Tetrapod phylogeny inferred from 18S and 28S ribosomal RNA sequences and a review of the evidence for amniote relationships [published erratum appears in Mol Biol Evol 1991 May;8(3):398]

The 18S ribosomal RNAs of 21 tetrapods were sequenced and aligned with five published tetrapod sequences. When the coelacanth was used as an outgroup, Lissamphibia (living amphibians) and Amniota (amniotes) were found to be statistically significant monophyletic groups. Although little resolution was obtained among the lissamphibian taxa, the amniote sequences support a sister-group relationship between birds and mammals. Portions of the 28S ribosomal RNA (rRNA) molecule in 11 tetrapods also were sequenced, although the phylogenetic results were inconclusive. In contrast to previous studies, deletion or down- weighting of base-paired sites were found to have little effect on phylogenetic relationships. Molecular evidence for amniote relationships is reviewed, showing that three genes (beta-hemoglobin, myoglobin, and 18S rRNA) unambiguously support a bird-mammal relationship, compared with one gene (histone H2B) that favors a bird- crocodilian clade. Separate analyses of four other genes (alpha- crystallin A, alpha-hemoglobin, insulin, and 28S rRNA) and a combined analysis of all sequence data are inconclusive, in that different groups are defined in different analyses and none are strongly supported. It is suggested that until sequences become available from a broader array of taxa, the molecular evidence is best evaluated at the level of individual genes, with emphasis placed on those studies with the greatest number of taxa and sites. When this is done, a bird-mammal relationship is most strongly supported. When regarded in combination with the morphological evidence for this association, it must be considered at least as plausible as a bird-crocodilian relationship.      

Mitochondrial Genetics of Chlamydomonas Reinhardtii: Resistance Mutations Marking the Cytochrome B Gene

We describe the genetic and molecular analysis of the first non-Mendelian mutants of Chlamydomonas reinhardtii resistant to myxothiazol, an inhibitor of the respiratory cytochrome bc1 complex. Using a set of seven oligonucleotide probes, restriction fragments containing the mitochondrial cytochrome b (cyt b) gene from C. reinhardtii were isolated from a mitochondrial DNA library. This gene is located adjacent to the gene for subunit 4 of the mitochondrial NADH-dehydrogenase (ND4), near one end of the 15.8-kb linear mitochondrial genome of C. reinhardtii. The algal cytochrome b apoprotein contains 381 amino-acid residues and exhibits a sequence similarity of about 59% with other plant cytochrome b proteins. The cyt b gene from four myxothiazol resistant mutants of C. reinhardtii was amplified for DNA sequence analysis. In comparison to the wild-type strain, all mutants contain an identical point mutation in the cyt b gene, leading to a change of a phenylalanine codon to a leucine codon at amino acid position 129 of the cytochrome b protein. Segregation analysis in tetrads from reciprocal crosses of mutants with wild type shows a strict uniparental inheritance of this mutation from the mating type minus parent (UP-). However, mitochondrial markers from both parents are recovered in vegetative diploids in variable proportions from one experiment to the next for a given cross. On the average, a strong bias is seen for markers inherited from the mating type minus parent.     

Odorant-stimulated phosphoinositide signaling in mammalian olfactory receptor neurons

Recent evidence has revived interest in the idea that phosphoinositides (PIs) may play a role in signal transduction in mammalian olfactory receptor neurons (ORNs). To provide direct evidence that odorants indeed activate PI signaling in ORNs, we used adenoviral vectors carrying two different fluorescently tagged probes, the pleckstrin homology (PH) domains of phospholipase Cδ1 (PLCδ1) and the general receptor of phosphoinositides (GRP1), to monitor PI activity in the dendritic knobs of ORNs in vivo. Odorants mobilized PI(4,5)P₂/IP₃ and PI(3,4,5)P₃, the substrates and products of PLC and PI3K. We then measured odorant activation of PLC and PI3K in olfactory ciliary-enriched membranes in vitro using a phospholipid overlay assay and ELISAs. Odorants activated both PLC and PI3K in the olfactory cilia within 2 s of odorant stimulation. Odorant-dependent activation of PLC and PI3K in the olfactory epithelium could be blocked by enzyme-specific inhibitors. Odorants activated PLC and PI3K with partially overlapping specificity. These results provide direct evidence that odorants indeed activate PI signaling in mammalian ORNs in a manner that is consistent with the idea that PI signaling plays a role in olfactory transduction.     

Nature and localization of avian lens glycogen by electron microscopy and Raman spectroscopy.

Electron microscopy confirms the presence of a high concentration of glycogen particles in the lens nuclear region of birds of flying habit such as the ring-neck dove and pigeon. This observation is consistent with Raman spectroscopy. The glycogen particles in the dove lens, which are approximately 35 nm in diameter, are classified as beta type particles. Although this type has been previously characterized by high rates of glycogen turnover in other tissues, its localization in the lens nucleus indicates that it may serve a structural function rather than as a storage depot of carbohydrate in the lens. In a comparative electron microscopy study, glycogen particles were not observed in the chicken lens.     

