Defect of sperm assembly in a neurological mutant of the mouse, wobbler (WR)

In the wobbler (WR) mouse, a neuromuscular mutant characterized by a motoneuron degeneration and male infertility, the cellular basis of the defect in spermiogenesis was studied by light and electron microscopy as well as by lectin binding. Spermatozoa of the wobbler mutant had rounded heads, and their motility was reduced. In histological sections of WR testes, spermatogenesis appeared normal up to the stage of round spermatids, but the elongation and flattening of the nucleus during late spermiogenesis did not occur. Numbers of spermatid nuclei in WR testes were reduced to 70%-80% of controls. The acrosomal marker glycoprotein, peanut agglutinin receptor, was synthesized, but the acrosomal membrane did not attach to the nucleus. The disturbance in spermiogenesis of the wobbler mouse is not due to impaired descent of the testis, nor to a lack of testosterone, and is distinct from that observed in other mouse mutants (quaking, QK; Purkinje cell degeneration, PCD) with combined neurological and spermiogenesis defects.     

Estrogen receptor expression in serially cultivated rat endometrial cells: stimulation by forskolin and cholera toxin

Serially propagated with 3T3 feeder layer support, epithelial cells derived from normal rat endometrium expressed estrogen receptor activity. Specific binding of 17-beta-estradiol was in the range of 30-60 fmol/mg of protein and was of high affinity (Kd = 0.3 nM). A survey of cell lines derived from several other normal epithelia showed that rat vaginal and human cervical cultures also had high-affinity estrogen receptors (6-13 fmol/mg of protein), while rat epidermal and esophageal cells had no detectable activity. In the endometrial cultures, receptor levels were elevated nearly two- to fourfold by cholera toxin or forskolin in the medium. This effect was detectable after 4 hr but not 1 hr of treatment and did not occur in the presence of cycloheximide. We conclude that serially cultivated rat endometrial cells retain hormonal properties expressed in vivo while exhibiting some keratinocyte character. These cells may provide a useful model for study of receptor modulation.     

Analysis of a tyrosine-specific protein kinase activity associated with the retroviral erbB oncogene product

The transforming protein erbB of avian erythroblastosis virus (AEV) has considerable sequence homology with the epidermal growth factor (EGF) and appears to represent a truncated form of this receptor. The sequence of the erbB gene is furthermore related to that of other viral transforming genes such as src, fps, yes or abl. The transforming proteins of these src-related oncogenes as well as receptors for EGF, platelet-derived growth factor (PDGF), and insulin are associated with tyrosine-specific protein kinases. It has been difficult to demonstrate this activity for the erbB protein. To analyze the erbB gene product, we prepared polyclonal antibodies against a bacterially expressed erbB DNA restriction fragment (BamHI/BamHI). The antiserum is shown to immunoprecipitate the erbB protein from AEV-transformed chicken fibroblasts and also recognizes the EGF receptor protein. Both proteins become phosphorylated in vitro on tyrosine residues upon the addition of [gamma-32P]ATP. The protein kinase activity is low compared to other oncogene-specific kinases. This is not due to kinase blocking by the serum, because erbB carboxyterminal synthetic peptide antibodies give rise to low levels of protein kinase activity as well indicating that this may be a characteristic property of erbB in vitro.     

A comparative description of mitochondrial DNA differentiation in selected avian and other vertebrate genera

Levels of mitochondrial DNA (mtDNA) sequence divergence between species within each of several avian (Anas, Aythya, Dendroica, Melospiza, and Zonotrichia) and nonavian (Lepomis and Hyla) vertebrate genera were compared. An analysis of digestion profiles generated by 13-18 restriction endonucleases indicates little overlap in magnitude of mtDNA divergence for the avian versus nonavian taxa examined. In 55 interspecific comparisons among the avian congeners, the fraction of identical fragment lengths (F) ranged from 0.26 to 0.96 (F = 0.46), and, given certain assumptions, these translate into estimates of nucleotide sequence divergence (p) ranging from 0.007 to 0.088; in 46 comparisons among the fish and amphibian congeners, F values ranged from 0.00 to 0.36 (F = 0.09), yielding estimates of P greater than 0.070. The small mtDNA distances among avian congeners are associated with protein-electrophoretic distances (D values) less than approximately 0.2, while the mtDNA distances among assayed fish and amphibian congeners are associated with D values usually greater than 0.4. Since the conservative pattern of protein differentiation previously reported for many avian versus nonavian taxa now appears to be paralleled by a conservative pattern of mtDNA divergence, it seems increasingly likely that many avian species have shared more recent common ancestors than have their nonavian taxonomic counterparts. However, estimates of avian divergence times derived from mtDNA- and protein-calibrated clocks cannot readily be reconciled with some published dates based on limited fossil remains. If the earlier paleontological interpretations are valid, then protein and mtDNA evolution must be somewhat decelerated in birds. The empirical and conceptual issues raised by these findings are highly analogous to those in the long-standing debate about rates of molecular evolution and times of separation of ancestral hominids from African apes.      

Methods for computing the standard errors of branching points in an evolutionary tree and their application to molecular data from humans and apes

Statistical methods for computing the standard errors of the branching points of an evolutionary tree are developed. These methods are for the unweighted pair-group method-determined (UPGMA) trees reconstructed from molecular data such as amino acid sequences, nucleotide sequences, restriction-sites data, and electrophoretic distances. They were applied to data for the human, chimpanzee, gorilla, orangutan, and gibbon species. Among the four different sets of data used, DNA sequences for an 895-nucleotide segment of mitochondrial DNA (Brown et al. 1982) gave the most reliable tree, whereas electrophoretic data (Bruce and Ayala 1979) gave the least reliable one. The DNA sequence data suggested that the chimpanzee is the closest and that the gorilla is the next closest to the human species. The orangutan and gibbon are more distantly related to man than is the gorilla. This topology of the tree is in agreement with that for the tree obtained from chromosomal studies and DNA-hybridization experiments. However, the difference between the branching point for the human and the chimpanzee species and that for the gorilla species and the human-chimpanzee group is not statistically significant. In addition to this analysis, various factors that affect the accuracy of an estimated tree are discussed.      

