures from the X-Y pairing pattern of eutherian mammals are discussed and assumed to present a special mechanism for holding the minute Y joined to the X chromosome in this marsupial.     

Morphological changes of the surface of the egg of Xenopus laevis in the course of development: III. Scanning electron microscopy of gastrulation

Cell surface changes occurring before and during gastrulation in Xenopus laevis embryos have been examined by scanning electron microscopy (SEM). Our study covers the period of development from very young blastulae (stage 7) to late gastrulae (stage $\text{12}\text{1}\text{2}$. Before the onset of the epibolic movement there is evidence of locomotory activity of the cells lining the blastocoel at the animal pole. In the medim- (stage 8) and small-cell (stage 9) blastula, when pregastrulation movements are progressing rapidly, microvilli appear in the interstices between cells, both at the animal and at the vegetal pole. In the gastrula, most of the cells close to the blastopore have either their entire exposed surface or part of it covered with microvilli. On the other hand, the cells that have just reached the blastopore and have become clubshaped do not display microvilli on their surfaces; microvilli are also absent on the surface of the cells that have undergone invagination. The invaginated chorda-mesoderm is made up of single fibroblastlike cells with long thin filopodia which are interwoven with those of nearby cells. The observations are discussed in relation to changes in cell-to-cell connections and to the role of cell surface organization in the morphogenetic movements of gastrulation.     

Melanoma RBPome identification reveals PDIA6 as an unconventional RNA-binding protein involved in metastasis

RNA-binding proteins (RBPs) have been relatively overlooked in cancer research despite their contribution to virtually every cancer hallmark. Here, we use RNA interactome capture (RIC) to characterize the melanoma RBPome and uncover novel RBPs involved in melanoma progression. Comparison of RIC profiles of a non-tumoral versus a metastatic cell line revealed prevalent changes in RNA-binding capacities that were not associated with changes in RBP levels. Extensive functional validation of a selected group of 24 RBPs using five different in vitro assays unveiled unanticipated roles of RBPs in melanoma malignancy. As proof-of-principle we focused on PDIA6, an ER-lumen chaperone that displayed a novel RNA-binding activity. We show that PDIA6 is involved in metastatic progression, map its RNA-binding domain, and find that RNA binding is required for PDIA6 tumorigenic properties. These results exemplify how RIC technologies can be harnessed to uncover novel vulnerabilities of cancer cells.     

Sequence dependence of transient Hoogsteen base pairing in DNA

Hoogsteen (HG) base pairing is characterized by a 180° rotation of the purine base with respect to the Watson-Crick-Franklin (WCF) motif. Recently, it has been found that both conformations coexist in a dynamical equilibrium and that several biological functions require HG pairs. This relevance has motivated experimental and computational investigations of the base-pairing transition. However, a systematic simulation of sequence variations has remained out of reach. Here, we employ advanced path-based methods to perform unprecedented free-energy calculations. Our methodology enables us to study the different mechanisms of purine rotation, either remaining inside or after flipping outside of the double helix. We study seven different sequences, which are neighbor variations of a well-studied A⋅T pair in A₆-DNA. We observe the known effect of A⋅T steps favoring HG stability, and find evidence of triple-hydrogen-bonded neighbors hindering the inside transition. More importantly, we identify a dominant factor: the direction of the A rotation, with the 6-ring pointing either towards the longer or shorter segment of the chain, respectively relating to a lower or higher barrier. This highlights the role of DNA’s relative flexibility as a modulator of the WCF/HG dynamic equilibrium. Additionally, we provide a robust methodology for future HG proclivity studies.     

Ultrahigh resolution drug design. II. Atomic resolution structures of human aldose reductase holoenzyme complexed with Fidarestat and Minalrestat: implications for the binding of cyclic imide inhibitors

The X-ray structures of human aldose reductase holoenzyme in complex with the inhibitors Fidarestat (SNK-860) and Minalrestat (WAY-509) were determined at atomic resolutions of 0.92 A and 1.1 A, respectively. The hydantoin and succinimide moieties of the inhibitors interacted with the conserved anion-binding site located between the nicotinamide ring of the coenzyme and active site residues Tyr48, His110, and Trp111. Minalrestat's hydrophobic isoquinoline ring was bound in an adjacent pocket lined by residues Trp20, Phe122, and Trp219, with the bromo-fluorobenzyl group inside the "specificity" pocket. The interactions between Minalrestat's bromo-fluorobenzyl group and the enzyme include the stacking against the side-chain of Trp111 as well as hydrogen bonding distances with residues Leu300 and Thr113. The carbamoyl group in Fidarestat formed a hydrogen bond with the main-chain nitrogen atom of Leu300. The atomic resolution refinement allowed the positioning of hydrogen atoms and accurate determination of bond lengths of the inhibitors, coenzyme NADP+ and active-site residue His110. The 1'-position nitrogen atom in the hydantoin and succinimide moieties of Fidarestat and Minalrestat, respectively, form a hydrogen bond with the Nepsilon2 atom of His 110. For Fidarestat, the electron density indicated two possible positions for the H-atom in this bond. Furthermore, both native and anomalous difference maps indicated the replacement of a water molecule linked to His110 by a Cl-ion. These observations suggest a mechanism in which Fidarestat is bound protonated and becomes negatively charged by donating the proton to His110, which may have important implications on drug design.     

Investigation of ubiquinol formation in isolated photosynthetic reaction centers by rapid-scan Fourier transform IR spectroscopy

Light-induced formation of ubiquinol-10 in Rhodobacter sphaeroides reaction centers was followed by rapid-scan Fourier transform IR difference spectroscopy, a technique that allows the course of the reaction to be monitored, providing simultaneously information on the redox states of cofactors and on protein response. The spectrum recorded between 4 and 29 ms after the second flash showed bands at 1,470 and 1,707 cm(-1), possibly due to a QH(-) intermediate state. Spectra recorded at longer delay times showed a different shape, with bands at 1,388 (+) and 1,433 (+) cm(-1) characteristic of ubiquinol. These spectra reflect the location of the ubiquinol molecule outside the Q(B) binding site. This was confirmed by Fourier transform IR difference spectra recorded during and after continuous illumination in the presence of an excess of exogenous ubiquinone molecules, which revealed the process of ubiquinol formation, of ubiquinone/ubiquinol exchange at the Q(B) site and between detergent micelles, and of Q(B)(-) and QH(2) reoxidation by external redox mediators. Kinetics analysis of the IR bands allowed us to estimate the ubiquinone/ubiquinol exchange rate between detergent micelles to approximately 1 s. The reoxidation rate of Q(B)(-) by external donors was found to be much lower than that of QH(2), most probably reflecting a stabilizing/protecting effect of the protein for the semiquinone form. A transient band at 1,707 cm(-1) observed in the first scan (4-29 ms) after both the first and the second flash possibly reflects transient protonation of the side chain of a carboxylic amino acid involved in proton transfer from the cytoplasm towards the Q(B) site.