An Enigmatic Hypoplastic Defect of the Maxillary Lateral Incisor in Recent and Fossil Orangutans from Sumatra (<Emphasis Type="Italic">Pongo abelii</Emphasis>) and Borneo (<Emphasis Type="Italic">Pongo pygmaeus</Emphasis>)

Developmental dental pathologies provide insight into health of primates during ontogeny, and are particularly useful for elucidating the environment in which extant and extinct primates matured. Our aim is to evaluate whether the prevalence of an unusual dental defect on the mesiolabial enamel of the upper lateral incisor, thought to reflect dental crowding during maturation, is lesser in female orangutans, with their smaller teeth, than in males; and in Sumatran orangutans, from more optimal developmental habitats, than in those from Borneo. Our sample includes 49 Pongo pygmaeus (87 teeth), 21 P. abelii (38 teeth), Late Pleistocene paleo-orangutans from Sumatra and Vietnam (67 teeth), Late Miocene catarrhines Lufengpithecus lufengensis (2 teeth), and Anapithecus hernyaki (7 teeth). Methods include micro-CT scans, radiography, and dental metrics of anterior teeth. We observed fenestration between incisor crypts and marked crowding of unerupted crowns, which could allow tooth-to-tooth contact. Tooth size does not differ significantly in animals with or without the defect, implicating undergrowth of the jaw as the proximate cause of dental crowding and defect presence. Male orangutans from both islands show more defects than do females. The defect is significantly more common in Bornean orangutans (71 %) compared to Sumatran (29 %). Prevalence among fossil forms falls between these extremes, except that all five individual Anapithecus show one or both incisors with the defect. We conclude that maxillary lateral incisor defect is a common developmental pathology of apes that is minimized in optimal habitats and that such evidence can be used to infer habitat quality in extant and fossil apes.     

Diversity of the human immunodeficiency virus type 1 (HIV-1) env sequence after vertical transmission in mother-child pairs infected with HIV-1 subtype A

Although several virologic and immunologic factors associated with an increased risk of perinatal human immunodeficiency virus type 1 (HIV-1) transmission have been described, the mechanism of mother-to-child transmission is still unclear. More specifically, the question of whether selective pressures influence the transmission remains unanswered. The aim of this study was to assess the genetic diversity of the transmitted virus after in utero transmission and after peripartum transmission and to compare the viral heterogeneity in the child with the viral heterogeneity in the mother. To allow a very accurate characterization of the viral heterogeneity in a single sample, limiting-dilution sequencing of a 1016-bp fragment of the env gene was performed. Thirteen children were tested, including 6 with in utero infections and 7 with peripartum infections. Samples were taken the day after birth and at the ages of 6 and 14 weeks. A homogeneous virus population was seen in six (46.2%) infants, of whom two were infected in utero and four were infected peripartum. A more heterogeneous virus population was detected in seven infants (53.8%), four infected in utero and three infected peripartum. The phylogenetic trees of the mother-child pairs presented a whole range of different tree topologies and showed infection of the child by one or more maternal variants. In conclusion, after HIV-1 transmission from mother to child a heterogeneous virus population was detected in approximately one-half of the children examined. Heterogeneous virus populations were found after peripartum infection as well as after in utero infection. Phylogenetic tree topologies argue against selection processes as the major mechanism driving mother-to-child transmission but support the hypothesis that virus variability is mainly driven by the inoculum level and/or exposure time.     

Characterization of the putative operon containing arylamine N-acetyltransferase (nat) in Mycobacterium bovis BCG

Mycobacterium bovis BCG and Mycobacterium tuberculosis possess a single arylamine N-acetyltransferase whose gene is predicted to occur within a six-gene operon. Deletion of the nat gene caused an extended lag phase in M. bovis BCG and a cell morphology associated with an altered pattern of cell wall mycolates. Analysis of cDNA from M. bovis BCG shows that during in vitro growth all the genes in the putative nat operon are expressed and the open reading frames are contiguous, supporting the existence of an operon. Two genes in the operon, Mb3599c and Mb3600c, are predicted to encode homologues of enzymes annotated as a 2,3-dihydroxybiphenyl 1,2-dioxygenase (bphC5) and a 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (bphD2), respectively, in Rhodococcus RHA1. As predicted, M. bovis BCG cell lysates metabolized the BphC substrate 2,3-dihydroxybiphenyl (2,3-DHB) to 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA), a BphD substrate, which was subsequently hydrolysed. Immunoprecipitation of the BphD homologue from these lysates led to an accumulation of HOPDA. M. bovis BCG growth on both solid and liquid media was inhibited with either 2,3-DHB or an inhibitor of BphC, 3-chlorocatechol (3-CC). In addition, incubation with 2,3-DHB affects the lipid composition of the cell wall resulting in a diminished level of mycolates and an altered cell morphology similar to the Deltanat strain. We propose the enzymes encoded by the putative operon have a similar endogenous role to that of the NAT enzyme and are part of a pathway important for cell wall synthesis.     

