R-type Ca2+ currents evoke transmitter release at a rat central synapse

Voltage-dependent Ca2+ currents evoke synaptic transmitter release. Of six types of Ca2+ channels, L-, N-, P-, Q-, R-, and T-type, only N- and P/Q-type channels have been pharmacologically identified to mediate action-potential-evoked transmitter release in the mammalian central nervous system. We tested whether Ca2+ channels other than N- and P/Q-type control transmitter release in a calyx-type synapse of the rat medial nucleus of the trapezoid body. Simultaneous recordings of presynaptic Ca2+ influx and the excitatory postsynaptic current evoked by a single action potential were made at single synapses. The R-type channel, a high-voltage-activated Ca2+ channel resistant to L-, N-, and P/Q-type channel blockers, contributed 26% of the total Ca2+ influx during a presynaptic action potential. This Ca2+ current evoked transmitter release sufficiently large to initiate an action potential in the postsynaptic neuron. The R-type current controlled release with a lower efficacy than other types of Ca2+ currents. Activation of metabotropic glutamate receptors and γ-aminobutyric acid type B receptors inhibited the R-type current. Because a significant fraction of presynaptic Ca2+ channels remains unidentified in many other central synapses, the R-type current also could contribute to evoked transmitter release in these synapses.

Down-regulation of transmembrane carbonic anhydrases in renal cell carcinoma cell lines by wild-type von Hippel-Lindau transgenes

To discover genes involved in von Hippel-Lindau (VHL)-mediated carcinogenesis, we used renal cell carcinoma cell lines stably transfected with wild-type VHL-expressing transgenes. Large-scale RNA differential display technology applied to these cell lines identified several differentially expressed genes, including an alpha carbonic anhydrase gene, termed CA12. The deduced protein sequence was classified as a one-pass transmembrane CA possessing an apparently intact catalytic domain in the extracellular CA module. Reintroduced wild-type VHL strongly inhibited the overexpression of the CA12 gene in the parental renal cell carcinoma cell lines. Similar results were obtained with CA9, encoding another transmembrane CA with an intact catalytic domain. Although both domains of the VHL protein contribute to regulation of CA12 expression, the elongin binding domain alone could effectively regulate CA9 expression. We mapped CA12 and CA9 loci to chromosome bands 15q22 and 17q21.2 respectively, regions prone to amplification in some human cancers. Additional experiments are needed to define the role of CA IX and CA XII enzymes in the regulation of pH in the extracellular microenvironment and its potential impact on cancer cell growth.

The type 2 iodothyronine deiodinase is expressed primarily in glial cells in the neonatal rat brain

Thyroid hormone plays an essential role in mammalian brain maturation and function, in large part by regulating the expression of specific neuronal genes. In this tissue, the type 2 deiodinase (D2) appears to be essential for providing adequate levels of the active thyroid hormone 3,5,3′-triiodothyronine (T3) during the developmental period. We have studied the regional and cellular localization of D2 mRNA in the brain of 15-day-old neonatal rats. D2 is expressed in the cerebral cortex, olfactory bulb, hippocampus, caudate, thalamus, hypothalamus, and cerebellum and was absent from the white matter. At the cellular level, D2 is expressed predominantly, if not exclusively, in astrocytes and in the tanycytes lining the third ventricle and present in the median eminence. These results suggest a close metabolic coupling between subsets of glial cells and neurons, whereby thyroxine is taken up from the blood and/or cerebrospinal fluid by astrocytes and tanycytes, is deiodinated to T3, and then is released for utilization by neurons.

Binding of high-risk human papillomavirus E6 oncoproteins to the human homologue of the Drosophila discs large tumor suppressor protein

In the majority of cervical cancers, DNAs of high-risk mucosotpropic human papillomaviruses (HPVs), such as type 16, are maintained so as to express two viral proteins, E6 and E7, suggesting an essential importance to carcinogenesis. The high-risk HPV E6 proteins are known to inactivate p53 tumor suppressor protein but appear to have an additional, molecularly unknown function(s). In this study, we demonstrate that these E6 proteins can bind to the second PDZ domain of the human homologue of the Drosophila discs large tumor suppressor protein (hDLG) through their C-terminal XS/TXV/L (where X represents any amino acid, S/T serine or threonine, and V/L valine or leucine) motif. This finding is similar to the interaction between the adenomatous polyposis coli gene product and hDLG. E6 mutants losing the ability to bind to hDLG are no longer able to induce E6-dependent transformation of rodent cells. These results suggest an intriguing possibility that interaction between the E6 protein and hDLG or other PDZ domain-containing proteins could be an underlying mechanism in the development of HPV-associated cancers.

