Potent, protective anti-HIV immune responses generated by bimodal HIV envelope DNA plus protein vaccination

It is generally thought that an effective vaccine to prevent HIV-1 infection should elicit both strong neutralizing antibody and cytotoxic T lymphocyte responses. We recently demonstrated that potent, boostable, long-lived HIV-1 envelope (Env)-specific cytotoxic T lymphocyte responses can be elicited in rhesus monkeys using plasmid-encoded HIV-1 env DNA as the immunogen. In the present study, we show that the addition of HIV-1 Env protein to this regimen as a boosting immunogen generates a high titer neutralizing antibody response in this nonhuman primate species. Moreover, we demonstrate in a pilot study that immunization with HIV-1 env DNA (multiple doses) followed by a final immunization with HIV-1 env DNA plus HIV-1 Env protein (env gene from HXBc2 clone of HIV IIIB; Env protein from parental HIV IIIB) completely protects monkeys from infection after i.v. challenge with a chimeric virus expressing HIV-1 env (HXBc2) on a simian immmunodeficiency virusmac backbone (SHIV-HXBc2). The potent immunity and protection seen in these pilot experiments suggest that a DNA prime/DNA plus protein boost regimen warrants active investigation as a vaccine strategy to prevent HIV-1 infection.

Iron accumulation in Alzheimer disease is a source of redox-generated free radicals

Damage from free radicals has been demonstrated in susceptible neuronal populations in cases of Alzheimer disease. In this study, we investigated whether iron, a potent source of the highly reactive hydroxyl radical that is generated by the Fenton reaction with H2O2, might contribute to the source of radicals in Alzheimer disease. We found, using a modified histochemical technique that relies on the formation of mixed valence iron complexes, that redox-active iron is associated with the senile plaques and neurofibrillary tangles—the pathological hallmark lesions of this disease. This lesion-associated iron is able to participate in in situ oxidation and readily catalyzes an H2O2-dependent oxidation. Furthermore, removal of iron was completely effected using deferoxamine, after which iron could be rebound to the lesions. Characterization of the iron-binding site suggests that binding is dependent on available histidine residues and on protein conformation. Taken together, these findings indicate that iron accumulation could be an important contributor toward the oxidative damage of Alzheimer disease.

A gene spans the pseudoautosomal boundary in mice

The X and Y chromosomes of the mouse, like those of other mammals, are heteromorphic over most of their length, but at the distal ends of the chromosomes is a region of sequence identity, the pseudoautosomal region (PAR), where the chromosomes pair and recombine during male meiosis. The point at which the PAR diverges into X- and Y-specific sequences is called the pseudoautosomal boundary. We have completed a genomic walk from the X-specific Amelogenin gene to the PAR. Analysis of this region revealed that the pseudoautosomal boundary of mice is located within an intron of a transcribed gene that encodes a novel RING finger protein. The first three of the exons of the gene are located on the X chromosome whereas the 3′ exons of the gene are located on both X and Y chromosomes. This unusual arrangement may indicate that the gene is in a state of transition from pseudoautosomal to X-unique and provides evidence for a process of attrition of the pseudoautosomal region on the Y chromosome.

EphA4 (Sek1) receptor tyrosine kinase is required for the development of the corticospinal tract

Members of the Eph family of tyrosine kinase receptors have been implicated in the regulation of developmental processes and, in particular, axon guidance in the developing nervous system. The function of the EphA4 (Sek1) receptor was explored through creation of a null mutant mouse. Mice with a null mutation in the EphA4 gene are viable and fertile but have a gross motor dysfunction, which is evidenced by a loss of coordination of limb movement and a resultant hopping, kangaroo-like gait. Consistent with the observed phenotype, anatomical studies and anterograde tracing experiments reveal major disruptions of the corticospinal tract within the medulla and spinal cord in the null mutant animals. These results demonstrate a critical role for EphA4 in establishing the corticospinal projection.

