DQ-haplotype analysis in rhesus macaques: implications for the evolution of these genes

The DQA1 and DQB1 alleles of 258 rhesus monkeys (Macaca mulatta) of different origin were typed by PCR-RFLP. Five novel MamuDQA1 and five novel -DQB1 alleles were detected and 15 Mamu-DQA1-DQB1 haplotypes were identified. Haplotype analysis confirmed the conservation of the DQA1*01-DQB1 *06 haplotypes in evolution. The most conspicuous finding was the tight linkage between the Mamu-DQA1 and -DQB1 alleles. Almost in every case the Mamu-DQA1 allele was linked to only one particular Mamu-DQB1 allele. Although there also are constraints in the formation of DQ haplotypes in humans, such tight linkages are not observed. These findings support the hypothesis of some kind of co-evolution between DQA1 and DQB1 alleles and may reflect a stronger force of natural selection in macaques than in humans.     

Tctex3, related to Drosophila polycomblike, is expressed in male germ cells and mapped to the mouse t-complex

Tctex3 showing restricted expression in male germ cells has been isolated during the process of chromosome walking in the mouse t-complex region. The total sequence of Tctex3 cDNA predicts a protein of 580 amino acids with two C4HC3 type PHD fingers. The region containing this conserved motif is shared among members of the Polycomblike proteins that include the mouse M96 and Drosophila Polycomblike. A partial cDNA for a human homolog of Tctex3, HUTEX3, has also been isolated. Mouse Tctex3 gene was mapped adjacent to Tsc2 gene on mouse Chromosome (Chr) 17, and HUTEX3 was located closely to HSET gene in the HLA class II region of chromosome 6.     

Common mutations in the fibroblast growth factor receptor 3 (FGFR 3) gene account for achondroplasia, hypochondroplasia, and thanatophoric dwarfism

A standard electrical stimulus applied to the posterior hypothalamus evoked cardiac arrhythmogenic responses in the spontaneously hypertensive rat. Isolated premature ventricular beats or doublets and nonsustained ventricular tachycardic salvos were observed. This effect was associated with a large rise in blood pressure (79 +/- 3 mm Hg). The same stimulus in normotensive Wistar-Kyoto rats produced no significant cardiac arrhythmias, and the rise in blood pressure was smaller (36 +/- 2 mm Hg). We investigated the influence of baclofen, a GABAB receptor agonist, and two N-methyl-D-aspartate receptor antagonists on the arrhythmogenic response to hypothalamic stimulation. Intravenous baclofen (3 mg/kg) had no effect in the normotensive Wistar-Kyoto rats, but in the spontaneously hypertensive rats it enhanced the adjusted mean value of the number of extrasystoles from 0.5 +/- 0.5 to 18 +/- 1 (P < .001). This value was also increased (from 3 +/- 1 to 17 +/- 1, P < .001) by an intracisternal injection of baclofen (1 micrograms/kg). This facilitatory effect of baclofen was prevented by treatment with atenolol (0.5 mg/kg). Two glutamate receptor antagonists, ketamine (7.5 mg/kg IV) and kynurenic acid (200 micrograms/kg intracerebroventricularly), prevented both the arrhythmogenic response to the hypothalamic stimulation and its facilitation by baclofen. The study confirms that hypothalamic stimulation facilitates the development of arrhythmias through a sympathetic drive and that these arrhythmias are easier to induce in spontaneously hypertensive rats than in normotensive Wistar-Kyoto rats. Both the central GABAergic and the glutamatergic systems are implicated in the development of these ventricular arrhythmias, since baclofen could disinhibit the glutamatergic central pathway. These results could account for the ability of the spontaneously hypertensive rats to develop ventricular arrhythmias of central origin.     