Rat esophageal and epidermal keratinocytes: intrinsic differences in culture and derivation of continuous lines

Serially cultivated with 3T3 feeder layer support as colonies of stratified squamous epithelium, rat epidermal and esophageal epithelial cells were readily distinguishable by three criteria. First, the epidermal colonies, exhibiting extensive piling up of squames in the centers, were more stratified than esophageal colonies. Second, in sparse culture 70 to 90% of the esophageal cells but as few as 1 to 5% of the epidermal cells were competent in cross-linked envelope formation upon treatment with the ionophore X537A. After reaching confluence, up to 90% of the cells of both types formed envelopes upon ionophore treatment. Third, epidermal cells in suspension culture reached maximal levels of spontaneously cross-linked envelopes in 1 day or less, while esophageal cells required about 4 days in suspension to reach maximal levels. A reproducible finding with both cell types was that initial colony-forming efficiencies of less than 1% increased to about 40% upon serial passage with consequent derivation of continuous lines. Sparse cultures of esophageal cells with high colony-forming ability retained a high degree of envelope competence (70 to 90%), indicating these two properties are not mutually exclusive. The derived lines exhibited reduced dependence upon feeder layer support at clonal density, but in suspension culture the cells did not grow and lost colony-forming ability with a half-time of several hours. We conclude that cells from these keratinized rat epithelia exhibit intrinsic differences in culture and become continuous lines expressing characteristic regulation of envelope competence and loss of germinative capability in suspension.     

Spinal metastasis of a thyroglobulin-rich Hürthle cell carcinoma detected by fine needle aspiration. Light and electron microscopic study of an unusual case

A prospective diagnosis of metastatic thyroid carcinoma was made on an aspirate of a spinal mass. While the cellular component of the aspirate was compatible with a renal or hepatic neoplasm, the recognition of colloid on air-dried smears defined the cells as Hürthle cells. Biopsy of the spinal tumor and subsequent thyroidectomy revealed a Hürthle cell carcinoma. Whereas the primary tumor showed typical Hürthle cell architecture, with solid tumor nests and microfollicle formation, the metastasis contained areas of macrofollicle formation with abundant colloid production and strong immunocytochemical reactivity for thyroglobulin. This change of histologic pattern from the primary tumor to the metastasis has not been previously reported in Hürthle cell lesions. The unusual light microscopic and ultrastructural aspects of this tumor are discussed.     

Anonymous nuclear DNA markers in the American oyster and their implications for the heterozygote deficiency phenomenon in marine bivalves

A puzzling population-genetic phenomenon widely reported in allozyme surveys of marine bivalves is the occurrence of heterozygote deficits relative to Hardy-Weinberg expectations. Possible explanations for this pattern are categorized with respect to whether the effects should be confined to protein-level assays or are genomically pervasive and expected to be registered in both protein- and DNA-level assays. Anonymous nuclear DNA markers from the American oyster were employed to reexamine the phenomenon. In assays based on the polymerase chain reaction (PCR), two DNA-level processes were encountered that can lead to artifactual genotypic scorings: (a) differential amplification of alleles at a target locus and (b) amplification from multiple paralogous loci. We describe symptoms of these complications and prescribe methods that should generally help to ameliorate them. When artifactual scorings at two anonymous DNA loci in the American oyster were corrected, Hardy-Weinberg deviations registered in preliminary population assays decreased to nonsignificant values. Implications of these findings for the heterozygote-deficit phenomenon in marine bivalves, and for the general development and use of PCR-based assays, are discussed.      

Pan-Arctic soil moisture control on tundra carbon sequestration and plant productivity

Long-term atmospheric CO₂ concentration records have suggested a reduction in the positive effect of warming on high-latitude carbon uptake since the 1990s. A variety of mechanisms have been proposed to explain the reduced net carbon sink of northern ecosystems with increased air temperature, including water stress on vegetation and increased respiration over recent decades. However, the lack of consistent long-term carbon flux and in situ soil moisture data has severely limited our ability to identify the mechanisms responsible for the recent reduced carbon sink strength. In this study, we used a record of nearly 100 site-years of eddy covariance data from 11 continuous permafrost tundra sites distributed across the circumpolar Arctic to test the temperature (expressed as growing degree days, GDD) responses of gross primary production (GPP), net ecosystem exchange (NEE), and ecosystem respiration (ER) at different periods of the summer (early, peak, and late summer) including dominant tundra vegetation classes (graminoids and mosses, and shrubs). We further tested GPP, NEE, and ER relationships with soil moisture and vapor pressure deficit to identify potential moisture limitations on plant productivity and net carbon exchange. Our results show a decrease in GPP with rising GDD during the peak summer (July) for both vegetation classes, and a significant relationship between the peak summer GPP and soil moisture after statistically controlling for GDD in a partial correlation analysis. These results suggest that tundra ecosystems might not benefit from increased temperature as much as suggested by several terrestrial biosphere models, if decreased soil moisture limits the peak summer plant productivity, reducing the ability of these ecosystems to sequester carbon during the summer.