Potentially predictive and manipulable blood serum correlates of aging in the healthy human male: Progressive decreases in bioavailable testosterone, dehydroepiandrosterone sulfate, and the ratio of insulin-like growth factor 1 to growth hormone

A cross-sectional survey was made in 56 exceptionally healthy males, ranging in age from 20 to 84 years. Measurements were made of selected steroidal components and peptidic hormones in blood serum, and cognitive and physical tests were performed. Of those blood serum variables that gave highly significant negative correlations with age (r > −0.6), bioavailable testosterone (BT), dehydroepiandrosterone sulfate (DHEAS), and the ratio of insulin-like growth factor 1 (IGF-1) to growth hormone (GH) showed a stepwise pattern of age-related changes most closely resembling those of the age steps themselves. Of these, BT correlated best with significantly age-correlated cognitive and physical measures. Because DHEAS correlated well with BT and considerably less well than BT with the cognitive and physical measures, it seems likely that BT and/or substances to which BT gives rise in tissues play a more direct role in whatever processes are rate-limiting in the functions measured and that DHEAS relates more indirectly to these functions. The high correlation of IGF-1/GH with age, its relatively low correlation with BT, and the patterns of correlations of IGF-1/GH and BT with significantly age-correlated cognitive and physical measures suggest that the GH–IGF-1 axis and BT play independent roles in affecting these functions. Serial determinations made after oral ingestion of pregnenolone and data from the literature suggest there is interdependence of steroid metabolic systems with those operational in control of interrelations in the GH–IGF-1 axis. Longitudinal concurrent measurements of serum levels of BT, DHEAS, and IGF-1/GH together with detailed studies of their correlations with age-correlated functional measures may be useful in detecting early age-related dysregulations and may be helpful in devising ameliorative approaches.

Pinus banksiana has at least seven expressed alcohol dehydrogenase genes in two linked groups

The alcohol dehydrogenase (Adh) gene family is much more complex in Pinus banksiana than in angiosperms, with at least seven expressed genes organized as two tightly linked clusters. Intron number and position are highly conserved between P. banksiana and angiosperms. Unlike angiosperm Adh genes, numerous duplications, as large as 217 bp, were observed within the noncoding regions of P. banksiana Adh genes and may be a common feature of conifer genes. A high frequency of duplication over a wide range of scales may contribute to the large genome size of conifers.

Glial cell line-derived neurotrophic factor promotes the development of adrenergic neurons in mouse neural crest cultures

Growth of mouse neural crest cultures in the presence of glial cell line-derived neurotrophic factor (GDNF) resulted in a dramatic dose-dependent increase in the number of tyrosine hydroxylase (TH)-positive cells that developed when 5% chicken embryo extract was present in the medium. In contrast, growth in the presence of bone morphogenetic protein (BMP)-2, BMP-4, BMP-6, transforming growth factor (TGF) β1, TGF-β2, and TGF-β3 elicited no increase in the number of TH-positive cells. The TH-positive cells that developed in the presence of GDNF had neuronal morphology and contained the middle and low molecular weight neurofilament proteins. Numerous TH-negative cells with the morphology of neurons also were observed in GDNF-treated cultures. Analysis revealed that the period from 6 to 12 days in vitro was the critical time for exposure to GDNF to generate the increase in TH-positive cell number. The growth factors neurotrophin-3 and fibroblast growth factor-2 elicited increases in the number of TH-positive cells similar to that seen in response to GDNF. In contrast, nerve growth factor was unable to substitute for GDNF. These findings extend the previously reported biological activities of GDNF by showing that it can act on mouse neural crest cultures to promote the development of neurons.

