Dynamics of the immune system response in cerebrospinal fluid and blood of SIVmac-infected rhesus monkeys

Paired sera and CSF samples were collected from SIV_mac-infected macaques. Animals infected with SIV_mac251 maintained low gag and high env-specific antibody levels in plasma. Increasing env-specific antibody titers in CSF were associated in one animal with strong intrathecal synthesis. SIV_mac239-infected monkeys revealed high antibody titers of gag and env-specificity, in one animal accompanied by weak intrathecal synthesis of virus-specific antibodies. In all animals, the CD4/CD8 ratio in CSF decreased faster compared to blood.     

A comparison of three packaging techniques using two extenders for the cryopreservation of canine semen

The progressive motility of frozen-thawed canine semen was used as a criterion to compare methods of semen cryopreservation. Twenty-one ejaculates from 7 dogs were frozen in 2 extenders, Tris-citrate (TC) and BES-lactose (BL), in each of 3 packaging techniques (pellets, 0.5-ml, and 2.5-ml straws). Duplicate samples were frozen on dry ice (pellets) or in liquid nitrogen vapor (straws). Least squares means for the percentage of post-thaw progressive motility (PTPM) were greater for TC (33.2 ± 1.7) than for BL (20.9 ± 1.7; P<0.0001). Freezing in pellets (PTPM = 34 ± 2.3) resulted in greater PTPM than freezing in either 0.5-ml (24.7 ± 1.6) or 2.5-ml (22.1 ± 2.3) straws (P<0.001). The percentage of PTPM of spermatozoa frozen in TC pellets was greater than that in TC 2.5-ml straws or in BL in any packaging method (P<0.05). The percentage of PTPM of spermatozoa from semen extended in BL was greater in pellets than in 0.5 or 2.5-ml straws (P<0.05).     

Neuronal responses to purinoceptor agonists in the leech central nervous system

Exracellular nucleotides like ATP and its derivatives are possible chemical messengers in vertebrate nervous systems. In invertebrate nervous systems, however, little is known about their role in neurotransmission. We have studied the reponse of identified neurones of the leech Hirudo medicinalis to the purinoceptor agonist ATP, ADP, AMP, and adenosine using conventional intracellular microelectrodes and whole-cell patch-clamp recording. Bath application of the agoinsts depolarized the different neurons, but not neuropil glial cells. The most effective responses (up to 10 mV) were observed with ATP (100 μM) or ADP (100 μM) in the noxious and touch cells. In most neurons the nonhydrolyzable ATP derivative ATP-γ-S (5 μM) induced larger depolarizations that 100 μM ATP, indicating that most of the potency of ATP is lost presumably due to its degradation by ectonucleotidases. In medial noxios cells, ATP (100 μM) induced an inward current of 1.7 ± 1.1 nA at a holding potential of ?60 mV. The ATP-induced current-voltage relationship showed an inward rectification and a reversal potential close to 0 m V. In a Na⁺-free extracellular solution, the ATP-induced inward current decreased and in a Na⁺- and Ca²⁺-free saline only a small residual current persisted. The possible P₂ purinoceptor antagonist suramin did not antagonize the ATP-induced current, but itself evoked an inward current and a conductance increase. We conclude that ATP activates nonselective cation channels in medial noxious cells of the leech with the order of potency of purinoceptor agonists ATP ≥ ADP > AMP. The results suggest that these cells express purinoceptors of the P₂ type. 1994 John Wiley & Sons, Inc.     

Comparative efficacy of amphotericin B,clotrimazole and itraconazole againstAspergillus spp.

The susceptibilities of two isolates ofAspergillus flavus, one from a human case of recalcitrant mycotic keratitis, and an environmental isolate ofA. fumigatus, to itraconazole, clotrimazole and amphotericin B were measured. Observations of macroscopic growth and microscopic evaluations of conidia germination both indicated that the two isolates ofA. flavus were markedly more resistant to amphotericin B than to itraconazole and clotrimazole. Itraconazole was more effective than clotrimazole for all isolates. Ourin vitro susceptibility results suggest the use of itraconazole should be a primary consideration in the treatment ofAspergillus keratitis.     

