Higher‐order assemblies of oligomeric cargo receptor complexes form the membrane scaffold of the Cvt vesicle

Selective autophagy is the mechanism by which large cargos are specifically sequestered for degradation. The structural details of cargo and receptor assembly giving rise to autophagic vesicles remain to be elucidated. We utilize the yeast cytoplasm‐to‐vacuole targeting (Cvt) pathway, a prototype of selective autophagy, together with a multi‐scale analysis approach to study the molecular structure of Cvt vesicles. We report the oligomeric nature of the major Cvt cargo Ape1 with a combined 2.8 Å X‐ray and negative stain EM structure, as well as the secondary cargo Ams1 with a 6.3 Å cryo‐EM structure. We show that the major dodecameric cargo prApe1 exhibits a tendency to form higher‐order chain structures that are broken upon interaction with the receptor Atg19 in vitro. The stoichiometry of these cargo–receptor complexes is key to maintaining the size of the Cvt aggregate in vivo. Using correlative light and electron microscopy, we further visualize key stages of Cvt vesicle biogenesis. Our findings suggest that Atg19 interaction limits Ape1 aggregate size while serving as a vehicle for vacuolar delivery of tetrameric Ams1.     

Molecular structure of an A-DNA decamer d(ACCGGCCGGT)

The molecular structure of the DNA decamer d(ACCGGCCGGT) has been solved and refined by single-crystal X-ray-diffraction analysis at 0.20 nm to a final R-factor of 18.0%. The decamer crystallizes as an A-DNA double helical fragment with unit-cell dimensions of a = b = 3.923 nm and c = 7.80 nm in the space group P6(1)22. The overall conformation of this A-DNA decamer is very similar to that of the fiber model for A-DNA which has a large average base-pair tilt and hence a wide and shallow minor groove. This structure is in contrast to that of several A-DNA octamers in which the molecules all have low base-pair-tilt angles (8-12 degrees) resulting in an appearance intermediate between B-DNA and A-DNA. The average helical parameters of this decamer are typical of A-DNA with 10.9 base pairs/turn of helix, an average helical twist angle of 33.1 degrees, and a base-pair-tilt angle of 18.2 degrees. However, the CpG step in this molecule has a low local-twist angle of 24.5 degrees, similar to that seen in other A-DNA oligomers, and therefore appears to be an intrinsic stacking pattern for this step. The molecules pack in the crystal using a recurring binding motif, namely, the terminal base pair of one helix abuts the surface of the shallow minor groove of another helix. In addition, the GC base pairs have large propeller-twist angles, unlike those found most other A-DNA structures.     

Gamete processing in an environmental chamber: Effect on in vitro fertilization outcome

A study of mouse gamete processing for in vitro fertilization (IVF) under various conditions showed that it is necessary to control the atmosphere if the temperature is raised from 22°C to 37°C. The data suggest that maximum IVF success is attained by processing the gametes at 37°C, under an atmosphere of 5% O₂ and 5% CO₂, and overlaying the medium with silicone oil.     

Endothelial cell junctions

In the course of a freeze-cleave study on intercellular junctions in the regenerating rat liver, we observed an unusual array of intramembranous particles located in regions of contact between endothelial cells lining the hepatic sinusoids. These arrays were characterized by an accumulation of particles which resembled a zonula occludens in their linear deployment but differed in that the contact regions were composed of individual particles which remained separated from each other by regular particle-free intervals.     

X-ray crystallographic studies of polymorphic forms of yeast phenylalanine transfer RNA

Yeast phenylalanine transfer RNA has been found to crystallize in five different crystal systems involving eight different space groups. The X-ray diffraction characteristics of these forms are described. One of the orthorhombic forms yields a diffraction pattern with higher resolution than either the hexagonal, the cubic or the monoclinic forms. One region of this orthorhombic diffraction pattern is particularly sensitive to X-ray exposure and to changes in the concentration of various solutes. The diffraction pattern from the cubic crystal form extends to a resolution of 3 Å, and there are a number of strong reflections in the 3 to 4 Å region which suggest that double-helical segments of the tRNA molecules are oriented along the 4-fold axes. Some comments are made regarding the nature of the polymorphism in the transfer RNA crystals.     

