Morphine tolerance in mice is independent of polymorphisms in opioid receptor sequences.

Pharmacogenomics links individual drug response variation to genetic differences, such as single nucleotide polymorphisms (SNPs). In particular, pharmacogenomics will allow clinicians to use genetic diagnostics to predict the response of a patient to a drug. We investigated whether SNPs in opioid receptors correlated with the development of morphine tolerance in mouse strains that showed either high or low tolerance to morphine. Sequencing identified five silent SNPs in the delta opioid receptor that varied from the published sequence in some strains, but which were found in both high and low tolerance strains. The mu and kappa opioid receptor sequences had no SNPs. Taken together, these data definitively demonstrate that morphine tolerance development in mice is independent of opioid receptor sequence.

Roles of LAP2 proteins in nuclear assembly and DNA replication: truncated LAP2beta proteins alter lamina assembly, envelope formation, nuclear size, and DNA replication efficiency in Xenopus laevis extracts.

Humans express three major splicing isoforms of LAP2, a lamin- and chromatin-binding nuclear protein. LAP2beta and gamma are integral membrane proteins, whereas alpha is intranuclear. When truncated recombinant human LAP2beta proteins were added to cell-free Xenopus laevis nuclear assembly reactions at high concentrations, a domain common to all LAP2 isoforms (residues 1-187) inhibited membrane binding to chromatin, whereas the chromatin- and lamin-binding region (residues 1-408) inhibited chromatin expansion. At lower concentrations of the common domain, membranes attached to chromatin with a unique scalloped morphology, but these nuclei neither accumulated lamins nor replicated. At lower concentrations of the chromatin- and lamin-binding region, nuclear envelopes and lamins assembled, but nuclei failed to enlarge and replicated on average 2. 5-fold better than controls. This enhancement was not due to rereplication, as shown by density substitution experiments, suggesting the hypothesis that LAP2beta is a downstream effector of lamina assembly in promoting replication competence. Overall, our findings suggest that LAP2 proteins mediate membrane-chromatin attachment and lamina assembly, and may promote replication by influencing chromatin structure.

Nuclear envelope and nuclear pore assembly: analysis of assembly intermediates by electron microscopy.

At mitosis, the nucleus of higher eukaryotic cells disassemblies into components which subsequently reform functional nuclear envelopes in the two daughter cells. The molecular mechanisms underlying this remarkable morphological reorganization are the focus of active investigation. Recent electron microscopy techniques have provided intriguing glimpses of intermediate structures in both nuclear envelope and nuclear pore complex reassembly.

ARF is not required for nuclear vesicle fusion or mitotic membrane disassembly in vitro: evidence for a non-ARF GTPase in fusion.

Xenopus laevis egg extracts are a well-characterized system for studying nuclear envelope dynamics in vitro. ADP-ribosylation factor (ARF), a 21 kDa GTPase involved in vesicular transport, inhibits nuclear vesicle fusion in vitro when membranes are preincubated with ARF and GTP gamma S (Boman et al., Nature 358, 512-514 (1992)). To test the hypothesis that ARF was required for nuclear envelope assembly or disassembly, we examined these events in cytosol from which ARF was depleted by size fractionation or inhibited with brefeldin A (BFA). In ARF-depleted extracts, vesicles bound chromatin and fused to enclose the chromatin, but the resulting enclosed nuclei lacked pore complexes and remained small. Further growth was not stimulated by adding ARF1, suggesting that fractionation removed other proteins required for pore complex assembly and nuclear growth. Nuclei assembled in ARF-depleted extracts, and rat liver nuclei, disassembled normally in mitotic ARF-depleted reactions. BFA, which inhibits ARF binding to membranes, had no effect on nuclear assembly or disassembly. We concluded that ARF is not essential for nuclear membrane dynamics. Nuclear vesicle fusion was still inhibited by GTP gamma S in ARF-depleted reactions and in reactions containing BFA, strongly suggesting that there is another unidentified GTPase that is either required for vesicle fusion or capable of inhibiting fusion in the presence of GTP gamma S.