Regulating the balance between differentiation and apoptosis: role of CREM in the male germ cells

Axon regeneration fails in the CNS because the glial environment is inhibitory, and because the regenerative response of CNS is poor. Regeneration can therefore be induced by removing the inhibitory effect of CNS glial molecules, by increasing the regenerative in animal models of spinal cord injury has recently been achieved by several strategies that apply these principles. The successful techniques have been to block inhibitory molecules made by astrocytes, to implant peripheral nerve grafts embedded in a bFGF-containing fibrin gel, to implant olfactory ensheathing cells, to graft embryonic spinal cord tissue, and to implant trophic factor-secreting fibroblasts. The next challenge is to prepare to apply these types of treatment to human patients with spinal cord injuries.     

Indirect regulation of translational termination efficiency at highly expressed genes and recoding sites by the factor recycling function of Escherichia coli release factor RF3

Prokaryotic release factor RF3 is a stimulatory protein that increases the rate of translational termination by the decoding release factors RF1 and RF2. The favoured model for RF3 function is the recycling of RF1 and RF2 after polypeptide release by displacing the factors from the ribosome. In this study, we have demonstrated that RF3 also plays an indirect role in the decoding of stop signals of highly expressed genes and recoding sites by accentuating the influence of the base following the stop codon (+4 base) on termination signal strength. The efficiency of decoding strong stop signals (e.g. UAAU and UAAG) in vivo is markedly improved with increased RF3 activity, while weak signals (UGAC and UAGC) are only modestly affected. However, RF3 is not responsible for the +4 base influence on termination signal strength, since prfC- strains lacking the protein still exhibit the same qualitative effect. The differential effect of RF3 at stop signals can be mimicked by modest overexpression of decoding RF. These findings can be interpreted according to current views of RF3 as a recycling factor, which functions to maintain the concentration of free decoding RF at stop signals, some of which are highly responsive to changes in RF levels.     

Molecular cloning and chromosomal mapping of olfactory receptor genes expressed in the male germ line: evidence for their wide distribution in the human genome

PURPOSE: The authors evaluated the effectiveness of ultrasound biomicroscopy to determine the condition of the ciliary body during perioperative examinations of patients with atopic dermatitis and retinal detachment. METHODS: The authors compared two groups of patients with atopic dermatitis and retinal detachment. Parameters included patient age, gender, eye, cataract, type and location of breaks, macular involvement, detachment of the ciliary epithelium, and preoperative and postoperative best-corrected visual acuities. Group 1 included six patients (nine eyes) who were examined before surgery and after surgery using ultrasound biomicroscopy, with which the authors also measured the maximum height of the detachment of the ciliary epithelium. Group 2 included 10 patients (13 eyes) who did not undergo ultrasound biomicroscopy. RESULTS: In group 1, ultrasound biomicroscopy showed ciliary epithelium detachment in all eyes before surgery and in eight of nine eyes after successful retinal reattachment. The height of the ciliary detachment, however, decreased dramatically after surgery. Although almost all the parameters between groups 1 and 2 were similar, the authors observed a significant difference in the incidence of preoperative diagnosis of ciliary detachment (P = 0.023). CONCLUSION: Ultrasound biomicroscopy is beneficial in detecting detachment of the ciliary epithelium. The residual shallow detachment that remains after successful surgery suggests the fragility of the ciliary body.     

Serum insulin-like growth factor-I, insulin-like growth factor binding protein-3, sex steroids, osteocalcin and bone mineral density in male and female rats