Mouse model of Sanfilippo syndrome type B produced by targeted disruption of the gene encoding α-N-acetylglucosaminidase

The Sanfilippo syndrome type B is an autosomal recessive disorder caused by mutation in the gene (NAGLU) encoding α-N-acetylglucosaminidase, a lysosomal enzyme required for the stepwise degradation of heparan sulfate. The most serious manifestations are profound mental retardation, intractable behavior problems, and death in the second decade. To generate a model for studies of pathophysiology and of potential therapy, we disrupted exon 6 of Naglu, the homologous mouse gene. Naglu−/− mice were healthy and fertile while young and could survive for 8–12 mo. They were totally deficient in α-N-acetylglucosaminidase and had massive accumulation of heparan sulfate in liver and kidney as well as secondary changes in activity of several other lysosomal enzymes in liver and brain and elevation of gangliosides GM2 and GM3 in brain. Vacuolation was seen in many cells, including macrophages, epithelial cells, and neurons, and became more prominent with age. Although most vacuoles contained finely granular material characteristic of glycosaminoglycan accumulation, large pleiomorphic inclusions were seen in some neurons and pericytes in the brain. Abnormal hypoactive behavior was manifested by 4.5-mo-old Naglu−/− mice in an open field test; the hyperactivity that is characteristic of affected children was not observed even in younger mice. In a Pavlovian fear conditioning test, the 4.5-mo-old mutant mice showed normal response to context, indicating intact hippocampal-dependent learning, but reduced response to a conditioning tone, perhaps attributable to hearing impairment. The phenotype of the α-N-acetylglucosaminidase-deficient mice is sufficiently similar to that of patients with the Sanfilippo syndrome type B to make these mice a good model for study of pathophysiology and for development of therapy.

Suppression of an absolute defect in Type IV pilus biogenesis by loss-of-function mutations in pilT, a twitching motility gene in Neisseria gonorrhoeae

Type IV pili of Neisseria gonorrhoeae, the Gram-negative etiologic agent of gonorrhea, facilitate colonization of the human host. Gonococcal PilT, a protein belonging to a large family of molecules sharing a highly conserved nucleotide binding domain motif, has been shown to be dispensable for organelle biogenesis but essential for twitching motility and competence for genetic transformation. Here, we show that the defect in pilus biogenesis resulting from mutations in the pilC gene, encoding a putative pilus-associated adhesin for human tissue, can be suppressed by the absence of functional PilT. These data conclusively demonstrate that PilT influences the Type IV pilus biogenesis pathway and strongly suggest that organelle expression is a dynamic process. In addition, these findings imply that PilT antagonizes the process of organelle biogenesis and provide the basis for a model for how the counteractive roles of PilT and PilC might relate mechanistically to the phenomenon of twitching motility.

On representations of finite type

Representations of the (infinite) canonical anticommutation relations and the associated operator algebra, the fermion algebra, are studied. A “coupling constant” (in (0,1]) is defined for primary states of “finite type” of that algebra. Primary, faithful states of finite type with arbitrary coupling are constructed and classified. Their physical significance for quantum thermodynamical systems at high temperatures is discussed. The scope of this study is broadened to include a large class of operator algebras sharing some of the structural properties of the fermion algebra.

Attenuated T2 relaxation by mutual cancellation of dipole–dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution

Fast transverse relaxation of 1H, 15N, and 13C by dipole-dipole coupling (DD) and chemical shift anisotropy (CSA) modulated by rotational molecular motions has a dominant impact on the size limit for biomacromolecular structures that can be studied by NMR spectroscopy in solution. Transverse relaxation-optimized spectroscopy (TROSY) is an approach for suppression of transverse relaxation in multidimensional NMR experiments, which is based on constructive use of interference between DD coupling and CSA. For example, a TROSY-type two-dimensional 1H,15N-correlation experiment with a uniformly 15N-labeled protein in a DNA complex of molecular mass 17 kDa at a 1H frequency of 750 MHz showed that 15N relaxation during 15N chemical shift evolution and 1HN relaxation during signal acquisition both are significantly reduced by mutual compensation of the DD and CSA interactions. The reduction of the linewidths when compared with a conventional two-dimensional 1H,15N-correlation experiment was 60% and 40%, respectively, and the residual linewidths were 5 Hz for 15N and 15 Hz for 1HN at 4°C. Because the ratio of the DD and CSA relaxation rates is nearly independent of the molecular size, a similar percentagewise reduction of the overall transverse relaxation rates is expected for larger proteins. For a 15N-labeled protein of 150 kDa at 750 MHz and 20°C one predicts residual linewidths of 10 Hz for 15N and 45 Hz for 1HN, and for the corresponding uniformly 15N,2H-labeled protein the residual linewidths are predicted to be smaller than 5 Hz and 15 Hz, respectively. The TROSY principle should benefit a variety of multidimensional solution NMR experiments, especially with future use of yet somewhat higher polarizing magnetic fields than are presently available, and thus largely eliminate one of the key factors that limit work with larger molecules.