Fractionation factors and activation energies for exchange of the low barrier hydrogen bonding proton in peptidyl trifluoromethyl ketone complexes of chymotrypsin

NMR investigations have been carried out of complexes between bovine chymotrypsin Aα and a series of four peptidyl trifluoromethyl ketones, listed here in order of increasing affinity for chymotrypsin: N-Acetyl-l-Phe-CF3, N-Acetyl-Gly-l-Phe-CF3, N-Acetyl-l-Val-l-Phe-CF3, and N-Acetyl-l-Leu-l-Phe-CF3. The D/H fractionation factors (φ) for the hydrogen in the H-bond between His 57 and Asp 102 (His 57-Hδ1) in these four complexes at 5°C were in the range φ = 0.32–0.43, expected for a low-barrier hydrogen bond. For this series of complexes, measurements also were made of the chemical shifts of His 57-Hɛ1 (δ2,2-dimethylsilapentane-5-sulfonic acid 8.97–9.18), the exchange rate of the His 57-Hδ1 proton with bulk water protons (284–12.4 s−1), and the activation enthalpies for this hydrogen exchange (14.7–19.4 kcal⋅mol−1). It was found that the previously noted correlations between the inhibition constants (Ki 170–1.2 μM) and the chemical shifts of His 57-Hδ1 (δ2,2-dimethylsilapentane-5-sulfonic acid 18.61–18.95) for this series of peptidyl trifluoromethyl ketones with chymotrypsin [Lin, J., Cassidy, C. S. & Frey, P. A. (1998) Biochemistry 37, 11940–11948] could be extended to include the fractionation factors, hydrogen exchange rates, and hydrogen exchange activation enthalpies. The results support the proposal of low barrier hydrogen bond-facilitated general base catalysis in the addition of Ser 195 to the peptidyl carbonyl group of substrates in the mechanism of chymotrypsin-catalyzed peptide hydrolysis. Trends in the enthalpies for hydrogen exchange and the fractionation factors are consistent with a strong, double-minimum or single-well potential hydrogen bond in the strongest complexes. The lifetimes of His 57-Hδ1, which is solvent shielded in these complexes, track the strength of the hydrogen bond. Because these lifetimes are orders of magnitude shorter than those of the complexes themselves, the enzyme must have a pathway for hydrogen exchange at this site that is independent of dissociation of the complexes.

Mice cloned from embryonic stem cells

Cloning allows the asexual reproduction of selected individuals such that the offspring have an essentially identical nuclear genome. Cloning by nuclear transfer thus far has been reported only with freshly isolated cells and cells from primary cultures. We previously reported a method of cloning mice from adult somatic cells after nuclear transfer by microinjection. Here, we apply this method to clone mice from widely available, established embryonic stem (ES) cell lines at late passage. With the ES cell line R1, 29% of reconstructed oocytes developed in vitro to the morula/blastocyst stage, and 8% of these embryos developed to live-born pups when transferred to surrogate mothers. We thus cloned 26 mice from R1 cells. Nuclei from the ES cell line E14 also were shown to direct development to term. We present evidence that the nuclei of ES cells at G1- or G2/M-phases are efficiently able to support full development. Our findings demonstrate that late-passage ES cells can be used to produce viable cloned mice and provide a link between the technologies of ES cells and animal cloning. It thus may be possible to clone from a single cell a large number of individuals over an extended period.

Peptide nucleic acid pre-gel hybridization: An alternative to Southern hybridization

We have found that it is possible to use labeled peptide nucleic acid (PNA)-oligomers as probes in pre-gel hybridization experiments, as an alternative for Southern hybridization. In this technique, the PNA probe is hybridized to a denatured DNA sample at low ionic strength and the mixture is loaded directly on to an electrophoresis system for size separation. Ensuing gel electrophoresis separates the single-stranded DNA fragments by length. The neutral backbone of PNA allows for hybridization at low ionic strength and imparts very low mobility to excess PNA. Detection of the bound PNA is possible by direct fluorescence detection with capillary electrophoresis, or the DNA/PNA hybrids can be blotted onto a membrane and detected with standard chemiluminescent techniques. Efficient single bp discrimination was achieved routinely using both capillary and slab-gel electrophoresis.

