Ultrastructural localization of laminin on in vivo embryonic, neonatal, and adult rat cardiac myocytes and in early rat embryos raised in whole-embryo culture.

The temporal and spatial distribution of the basement membrane component laminin was examined in vivo in developing rat hearts at 11.5 and 15 days of embryonic development (ED), and in neonates and adults, by pre-embedding ultrastructural immunocytochemistry. In addition, the patterns observed at 11.5 days ED were compared to the distribution of laminin in embryos maintained in whole-embryo culture. At 11.5 days ED laminin was localized in punctate patches on the surface of the plasma membrane, with large gaps between areas of staining. The development of myocytes and localization of laminin in the whole embryo-cultured embryos was similar to that found in the in vivo embryos. At 15 days ED, laminin localization was limited to distinct patches of developing extracellular matrix material associated with the sarcolemma. Gaps between areas of localization were shorter than in the 11.5-day hearts. In neonates, distribution of laminin localization was more extensive with fewer gaps and was associated with the developing basement membrane. In adult hearts, laminin was localized along the entire length of the basement membrane and was heaviest in areas of morphological specialization, such as Z-bands, where collagen bundles contacted the sarcolemma.     

Complete nucleotide sequence, genome organization, and biological properties of human immunodeficiency virus type 1 in vivo: evidence for limited defectiveness and complementation.

Previous studies of the genetic and biologic characteristics of human immunodeficiency virus type 1 (HIV-1) have by necessity used tissue culture-derived virus. We recently reported the molecular cloning of four full-length HIV-1 genomes directly from uncultured human brain tissue (Y. Li, J. C. Kappes, J. A. Conway, R. W. Price, G. M. Shaw, and B. H. Hahn, J. Virol. 65:3973-3985, 1991). In this report, we describe the biologic properties of these four clones and the complete nucleotide sequences and genome organization of two of them. Clones HIV-1YU-2 and HIV-1YU-10 were 9,174 and 9,176 nucleotides in length, differed by 0.26% in nucleotide sequence, and except for a frameshift mutation in the pol gene in HIV-1YU-10, contained open reading frames corresponding to 5'-gag-pol-vif-vpr-tat-rev-vpu-env-nef-3' flanked by long terminal repeats. HIV-1YU-2 was fully replication competent, while HIV-1YU-10 and two other clones, HIV-1YU-21 and HIV-1YU-32, were defective. All three defective clones, however, when transfected into Cos-1 cells in any pairwise combination, yielded virions that were replication competent and transmissible by cell-free passage. The cellular host range of HIV-1YU-2 was strictly limited to primary T lymphocytes and monocyte-macrophages, a property conferred by its external envelope glycoprotein. Phylogenetic analyses of HIV-1YU-2 gene sequences revealed this virus to be a member of the North American/European HIV-1 subgroup, with specific similarity to other monocyte-tropic viruses in its V3 envelope amino acid sequence. These results indicate that HIV-1 infection of brain is characterized by the persistence of mixtures of fully competent, minimally defective, and more substantially altered viral forms and that complementation among them is readily attainable. In addition, the limited degree of genotypic heterogeneity observed among HIV-1YU and other brain-derived viruses and their preferential tropism for monocyte-macrophages suggest that viral replication within the central nervous system may differ from that within the peripheral lymphoid compartment in significant and clinically important ways. The availability of genetically and biologically well characterized HIV-1 clones from uncultured human tissue should facilitate future studies of virus-cell interactions relevant to viral pathogenesis and drug and vaccine development.     

Macrophage tropism determinants of human immunodeficiency virus type 1 in vivo.

Strains of human immunodeficiency virus type 1 differ in their abilities to infect and replicate in primary human macrophages. Chimeric clones were constructed from a provirus unable to infect macrophages (NLHX) and envelope sequences (V3 loop) of viruses derived without cultivation from brain (YU2 and w1-1c1) or spleen (w2-1b4) tissues. The substituted V3 loop sequences in each case were sufficient to confer upon NLHX the ability to infect macrophages. Furthermore, an envelope domain immediately N terminal to the V3 loop also was found to modulate the level of replication in macrophages. These results demonstrate that an envelope determinant derived directly from patients with AIDS confers HIV-1 tropism for macrophages.     

The adaptive significance of insect gall distribution: survivorship of species in xeric and mesic habitats

Summary We studied the relationship between habitat moisture and gall-forming insect populations. Population sizes for most galling taxa were significantly larger in xeric habitats compared with mesic habitats. Our results indicate that the differential abundance of galling insects in these habitats is due primarily to differential mortality and survivorship. Mortality factors acting upon eight insect galling species (belonging to eight genera and four families) were measured on six species (five genera and five families) of host plants. Survival was significantly higher for galling populations inhabiting xeric habitats compared with mesic habitats. Parasitism was higher in mesic habitats in seven of eight habitats and fungus-induced diseases were higher in five of seven habitats. Mortality due to predation and other (unknown) factors showed no clear trends. Overall, there was a tendency towards lower mortality and consequently higher survival for populations inhabiting xeric habitats. We hypothesize that reduced mortality caused by natural enemies and endophytic fungi has contributed to the speciation and radiation of galling insects in apparently harsh environments.     