A targeted mutation in the murine gene encoding the high density lipoprotein (HDL) receptor scavenger receptor class B type I reveals its key role in HDL metabolism

Plasma high density lipoprotein (HDL), which protects against atherosclerosis, is thought to remove cholesterol from peripheral tissues and to deliver cholesteryl esters via a selective uptake pathway to the liver (reverse cholesterol transport) and steroidogenic tissues (e.g., adrenal gland for storage and hormone synthesis). Despite its physiologic and pathophysiologic importance, the cellular metabolism of HDL has not been well defined. The class B, type I scavenger receptor (SR-BI) has been proposed to play an important role in HDL metabolism because (i) it is a cell surface HDL receptor which mediates selective cholesterol uptake in cultured cells, (ii) its physiologically regulated expression is most abundant in the liver and steroidogenic tissues, and (iii) hepatic overexpression dramatically lowers plasma HDL. To test directly the normal role of SR-BI in HDL metabolism, we generated mice with a targeted null mutation in the SR-BI gene. In heterozygous and homozygous mutants relative to wild-type controls, plasma cholesterol concentrations were increased by ≈31% and 125%, respectively, because of the formation of large, apolipoprotein A-I (apoA-I)-containing particles, and adrenal gland cholesterol content decreased by 42% and 72%, respectively. The plasma concentration of apoA-I, the major protein in HDL, was unchanged in the mutants. This, in conjunction with the increased lipoprotein size, suggests that the increased plasma cholesterol in the mutants was due to decreased selective cholesterol uptake. These results provide strong support for the proposal that in mice the gene encoding SR-BI plays a key role in determining the levels of plasma lipoprotein cholesterol (primarily HDL) and the accumulation of cholesterol stores in the adrenal gland. If it has a similar role in controlling plasma HDL in humans, SR-BI may influence the development and progression of atherosclerosis and may be an attractive candidate for therapeutic intervention in this disease.

The Activity of Differentiation Factors Induces Apoptosis in Polyomavirus Large T-Expressing Myoblasts

It is commonly accepted that pathways that regulate proliferation/differentiation processes, if altered in their normal interplay, can lead to the induction of programmed cell death. In a previous work we reported that Polyoma virus Large Tumor antigen (PyLT) interferes with in vitro terminal differentiation of skeletal myoblasts by binding and inactivating the retinoblastoma antioncogene product. This inhibition occurs after the activation of some early steps of the myogenic program. In the present work we report that myoblasts expressing wild-type PyLT, when subjected to differentiation stimuli, undergo cell death and that this cell death can be defined as apoptosis. Apoptosis in PyLT-expressing myoblasts starts after growth factors removal, is promoted by cell confluence, and is temporally correlated with the expression of early markers of myogenic differentiation. The block of the initial events of myogenesis by transforming growth factor β or basic fibroblast growth factor prevents PyLT-induced apoptosis, while the acceleration of this process by the overexpression of the muscle-regulatory factor MyoD further increases cell death in this system. MyoD can induce PyLT-expressing myoblasts to accumulate RB, p21, and muscle- specific genes but is unable to induce G00 arrest. Several markers of different phases of the cell cycle, such as cyclin A, cdk-2, and cdc-2, fail to be down-regulated, indicating the occurrence of cell cycle progression. It has been frequently suggested that apoptosis can result from an unbalanced cell cycle progression in the presence of a contrasting signal, such as growth factor deprivation. Our data involve differentiation pathways, as a further contrasting signal, in the generation of this conflict during myoblast cell apoptosis.

Presence of a mitochondrial-type 70-kDa heat shock protein in Trichomonas vaginalis suggests a very early mitochondrial endosymbiosis in eukaryotes

Molecular phylogenetic analyses, based mainly on ribosomal RNA, show that three amitochondriate protist lineages, diplomonads, microsporidia, and trichomonads, emerge consistently at the base of the eukaryotic tree before groups having mitochondria. This suggests that these groups could have diverged before the mitochondrial endosymbiosis. Nevertheless, since all these organisms live in anaerobic environments, the absence of mitochondria might be due to secondary loss, as demonstrated for the later emerging eukaryote Entamoeba histolytica. We have now isolated from Trichomonas vaginalis a gene encoding a chaperone protein (HSP70) that in other lineages is addressed to the mitochondrial compartment. The phylogenetic reconstruction unambiguously located this HSP70 within a large set of mitochondrial sequences, itself a sister-group of α-purple bacteria. In addition, the T. vaginalis protein exhibits the GDAWV sequence signature, so far exclusively found in mitochondrial HSP70 and in proteobacterial dnaK. Thus mitochondrial endosymbiosis could have occurred earlier than previously assumed. The trichomonad double membrane-bounded organelles, the hydrogenosomes, could have evolved from mitochondria.