Although it has been reported that the rate of weight gain and linear growth increases markedly during puberty in rats, little is known about the relationship between endocrine changes and bone mineral density (BMD) changes upon sexual maturation in these animals. The aim of this study was to examine the levels of serum insulin-like growth factor-I (IGF-I), IGF binding protein (IGFBP)-3, sex steroids and osteocalcin, and the changes in BMD in normal aging male and female rats. Male rats exhibited increases in serum IGF-I and IGFBP-3 concentrations before increases in serum testosterone levels. IGF-I and testosterone peaked at 9 weeks of age, and thereafter remained in a steady state, whereas IGFBP-3 reached a peak at 7 weeks of age, and then gradually declined. A strong correlation between serum IGF-I and IGFBP-3 levels was found in subjects 3-9 weeks old. A highly significant correlation between serum IGF-I and testosterone levels was also found. In females, serum 17 beta-estradiol, IGF-I and IGFBP-3 levels increased gradually from 3 to 5 weeks old, peaked at 9 weeks, and then decreased slowly thereafter. The correlation coefficient between serum IGF-I and IGFBP-3 was highly significant. The correlation coefficient between serum IGF-I and 17 beta-estradiol levels was weak, although it was strongest when the subjects were 3-9 weeks old. Serum osteocalcin is a marker of bone formation; its level remained relatively high from 3 to 9 and from 3 to 7 weeks of age in males and females, respectively, although osteocalcin in both sexes declined gradually with age. As for bone mass, sharp increases in BMD in the tibia, femur and lumbar vertebrae appeared earlier in female than in male rats, and the BMD in females tended to be higher than in males between 5 and 9 weeks old. After 9 weeks of age, BMD in males was higher than that in females, as BMD in males continued to increase whereas females tended to remain in a steady state after this stage. The correlation coefficients between tibial BMD and serum IGF-I or IGFBP-3 levels were highly significant when the subjects were from 3 to 9 weeks old. Taken together, these results suggest that BMD development occurs earlier in female than in male rats. This sex-related difference in changes in the BMD pattern may result from the earlier onset of puberty in females, and from sex-specific differences in concentrations of IGF-I, IGFBP-3 and sex steroids during maturation.     

Heat shock induces rapid dephosphorylation of tau in both female and male rats followed by hyperphosphorylation only in female rats: implications for Alzheimer's disease

Female and male 2-3-month-old Sprague-Dawley rats were heat shocked at 42 degrees C for 15 min. At 0, 3, 6, 12 and 24 h after heat shock, qualitative and quantitative immunoblot analysis of cerebral extracts and immunohistochemistry were performed using monoclonal anti-tau antibodies that recognize nonphosphorylated (Tau-1), phosphorylated (PHF-1), and phosphate-independent (Tau-5 and Tau-46) epitopes. At 0 h after heat shock, there was dephosphorylation of tau in both female and male rats as evidenced by (1) accentuation and attenuation of tau isoforms recognized by Tau-1 and PHF-1, respectively, and recognition of additional tau polypeptides by Tau-1, Tau-5, and Tau-46 but not PHF-1; (2) significant increase in the nonphosphorylated Tau-1 epitope with resultant decrease in the ratio of total (phosphorylated plus nonphosphorylated) tau to nonphosphorylated tau; and (3) dephosphorylation of the Tau-1 epitope in the somatodendritic compartment. By 6 h after heat shock, there was progressive hyperphosphorylation of tau in female but not male rats exemplified by (1) upward gel mobility shift recognized by PHF-1, Tau-5, and Tau-46, and by Tau-1 after dephosphorylation; (2) significant increase in the ratio of total tau to nonphosphorylated tau; and (3) rephosphorylation of the Tau-1 epitope in the somatodendritic compartment. Two-dimensional electrophoresis showed shifts to basic and acidic tau polypeptides at 0 and 6 h after heat shock, respectively. Hyperphosphorylation of tau also occurred after multiple heat-shock episodes. Microtubules were present at 6 h after heat shock. There were no differences between control and heat-shocked rats in extracts from peripheral nerves. Thus, we now have a simple rat model to study within 6 h the processes of dephosphorylation and hyperphosphorylation of tau, which are altered in Alzheimer's disease.     

