Different rates of substitution may produce different phylogenies of the eutherian mammals

Summary In an attempt to resolve some points of branching order in the phylogeny of the eutherian mammals, a phylogenetic analysis of 26 nuclear and 6 mitochondrial genes was undertaken using a maximum likelihood method on a constant rate stochastic model of molecular evolution. Seventeen of the nuclear genes gave a primates/artiodactyls grouping highest support whereas three of the mitochondrial genes found a rodents/artiodactyls grouping to be best supported. The primates/rodents grouping was never the best supported. On the assumption that rodents are indeed an outgroup to primates and artiodactyls and that the latter taxa diverged 70 million years ago, an estimation was made, for each gene, of the time of divergence of the rodent lineage. In most cases such estimates were beyond the limits set by present interpretations of the paleontological record as were many estimates of the divergence time of mouse and rat. These results suggest that, although there is locus variation, the divergent position of the rodent lineage may be an artifact of an elevated rate of nucleotide substitution in this order.     

Effects of Handling or Immobilization on Plasma Levels of 3,4-Dihydroxyphenylalanine, Catecholamines, and Metabolites in Rats

In conscious animals, handling and immobilization increase plasma levels of the catecholamines norepinephrine (NE) and epinephrine (EPI). This study examined plasma concentrations of endogenous compounds related to catecholamine synthesis and metabolism during and after exposure to these stressors in conscious rats. Plasma levels of 3,4-dihydroxyphenylalanine (DOPA), NE, EPI, and dopamine (DA), the deaminated catechol metabolites 3,4-dihydroxyphenylglycol (DHPG), and 3,4-dihydroxyphenylacetic acid (DOPAC), and their O-methylated derivatives methoxyhydroxyphenylglycol (MHPG) and homovanillic acid (HVA) were measured using liquid chromatography with electrochemical detection at 1, 3, 5, 20, 60, and 120 min of immobilization. By 1 min of immobilization, plasma NE and EPI levels had already reached peak values, and plasma levels of DOPA, DHPG, DOPAC, and MHPG were increased significantly from baseline, whereas plasma DA and HVA levels were unchanged. During the remainder of the immobilization period, the increased levels of DOPA, NE, and EPI were maintained, whereas levels of the metabolites progressively increased. In animals immobilized briefly (5 min), elevated concentrations of the metabolites persisted after release from the restraint, whereas DOPA and catecholamine levels returned to baseline. Gentle handling for 1 min also significantly increased plasma levels of DOPA, NE, EPI, and the NE metabolites DHPG and MHPG, without increasing levels of DA or HVA. The results show that in conscious rats, immobilization or even gentle handling rapidly increases plasma levels of catecholamines, the catecholamine precursor DOPA, and metabolites of NE and DA, indicating rapid increases in the synthesis, release, reuptake, and metabolism of catecholamines.     

Coronavirus species specificity: murine coronavirus binds to a mouse-specific epitope on its carcinoembryonic antigen-related receptor glycoprotein.

Like most coronaviruses, the coronavirus mouse hepatitis virus (MHV) exhibits strong species specificity, causing natural infection only in mice. MHV-A59 virions use as a receptor a 110- to 120-kDa glycoprotein (MHVR) in the carcinoembryonic antigen (CEA) family of glycoproteins (G. S. Dveksler, M. N. Pensiero, C. B. Cardellichio, R. K. Williams, G. S. Jiang, K. V. Holmes, and C. W. Dieffenbach, J. Virol. 65:6881-6891, 1991; and R. K. Williams, G. S. Jiang, and K. V. Holmes, Proc. Natl. Acad. Sci. USA 88:5533-5536, 1991). The role of virus-receptor interactions in determining the species specificity of MHV-A59 was examined by comparing the binding of virus and antireceptor antibodies to cell lines and intestinal brush border membranes (BBM) from many species. Polyclonal antireceptor antiserum (anti-MHVR) raised by immunization of SJL/J mice with BALB/c BBM recognized MHVR specifically in immunoblots of BALB/c BBM but not in BBM from adult SJL/J mice that are resistant to infection with MHV-A59, indicating a major difference in epitopes between MHVR and its SJL/J homolog which does not bind MHV (7). Anti-MHVR bound to plasma membranes of MHV-susceptible murine cell lines but not to membranes of human, cat, dog, monkey, or hamster cell lines. Cell lines from these species were resistant to MHV-A59 infection, and only the murine cell lines tested were susceptible. Pretreatment of murine fibroblasts with anti-MHVR prevented binding of radiolabeled virions to murine cells and prevented virus infection. Solid-phase virus-binding assays and virus overlay protein blot assays showed that MHV-A59 virions bound to MHVR on intestinal BBM from MHV-susceptible mouse strains but not to proteins on intestinal BBM from humans, cats, dogs, pigs, cows, rabbits, rats, cotton rats, or chickens. In immunoblots of BBM from these species, both polyclonal and monoclonal antireceptor antibodies that block MHV-A59 infection of murine cells recognized only the murine CEA-related glycoprotein and not homologous CEA-related glycoproteins of other species. These results suggest that MHV-A59 binds to a mouse-specific epitope of MHVR, and they support the hypothesis that the species specificity of MHV-A59 infection may be due to the specificity of the virus-receptor interaction.     