Spotting optimization for oligo microarrays on aldehyde-glass

Low-density microarrays that utilize short oligos (<100 nt) for capture are highly attractive for use in diagnostic applications, yet these experiments require strict quality control and meticulous reproducibility. However, a survey of current literature indicates vast inconsistencies in the spotting and processing procedures. In this study, spotting and processing protocols were optimized for aldehyde-functionalized glass substrates. Figures of merit were developed for quantitative comparison of spot quality and reproducibility. Experimental variables examined included oligo concentration in the spotting buffer, composition of the spotting buffer, postspotting "curing" conditions, and postspotting wash conditions. Optimized conditions included the use of 3-4 microM oligo in a 3x standard saline citrate/0.05% sodium dodecyl sulfate/0.001% (3-[(3-cholamidopropyl) dimethylammonia]-1-propane sulfonate) spotting buffer, 24-h postspotting reaction at 100% relative humidity, and a four-step wash procedure. Evaluation of six types of aldehyde-functionalized glass substrates indicated that those manufactured by CEL Associates, Inc. yield the highest oligo coverage.     

The cytokine and chemokine expression profile of nucleus pulposus cells: implications for degeneration and regeneration of the intervertebral disc

Introduction

The aims of these studies were to identify the cytokine and chemokine expression profile of nucleus pulposus (NP) cells and to determine the relationships between NP cell cytokine and chemokine production and the characteristic tissue changes seen during intervertebral disc (IVD) degeneration.

Methods

Real-time q-PCR cDNA Low Density Array (LDA) was used to investigate the expression of 91 cytokine and chemokine associated genes in NP cells from degenerate human IVDs. Further real-time q-PCR was used to investigate 30 selected cytokine and chemokine associated genes in NP cells from non-degenerate and degenerate IVDs and those from IVDs with immune cell infiltrates (‘infiltrated’). Immunohistochemistry (IHC) was performed for four selected cytokines and chemokines to confirm and localize protein expression in human NP tissue samples.

Results

LDA identified the expression of numerous cytokine and chemokine associated genes including 15 novel cytokines and chemokines. Further q-PCR gene expression studies identified differential expression patterns in NP cells derived from non-degenerate, degenerate and infiltrated IVDs. IHC confirmed NP cells as a source of IL-16, CCL2, CCL7 and CXCL8 and that protein expression of CCL2, CCL7 and CXCL8 increases concordant with histological degenerative tissue changes.

Conclusions

Our data indicates that NP cells are a source of cytokines and chemokines within the IVD and that these expression patterns are altered in IVD pathology. These findings may be important for the correct assessment of the ‘degenerate niche’ prior to autologous or allogeneic cell transplantation for biological therapy of the degenerate IVD.     

Pregnancy outcomes in women with repeated implantation failures after intracytoplasmic morphologically selected sperm injection (IMSI)

Background

The role of serum anti-Müllerian hormone (AMH) as predictor of in-vitro fertilization outcomes has been much debated. The aim of the present study is to investigate the practicability of combining serum AMH level with biological age as a simple screening method for counseling IVF candidates of advanced reproductive age with potential poor outcomes prior to treatment initiation.

Methods

A total of 1,538 reference patients and 116 infertile patients aged greater than or equal to 40 years enrolled in IVF/ICSI cycles were recruited in this retrospective analysis. A reference chart of the age-related distribution of serum AMH level for Asian population was first created. IVF/ICSI patients aged greater than or equal to 40 years were then divided into three groups according to the low, middle and high tertiles the serum AMH tertiles derived from the reference population of matching age. The cycle outcomes were analyzed and compared among each individual group.

Results

For reference subjects aged greater than or equal to 40 years, the serum AMH of the low, middle and high tertiles were equal or lesser than 0.48, 0.49-1.22 and equal or greater than 1.23 ng/mL respectively. IVF/ICSI patients aged greater than or equal to 40 years with AMH levels in the low tertile had the highest cycle cancellation rate (47.6%) with zero clinical pregnancy. The nadir AMH level that has achieved live birth was 0.56 ng/mL, which was equivalent to the 36.4th percentile of AMH level from the age-matched reference group. The optimum cut-off levels of AMH for the prediction of nonpregnancy and cycle cancellation were 1.05 and 0.68 ng/mL, respectively.

Conclusions

Two criteria: (1) age greater than or equal to 40 years and (2) serum AMH level in the lowest tertile (equal or lesser than 33.3rd percentile) of the matching age group, may be used as markers of futility for counseling IVF/ICSI candidates.     

Real-time quantification of RNA polymerase activity using a "broken beacon"

A novel assay using a hybridization-based method was developed for real-time monitoring of RNA synthesis. In this work, a "broken beacon" in which the fluor and quencher were located on separate but complementary oligonucleotides was used to quantify the amount of RNA production by T7 polymerase. The relative lengths of the fluor-oligo and quencher-oligo, and their relative concentrations were optimized. The experimentally determined limit-of-detection was approximately 45 nM. The new assay was compared to the "gold-standard" radiolabel ([(32)P]NTP incorporation) assay for RNA quantification. While the broken beacon assay exhibited a higher limit of detection, it provided an accurate measure of RNA production rates. However, the broken beacon assay provided the significant analytical advantages of (i) a real-time and continuous measurement, (ii) no requirement for the use of radiolabels or gel-based analysis, and (iii) substantial time and labor savings.