Properties of Bacteria Isolated from Deep-Sea Sediments

Thirty-eight isolates were subjected to taxonomic analysis by computer. Of the 38 isolates, 31 were from sediment samples collected at depths from 9,400 to 10,400 meters in the Philippine and Marianas Trenches of the Pacific Ocean, and 7 cultures were from seawater samples collected at various depths from surface to 4,000 meters and from several locations in the Pacific Ocean. A total of 116 characteristics were determined for each isolate, coded, and transferred to punch cards. Similarity values were obtained by computer analysis, with the use of two recently developed computer programs. Five distinct phenetic clusters were observed from the numerical analyses. Four of the clusters were identified as species of the genus Pseudomonas, and one, as an aerogenic species of Aeromonas. Group IV was identified as pigmented Pseudomonas fluorescens, and the major cluster, consisting of groups I and II, which merged at a species level of similarity, was treated as a new species of Pseudomonas. The 38 strain data were compared with data for 132 marine and nonmarine strains previously subjected to computer taxonomic analysis. The barotolerant deep-sea strains, with the exception of the deep-sea P. fluorescens isolates, clustered separately from all other marine strains.     

Structure of alpha 2-macroglobulin from the arthropod Limulus polyphemus.

A structural and functional homologue of vertebrate alpha 2-macroglobulin (alpha 2M) has been identified in the hemolymph and blood cells of the arthropod Limulus polyphemus, one of the oldest living fossil invertebrates (Quigley, J. P., and Armstrong, P. B. (1985) J. Biol. Chem. 260, 12715-12719). The subunit molecular mass is 185 kDa. The native molecular mass, determined by scanning transmission electron microscopy (STEM) under conditions in which the linear relationship between the STEM large angle detector signal and specimen mass thickness allows the determination of the total macromolecular mass, was 354 +/- 35 kDa. Sedimentation equilibrium measurements gave a value of 366 kDa, independent of solute concentration. Sedimentation velocity experiments indicated a homogeneous component with a frictional ratio of 1.41. Thus, the native structure appears to be a dimer, with a somewhat extended conformation. The behavior during gel permeation chromatography was anomalous, yielding an apparent molecular mass approximately half-way between that expected for the dimeric and tetrameric configurations. Transmission electron microscopy of negatively stained preparations revealed a dimeric butterfly-like structure that collapsed following reaction with chymotrypsin.     

Sorbose-resistant mutants ofNeurospora crassa do not have alteredβ(1–3)glucan synthase activity

A number of mutants ofNeurospora crassa (sor-1, sor-3, sor-4, sor-5, sor-6, sor-15, sor-T9, andpatch) were found to be resistant to the growth-inhibiting effect of sorbose.(1–3)-Glucan synthase activity from each strain was found to be as sensitive to sorbose as wild-type enzyme activity. Four of these strains (sor-1, sor-4, sor-5, sor-T9) had altered sorbose transport; the remaining strains tested had normal sorbose transport. All of these strains (except forsor-3) were found to metabolize sorbose to glucose (and other compounds). This may explain their sorbose resistance.     

Molecular mechanics modeling of oligonucleotide adducts of the antitumor drug cis-diamminedichloroplatinum(II)

In order to assess the geometric changes caused when the antitumor drug cis-diammine-dichloroplatinum(II) (cis-DDP) binds to DNA, molecular mechanics calculations were performed on two double-stranded and two single-stranded oligonucleotides and their adducts with cis-{Pt(NH₃)₂}²⁺. For the platinated duplexes, three model structures have been derived, one involving only local disruption of base pairing with retention of the helix directionality, and two models showing pronounced kinking of the double helix. One of the kinked models is stabilized by bridging sodium ions. The other kinked duplex model shows retention of all Watson–Crick base pairing, including that of the coordinated guanines. All models exhibit hydrogen bonds connecting one ammine ligand of platinum with one or two phosphate groups located at the 5′ side of the platinated strand.     

The dopaminergic inhibition of LH secretion during the menstrual cycle

To assess the potential inhibitory role of hypothalamic dopaminergic input on the LRF-LH system, the gonadotropin response to a dopamine receptor antagonist, metoclopramid (MCP, 10 mg iv bolus) was examined during different phases of the menstrual cycle in 12 women. In addition, the role of dopamine infusion on naloxone (opiate receptor antagonist) induced LH increments was examined. MCP induced an abrupt increase in circulating LH levels in the mid-luteal phases but not in the early and late follicular phase subjects. No significant changes in serum FSH levels were observed. Dopamine, when infused concomitantly with naloxone, completely suppressed the naloxone induced pulsatile increments of LH in mid-luteal subjects. These findings support the contention that an increased dopaminergic inhibition of LRF-LH system occurs during the high estrogen-progesterone phase of the menstrual cycle, and provide preliminary evidence that the inhibitory role of endogenous opioids on LRF release may involve the dopaminergic system.