Epsilon substituted lysinol derivatives as HIV-1 protease inhibitors

A series of HIV-1 protease inhibitors containing an epsilon substituted lysinol backbone was synthesized. Two novel synthetic routes using N-boc-l-glutamic acid alpha-benzyl ester and 2,6-diaminopimelic acid were developed. Incorporation of this epsilon substituent enabled access to the S2 pocket of the enzyme, affording high potency inhibitors. Modeling studies and synthetic efforts suggest the potency increase is due to both conformational bias and van der Waals interactions with the S2 pocket.     

Serotonin increases phosphorylation of synaptic 4EBP through TOR, but eukaryotic initiation factor 4E levels do not limit somatic cap-dependent translation in aplysia neurons

The target of rapamycin (TOR) plays an important role in memory formation in Aplysia californica. Here, we characterize one of the downstream targets of TOR, the eukaryotic initiation factor 4E (eIF4E) binding protein (4EBP) from Aplysia. Aplysia 4EBP contains the four critical phosphorylation sites regulated by TOR as well as an N-terminal RAIP motif and a C-terminal TOS site. Aplysia 4EBP was hypophosphorylated in synaptosomes, and serotonin addition caused a rapamycin-sensitive increase in 4EBP phosphorylation both in synaptosomes and in isolated neurites. Aplysia 4EBP was regulated in a fashion similar to that of mammalian 4EBPs, binding to eIF4E when dephosphorylated and releasing eIF4E after phosphorylation. Overexpression of 4EBP in the soma of Aplysia neurons caused a specific decrease in cap-dependent translation that was rescued by concomitant overexpression of eIF4E. However, eIF4E overexpression by itself did not increase cap-dependent translation, suggesting that increasing levels of free eIF4E by phosphorylating 4EBP is not important in regulating cap-dependent translation in the cell soma. Total levels of eIF4E were also regulated by 4EBP, suggesting that 4EBP can also act as an eIF4E chaperone. These studies demonstrate the conserved nature of 4EBP regulation and its role in cap-dependent translation and suggest differential roles of 4EBP phosphorylation in the soma and synapse.     

Calcium- and phosphate-dependent release and loading of glutathione by liver mitochondria

The status of glutathione (GSH) was studied in isolated rat liver mitochondria under conditions which induce a permeability transition. This transition, which is inhibited by cyclosporin A (CyA), requires the presence of Ca2+ and an inducing agent such as near physiological levels (3 mM) of inorganic phosphate (Pi). The transition is characterized by an increased inner membrane permeability to some low molecular weight solutes and by large amplitude swelling under some experimental conditions. Addition of 70 microM Ca2+ and 3 mM Pi to mitochondria resulted in mitochondrial swelling and extensive release of GSH that was recovered in the extramitochondrial medium as GSH. Both swelling and the efflux of mitochondrial GSH were prevented by CyA. Incubation of mitochondria in the presence of Ca2+, Pi, and GSH followed by addition of CyA provided a mechanism to load mitochondria with exogenous GSH that was greater than the rate of uptake by untreated mitochondria. Thus, GSH efflux from mitochondria may occur under toxicological and pathological conditions in which mitochondria are exposed to elevated Ca2+ in the presence of near physiological concentrations of Pi through a nonspecific pore. Cyclical opening and closing of the pore could also provide a mechanism for uptake of GSH by mitochondria.     

Role of the DNA Base Excision Repair Protein,APE1 in Cisplatin,Oxaliplatin, or Carboplatin Induced Sensory Neuropathy