Overexpression of stromelysin-3, BM-40/SPARC, and MET genes in human esophageal carcinoma: implications for prognosis

We estimated the energy expenditures resulting from helping behaviour in the cooperatively breeding cichlid N. pulcher by measuring the metabolic rates directly associated with specific social and territory maintenance behaviours of individual pair males and females, and their helpers, in a respirometer. In pair males, pair females and helpers, routine metabolism was raised on average 4.4, 3.8 and 3.6 times, respectively, during agonistic behaviour. Helpers spent 3.3 and 6.1 times routine metabolism, respectively, on submissive behaviour (tail quivering) and digging. These estimates of energy expenditure were combined with laboratory time budgets, obtained previously, to calculate behavioural time-energy budgets for pair members and helpers. Both groups spent on average 98.5% of total metabolism on routine and standard metabolism. With regard to the energy expended on specific behaviours, pair males invested almost exclusively in intrafamily agonistic behaviour, while pair females and helpers shared the investment in territory maintenance and direct brood care. The behavioural energy budget of helpers was strongly determined by their submissive behaviour. This serves to maintain the social status of the helpers within the family hierarchy and may therefore be regarded as 'paying for staying', which may also be true for direct brood care and other helper duties. We conclude that the substantial energy expenditures associated with helping behaviours are probably partly responsible for the reduced growth rates of helpers. This is the first study in which energy expenditures associated with specific helping behaviours have been measured in brood care helpers, and it provides the first estimate of total behavioural energy expenditure in a cooperatively breeding fish. (c) 1998 The Association for the Study of Animal Behaviour.     

Analysis of ecdysteroid action in Malacosoma disstria cells: cloning selected regions of E75- and MHR3-like genes

IPRI-MD-66 (MD-66) cells respond to 20-hydroxyecdysone (20E, 4 x 10(-6) M) in the medium by producing cytoplasmic extensions, clumping and attaching themselves to the substrate. These morphological changes are at a maximum by 6 days post treatment. Degenerate oligonucleotides, designed on the basis of conserved amino acid sequences in the DNA and ligand binding regions of the members of the steroid hormone receptor superfamily, were used in RNA-PCR to isolate two cDNA fragments, Malacosoma disstria hormone receptor 2 (MdHR2) and Malacosoma disstria hormone receptor 3 (MdHR3) from the MD-66 cells. Comparison of deduced amino acid sequences of these cDNA fragments with the members of the steroid hormone receptor superfamily showed that MdHR2 is most closely related to E75 proteins of Manduca sexta, Galleria mellonella and Drosophila melanogaster. The MdHR3 is most closely related to Manduca hormone receptor 3 (MHR3), Galleria hormone receptor 3 (GHR3) and Drosophila hormone receptor 3 (DHR3) proteins. At a concentration of 4 x 10(-6) M, 20E induces the expression of MdHR2 and MdHR3 beginning at 3 h, reaching maximum levels in 12 h and declining in 24 h. MdHR2 binds to a 2.5 kb mRNA, whereas MdHR3 binds to a 4.5 kb mRNA. Based on sequence similarity, RNA size and ecdysone inducibility, we conclude that these cDNA fragments, cloned from MD-66 cells, are regions of E75- (MdHR2) and MHR3- (MdHR3) like genes.     

The DNA/RNA-binding protein, TB-RBP, moves from the nucleus to the cytoplasm and through intercellular bridges in male germ cells

In male infants, traumatic ablation of the penis, with or without loss of the testicles may occur as a sequel to mutilatory violence, accidental injury, or circumcision error. Post-traumatically, one program of case management is surgical sex reassignment to live as a girl, with female hormonal therapy at the age of puberty. The other program is genital reconstructive surgery to live as a boy, with male hormonal therapy at puberty if the testicles are missing. In both programs, the long term outcome is less than perfect and is contingent on intervening variables that include societal ideology; surgical technology; juvenile and adolescent timing and frequency of hospital admissions construed by the child as nosocomial abuse; development of body image; health and sex education; fertility versus sterility; coitus and orgasm; possible lesbian orientation if living as a girl; and long-term cost accounting, including the psychic cost of being a pawn in possible malpractice litigation on whose disability a very large fortune in compensation may devolve. There is, as yet, no unanimously endorsed set of guidelines for the treatment of genital trauma and mutilation in infancy, and no provision for a statistical depository for outcome data.     