Intermolecular interaction of voriconazole analogues with model membrane by DSC and NMR,and their antifungal activity using NMR based metabolic profiling

The development of novel antifungal agents with high susceptibility and increased potency can be achieved by increasing their overall lipophilicity. To enhance the lipophilicity of voriconazole, a second generation azole antifungal agent, we have synthesized its carboxylic acid ester analogues, namely p-methoxybenzoate (Vpmb), toluate (Vtol), benzoate (Vbz) and p-nitrobenzoate (Vpnb). The intermolecular interactions of these analogues with model membrane have been investigated using nuclear magnetic resonance (NMR) and differential scanning calorimetric (DSC) techniques. The results indicate varying degree of changes in the membrane bilayer’s structural architecture and physico-chemical characteristics which possibly can be correlated with the antifungal effects via fungal membrane. Rapid metabolite profiling of chemical entities using cell preparations is one of the most important steps in drug discovery. We have evaluated the effect of synthesized analogues on Candida albicans. The method involves real time ¹H NMR measurement of intact cells monitoring NMR signals from fungal metabolites which gives Metabolic End Point (MEP). This is then compared with Minimum Inhibitory Concentration (MIC) determined using conventional methods. Results indicate that one of the synthesized analogues, Vpmb shows reasonably good activity.     

Restricted spacer tolerance of a zinc finger nuclease with a six amino acid linker

Zinc finger nucleases can be engineered to create highly efficient and precise changes to the genetic information within living cells. We report the investigation of an important parameter that defines the type of target site the nuclease can cleave. The active nuclease is a dimer, requiring that the DNA target site contain two zinc finger binding sites separated by a short spacer. Using a plasmid-based recombination assay in HEK 293T cells, we show that a 6 amino acid linker between the zinc finger DNA-binding domain and the FokI cleavage domain restricts nuclease activity to sites containing a 6 bp spacer. These observations concur with other recent studies, suggesting this information will be useful in the design of new potent and accurate zinc finger nucleases.     

The insulin-like growth factors (IGFs) I and II bind to articular cartilage via the IGF-binding proteins

Bovine articular cartilage discs (3 mm diameter x 400 micrometer thick) were equilibrated in buffer containing (125)I-insulin-like growth factor (IGF)-I (4 degrees C) +/- unlabeled IGF-I or IGF-II. Competition for binding to cartilage discs by each unlabeled IGF was concentration-dependent, with ED(50) values for inhibition of (125)I-IGF-I binding of 11 and 10 nM for IGF-I and -II, respectively, and saturation by 50 nM. By contrast, an analog of IGF-I with very low affinity for the insulin-like growth factor-binding proteins (IGF-BPs), des-(1-3)-IGF-I, was not competitive with (125)I-IGF-I for cartilage binding even at 100-400 nM. Binding of the (125)I-labeled IGF-II isoform to cartilage was competed for by unlabeled IGF-I or -II, with ED(50)s of 160 and 8 nM, respectively. This probably reflected the differential affinities of the endogenous IGF-BPs (IGF-BP-6 and -2) for IGF-II/IGF-I. Transport of (125)I-IGF-I was also measured in an apparatus that allows diffusion only across the discs (400 micrometer), by addition to one side and continuous monitoring of efflux on the other side. The time lag for transport of (125)I-IGF was 266 min, an order of magnitude longer than the theoretical prediction for free diffusion in the matrix. (125)I-IGF-I transport then reached a steady state rate (% efflux of total added (125)I-IGF/unit time), which was subsequently accelerated approximately 2-fold by addition of an excess of unlabeled IGF-I. Taken together, these results indicate that IGF binding to cartilage, mostly through the IGF-BPs, regulates the transport of IGFs in articular cartilage, probably contributing to the control of their paracrine activities.     

