Tracking Members of the Simian Immunodeficiency Virus deltaB670 Quasispecies Population In Vivo at Single-Cell Resolution

Genetically distinct lentiviruses constitute a quasispecies population that can evolve in response to selective forces. To move beyond characterization of the population as a whole to the behavior of individual members, we devised an in situ hybridization approach that uses genotype-specific probes. We used probes that detect simian immunodeficiency viruses (SIV) that differ in sequence in the V1 region of the surface envelope glycoprotein (env) gene to investigate the replication and cellular tropisms of four viral variants in the tissues of infected rhesus macaques. We found that the V1 genotypic variants replicated in spatially defined patterns and to different extents at each anatomic site. The two variants that replicated most extensively in animals with AIDS were detected in both macrophages and T lymphocytes in tissues. By extension of this approach, it will be possible to investigate the role of individual lentiviruses in a quasispecies in pathogenesis and to evaluate the effects of antiviral or immunotherapeutic treatment on select members of a quasispecies.     

Habituating pigs for in-pen, non-invasive biophysical skin analysis.

The purpose of this study was to develop a method for habituating pigs (Sus scrofa domestica, middle white strain) to enable non-invasive, biophysical measurements of dorsal skin to be obtained on a daily basis over a 7-week period, thus eliminating the need for anaesthesia or restraint. This was accomplished by associating measurements of transepidermal water loss (TEWL) and skin reflectance spectroscopy (SRS) with feeding times, and with positive reinforcement by allowing exercise outside the home pen. During the pig habituation period, a well-defined series of behavioural changes were observed that included dominant/ submissive leadership changes. Values of TEWL (6.29 +/- 1.25g.m(-2) x h(-1)) were in agreement with previous studies (7.56+/-2.90g.m 2 x h(-1)) obtained from unrestrained Yucatan hairless micro-pigs (Gabard et al. 1995). The coefficient of variance of TEWL and SRS measurements were comparable with those reported previously using anaesthetized pigs (Chilcott et al. 2000). These data imply that biophysical skin measurements obtained from unrestrained, conscious animals are comparable to those obtained from anaesthetized pigs and therefore, support the use of unrestrained pigs for non-invasive biophysical skin measurements. Habituating animals for in-pen, non-invasive, biophysical measurements has substantial implications for reducing and refining laboratory animal experiments in dermatological research without compromising animal welfare.     

Molecular Analysis of the Gal/GalNAc Adhesin of Entamoeba histolytica1

Attachment of Entamoeba histolytica to colonic epithelium and a variety of other target cells is mediated by a galactosc/N-acetyl D-galactosamine (Gal/GalNAc) inhibitable adhesin. Seven monoclonal antibodies specific for nonoverlapping epitopes of the 170 kDa subunit have been shown to have distinct effects on adherence. Four of these monoclonal antibodies inhibit or have no effect on amebic adherence while two others enhance amebic adherence. The epitopes recognized by these seven monoclonal antibodies have been mapped to the extracellular cysteine rich region of the 170 kDa subunit. The conformational nature of the epitopes was examined by testing monoclonal antibody reactivity with isolated regions of the 170 kDa subunit expressed as fusion proteins in E. coli and also with denatured native adhesin. These analyses suggested that three of monoclonal antibodies recognized conformational epitopes while the remaining four recognized linear epitopes. The mapping of these monoclonal antibodies have identified functionally important regions of the Gal/GalNAc adhesin and have also shown that recombinant Gal/GalNAc adhesin, when expressed in E. coli, retained at least some of its native conformation.     

Direct evidence for suppression of recombination within two pericentric inversions in humans: a new sperm-FISH technique.

Crossover within a pericentric inversion produces reciprocal recombinant chromosomes that are duplicated/deficient for all chromatin distal to the breakpoints. In view of this fact, a new technique is presented for estimating the frequency of recombination within pericentric inversions. YAC probes were selected from within the q- and p-arm flanking regions of two human inversions, and two-color FISH analysis was performed on sperm from heterozygous inversion carriers. A total of 6,006 sperm were analyzed for chromosome 1 inversion (p31q12), and 3,168 were analyzed for chromosome 8 inversion (p23q22). Both inversions displayed suppression of crossing-over, although the amount of suppression differed between the two inversions. The recombination frequency of 13.1% recorded for chromosome 8 inversion was similar to the frequency of 11.4% previously estimated by the human/hamster-fusion method. For chromosome 1 inversion, the recombination frequency of 0. 4% reported here was below the limits of detection of the fusion technique. The simplicity of the FISH technique and the ease of scoring facilitate analysis of a sample-population size much larger than previously had been possible.     