Cytoplasmically sequestered wild-type p53 protein in neuroblastoma is relocated to the nucleus by a C-terminal peptide

Cytoplasmic sequestration of wild-type p53 protein occurs in a subset of primary human tumors including breast cancer, colon cancer, and neuroblastoma (NB). The sequestered p53 localizes to punctate cytoplasmic structures that represent large protein aggregates. One functional consequence of this blocked nuclear access is impairment of the p53-mediated G1 checkpoint after DNA damage. Here we show that cytoplasmic p53 from NB cells is incompetent for specific DNA binding, probably due to its sequestration. Importantly, the C-terminal domain of sequestered p53 is masked, as indicated by the failure of a C-terminally directed antibody to detect p53 in these structures. To determine (i) which domain of p53 is involved in the aggregation and (ii) whether this phenotype is potentially reversible, we generated stable NB sublines that coexpress the soluble C-terminal mouse p53 peptide DD1 (amino acids 302–390). A dramatic phenotypic reversion occurred in five of five lines. The presence of DD1 blocked the sequestration of wild-type p53 and relocated it to the nucleus, where it accumulated. The nuclear translocation is due to shuttling of wild-type p53 by heteroligomerization to DD1, as shown by coimmunoprecipitation. As expected, the nuclear heterocomplexes were functionally inactive, since DD1 is a dominant negative inhibitor of wild-type p53. In summary, we show that nuclear access of p53 can be restored in NB cells.

Ubiquitination of RNA polymerase II large subunit signaled by phosphorylation of carboxyl-terminal domain

A sensitive assay using biotinylated ubiquitin revealed extensive ubiquitination of the large subunit of RNA polymerase II during incubations of transcription reactions in vitro. Phosphorylation of the repetitive carboxyl-terminal domain of the large subunit was a signal for ubiquitination. Specific inhibitors of cyclin-dependent kinase (cdk)-type kinases suppress the ubiquitination reaction. These kinases are components of transcription factors and have been shown to phosphorylate the carboxyl-terminal domain. In both regulation of transcription and DNA repair, phosphorylation of the repetitive carboxyl-terminal domain by kinases might signal degradation of the polymerase.

Elucidating the mechanism of familial amyloidosis– Finnish type: NMR studies of human gelsolin domain 2

Familial amyloidosis–Finnish type (FAF) results from a single mutation at residue 187 (D187N or D187Y) within domain 2 of the actin-regulating protein gelsolin. The mutation somehow allows a masked cleavage site to be exposed, leading to the first step in the formation of an amyloidogenic fragment. We have performed NMR experiments investigating structural and dynamic changes between wild-type (WT) and D187N gelsolin domain 2 (D2). On mutation, no significant structural or dynamic changes occur at or near the cleavage site. Areas in conformational exchange are observed between β-strand 4 and α-helix 1 and within the loop region following β-strand 5. Chemical shift differences are noted along the face of α-helix 1 that packs onto the β-sheet, suggesting an altered conformation. Conformational changes within these areas can have an effect on actin binding and may explain why D187N gelsolin is inactive. {1H-15N} nuclear Overhauser effect and chemical shift data suggest that the C-terminal tail of D187N gelsolin D2 is less structured than WT by up to six residues. In the crystal structure of equine gelsolin, the C-terminal tail of D2 lies across a large cleft between domains 1 and 2 where the masked cleavage site sits. We propose that the D187N mutation destabilizes the C-terminal tail of D2 resulting in a more exposed cleavage site leading to the first proteolysis step in the formation of the amyloidogenic fragment.

The barrier-to-autointegration protein is a host factor for HIV type 1 integration

In vivo, retroviral integration is mediated by a large nucleoprotein complex, termed the preintegration complex (PIC). PICs isolated from infected cells display in vitro integration activity. Here, we analyze the roles of different host cell factors in the structure and function of HIV type 1 (HIV-1) PICs. PICs purified by size exclusion after treatment with high salt lost their integration activity, and adding back an extract from uninfected cells restored this activity. In parallel, the native protein–DNA intasome structure detected at the ends of HIV-1 by Mu-mediated PCR footprinting was abolished by high salt and restored by the crude cell extract. Various purified proteins previously implicated in retroviral PIC function then were analyzed for their effects on the structure and function of salt-treated HIV-1 PICs. Whereas relatively low amounts (5–20 nM) of human barrier-to-autointegration factor (BAF) protein restored integration activity, substantially more (5–10 μM) human host factor HMG I(Y) was required. Similarly high levels (3–8 μM) of bovine RNase A, a DNA-binding protein used as a nonspecific control, also restored activity. Mu-mediated PCR footprinting revealed that of these three purified proteins, only BAF restored the native structure of the HIV-1 protein–DNA intasome. We suggest that BAF is a natural host cofactor for HIV-1 integration.