A strategy for the characterization of minute chromosome rearrangements using multiple color fluorescence in situ hybridization with chromosome-specific DNA libraries and YAC clones

The identification of marker chromosomes in clinical and tumor cytogenetics by chromosome banding analysis can create problems. In this study, we present a strategy to define minute chromosomal rearrangements by multicolor fluorescence in situ hybridization (FISH) with whole chromosome painting probes derived from chromosome-specific DNA libraries and Alu-polymerase chain reaction (PCR) products of various region-specific yeast artificial chromosome (YAC) clones. To demonstrate the usefulness of this strategy for the characterization of chromosome rearrangements unidentifiable by banding techniques, an 8p+ marker chromosome with two extra bands present in the karyotype of a child with multiple anomalies, malformations, and severe mental retardation was investigated. A series of seven-color FISH experiments with sets of fluorochrome-labeled DNA library probes from flow-sorted chromosomes demonstrated that the additional segment on 8p+ was derived from chromosome 6. For a more detailed characterization of the marker chromosome, three-color FISH experiments with library probes specific to chromosomes 6 and 8 were performed in combination with newly established telomeric and subtelomeric YAC clones from 6q25, 6p23, and 8p23. These experiments demonstrated a trisomy 6pter6p22 and a monosomy 8pter8p23 in the patient. The present limitations for a broad application of this strategy and its possible improvements are discussed.Dedicated to Professor Dr. U. Wolf on the occasion of his 60th birthday     

Faster induction of photosynthesis increases biomass and grain yield in glasshouse-grown transgenic Sorghum bicolor overexpressing Rieske FeS

Sorghum is one of the most important crops providing food and feed in many of the world's harsher environments. Sorghum utilizes the C₄ pathway of photosynthesis in which a biochemical carbon-concentrating mechanism results in high CO₂ assimilation rates. Overexpressing the Rieske FeS subunit of the Cytochrome b₆f complex was previously shown to increase the rate of photosynthetic electron transport and stimulate CO₂ assimilation in the model C₄ plant Setaria viridis. To test whether productivity of C₄ crops could be improved by Rieske overexpression, we created transgenic Sorghum bicolor Tx430 plants with increased Rieske content. The transgenic plants showed no marked changes in abundances of other photosynthetic proteins or chlorophyll content. The steady-state rates of electron transport and CO₂ assimilation did not differ between the plants with increased Rieske abundance and control plants, suggesting that Cytochrome b₆f is not the only factor limiting electron transport in sorghum at high light and high CO₂. However, faster responses of non-photochemical quenching as well as an elevated quantum yield of Photosystem II and an increased CO₂ assimilation rate were observed from the plants overexpressing Rieske during the photosynthetic induction, a process of activation of photosynthesis upon the dark–light transition. As a consequence, sorghum with increased Rieske content produced more biomass and grain when grown in glasshouse conditions. Our results indicate that increasing Rieske content has potential to boost productivity of sorghum and other C₄ crops by improving the efficiency of light utilization and conversion to biomass through the faster induction of photosynthesis.     

Protective immunity and granulomatous inflammation is mediated in vivo by T cells reactive to epitopes common to avirulent and listeriolysin-negative mutants of Listeria monocytogenes.

The ability of several listeriolysin O-negative mutants of the EGD and NCTC 7973 strains of Listeria monocytogenes to activate specific T cell responses in vitro and in vivo was determined. T cell lines from different inbred mouse strains and derived T cell clones elicited by L. monocytogenes, strain EGD, which are able to adoptively transfer protection and granuloma formation were examined. Specificity testing revealed no differences between listeriolysin-positive and -negative strains to induce proliferation of the T cell lines and clones. Similar results were obtained when we examined CD4+ T cell-mediated granuloma formation in the livers of mice previously immunized with viable bacteria of the virulent strain. Granulomatous inflammation could be elicited by iv application of heat-killed bacteria of listeriolysin-positive and of -negative bacteria. Protective immunity to listerial infections and granulomatous inflammation therefore appears to be mediated by T cells recognizing epitopes on listerial antigens that are shared by both pathogenic and nonpathogenic Listeria strains.     