Point mutations in the DNA polymerase gene of human cytomegalovirus that result in resistance to antiviral agents.

Three independently isolated mutants of human cytomegalovirus strain AD169 were found to be resistant to ganciclovir at a 50% effective dose of 200 microM. Phosphorylation of ganciclovir was reduced 10-fold in mutant-infected cells compared with AD169-infected cells. All three mutants were also determined to be resistant to the nucleotide analogs (S)-1-[(3-hydroxy-2- phosphonylmethoxy)propyl]adenine (HPMPA) and (S)-1-[(3-hydroxy-2-phosphonylmethoxy)propyl]cytosine (HPMPC) and hypersensitive to thymine-1-D-arabinofuranoside (AraT). Single base changes resulting in amino acid substitutions were demonstrated in the nucleotide sequence of the DNA polymerase gene of each mutant. The polymerase mutation contained in one of the mutants was transferred to the wild-type AD169 background. Ganciclovir phosphorylation in cells infected with the recombinant virus produced by this transfer was found to be equivalent to that of AD169-infected cells. The ganciclovir resistance of the recombinant was reduced fourfold compared with that of the parental mutant; however, the recombinant remained resistant to HPMPA and HPMPC and hypersensitive to AraT. The ganciclovir resistance of the mutants therefore appears to result from mutations in two genes: (i) a kinase which phosphorylates ganciclovir and (ii) the viral DNA polymerase.     

Ehlers Danlos syndrome type VIIB. Incomplete cleavage of abnormal type I procollagen by N-proteinase in vitro results in the formation of copolymers of collagen and partially cleaved pNcollagen that are near circular in cross-section.

We have shown that a child with Ehlers Danlos syndrome (EDS) type VII has a G to A transition at the first nucleotide of intron 6 in one of her COL1A2 alleles. Half of the cDNA clones prepared from the proband's pro alpha 2(I) mRNA lacked exon 6. The type I procollagen secreted by the proband's dermal fibroblasts in culture was purified, and collagen fibrils were generated in vitro by cleavage of the procollagen with the procollagen N- and C-proteinases. Incubation of the procollagen with N-proteinase resulted in a 1:1 mixture of pCcollagen and uncleaved procollagen. Incubation of this mixture with C-proteinase generated collagen and abnormal pNcollagen (pNcollagen-ex6) that readily copolymerized into fibrils. By electron microscopy these fibrils resembled the hieroglyphic fibrils seen in the N-proteinase-deficient skin of dermatosparactic animals and humans and were distinct from the near circular cross-section fibrils seen in the tissues of individuals with EDS type VII. Further incubation of the hieroglyphic fibrils with N-proteinase resulted in partial cleavage of the pNcollagen-ex6 in which the abnormal pN alpha 2(I) chains remained intact. These fibrils were not hieroglyphic but were near circular in cross-section. Fibrils formed from collagen and pNcollagen-ex6 that had been partially cleaved with elevated amounts of N-proteinase prior to fibril formation were also near circular in cross-section. The results are consistent with a model of collagen fibril formation in which the intact N-propeptides are located exclusively at the surface of the hieroglyphic fibrils. Partial cleavage of the pNcollagen-ex6 by N-proteinase allows the N-propeptides to be incorporated within the body of the fibrils. The model provides an explanation for the morphology and molecular composition of collagen fibrils in the tissues of patients with EDS type VII.     

Role of antigen-specific T cell help in the generation of in vivo antibody responses. II. Sustained antigen-specific T cell help is required to induce a specific antibody response.

The injection of mice with a goat or rabbit antibody to mouse IgD stimulates a large polyclonal IgG response, approximately 10% of which is specific for antigenic determinants on the anti-IgD antibody molecule. The large goat IgG (GIgG)-specific antibody response in mice injected with goat antibody to mouse IgD requires that GIgG-specific B cells undergo much greater clonal expansion than B cells specific for other Ag. One possible explanation for the greater clonal expansion of GIgG-specific B cells is that B cells that lack GIgG specificity can only be stimulated with GIgG-specific T help during the relatively short time that anti-IgD binds to, and is processed and presented by, these B cells before they cease to express membrane mIgD. In contrast, GIgG-specific B cells can continue to bind, process, and present GIgG through mIgM after they lose mIgD. To test the hypothesis that extended stimulation with Ag-specific T help is required to generate a specific antibody response, we determined time requirements for Ag-specific T cell help for the development of such a response. Mice were injected with rabbit antibody to mouse IgD plus one or more daily injections of FITC conjugated to a F(ab')2 fragment of rabbit IgG (FITC-(Fab')2), which has a short in vivo half-life, and IgG1 anti-FITC antibody production was analyzed. In this system, each additional injection of FITC-F(ab')2 extends the period during which FITC-specific B cells can process this Ag and present it to rabbit IgG-specific T cells. Each additional injection of FITC-F(ab')2 stimulated a several-fold increase in IgG1 anti-FITC antibody levels, and injections on 5 consecutive days were required to induce a maximal anti-FITC response. These observations provide evidence that sustained Ag-specific T cell help is required to stimulate the degree of B cell clonal expansion that characterizes a specific antibody response.     

