Species-specific diversity among simian immunodeficiency viruses from African green monkeys

The prevalence, natural history, and genetic characteristics of simian immunodeficiency virus (SIV) infections in most feral African monkey species are presently unknown, yet this information is essential to elucidate their origin and relationship to other simian and human immunodeficiency viruses. In this study, a combination of classical and molecular approaches were used to identify and characterize SIV isolates from West African green monkeys (Cercopithecus sabaeus) (SIVagm isolates). Four SIVagm viruses from wild-caught West African green monkeys were isolated and analyzed biologically and molecularly. Amplification, cloning, and sequencing of a 279-bp polymerase fragment directly from uncultured peripheral blood mononuclear cells was facilitated by the use of nested polymerase chain reaction. The results indicated that West African green monkeys are naturally infected with SIVs which are closely related to East African SIVagm isolates. However, structural, antigenic, and genetic differences were observed which strongly suggest that the West African green monkey viruses comprise a phylogenetically distinct subgroup of SIVagm. These findings support our previous hypothesis that SIVagm viruses may have evolved and diverged coincident with the evolution and divergence of their African green monkey host. In addition, this study describes a polymerase chain reaction-based approach that allows the identification and molecular analysis of divergent SIV strains directly from primary monkey tissue. This approach, which does not depend on virus isolation methods, should facilitate future studies aimed at elucidating the origins and natural history of SIVs in feral African green monkey populations.     

DNA probes for the Y-chromosome ofSilene latifolia,a dioecious angiosperm

Summary In order to obtain markers for the Y chromosome ofSilene latifolia, we pooled equal weights of leaf tissue from 18 female siblings into one sample and repeated the process with 18 male siblings. Pooling was intended to provide a common genetic background for each sample, leaving the absence or presence of the Y chromosome as the primary difference between the two samples. DNA was extracted from each sample and subjected to polymerase chain reaction (PCR) amplification with arbitrary 10 bp primers. Four of 60 primers used gave an amplification with the male DNA not found among those from the female DNA. Each of these was subsequently shown to provide a reliable marker for the Y chromosome.     

Regulation of proneural gene expression and cell fate during neuroblast segregation in the Drosophila embryo.

The Drosophila embryonic central nervous system develops from sets of progenitor neuroblasts which segregate from the neuroectoderm during early embryogenesis. Cells within this region can follow either the neural or epidermal developmental pathway, a decision guided by two opposing classes of genes. The proneural genes, including the members of the achaete-scute complex (AS-C), promote neurogenesis, while the neurogenic genes prevent neurogenesis and facilitate epidermal development. To understand the role that proneural gene expression and regulation play in the choice between neurogenesis and epidermogenesis, we examined the temporal and spatial expression pattern of the achaete (ac) regulatory protein in normal and neurogenic mutant embryos. The ac protein is first expressed in a repeating pattern of four ectodermal cell clusters per hemisegment. Even though 5-7 cells initially express ac in each cluster, only one, the neuroblast, continues to express ac. The repression of ac in the remaining cells of the cluster requires zygotic neurogenic gene function. In embryos lacking any one of five genes, the restriction of ac expression to single cells does not occur; instead, all cells of each cluster continue to express ac, enlarge, delaminate and become neuroblasts. It appears that one key function of the neurogenic genes is to silence proneural gene expression within the nonsegregating cells of the initial ectodermal clusters, thereby permitting epidermal development.     

Postnatal expression of glutamate decarboxylases in developing rat cerebellum

The recent identification of two genes encoding distinct forms of the GABA synthetic enzyme, glutamate decarboxylase (GAD), raises the possibility that varying expression of the two genes may contribute to the regulation of GABA production in individual neurons. We investigated the postnatal development the two forms of GAD in the rat cerebellum. The mRNA for GAD₆₇, the form which is less dependent on the presence of the cofactor, pyridoxal phosphate (PLP), is present at birth in presumptive Purkinje cells and increases during postnatal development. GAD₆₇ mRNA predominates in the cerebellum. The mRNA for GAD₆₅, which displays marked PLP-dependence for enzyme activity, cannot be detected in cerebellar cortex by in situ hybridization until P7 in Purkinje cells, and later in other GABA neurons. In deep cerebellar nuclei, which mature prenatally, both forms of GAD mRNA can be detected at birth. The amounts of immunoreactice GAD and GAD enzyme activity parallel changes in mRNA levels. We suggest that the delayed appearance of GAD₆₅ is coincident with synapse formation between GABA neurons and their targets during the second postnatal week. GAD₆₇ mRNA may be present prior to synaptogenesis to produce GABA for trophic and metabolic functions.Special issue dedicated to Dr. Eugene Roberts.     

