An improved method to detect β- galactosidase activity in transgenic mice: a post-staining procedure on paraffin embedded tissue sections

The Escherichia coli -galactosidase gene is frequently used as a reporter gene in transgenic studies because its activity can be easily detected at the cellular level. Here we report a procedure for monitoring -galactosidase activity directly in tissue sections, which involves the use of a mixture of ethanol and poly-ethylene-glycol as a fixative (Kryofix) and a special paraffin characterized by a lower fusion point of 42 °C. After embedding and cutting, the sections are stained by the chromogenic substrate 5-bromo-4-chloro-3-indoyl--d galactopyranoside (X-Gal). This procedure allows both the retention of a high level of -galactosidase activity and the preservation of good tissue morphology. Furthermore, it can be combined with immunohistochemical methods to detect other cellular components without compromising reporter gene detection     

Testing for heterogeneity among phenotypic correlations: a comparison of methods using Monte Carlo simulations

Heterogeneous phenotypic correlations may be suggestive of underlying changes in genetic covariance among life-history, morphology, and behavioural traits, and their detection is therefore relevant to many biological studies. Two new statistical tests are proposed and their performances compared with existing methods. Of all tests considered, the existing approximate test of homogeneity of product-moment correlations provides the greatest power to detect heterogeneous correlations, when based on Hotelling's z*-transformation. The use of this transformation and test is recommended under conditions of bivariate normality. A new distribution-free randomisation test of homogeneity of Spearman's rank correlations is described and recommended for use when the bivariate samples are taken from populations with non-normal or unknown distributions. An alternative randomisation test of homogeneity of product-moment correlations is shown to be a useful compromise between the approximate tests and the randomisation tests on Spearman's rank correlations: it is not as sensitive to departures from normality as the approximate tests, but has greater power than the rank correlation test. An example is provided that shows how choice of test will have a considerable influence on the conclusions of a particular study. This revised version was published online in July 2006 with corrections to the Cover Date.     

Directional and anteroposterior asymmetry of common white markings in the legs of the Arabian horse: response to selection

Arabian bay horses manifest, on the average, more common white markings in their hind legs than their forelegs (anteroposterior asymmetry) and more common white markings in their left legs than their right legs (directional asymmetry). To determine if genetic variation exists for these types of asymmetry, the phenotypic response was studied in bay foals when their dams and sires were selected for the directions of fore-hind and left-right differences. In the fore-hind studies, the quantitative shifts in the bay foals were in the direction specified by the selection scheme and the observed deviations were all statistically significant. The shifts were also consistently in the direction favored by selection in the left-right studies, but only two of six observed deviations were statistically significant using a one-tailed test of significance. Thus, only marginal statistical evidence is available to support the observed consistent responses to selection in the left-right studies. These differential responses are reflected in the magnitudes of the heritability estimates. Based on the overall results, it is concluded that both types of asymmetry have a genetic basis in the Arabian horse, but much more genetic variation is present for anteroposterior asymmetry than for directional asymmetry. This revised version was published online in July 2006 with corrections to the Cover Date.     

A Cluster of Cuticle Protein Genes of Drosophila Melanogaster at 65a: Sequence, Structure and Evolution

A 36-kb genomic DNA segment of the Drosophila melanogaster genome containing 12 clustered cuticle genes has been mapped and partially sequenced. The cluster maps at 65A 5-6 on the left arm of the third chromosome, in agreement with the previously determined location of a putative cluster encompassing the genes for the third instar larval cuticle proteins LCP5, LCP6 and LCP8. This cluster is the largest cuticle gene cluster discovered to date and shows a number of surprising features that explain in part the genetic complexity of the LCP5, LCP6 and LCP8 loci. The genes encoding LCP5 and LCP8 are multiple copy genes and the presence of extensive similarity in their coding regions gives the first evidence for gene conversion in cuticle genes. In addition, five genes in the cluster are intronless. Four of these five have arisen by retroposition. The other genes in the cluster have a single intron located at an unusual location for insect cuticle genes.     