X-ray diffraction studies on oriented gels of vertebrate smooth muscle thin filaments.

The ATPase activity of acto-myosin subfragment 1 (S1) at low ratios of S1 to actin in the presence of tropomyosin is dependent on the tropomyosin source and ionic conditions. Whereas skeletal muscle tropomyosin causes a 60% inhibitory effect at all ionic strengths, the effect of smooth muscle tropomyosin was found to be dependent on the ionic strength. At low ionic strength (20 mM) smooth muscle tropomyosin inhibits the ATPase activity by 60%, while at high ionic strength (120 mM) it potentiates the ATPase activity three- to five-fold. Therefore, the difference in the effect of smooth muscle and skeletal muscle tropomyosin on the acto-S1 ATPase activity was due to a greater fraction of the tropomyosin-actin complex being turned on in the absence of S1 with smooth muscle tropomyosin than with skeletal muscle tropomyosin. Using well-oriented gels of actin and of reconstituted specimens from vertebrate smooth muscle thin filament proteins suitable for X-ray diffraction, we localized the position of tropomyosin on actin under different levels of acto-S1 ATPase activity. By analysing the equatorial X-ray pattern of the oriented specimens in combination with solution scattering experiments, we conclude that tropomyosin is located at a binding radius of about 3.5 nm on the f-actin helix under all conditions studied. Furthermore, we find no evidence that the azimuthal position of tropomyosin is different for smooth muscle tropomyosin at various ionic strengths, or vertebrate tropomyosin, since the second actin layer-line intensity (at 17.9 nm axial and 4.3 nm radial spacing), which was shown in skeletal muscle to be a sensitive measure of this parameter, remains strong and unchanged. Differences in the ATPase activity are not necessarily correlated with different positions of tropomyosin on f-actin. The same conclusion is drawn from our observations that, although the regulatory protein caldesmon inhibits the ATPase activity in native and reconstituted vertebrate smooth muscle thin filaments at a molar ratio of actin/tropomyosin/caldesmon of 28:7:1, the second actin layer-line remains strong. Only adding caldesmon in excess reduces the intensity of the second actin layer-line, from which the binding radius of caldesmon can be estimated to be about 4 nm. The lack of predominant meridional reflections in oriented specimens, with caldesmon present, suggests that caldesmon does not project away from the thin filament as troponin molecules in vertebrate striated muscle in agreement with electron micrographs of smooth muscle thin filaments. In freshly prepared native smooth muscle thin filaments we observed a Ca(2+)-sensitive reversible bundling effect.(ABSTRACT TRUNCATED AT 400 WORDS)     

Aldehyde dehydrogenase (ALDH) isozymes in the gray short-tailed opossum (Monodelphis domestica): Tissue and subcellular distribution and biochemical genetics of ALDH3

Polyacrylamide gel isoelectric focusing (PAGE-IEF), cellulose acetate electrophoresis, and histochemical techniques were used to examine the tissue and subcellular distribution, genetics and biochemical properties of aldehyde dehydrogenase (ALDH) isozymes in a didelphid marsupial, the gray short-tail opossum (Monodelphis domestica). At least 14 zones of activity were resolved by PAGE-IEF and divided into five isozyme groups and three ALDH classes, based upon comparisons with properties previously reported for human, baboon, rat, and mouse ALDHs. Opossum liver ALDHs were distributed among cytosol (ALDHs 1 and 5) and large granular (mitochondrial) fractions (ALDHs 2 and 5). Similarly, kidney ALDHs were distributed between the cytosol (ALDH5) and the mitochondrial fractions (ALDHs 2, 4, and 5), whereas a major isozyme (ALDH3), found in high activity in cornea, esophagus, ear pinna, tail, and stomach extracts, was localized predominantly in the cytosol fraction. Phenotypic variants of the latter enzyme were shown to be inherited in a normal Mendelian fashion, with two alleles at a single locus (ALDH3) showing codominant expression. The data provided evidence for genetic identity of corneal, ear pinna, tail, and stomach ALDH3 and supported biochemical evidence from other mammalian species that this enzyme has a dimeric subunit structure.