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.     

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.     

Mesodern-inducing factors and mesodermal patterning

Identification of the signalling molecules involved in mesoderm formation in amphibian embryos still presents problems. None of the original candidates, such as activin, have been definitively ruled out, and the new factors, such as the nodal-related genes, have come on to the scene. Of the original candidates, activin has been definitively shown to act as a morphogen, whereas bone morphogenetic protein (BMP)-4 has emerged as a ventral inducer and an inhibitor of neural differentiation. The effects of BMP-4 are antagonized by chordin, a molecule related to the product of the Drosophila gene short gastrulation.     

Function of bovine CD46 as a cellular receptor for bovine viral diarrhea virus is determined by complement control protein 1

The pestivirus bovine viral diarrhea virus (BVDV) was shown to bind to the bovine CD46 molecule, which subsequently promotes entry of the virus. To assess the receptor usage of BVDV type 1 (BVDV-1) and BVDV-2, 30 BVDV isolates including clinical samples were assayed for their sensitivity to anti-CD46 antibodies. With a single exception the infectivity of all tested strains of BVDV-1 and BVDV-2 was inhibited by anti-CD46 antibodies, which indicates the general usage of CD46 as a BVDV receptor. Molecular analysis of the interaction between CD46 and the BVD virion was performed by mapping the virus binding site on the CD46 molecule. Single complement control protein modules (CCPs) within the bovine CD46 were either deleted or replaced by analogous CCPs of porcine CD46, which does not bind BVDV. While the epitopes recognized by anti-CD46 monoclonal antibodies which block BVDV infection were attributed to CCP1 and CCP2, in functional assays only CCP1 turned out to be essential for BVDV binding and infection. Within CCP1 two short peptides on antiparallel beta strands were identified as crucial for the binding of BVDV. Exchanges of these two peptide sequences were sufficient for a loss of function in bovine CD46 as well as a gain of function in porcine CD46. Determination of the size constraints of CD46 revealed that a minimum length of four CCPs is essential for receptor function. An increase of the distance between the virus binding domain and the plasma membrane by insertion of one to six CCPs of bovine C4 binding protein exhibited only a minor influence on susceptibility to BVDV.     

Methanosarcina spp. drive vinyl chloride dechlorination via interspecies hydrogen transfer

Two highly enriched cultures containing Dehalococcoides spp. were used to study the effect of aceticlastic methanogens on reductive vinyl chloride (VC) dechlorination. In terms of aceticlastic methanogens, one culture was dominated by Methanosaeta, while the other culture was dominated by Methanosarcina, as determined by fluorescence in situ hybridization. Cultures amended with 2-bromoethanesulfonate (BES), an efficient inhibitor of methanogens, exhibited slow VC dechlorination when grown on acetate and VC. Methanogenic cultures dominated by Methanosaeta had no impact on dechlorination rates, compared to BES-amended controls. In contrast, methanogenic cultures dominated by Methanosarcina displayed up to sevenfold-higher rates of VC dechlorination than their BES-amended counterparts. Methanosarcina-dominated cultures converted a higher percentage of [2-(14)C]acetate to (14)CO(2) when concomitant VC dechlorination took place, compared to nondechlorinating controls. Respiratory indices increased from 0.12 in nondechlorinating cultures to 0.51 in actively dechlorinating cultures. During VC dechlorination, aqueous hydrogen (H(2)) concentrations dropped to 0.3 to 0.5 nM. However, upon complete VC consumption, H(2) levels increased by a factor of 10 to 100, indicating active hydrogen production from acetate oxidation. This process was thermodynamically favorable by means of the extremely low H(2) levels during dechlorination. VC degradation in nonmethanogenic cultures was not inhibited by BES but was limited by the availability of H(2) as electron donor, in cultures both with and without BES. These findings all indicate that Methanosarcina (but not Methanosaeta), while cleaving acetate to methane, simultaneously oxidizes acetate to CO(2) plus H(2), driving hydrogenotrophic dehalorespiration of VC to ethene by Dehalococcoides.