Although chemotherapy-induced peripheral neuropathy (CIPN) is a dose-limiting side effect of platinum drugs, the mechanisms of this toxicity remain unknown. Previous work in our laboratory suggests that cisplatin-induced CIPN is secondary to DNA damage which is susceptible to base excision repair (BER). To further examine this hypothesis, we studied the effects of cisplatin, oxaliplatin, and carboplatin on cell survival, DNA damage, ROS production, and functional endpoints in rat sensory neurons in culture in the absence or presence of reduced expression of the BER protein AP endonuclease/redox factor-1 (APE1). Using an in situ model of peptidergic sensory neuron function, we examined the effects of the platinum drugs on hind limb capsaicin-evoked vasodilatation. Exposing sensory neurons in culture to the three platinum drugs caused a concentration-dependent increase in apoptosis and cell death, although the concentrations of carboplatin were 10 fold higher than cisplatin. As previously observed with cisplatin, oxaliplatin and carboplatin also increased DNA damage as indicated by an increase in phospho-H2AX and reduced the capsaicin-evoked release of CGRP from neuronal cultures. Both cisplatin and oxaliplatin increased the production of ROS as well as 8-oxoguanine DNA adduct levels, whereas carboplatin did not. Reducing levels of APE1 in neuronal cultures augmented the cisplatin and oxaliplatin induced toxicity, but did not alter the effects of carboplatin. Using an in vivo model, systemic injection of cisplatin (3 mg/kg), oxaliplatin (3 mg/kg), or carboplatin (30 mg/kg) once a week for three weeks caused a decrease in capsaicin-evoked vasodilatation, which was delayed in onset. The effects of cisplatin on capsaicin-evoked vasodilatation were attenuated by chronic administration of E3330, a redox inhibitor of APE1 that serendipitously enhances APE1 DNA repair activity in sensory neurons. These outcomes support the importance of the BER pathway, and particularly APE1, in sensory neuropathy caused by cisplatin and oxaliplatin, but not carboplatin and suggest that augmenting DNA repair could be a therapeutic target for CIPN.     

Low-molecular mass carbohydrate accumulation in cyanobacteria from a marine microbial mat in response to salt

Forty seven strains of cyanobacteria, all isolated from microbial mats of intertidal sediments of the island of Mellum (North Sea), were analyzed for the presence of organic osmotica. The cyanobacteria examined belonged to taxonomically different groups and were classified according to their salt optimum and salt tolerance as either freshwater, brackish or marine. Except betaine, all organic osmotica known to occur in cyanobacteria, were found. The results showed no clear correlation between the chemical nature of the organic solute and the salt optimum or salt tolerance of the cyanobacteria examined, indicating that these solutes are not specific to this marine habitat. All strains belonging to the Nostoc/Anabea-group accumulated sucrose as the sole organic osmoticum. The marine, heterocystous Calothrix sp. accumulated trehalose. All strains of the LPP-group (Lyngbya, Plectonema, Phormidium) accumulated glucosylglycerol as sole or primary organic solute. Some LPP-strains accumulated a disaccharide as a secundary solute, e.g. sucrose or trehalose. Gloeocapsa, Synechocystis and Spirulina accumulated glucosylglycerol. Two marine Oscillatoria accumulated trehalose, whereas a freshwater Oscillatoria with a broad salinity tolerance, accumulated sucrose.Analysis of field samples of the microbial mats demonstrated the presence of glycerol, glucosylglycerol, sucrose and trehalose. The relative abundance of the different compounds was related to the species composition as could be predicted from laboratory observations. These data suggest that these carbohydrates have a function in maintaining osmotic balance in the organisms within the microbial mat.     

Motor cortex excitability does not increase during sustained cycling exercise to volitional exhaustion

The excitability of the motor cortex increases as fatigue develops during sustained single-joint contractions, but there are no previous reports on how corticospinal excitability is affected by sustained locomotor exercise. Here we addressed this issue by measuring spinal and cortical excitability changes during sustained cycling exercise. Vastus lateralis (VL) and rectus femoris (RF) muscle responses to transcranial magnetic stimulation of the motor cortex (motor evoked potentials, MEPs) and electrical stimulation of the descending tracts (cervicomedullary evoked potentials, CMEPs) were recorded every 3 min from nine subjects during 30 min of cycling at 75% of maximum workload (W(max)), and every minute during subsequent exercise at 105% of W(max) until subjective task failure. Responses were also measured during nonfatiguing control bouts at 80% and 110% of W(max) prior to sustained exercise. There were no significant changes in MEPs or CMEPs (P > 0.05) during the sustained cycling exercise. These results suggest that, in contrast to sustained single-joint contractions, sustained cycling exercise does not increase the excitability of motor cortical neurons. The contrasting corticospinal responses to the two modes of exercise may be due to differences in their associated systemic physiological consequences.     