Delayed symptom onset and increased life expectancy in Sandhoff disease mice treated with N-butyldeoxynojirimycin

Sandhoff disease is a neurodegenerative disorder resulting from the autosomal recessive inheritance of mutations in the HEXB gene, which encodes the β-subunit of β-hexosaminidase. GM2 ganglioside fails to be degraded and accumulates within lysosomes in cells of the periphery and the central nervous system (CNS). There are currently no therapies for the glycosphingolipid lysosomal storage diseases that involve CNS pathology, including the GM2 gangliosidoses. One strategy for treating this and related diseases is substrate deprivation. This would utilize an inhibitor of glycosphingolipid biosynthesis to balance synthesis with the impaired rate of catabolism, thus preventing storage. One such inhibitor is N-butyldeoxynojirimycin, which currently is in clinical trials for the potential treatment of type 1 Gaucher disease, a related disease that involves glycosphingolipid storage in peripheral tissues, but not in the CNS. In this study, we have evaluated whether this drug also could be applied to the treatment of diseases with CNS storage and pathology. We therefore have treated a mouse model of Sandhoff disease with the inhibitor N-butyldeoxynojirimycin. The treated mice have delayed symptom onset, reduced storage in the brain and peripheral tissues, and increased life expectancy. Substrate deprivation therefore offers a potentially general therapy for this family of lysosomal storage diseases, including those with CNS disease.

β-Arrestin 1 and 2 differentially regulate heptahelical receptor signaling and trafficking

The two widely coexpressed isoforms of β-arrestin (termed βarrestin 1 and 2) are highly similar in amino acid sequence. The β-arrestins bind phosphorylated heptahelical receptors to desensitize and target them to clathrin-coated pits for endocytosis. To better define differences in the roles of β-arrestin 1 and 2, we prepared mouse embryonic fibroblasts from knockout mice that lack one of the β-arrestins (βarr1-KO and βarr2-KO) or both (βarr1/2-KO), as well as their wild-type (WT) littermate controls. These cells were analyzed for their ability to support desensitization and sequestration of the β2-adrenergic receptor (β2-AR) and the angiotensin II type 1A receptor (AT1A-R). Both βarr1-KO and βarr2-KO cells showed similar impairment in agonist-stimulated β2-AR and AT1A-R desensitization, when compared with their WT control cells, and the βarr1/2-KO cells were even further impaired. Sequestration of the β2-AR in the βarr2-KO cells was compromised significantly (87% reduction), whereas in the βarr1-KO cells it was not. Agonist-stimulated internalization of the AT1A-R was only slightly reduced in the βarr1-KO but was unaffected in the βarr2-KO cells. In the βarr1/2-KO cells, the sequestration of both receptors was dramatically reduced. Comparison of the ability of the two β-arrestins to sequester the β2-AR revealed β-arrestin 2 to be 100-fold more potent than β-arrestin 1. Down-regulation of the β2-AR was also prevented in the βarr1/2-KO cells, whereas no change was observed in the single knockout cells. These findings suggest that sequestration of various heptahelical receptors is regulated differently by the two β-arrestins, whereas both isoforms are capable of supporting receptor desensitization and down-regulation.

ALFRED: an allele frequency database for diverse populations and DNA polymorphisms—an update

ALFRED (the ALelle FREquency Database) is designed to store and disseminate frequencies of alleles at human polymorphic sites for multiple populations, primarily for the population genetics and molecular anthropology communities. Currently ALFRED has information on over 180 polymorphic sites for more than 70 populations. Since our initial release of the database we have focussed on increasing the quantity and quality of data, making reciprocal links between ALFRED and other related databases, and providing useful tools to make the data more comprehensible to the end user. ALFRED is accessible from the Kidd Lab home page (http://info.med.yale.edu/genetics/kkidd/) or from ALFRED directly (http://alfred.med.yale.edu/alfred/index.asp).