Genetic load caused by variation in the amount of rDNA in a wasp

Extensive variation in the size of the short (heterochromatic) arm of chromosome 14 was found in the wasp Trypoxylon (Trypargilum) albitarse. Ten different variants were differentiated by size and C-banding pattern. Fluorescent in-situ hybridization (FISH) revealed that ribosomal DNA in this species is clustered in the darkly C-banded parts of the heterochromatic short arm of chromosome 14. On this basis, we got an indirect estimate of the amount of rDNA from the area of these dark C-bands. The significant absence in males of the three chromosome variants with lower amounts of rDNA indicates that these three variants are lethal in this sex, and suggests the existence of a threshold marking the minimum amount of rDNA which is tolerable in haploidy. This implies about 4% genetic load in the population caused by variation in rDNA amount. This revised version was published online in July 2006 with corrections to the Cover Date.     

Short-term pharmacologically induced growth study of ontogenetic allometry of oxygen uptake in children

BACKGROUND: A range of allometric coefficients have been proposed in describing the maximal oxygen uptake (VO2max): body mass relation in children using weight-bearing ergometry. However, a wide deviation in the allometric coefficients for VO2max may be apparent when selected pediatric cohorts are studied in conjunction with clinical intervention for growth abnormalities. AIM: The purpose of this study was to determine the allometric coefficients for VO2max after short-term pharmacologically induced growth in pre- and early pubescent children. SUBJECTS AND METHODS: The treatment group consisted of nine subjects with non-growth hormone (GH)-deficient short stature and one with GH-deficient short stature (mean age: 13.7+/-1.7 years). Ten pre- and early pubescent children matched for age, height, weight, VO2max and body mass index (BMI) were controls. The treatment group were evaluated before (Pre-GH) and after (Post-GH) 4 months of subcutaneous GH therapy (0.05 mgkg(-1)day(-1) x 6 days week(-1)). RESULTS: The mean ontogenetic coefficient for the treatment group was 1.50+/-0.20 and for the control group was 0.77+/-0.34. The mean allometric coefficient for body mass relative to VO2max was significantly higher in the treatment group compared with the control group (p<0.05). Height, weight, fat free mass (FFM), VO2max indexed to body mass (mLkg(-1)min(-1)) and FFM (mLkgFFM(-1)min(-1)) increased (p<0.05) with GH therapy. GH therapy also increased insulin-like growth factor-I (IGF-I) and served as a biochemical marker of GH therapy (p<0.05). The control group had no significant differences in all the variables tested (p<0.05). CONCLUSION: The scaling for oxygen uptake (VO2) for body mass varies with GH treatment and the increase in VO2max that commonly occurs in conjunction with physical growth in the pre-and early pubescent individual may be linked to an increase in FFM and linear size.     

Solubilization and immunological identification of presynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors in the rat hippocampus

Alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors have been identified mostly as postsynaptic receptors mediating fast glutamatergic synaptic transmission. However, neurochemical studies based on the modulation of neurotransmitter release have suggested the existence of presynaptic AMPA receptors. We have used a recently described technique that allows a high-purity fractionation of the pre- and postsynaptic proteins of synaptic junctions to evaluate the distribution of the different AMPA receptor subunits in rat hippocampal synapses. Surprisingly, we found very high levels of GluR1- and GluR2/3-like immunoreactivity in the presynaptic fraction, but also in the postsynaptic and extrasynaptic fractions. GluR4-like immunoreactivity was much less abundant but was still detected, predominantly in the postsynaptic fraction. This methodology appears to be far more sensitive than the classical immunogold electron microscopy to determine the localization of synaptic receptors.     

Characterization of Paracoccidioides brasiliensis isolates by random amplified polymorphic DNA analysis.

We initially used 25 different random primers in order to test their ability to generate random amplified polymorphic DNA fragments from the dimorphic human pathogenic fungus Paracoccidioides brasiliensis. From the tested primers we chose five to distinguish between seven isolates of this microorganism. The DNA amplification patterns allowed clear differentiation of the seven isolates into two distinct groups with only 35% genomic identity. One of these groups contained two subgroups with 81% genetic similarity. The random amplified polymorphic DNA analysis method proved to be a good tool for analyzing and comparing different genomes of P. brasiliensis isolates.     