An antibody Fab selected from a recombinant phage display library detects deesterified pectic polysaccharide rhamnogalacturonan II in plant cells.

Rhamnogalacturonan II (RG-II) is a structurally complex, low molecular weight pectic polysaccharide that is released from primary cell walls of higher plants by treatment with endopolygalacturonase and is chromatographically purified after alkaline deesterification. A recombinant monovalent antibody fragment (Fab) that specifically recognizes RG-II has been obtained by selection from a phage display library of mouse immunoglobulin genes. By itself, RG-II is not immunogenic. Therefore, mice were immunized with a neoglycoprotein prepared by covalent attachment of RG-II to modified BSA. A cDNA library of the mouse IgG1/kappa antibody repertoire was constructed in the phage display vector pComb3. Selection of antigen-binding phage particles resulted in the isolation of an antibody Fab, CCRC-R1, that binds alkali-treated RG-II with high specificity. CCRC-R1 binds an epitope found primarily at sites proximal to the plasma membrane of suspension-cultured sycamore maple cells. In cells deesterified by alkali, CCRC-R1 labels the entire wall, suggesting that the RG-II epitope recognized by CCRC-R1 is masked by esterification in most of the wall and tha such RG-II esterification is absent near the plasma membrane.     

Targeting foreign proteins to human immunodeficiency virus particles via fusion with Vpr and Vpx.

The human immunodeficiency virus type 1 (HIV-1) and HIV-2 Vpr and Vpx proteins are packaged into virions through virus type-specific interactions with the Gag polyprotein precursor. To examine whether HIV-1 Vpr (Vpr1) and HIV-2 Vpx (Vpx2) could be used to target foreign proteins to the HIV particle, their open reading frames were fused in frame with genes encoding the bacterial staphylococcal nuclease (SN), an enzymatically inactive mutant of SN (SN*), and chloramphenicol acetyltransferase (CAT). Transient expression in a T7-based vaccinia virus system demonstrated the synthesis of appropriately sized Vpr1-SN/SN* and Vpx2-SN/SN* fusion proteins which, when coexpressed with their cognate p55Gag protein, were efficiently incorporated into virus-like particles. Packaging of the fusion proteins was dependent on virus type-specific determinants, as previously seen with wild-type Vpr and Vpx proteins. Particle-associated Vpr1-SN and Vpx2-SN fusion proteins were enzymatically active, as determined by in vitro digestion of lambda phage DNA. To determine whether functional Vpr1 and Vpx2 fusion proteins could be targeted to HIV particles, the gene fusions were cloned into an HIV-2 long terminal repeat/Rev response element-regulated expression vector and cotransfected with wild-type HIV-1 and HIV-2 proviruses. Western blot (immunoblot) analysis of sucrose gradient-purified virions revealed that both Vpr1 and Vpx2 fusion proteins were efficiently packaged regardless of whether SN, SN*, or CAT was used as the C-terminal fusion partner. Moreover, the fusion proteins remained enzymatically active and were packaged in the presence of wild-type Vpr and Vpx proteins. Interestingly, virions also contained smaller proteins that reacted with antibodies specific for the accessory proteins as well as SN and CAT fusion partners. Since similar proteins were absent from Gag-derived virus-like particles and from virions propagated in the presence of an HIV protease inhibitor, they must represent cleavage products produced by the viral protease. Taken together, these results demonstrate that Vpr and Vpx can be used to target functional proteins, including potentially deleterious enzymes, to the human or simian immunodeficiency virus particle. These properties may be exploitable for studies of HIV particle assembly and maturation and for the development of novel antiviral strategies.