Plant size,geitonogamy and seed set in Ipomopsis aggregata

Summary We used powdered fluorescent dyes to estimate receipt of self vs. outcross pollen in the self-incompatible species Ipomopsis aggregata (Polemoniaceae). Flowers on small and large plants received equal amounts of outcross pollen, whereas flowers on large plants received more self pollen, so the proportion of self pollen delivered through geitonogamy increased with plant size. In natural populations emasculation of all flowers on a plant raised average seed set per flower from 5.19 to 6.99 and also raised fruit set, though not significantly. From these results one expects a negative correlation between plant size and seeds per flower. The opposite trend was observed in a sample of plants in the field, suggesting that deleterious effects of geitonogamy on female fecundity in large plants can be overruled by other factors such as size-related fruit or seed abortion. Results are discussed in relation to the evolution of gynodioecy.     

Developmental stages of the CD (Sprague-Dawley) rat skeleton after maternal exposure to ethylene glycol.

Ethylene glycol (EG), a chemical which causes skeletal malformations in rats, was administered by gavage to sperm positive CD rats on gestational days (gd) 6 through 15 at doses of 0 or 2,500 mg/kg/day to assess its effects on the pre- and postnatal skeletal development. Dams and fetuses/pups were killed on gd 18, 20, postnatal day (pnd) 1, 4, 14, 21, or 63, and offspring were double-stained for examination of skeletal malformations and degree of ossification of rapidly developing skeletal districts. No difference in gestational day of delivery between controls and the EG-treated dams was seen. Fetal weights per litter were significantly decreased with EG treatment in both the gd 18 and 20 groups. Pup body weight on pnd 1 was significantly below controls; however, EG had no effect on pup body weight on pnd 4-63. The percentage of fetuses/pups with skeletal malformations per litter was significantly increased after EG exposure for all time points except at pnd 63, with a predominance of axial skeletal defects. The percentages of total ossification, of sternabrae ossified, and of vertebral centra ossified were significantly reduced in the EG groups on gd 20 and on pnd 1-21, but not on gd 18 or on pnd 63. When the ossification data were subjected to statistical analysis with fetal/pup weights as a covariate, the values for EG-exposed pups on gd 20 were not statistically significantly different from the control values. The reduced ossification values for EG-exposed pups on pnd 1-21 retained statistical significance even after covariate analysis. There was no effect of dose or body weight on ossification of fore- or hindlimb digits. In conclusion, the differences in incidence of skeletal alterations observed prenatally and through pnd 21 were not evident by pnd 63, suggesting that perinatal skeletal abnormalities may not always be permanent.     

Comparison of proteoglycans synthesized by porcine normal and polycystic renal tubular epithelial cells in vitro

Newly synthesized porcine tubular epithelial cell proteoglycans were labeled in vitro with Na2[35S]SO4. At the beginning of the labeling period (24 h) [35S] sulfate incorporated into macromolecules was measured following PD-10 chromatography. There was a significant reduction in the amount of 35S-labeled macromolecules isolated from polycystic cells compared to that from normal cells. The distribution of recovered radiolabeled material among the medium, cell surface, and intracellular fractions was similar for both normal and polycystic cells. Analysis of the proteoglycans in polycystic cells demonstrated that 86 and 73% of 35S-labeled macromolecules were of the heparan sulfate type in polycystic and normal cells, respectively. The remainder was chondroitin sulfate. Proteoglycans were characterized using DEAE-Sephacel ion-exchange chromatography, chondroitinase ABC, heparitinase, and nitrous acid digestion followed by Sepharose CL-4B gel permeation chromatography. The majority of radiolabeled material in the medium, cell surface, and intracellular fractions eluted between 0.35 and 0.39 M NaCl. However, a second peak (peak II) that eluted at 0.25 M NaCl was found in the medium from polycystic cells. This peak accounted for 27% of the total macromolecules secreted into the medium. Proteoglycans in the major peak were susceptible to nitrous acid and chondroitinase ABC digestion. A similar proportion of peak II was degraded by chondroitinase ABC. However, the remainder was only slightly susceptible to treatment with nitrous acid or heparitase. In normal cells a small amount of material eluted at a similar low charge; the proteoglycans were the same as those found in the major peak and appeared as a shoulder on this peak.     

Multiple forms of Go alpha mRNA: analysis of the 3'-untranslated regions

Go, a guanine nucleotide binding protein found predominantly in neural tissues, interacts in vitro with rhodopsin, muscarinic, and other receptors and has been implicated in the regulation of ion channels. Despite the virtual identity of reported cDNA sequences for the alpha subunit of Go (Go alpha), multiple molecular weight forms of mRNA have been identified in tissues from all species examined. To investigate the molecular basis for the size heterogeneity of Go alpha mRNAs, four cDNA clones were isolated from the same retinal lambda gt10 cDNA library that was used earlier to isolate lambda GO9, a clone encompassing the complete coding region of Go alpha. These clones were identified as Go alpha clones based on nucleotide sequence identity with lambda GO9 in the coding region; they diverge, however, from lambda GO9 in the 3'-untranslated region 28 nucleotides past the stop codon. An oligonucleotide probe complementary to a portion of the 3'-untranslated region of lambda GO9 that differs from the newly isolated clones hybridized with 3.0- and 4.0-kb mRNAs present in bovine brain and retina whereas a similar probe for the unique region of the new clones hybridized with a 4.0-kb mRNA in both tissues and with a 2.0-kb mRNA found predominantly in retina. A similar hybridization pattern was observed when brain poly(A+) RNA from other species was hybridized with the different 3'-untranslated region probes. It appears that differences in the 3'-untranslated regions could, in part, be the basis for the observed heterogeneity in Go alpha mRNAs.