Metacyclogenesis is a major determinant of Leishmania promastigote virulence and attenuation.

The in vivo virulence patterns of promastigote populations defined on the basis of agglutination by the lectin peanut agglutinin (PNA) were studied for various cloned lines of Leishmania major. Promastigotes derived from logarithmic-phase cultures, which were routinely 100% agglutinated at 100 micrograms of PNA per ml, were relatively avirulent for BALB/c mice. The relative virulence of stationary-phase promastigotes appeared to be attributable to the proportion of nonagglutinable (PNA-) promastigotes contained within these populations. Purification of PNA- organisms from stationary cultures provided for each clone the most virulent inoculum, supporting the view that this change in lectin binding accurately reflects the development of infective metacyclic stage promastigotes. By studying this marker, we found that there was considerable variation in the degree to which different strains and clones underwent metacyclogenesis during growth. Examination of a reportedly avirulent L. major clone revealed that metacyclogenesis was unusually delayed and inefficient for this clone, but that those PNA- promastigotes which could be recovered from late-stationary-phase cultures were virulent for BALB/c mice. The loss of virulence associated with frequent subculture could also be attributed to a drastic diminution in metacyclogenesis potential over time. A clone which yielded over 90% PNA- promastigotes during growth within passage 1 generated fewer than 10% PNA- promastigotes during growth by passage 94. Subcloning of late-passage attenuated promastigotes yielded a clone for which no PNA- promastigotes could be generated during growth, and an infective population could not be derived from this clone. Thus, metacyclogenesis does not appear to be stable for even cloned lines of Leishmania promastigotes, and virulence comparisons between different strains and clones can be meaningfully made only if the metacyclic populations contained within the respective inocula are determined.     

Molecular cloning and characterization of actin genes from Echinococcus granulosus

An Echinococcus granulosus genomic library has been screened with a mouse β-actin cDNA probe. Two clones carrying DNA fragments of about 15 kb, possibly derived from the same genome region, have been isolated. This 15-kb genomic region includes 2 actin-related sequences (EgactI and EgactII) separated by about 4 kb. The nucleotide sequences of both genes were determined. The EgactI sequence presents no introns, but an intron of 591 bp was observed in the EgactII sequence. The genes potentially encode 375 and 376 amino-acid-long actins, respectively, with a homology of 85.3%. The deduced amino acid sequences from both genes were compared to the actin sequences from other organisms, showing similarities ranging from 63.5% to 90.6%. The nucleotide sequence of a partial actin cDNA clone has been determined. The deduced amino acids sequence showed a homology of 90.3% and 88.0% in relation to the EgactI and EgactII sequences respectively, suggesting the existence of at least one more actin gene in E. granulosus. This hypothesis is reinforced by the number of bands detected in the Southern blot analysis. Experiments based on the amplification of DNA segments using 3′-specific actin primers indicate that the EgactI gene is transcribed in protoscoleces.     

Nucleotide sequence and in vitro expression of the capsid protein gene of tobacco ringspot virus

The nucleotide sequence of the 3' terminal 2022 nucleotides (nt) of tobacco ringspot virus (TobRV) RNA 2 has been determined. Protein microsequence analysis of the amino-terminal residues of purified capsid protein localized the capsid protein gene between nt 2014 and 583 (from the 3' terminus) of this sequence. The proteolytic cleavage site that is processed to liberate the capsid protein from the RNA 2-encoded polyprotein was identified as Cys-Ala. The predicted translation product from the gene is a 477 amino acid long polypeptide with a calculated MW of 53 kDa. The gene was modified at the 5' end to facilitate sub-cloning, and to provide it with a methionine initiation codon. The modified gene was sub-cloned, transcribed in vitro and expressed in a rabbit reticulocyte lysate translation system, where it directed the synthesis of a 53 kDa polypeptide. Garnier-Osguthorpe-Robson analyses of the secondary structure of the capsid protein predicted the presence of three beta sheet domains, which suggests that this nepovirus capsid may be structurally analogous to those of the como- and picornaviruses. These and other results from computer analyses of the nucleic acid and amino acid sequences, and comparisons with the capsid proteins of nepoviruses and other related viruses are discussed.     