The initial characterization of the iron environment in lipoxygenase by M?ssbauer spectroscopy

The incorporation of 57Fe into two lipoxygenase isoenzymes from soybeans has been achieved making possible the first observations of the iron environment in these proteins using M?ssbauer spectroscopy. Immature soybean seeds were grown in tissue culture medium supplied with 57Fe. The iron in the active lipoxygenases that were isolated from the cultured seeds was readily detected in M?ssbauer measurements. It was unequivocally demonstrated that the native enzyme contains high-spin Fe(II). Based on the sign of the electric field gradient, the most likely ligand sphere for the iron in native lipoxygenase consists of oxygen and nitrogen ligands in a roughly octahedral field of symmetry. It was possible to detect M?ssbauer signals in highly concentrated samples of native lipoxygenases containing 57Fe at natural abundance. The spectra obtained for enriched and natural abundance native enzyme had the same high-spin Fe(II) M?ssbauer parameters. This confirmed that the environment of the iron in enzymes isolated from cultured seeds and dry soybeans were the same. The M?ssbauer spectra (4.2-250 K) for samples of both isoenzymes after oxidation of the iron in native enzyme by the product of lipoxygenase catalysis were extremely broad (20 mm/s) with no obvious narrow resonance lines. This was the result of the existence of paramagnetically broadened spectra for such samples even at relatively high temperature as evidenced by the appropriate EPR signal. A small molecule containing an iron site sharing many of these M?ssbauer and electron paramagnetic resonance properties with lipoxygenase was identified: Fe(II)/(III).diethylenetriaminepentaacetic acid.     

Missense Mutations Allow a Sequence-Blind Mutant of SpoIIIE to Successfully Translocate Chromosomes during Sporulation

SpoIIIE directionally pumps DNA across membranes during Bacillus subtilis sporulation and vegetative growth. The sequence-reading domain (γ domain) is required for directional DNA transport, and its deletion severely impairs sporulation. We selected suppressors of the spoIIIEΔγ sporulation defect. Unexpectedly, many suppressors were intragenic missense mutants, and some restore sporulation to near-wild-type levels. The mutant proteins are likely not more abundant, faster at translocating DNA, or sequence-sensitive, and rescue does not involve the SpoIIIE homolog SftA. Some mutants behave differently when co-expressed with spoIIIEΔγ, consistent with the idea that some, but not all, variants may form mixed oligomers. In full-length spoIIIE, these mutations do not affect sporulation, and yet the corresponding residues are rarely found in other SpoIIIE/FtsK family members. The suppressors do not rescue chromosome translocation defects during vegetative growth, indicating that the role of the γ domain cannot be fully replaced by these mutations. We present two models consistent with our findings: that the suppressors commit to transport in one arbitrarily-determined direction or delay spore development. It is surprising that missense mutations somehow rescue loss of an entire domain with a complex function, and this raises new questions about the mechanism by which SpoIIIE pumps DNA and the roles SpoIIIE plays in vivo.     

An asymptotic theory for model selection inference in general semiparametric problems

Hjort & Claeskens (2003) developed an asymptotic theoryfor model selection, model averaging and subsequent inferenceusing likelihood methods in parametric models, along with associatedconfidence statements. In this article, we consider a semiparametricversion of this problem, wherein the likelihood depends on parametersand an unknown function, and model selection/averaging is tobe applied to the parametric parts of the model. We show thatall the results of Hjort & Claeskens hold in the semiparametriccontext, if the Fisher information matrix for parametric modelsis replaced by the semiparametric information bound for semiparametricmodels, and if maximum likelihood estimators for parametricmodels are replaced by semiparametric efficient profile estimators.Our methods of proof employ Le Cam's contiguity lemmas, leadingto transparent results. The results also describe the behaviourof semiparametric model estimators when the parametric componentis misspecified, and also have implications for pointwise-consistentmodel selectors.     

The tal gene undergoes chromosome translocation in T cell leukemia and potentially encodes a helix-loop-helix protein.

We have analyzed t(1;14)(p32;q11) chromosome translocations from two patients with T cell acute lymphocytic leukemia. The chromosome 1 breakpoints of these patients lie within a kilobasepair of each other, and thus define a genetic locus (designated tal) involved in T cell oncogenesis. Moreover, we have identified sequences within tal that potentially encode an amphipathic helix-loop-helix motif, a DNA-binding domain found in a variety of proteins that control cell growth and differentiation. The homology domain of tal is especially related to that of lyl-1, a gene on chromosome 19 that has also been implicated in T cell oncogenesis. Hence, tal and lyl-1 encode a distinct family of helix-loop-helix proteins involved in the malignant development of lymphocytes.