Lattice spacing changes accompanying isometric tension development in intact single muscle fibers.

The myosin lattice spacing of single intact muscle fibers of the frog, Rana temporaria, was studied in Ringer's solution (standard osmolarity 230 mOsm) and hyper- and hypotonic salines (1.4 and 0.8 times standard osmolarity respectively) in the relaxed state, during "fixed end" tetani, and during shortening, using synchrotron radiation. At standard tonicity, a tetanus was associated with an initial brief lattice expansion (and a small amount of sarcomere shortening), followed by a slow compression (unaccompanied by sarcomere length changes). In hypertonic saline (myosin lattice compressed by 8.1%), these spacing changes were suppressed, in hypotonic saline (lattice spacing increased by 7.5%), they were enhanced. During unloaded shortening of activated fibers, a rapid lattice expansion occurred at all tonicities, but became larger as tonicity was reduced. This expansion was caused in part by the change in length of the preparation, but also by a recoil of a stressed radial compliance associated with axial force. The lattice spacing during unloaded shortening was equal to or occasionally greater than predicted for a relaxed fiber at that sarcomere length, indicating that the lattice compression associated with activation is rapidly reversed upon loss of axial force. Lattice recompression occurred upon termination of shortening under standard and hypotonic conditions, but was almost absent under hypertonic conditions. These observations indicate that axial cross-bridge tension is associated with a compressive radial force in intact muscle fibers at full overlap; however, this radial force exhibits a much greater sensitivity to lattice spacing than does the axial force.     

A comparison of the mouth morphology of three co-occurring species of atherinid

The mouth morphology of three species of atherinids, which feed at different levels in the water column (benthos, plankton and water surface) were compared. These three species, which all grow to less than 100 mm in length, inhabit the shallows (<2m) of Wilson Inlet, a temperate south-western Australian estuary. The species could be distinguished primarily on the basis of the extent to which they can protrude their jaws. Thus, whereas Leptatherina presbyteroides feeds highest in the water column, including at the water surface on terrestrial insects, and has the most protrusible jaws, Atherinosonia elongata feeds predominantly at or near the benthos and has the least protrusible jaws. Leptatherina wallacei , which ingests prey from the plankton and near the benthos, is intermediate in the degree to which it can protrude its jaws. Other characters of the three species which are associated with feeding, such as the number of gill rakers and the size of teeth, show consistent trends with the degree of jaw protrusion in relation to the type of prey consumed.     

Structural features of a multisubstate cardiac mitoplast anion channel: Inferences from single channel recording

Ion channels from sheep cardiac mitoplast (inverted inner mitochondrial membrane vesicle) preparations were incorporated into voltage-clamped planar lipid bilayers. A low-conductance anion channel (～40 or ～85 pS in symmetric 300 or 550 mM choline Cl, respectively), characterized by the presence of two well-defined substates, at ～25 and ～50% of the fully open level, was studied in detail. The substate behavior was consistent with a multibarrelled channel containing four functionally coupled pores. At negative (cis-trans) membrane potentials, the putative portomers appeared to gate with substantial positive cooperativity, accounting for the apparent absence of a ～75% sublevel. At positive holding potentials, allosteric protomer interactions were more complicated, and the channel complex could be modeled as a dimer of dimers. The protochannels in one dimer (“dimer A”) appeared to open independently of each other, and with a relatively high probability, while the monomers comprising the second dimer (“dimer B”) were functionally coupled, could only open if both protomers in dimer A were open, and closed as soon as one of the monomers in dimer A shut. The channels also displayed Ca²⁺- (and Mg²⁺-) sensitive rectification related to bilayer lipid surface charge. By assuming that Ca²⁺ acted solely by screening surface charge, the membrane surface potential profile was used as a “microscopic ruler” to place one mouth of the channel within 10–11 Å of the bilayer surface.