Responses of benthic macroinvertebrates in small intermittent streams to silvicultural practices

We examined macroinvertebrate communities in small(0.1-1.0 m²) pools of intermittent streams (alwayscontainingsome water but without perennial flow) with small watersheds(2-6 ha) subjected to five types of forest harvest to assesspotential impacts of the different harvest methods. Bufferstrips10 m wide were left on each side of the streams. Each harvesttreatment was coupled with a similar unharvested referencestand.An incomplete block design included three 0.05 m² vacuumsamples from each treatment paired with three from theadjacentreferences. There was a high degree of similarity amongreferencesfor parameters other than taxonomic composition (e.g.macroinvertebrate density, number of species, Shannondiversity,functional groups, etc.). Statistically significantdifferenceswere found between references and treatments and among harvestmethods but the responses varied among response variables(density,Shannon-Weiner diversity, species composition), differentspeciesassemblages (all invertebrates, chironomids,Ephemeroptera-Plecoptera-Trichoptera [EPT], isopods), andfunctional group categories (shredders, collector-gatherers).Wecollected 56 taxa, 7–16 per site, with low communitysimilarity(mean Jaccards=0.18, mean Bray-Curtis percentdissimilarity=81). The most severe harvest treatmentsresultedin the highest diversities of total invertebrates in thesesmallspring pool communities.     

Effects of asexual reproduction on the neighborhood area of cyclical parthenogens

The habitat occupied by a subpopulation and withinwhich there is random mating is known as itsneighborhood area. Neighborhood area is dependenton dispersal rates and organisms with low rates ofdispersal are expected to have small neighborhoodareas. In the absence of evolutionary forces,neighborhood areas under sexual reproduction will beconstant in size as long as dispersal patterns do notchange. This scenario differs when reproduction is bycyclical parthenogenesis since recombination anddispersal may occur in different generations. Ingeneral, dispersal distances increase with the numberof parthenogenetic generations. We show that cyclicalparthenogenesis increases neighborhood area which,concomitantly, decreases the potential for geneticsubdivision. It is noteworthy, however, that theincrease in neighborhood area is a decreasing functionof the number of parthenogenetic generations.This mechanism may have important implications for thepopulation structure of planktonic rotifers living ina horizontally undifferentiated habitat. In suchhabitats organisms are effectively unrestricted intheir lateral movements. Because rotifers typicallyhave low dispersal rates spatial geneticdiscontinuities may develop that divide the populationinto genetically distinct subpopulations. Counteringthis tendency is the increased neighborhood areaproduced by dispersal during the parthenogeneticphase. Thus cyclical parthenogenesis in organismslike rotifers may have important and previouslyunreported effects on the population's geneticstructure.     

3-hydroxy-3-methylglutaryl coenzyme A reductase of Sulfolobus solfataricus: DNA sequence, phylogeny, expression in Escherichia coli of the hmgA gene, and purification and kinetic characterization of the gene product.

The gene (hmgA) for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) from the thermophilic archaeon Sulfolobus solfataricus P2 was cloned and sequenced. S. solfataricus HMG-CoA reductase exhibited a high degree of sequence identity (47%) to the HMG-CoA reductase of the halophilic archaeon Haloferax volcanii. Phylogenetic analyses of HMG-CoA reductase protein sequences suggested that the two archaeal genes are distant homologs of eukaryotic genes. The only known bacterial HMG-CoA reductase, a strictly biodegradative enzyme from Pseudomonas mevalonii, is highly diverged from archaeal and eukaryotic HMG-CoA reductases. The S. solfataricus hmgA gene encodes a true biosynthetic HMG-CoA reductase. Expression of hmgA in Escherichia coli generated a protein that both converted HMG-CoA to mevalonate and cross-reacted with antibodies raised against rat liver HMG-CoA reductase. S. solfataricus HMG-CoA reductase was purified in 40% yield to a specific activity of 17.5 microU per mg at 50 degrees C by a sequence of steps that included heat treatment, ion-exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography. The final product was homogeneous, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The substrate was (S)- not (R)-HMG-CoA; the reductant was NADPH not NADH. The Km values for HMG-CoA (17 microM) and NADPH (23 microM) were similar in magnitude to those of other biosynthetic HMG-CoA reductases. Unlike other HMG-CoA reductases, the enzyme was stable at 90 degrees C and was optimally active at pH 5.5 and 85 degrees C.     