Haptic selective attention by foot and by hand

Nonvisual perceptions of a wielded object's spatial properties are based on the quantities expressing the object's mass distribution, quantities that are invariant during the wielding. The mechanoreceptors underlying the kind of haptic perception involved in wielding – referred to as effortful, kinesthetic, or dynamic touch – are those embedded in the muscles, tendons, and ligaments. The present experiment's focus was the selectivity of this muscle-based form of haptic perception. For an occluded rod grasped by the hand at some intermediate position along its length, participants can attend to and report selectively the rod's full length, its partial lengths (fore or aft of the hand), and the position of the grip. The present experiment evaluated whether participants could similarly attend selectively when wielding by foot. For a given rod attached to and wielded by foot or attached to (i.e. grasped) and wielded by hand, participants reported (by magnitude production) the rod's whole length or fractional length leftward of the point of attachment. On measures of mean perceived length, accuracy, and reliability, the degree of differentiation of partial from full extent achieved by means of the foot matched that achieved by means of the hand. Despite their neural, anatomical, and experiential differences, the lower and upper limbs seem to abide by the same principles of selective muscle-based perception and seem to express this perceptual function with equal facility.     

Mycobacteria and human autoimmune disease: direct evidence of cross-reactivity between human lactoferrin and the 65-kilodalton protein of tubercle and leprosy bacilli.

We document here by Western immunoblotting and immunogold ultracytochemistry that polyclonal antibodies against human lactoferrin (Lf) bind to tubercle and leprosy bacilli. In situ immunogold labeling of Mycobacterium leprae (present in armadillo liver and in human skin) and of Mycobacterium tuberculosis indicated that receptors for anti-Lf antibodies were present both on the cytoplasm and on the envelope of the bacilli. We found by immunoblotting that the 65-kDa heat shock protein is the major component of M. leprae and M. tuberculosis that is responsible for the binding of the anti-Lf probe. Furthermore, we show that anti-Lf immunoglobulin G eluted from the nitrocellulose-transferred mycobacterial 65-kDa protein band did bind back to Lf. Ultracytochemistry of biopsy samples of human lepromas showed that dead or severely damaged M. leprae was strongly marked by the anti-Lf antibodies; a similar pattern of immunogold marking was observed on M. leprae when antibodies against the 65-kDa mycobacterial protein were used. Our results offer direct evidence that the 65-kDa protein of leprosy and tubercle bacilli is recognized with specificity by antibodies against the human protein Lf. The Lf-65-kDa protein antigenic cross-reactivity may contribute to the formation of autoantibodies and immune complexes as well as to other autoimmune events that are frequent in tuberculosis and leprosy. Our immunocytochemical data also suggest that the cross-reactivity may persist for some time after the death of mycobacteria in infected hosts.     

Intermediate- and high-affinity interleukin-2 receptors expressed in an IL-4-dependent T-cell line induce different signals.

We have previously described a synergism between the two physiological hormones, retinoic acid (RA) and 1 alpha,25-dihydroxyvitamin D3 (VD) in the induction of U937 cell differentiation towards a more mature state. Herein, we investigated the regulation of cytokine production during RA and/or VD treatment of U937 cells. Cell differentiation was followed by measurement of their capacity to give oxidative responses, and interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF-alpha) and IL-6 gene and protein expression were determined in RA/VD-treated cells, activated or not with lipopolysaccharide (LPS). The undifferentiated and RA-treated U937 cells were unable to produce monokines even when they were stimulated by LPS. VD induced the monokine mRNA expression in U937 cells but failed to induce protein release. However, unlike RA, it primed the cells to secrete monokines upon endotoxin stimulation. A large enhancement of the production of the monokines both at mRNA and protein levels was observed in the U937 cells exposed to the combination of RA + VD. Nevertheless, protein release required a further step of activation of the RA + VD-primed cells. The co-inducer effect of RA and VD was not observed in HL-60 or THP-1 cells and seems to be restricted to U937 cells. These results on cytokine expression support our previous finding that a combination of RA and VD brings the U937 cells to a high stage of myeloid differentiation with major characteristics of monocytes/macrophages.