A 24-Residue Peptide (p5), Derived from p35, the Cdk5 Neuronal Activator,Specifically Inhibits Cdk5-p25 Hyperactivity and Tau Hyperphosphorylation

The activity of Cdk5-p35 is tightly regulated in the developing and mature nervous system. Stress-induced cleavage of the activator p35 to p25 and a p10 N-terminal domain induces deregulated Cdk5 hyperactivity and perikaryal aggregations of hyperphosphorylated Tau and neurofilaments, pathogenic hallmarks in neurodegenerative diseases, such as Alzheimer disease and amyotrophic lateral sclerosis, respectively. Previously, we identified a 125-residue truncated fragment of p35 called CIP that effectively and specifically inhibited Cdk5-p25 activity and Tau hyperphosphorylation induced by Aβ peptides in vitro, in HEK293 cells, and in neuronal cells. Although these results offer a possible therapeutic approach to those neurodegenerative diseases assumed to derive from Cdk5-p25 hyperactivity and/or Aβ induced pathology, CIP is too large for successful therapeutic regimens. To identify a smaller, more effective peptide, in this study we prepared a 24-residue peptide, p5, spanning CIP residues Lys²⁴⁵–Ala²⁷⁷. p5 more effectively inhibited Cdk5-p25 activity than did CIP in vitro. In neuron cells, p5 inhibited deregulated Cdk5-p25 activity but had no effect on the activity of endogenous Cdk5-p35 or on any related endogenous cyclin-dependent kinases in HEK293 cells. Specificity of p5 inhibition in cortical neurons may depend on the p10 domain in p35, which is absent in p25. Furthermore, we have demonstrated that p5 reduced Aβ(1–42)-induced Tau hyperphosphorylation and apoptosis in cortical neurons. These results suggest that p5 peptide may be a unique and useful candidate for therapeutic studies of certain neurodegenerative diseases.     

Analysis of variance components in gene expression data

MOTIVATION: A microarray experiment is a multi-step process, and each step is a potential source of variation. There are two major sources of variation: biological variation and technical variation. This study presents a variance-components approach to investigating animal-to-animal, between-array, within-array and day-to-day variations for two data sets. The first data set involved estimation of technical variances for pooled control and pooled treated RNA samples. The variance components included between-array, and two nested within-array variances: between-section (the upper- and lower-sections of the array are replicates) and within-section (two adjacent spots of the same gene are printed within each section). The second experiment was conducted on four different weeks. Each week there were reference and test samples with a dye-flip replicate in two hybridization days. The variance components included week-to-week, animal-to-animal and between-array and within-array variances. RESULTS: We applied the linear mixed-effects model to quantify different sources of variation. In the first data set, we found that the between-array variance is greater than the between-section variance, which, in turn, is greater than the within-section variance. In the second data set, for the reference samples, the week-to-week variance is larger than the between-array variance, which, in turn, is slightly larger than the within-array variance. For the test samples, the week-to-week variance has the largest variation. The animal-to-animal variance is slightly larger than the between-array and within-array variances. However, in a gene-by-gene analysis, the animal-to-animal variance is smaller than the between-array variance in four out of five housekeeping genes. In summary, the largest variation observed is the week-to-week effect. Another important source of variability is the animal-to-animal variation. Finally, we describe the use of variance-component estimates to determine optimal numbers of animals, arrays per animal and sections per array in planning microarray experiments.     

The transferable tail: fusion of the N-terminal acidic extension of heparin cofactor II to alpha1-proteinase inhibitor M358R specifically increases the rate of thrombin inhibition

The conversion of the reactive center bond of the serpin alpha1-proteinase inhibitor (alpha1-PI, also known as alpha1-antitrypsin) from Met-Ser to Arg-Ser decreases the rate at which it inhibits neutrophil elastase and endows it with the ability to inhibit thrombin and activated protein C (APC). Another serpin, heparin cofactor II (HCII), contains a unique N-terminal extension that binds thrombin exosite 1. We fused residues 1-75 of HCII to the N-terminus of alpha1-PI M358R, forming an HCII-alpha1-PI chimera (HAPI M358R). It inhibited alpha-thrombin 21-fold faster than alpha1-PI M358R, with second-order rate constants of 2.3 x 10(8) M(-1) min(-1) versus 1.1 x 10(7) M(-1) min(-1), respectively. When gammaT-thrombin, which lacks an intact exosite 1, was substituted for alpha-thrombin, the kinetic advantage of HAPI M358R over alpha1-PI M358R was reduced to 9-fold, whereas APC and trypsin, proteases lacking exosite 1-like regions, were inhibited only 1.3- and 2-fold more rapidly by HAPI M358R than by alpha1-PI M358R, respectively. Maximal enhancement of alpha1-PI M358R activity required the acidic residues found between HCII residues 55 and 75, because no enhancement was observed either by fusion of residues 1-54 alone or by fusion of a mutated HCII acidic extension in which all Glu and Asp residues between positions 55 and 75 were neutralized by mutation. Fusing residues 55-75 to alpha1-PI M358R yielded a relative rate enhancement of only 6-fold, suggesting a need for the full tail region to achieve maximal enhancement. Our results suggest that transfer of the N-terminal acidic extension of HCII to alpha1-PI M358R enhanced its inhibition of thrombin by conferring the ability to bind exosite 1 on HAPI M358R. This enhancement may aid in efforts to tailor this inhibitor for therapeutic use.