Neurotrophin-3–enhanced Nerve Regeneration Selectively Improves Recovery of Muscle Fibers Expressing Myosin Heavy Chains 2b

The purpose of this study was to evaluate the effect of neurotrophin 3 (NT-3) enhanced nerve regeneration on the reinnervation of a target muscle. Muscle fibers can be classified according to their mechanical properties and myosin heavy chain (MHC) isoform composition. MHC1 containing slow-type and MHC2a or 2b fast-type fibers are normally distributed in a mosaic pattern, their phenotype dictated by motor innervation. After denervation, all fibers switch to fast-type MHC2b expression and also undergo atrophy resulting in loss of muscle mass. After regeneration, discrimination between fast and slow fibers returns, but the distribution and fiber size change according to the level of reinnervation. In this study, rat gastrocnemius muscles (ipsilateral and contralateral to the side of nerve injury) were collected up to 8 mo after nerve repair, with or without local delivery of NT-3. The phenotype changes of MHC1, 2a, and 2b were analyzed by immunohistochemistry, and fiber type proportion, diameter, and grouping were assessed by computerized image analysis. At 8 mo, the local delivery of NT-3 resulted in significant improvement in gastrocnemius muscle weight compared with controls (NT-3 group 47%, controls 39% weight of contralateral normal muscle; P < 0.05). NT-3 delivery resulted in a significant increase in the proportion (NT-3 43.3%, controls 35.7%; P < 0.05) and diameter (NT-3 87.8 μm, controls 70.8 μm; P < 0.05) of fast type 2b fibers after reinnervation. This effect was specific to type 2b fibers; no normalization was seen in other fiber types.This study indicates that NT-3–enhanced axonal regeneration has a beneficial effect on the motor target organ. Also, NT-3 may be specifically affecting a subset of motoneurons that determine type 2b muscle fiber phenotype. As NT-3 was topically applied to cut nerves, our data suggest a discriminating effect of the neurotrophin on neuro–muscular interaction. These results would imply that muscle fibers may be differentially responsive to other neurotrophic factors and indicate the potential clinical role of NT-3 in the prevention of muscle atrophy after nerve injury.There has been much recent interest in the use of growth factors to augment peripheral nerve regeneration. A family of growth factors collectively known as the neurotrophins are now considered critical for the development, maintenance, and regeneration of the nervous system. The neurotrophin family includes NGF, brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3),¹ and neurotrophin-4/5 (NT-4/5) (Lewin and Barde, 1996; Lindsay, 1996). Little is known of their effect on regeneration of the peripheral nervous system.NT-3 has been shown to act on a subpopulation of muscle sensory neurons innervating muscle spindles and Golgi tendon organs, and there is also evidence of its effect on a subpopulation of cutaneous afferents (Ernfors et al., 1994; Tessarolo et al., 1994; Airaksinen et al., 1996). NT-3 has shown various effects on motor nerve regeneration, including differentiation of motoneurons from avian neural tube progenitor cells (Averbuch-Heller et al., 1994) and survival of neonatal and adult motoneurons in vitro (Hughes et al., 1993) and of neonatal motoneurons in vivo (Li et al., 1994; Vejsada et al., 1995), although the evidence is sometimes contradictory (Eriksson et al., 1994). In cocultures of adult muscle and embryonic motoneurons, NT-3 enhances the number and length of neurite outgrowths, the density of endplates per muscle fiber, and the amount of muscle innervation (Braun et al., 1996). NT-3 also plays a role in functional maturation of neuromuscular synapses (Lohoff et al., 1993; Wang et al., 1995) and regulates the cholinergic phenotype of developing motoneurons (Wong et al., 1993; Kato and Lindsay, 1994). NT-3 knockout mice show a loss of all muscle spindle afferent innervation and fusimotor neurons to the muscle but lose only few skeletomotor nerve fibers (Kucera et al., 1995a ). About 80% of adult motoneurons express the NT-3–specific trkC receptor (Henderson et al., 1993; Griesbeck et al., 1995), and NT-3 is the predominant neurotrophin expressed in skeletal muscle (Griesbeck et al., 1995). Furthermore, NT-3 is internalized and retrogradely transported from the periphery to motoneuron cell bodies (Di Stefano et al., 1992). Thus, there is experimental and circumstantial evidence to suggest that NT-3 may play a role in adult motoneurons, although in vivo data on the survival effect of NT-3 on adult motoneurons is still lacking. Furthermore, there is no evidence of an NT-3–dependent effect on neuro–muscular interaction.When a skeletal muscle is denervated and subsequently reinnervated, a characteristic sequence of events ensues. The muscle rapidly loses weight as the muscle fibers atrophy (Pellegrino and Franzini, 1963), but after reinnervation, it gradually recovers mass to a variable extent, depending upon the degree of reinnervation (Bertelli and Mira, 1995) and correlating with the maximum force of contraction (Gillespie et al., 1987). The fibers within an individual skeletal muscle do not exist as a homogenous population but can be classified according to their different metabolic and contractile properties (Burke et al., 1971; Peter et al., 1972). Also, they can be identified morphologically according to differential expression of specific myosin heavy chain isoforms. Slow, oxidative type 1 muscle fibers contain myosin heavy chain 1 (MHC 1), fast oxidative glycolytic type 2a fibers contain myosin heavy chain 2a (MHC 2a), while fast glycolytic type 2b fibers contain myosin heavy chain 2b (MHC 2b) (Bar and Pette, 1988). Muscle fiber phenotype is conferred by its innervation, and changes of neuro–muscular interaction lead to alteration of muscle fiber phenotype (Romanul and Van der Meulen, 1966; Fex and Sonneson, 1970; Salmons and Sreter, 1975). The relative proportions of fiber types vary with age, sex, strain, species, and muscle type (Maltin et al., 1989). Generally, there is a high proportion of type 1 fibers in postural muscles (e.g., soleus) and of type 2 fibers in fast muscles (e.g., extensor digitorum longus), while in mixed muscles (e.g., gastrocnemius) there are varying proportions of each type. Muscle fiber type proportion also varies dynamically with physiological and pathological parameters (Jansson et al., 1978; Green et al., 1983; Izumo et al., 1986; Goldspink et al., 1992; Pette and Vrbova, 1992). For example, the distribution of fiber types in normal muscle is dispersed in a “mosaic pattern,” but after denervation and reinnervation of the muscle there is a shift to grouping (Karpati and Engel, 1968). Also, there is a change in the proportions of fiber types, and in the rat the majority of fibers become initially fast with denervation, with subsequent fiber specialization being dictated by patterns of reinnervation (Fields and Ellisman, 1986). This plastic nature of muscle makes it an interesting model to investigate reinnervation changes that may occur after NT-3 administration.We have recently demonstrated that the local delivery of NT-3 to rat sciatic nerve enhances the rate and amount of nerve regeneration, and at 8 mo postoperative, there was a 40% increase in the myelinated fiber count (Sterne et al., 1997). However, enhanced regeneration by itself is not necessarily indicative of a beneficial effect on the target muscles, such as reacquisition of more normal physiological function. Therefore, the aim of this study was to assess whether NT-3–enhanced nerve regeneration resulted in biochemical or morphological changes in a target muscle (gastrocnemius), which would be suggestive of significant improvement of physiological function above that seen after nerve repair without administration of neurotrophin. Immunohistochemistry, in conjunction with computerized quantification and morphometrical analysis, was used to analyze the number, size, and pattern of distribution of the MHC fiber types after denervation and reinnervation of the gastrocnemius muscle.     