Experimental documentation of natural hybridization inCactaceae: Origin of Lloyd's Hedgehog Cactus,Echinocereus ×lloydii

The origin ofEchinocereus ×lloydii Britt. & Rose, pro sp. (Lloyd's Hedgehog Cactus) was investigated using comparative morphology, cytology, biochemistry, and particularly, artificial hybridization. Numerous artificial crosses between the putative parentsE. coccineus Engelm. (a species of claret-up cactus) andE. dasyacanthus Engelm. (Texas Rainbow Cactus) were successful, resulting in the production of hundreds of seeds with hybrid embryos. The F₁ hybrid progeny (i.e., syntheticE. ×lloydii) grew to sexual maturity in about four and one-half years, whereupon successful backcrosses and F₂ generation hybrids were also obtained. The known F₁ hybrids closely approximated naturalE. ×lloydii. The fertility of these syntheticE. ×lloydii was high, like their natural counterparts. The populations ofE. ×lloydii in Pecos County, Texas are inferred to have originated as the result of natural interspecific hybridization. It is assumed thatE. ×lloydii or similar plants may arise wherever the parental taxa grow sympatrically.     

The ATP-sensitive potassium channel in pancreatic B-cells is inhibited in physiological bicarbonate buffer

The effects of bicarbonate buffer (HCO3-/CO2) on the activity of the two K+ channels proposed by some to control the pancreatic B-cell membrane response to glucose were studied. Single K+-channel records from membrane patches of cultured B-cells dissociated from adult rat islets exposed to a glucose- and bicarbonate-free medium (Na-Hepes in place of bicarbonate) exhibit the activity of both the ATP-sensitive as well as the [Ca2+]i-activated K+ channels. However, in the presence of bicarbonate-buffered Krebs solution, the activity of the ATP-sensitive K+ channel is inhibited leaving the activity of the K+ channel activated by intracellular [Ca2+]i unaffected. In the absence of bicarbonate (Hepes/NaOH in place of bicarbonate), lowering the external pH from 7.4 to 7.0 also has differential effects on the two K+ channels. While the K+ channel sensitive to ATP is inhibited, the K+ channel activated by a rise in [Ca2+]i is not affected. To determine whether the response of the B-cell in culture to bicarbonate is also present when the B-cell is functioning within the islet syncytium, the effects of bicarbonate removal on membrane potential of B-cells from intact mouse islets were compared. These studies showed that glucose-evoked electrical activity is also blocked in bicarbonate-free Krebs solution. Furthermore, in the absence of bicarbonate and presence of glucose (11 mM), electrical activity was recovered by lowering the pHo from 7.4 to 7.0. The ATP-sensitive K+-channel activity is greatly reduced by physiologically buffered solutions in pancreatic B-cells in culture. The most likely explanation for the bicarbonate effects is that they are mediated by cytosolic pH changes. Removal of bicarbonate (keeping the external pH at 7.4 with Hepes/NaOH as buffer) would increase the pHi. Since the activity of the [Ca2+]i-dependent K+ channels is not affected by the removal of the bicarbonate buffer, our patch-clamp data in cultured B-cells indicate an involvement of [Ca2+]i-activated K+ channels in the control of the membrane potential. For the B-cell in the islet, we propose that the burst pattern of electrical activity (Ca2+ entry) is controlled, at least in part, by the [Ca2+]i-activated K+ channel.     

Effect of monensin on receptor recycling during continuous endocytosis of asialoorosomucoid

The binding of asialoglycoproteins to their liver cell receptor results in internalization of the ligand-receptor complex. These complexes rapidly appear in intracellular compartments termed endosomes whose acidification results in ligand-receptor dissociation. Ligand and receptor subsequently segregate: ligand is transported to lysosomes and is degraded while receptor recycles to the cell surface. The proton ionophore monensin prevents acidification of endosomes and reversibly inhibits this acid-dependent dissociation of ligand from receptor. The present study determined the effect of monensin treatment of short-term cultured rat hepatocytes on cell-surface-receptor content, determined both by their binding activity and immunologically, following continuous endocytosis of asialoorosomucoid. Inclusion of 5 microM monensin in the incubation medium reduced the number of immunologically detectable cell-surface receptors by 20% in the absence of ligand. During continuous endocytosis of asialoorosomucoid, inclusion of monensin resulted in a 30-40% reduction of cell-surface receptor detectable either by ligand binding or immunologically. These results suggest that the reduced liver-cell-surface content of receptor in monensin is due to intracellular trapping of ligand-receptor complexes. The reduction of surface receptor during monensin incubation in the absence of ligand suggests that "constitutive recycling" of plasma membrane components also requires intracellular acidification.