Ionophore-releasable lumenal Ca2+ stores are not required for nuclear envelope assembly or nuclear protein import in Xenopus egg extracts.

The nuclear envelope of higher eukaryotes disassembles early in mitosis and reassembles later around the daughter chromosomes. Previous in vitro work supported the hypothesis that the release of lumenal Ca2+ stores via inositol 1,4,5-trisphosphate-gated Ca2+ channels is required for nuclear assembly in Xenopus egg extracts. Other work suggested that lumenal Ca2+ stores are required for nuclear protein import in mammalian cells in vivo, but not in vitro. Here, we rigorously tested the role of lumenal Ca2+ stores in nuclear assembly and nuclear protein import using Xenopus egg extracts. Lumenal Ca2+ stores were depleted by pretreating the extracts with Ca2+ ionophores (ionomycin, A23187) or inhibitors of Ca(2+)-sequestering pumps (thapsigargin, cyclopiazonic acid). Extracts depleted of lumenal Ca2+ stores assembled nuclei around demembranated sperm chromatin. These nuclei were morphologically indistinguishable from control nuclei when viewed by light or electron microscopy. Nuclei lacking lumenal Ca2+ stores excluded membrane-impermeant fluorescent dextrans, indicating the formation of a sealed nuclear envelope, and they accumulated a fluorescent nucleophilic protein, nucleoplasmin, indicating that nuclear pore complexes were functional. DNA replication occurred in the lumenal-Ca(2+)-depleted nuclei, though less efficiently than control nuclei. Our demonstration that in vitro nuclear import does not depend on lumenal Ca2+ stores confirms a previous unpublished observation by Greber and Gerace, and suggests that import defects seen in ionophore-treated living cells are not directly due to the loss of lumenal Ca2+. Finally, we concluded that, contrary to our expectations, lumenal Ca2+ stores are not required for nuclear envelope assembly in Xenopus egg extracts.

Nuclear assembly.

We review old and new insights into the structure of the nuclear envelope and the components responsible for its dynamic reassembly during mitosis. New information is coming to light about several of the proteins that mediate nuclear reassembly. These proteins include the lamins and their emerging relationship with proteins such as otefin and the MAN antigens: peripheral proteins that might participate in lamina structure. There are four identified proteins localized to the inner nuclear membrane: the lamina-associated proteins LAP1 and LAP2, emerin, and the lamin B receptor (LBR). LBR can interact independently with lamin B and a chromodomain protein, Hp1, and appears to be a central player in targeting nuclear membranes to chromatin. Intermediates in the assembly of nuclear pore complexes (NPCs) can now be studied biochemically and visualized by high resolution scanning electron microscopy. We discuss the possibility that the filament-forming proteins Tpr/p270, NuMA, and perhaps actin may have roles in nuclear assembly.

cDNA cloning and bacterial expression of the human type I keratin 16.

The human type I keratin 16 is constitutively expressed in a number of complex epithelial tissues, including skin, but is better known for its induction under conditions favoring enhanced proliferation or abnormal differentiation, including wound healing, psoriasis, and cancer. We cloned the coding sequence of human K16 by applying a coupled reverse transcription-polymerase chain reaction procedure to mRNAs prepared from cultured human skin keratinocytes. We then expressed the human K16 coding sequence in E. coli and purified the solubilized protein by anion-exchange chromatography. The recombinant protein recovered behaves similarly to human K14 (a related acidic keratin) on the anion-exchanger, co-migrates with native human K16 on SDS-PAGE (M(r) 48 kD), and reacts with antisera directed against human K16. Based on the nucleotide sequence obtained and the properties of the corresponding recombinant protein, we conclude that we have cloned the coding portion of the human K16 cDNA. The sequence data obtained in this study is compared to earlier reports of the human K16 sequence, which are conflicting in many respects. The availability of K16 in a purified recombinant form will allow us to study how its properties may relate to its function during wound healing and in skin diseases.