Role for a Glycan Phosphoinositol Anchor in Fcγ Receptor Synergy

While many cell types express receptors for the Fc domain of IgG (FcγR), only primate polymorphonuclear neutrophils (PMN) express an FcγR linked to the membrane via a glycan phosphoinositol (GPI) anchor. Previous studies have demonstrated that this GPI-linked FcγR (FcγRIIIB) cooperates with the transmembrane FcγR (FcγRIIA) to mediate many of the functional effects of immune complex binding. To determine the role of the GPI anchor in Fcγ receptor synergy, we have developed a model system in Jurkat T cells, which lack endogenously expressed Fcγ receptors. Jurkat T cells were stably transfected with cDNA encoding FcγRIIA and/or FcγRIIIB. Cocrosslinking the two receptors produced a synergistic rise in intracytoplasmic calcium ([Ca²⁺]_i) to levels not reached by stimulation of either FcγRIIA or FcγRIIIB alone. Synergy was achieved by prolonged entry of extracellular Ca²⁺. Cocrosslinking FcγRIIA with CD59 or CD48, two other GPI-linked proteins on Jurkat T cells also led to a synergistic [Ca²⁺]_i rise, as did crosslinking CD59 with FcγRIIA on PMN, suggesting that interactions between the extracellular domains of the two Fcγ receptors are not required for synergy. Replacement of the GPI anchor of FcγRIIIB with a transmembrane anchor abolished synergy. In addition, tyrosine to phenylalanine substitutions in the immunoreceptor tyrosine-based activation motif (ITAM) of the FcγRIIA cytoplasmic tail abolished synergy. While the ITAM of FcγRIIA was required for the increase in [Ca²⁺]_i, tyrosine phosphorylation of crosslinked FcγRIIA was diminished when cocrosslinked with FcγRIIIB. These data demonstrate that FcγRIIA association with GPI-linked proteins facilitates FcγR signal transduction and suggest that this may be a physiologically significant role for the unusual GPI-anchored FcγR of human PMN.