A gene fusion at a homeobox locus: alterations in leaf shape and implications for morphological evolution

Compound leaves are seen in many angiosperm genera and are thought to be either fundamentally different from simple leaves or elaborations of simple leaves. The knotted1-like homeobox (knox) genes are known to regulate plant development. When overexpressed in homologous or heterologous species, this family of genes can cause changes in leaf morphology, including excessive leaf compounding in tomato. We describe here an instance of a spontaneously arisen fusion between a gene encoding a metabolic enzyme and a homeodomain protein. We show that the fusion results in overexpression of the homeodomain protein and a change in morphology that approximates the changes caused by overexpression of the same gene under the control of the cauliflower mosaic virus 35S promoter in transgenic plants. Exon-shuffling events can account for the modularity of proteins. If the shuffled exons are associated with altered promoters, changes in gene expression patterns can result. Our results show that gene fusions of this nature can cause changes in expression patterns that lead to altered morphology. We suggest that such phenomena may have played a role in the evolution of form.     

Analysis of the F8 gene in individuals with high plasma factor VIII: C levels and associated venous thrombosis

High FVIII:C levels have previously been shown to be an independent risk factor for thrombosis with 4.8 times higher potential risk of thrombosis in individuals with FVIII:C levels greater than 1.5 u/ml. Recently, we found that raised FVIII:C levels are largely attributable to elevated FVIII:Ag levels. The determinants of FVIII:Ag levels are unclear and might be partly genetic. The promoter of the F8 gene has recently been characterised we therefore investigated the promoter and the 3' terminus of the F8 gene for possible polymorphisms associated with raised FVIII:Ag levels in 62 selected individuals with a thrombotic tendency. We confirm previous reports that raised FVIII:C levels are largely attributable to an elevation in FVIII:Ag and this is also associated with elevation of vWF; non-O blood group: relatively short APTT and relatively low APC ratio. We screened 1140 bp of the proximal promoter including the protein binding sites identified by DNase I footprint analysis by SSCP, however no polymorphisms were identified. Direct DNA sequence analysis of the region -542 to +165 failed to identify any sequence polymorphisms. The recently described polymorphism in the polyadenylation cleavage site in the prothrombin gene associated with increased prothrombin activity prompted us to screen the region surrounding the 3' terminus of the F8 gene for polymorphisms but we found none.     

Evidence for an inducible repair-recombination system in the female germ line of Drosophila melanogaster. III. Correlation between reactivity levels, crossover frequency and repair efficiency

We previously reported evidence that the so-called reactivity level, a peculiar cellular state of oocytes that regulates the frequency of transposition of I factor, a LINE element-like retrotransposon, might be one manifestation of a DNA repair system. In this article, we report data showing that the reactivity level is correlated with the frequency of crossing over, at least on the X chromosome and on the pericentromeric region of the third chromosome. Moreover, a check for X-chromosome losses and recessive lethals produced after gamma irradiation in flies with different reactivity levels, but common genetic backgrounds, brings more precise evidence for the relationship between reactivity levels and DNA repair. Those results support the existence of a repair-recombination system whose efficiency is modulated by endogenous and environmental factors. The implications of this biological system in connecting genomic variability and environment may shed new lights on adaptative mechanisms. We propose to call it VAMOS for variability modulation system.     

Identification of constitutive and ras-inducible phosphorylation sites of KSR: implications for 14-3-3 binding, mitogen-activated protein kinase binding, and KSR overexpression

The chromatin elements targeted by the ATPdependent, Swi-Snf nucleosome-remodeling complex are unknown. To address this question, we generated mutations in yeast histone H2B that suppress phenotypes associated with the absence of Swi-Snf. Sin- (Swi-Snf-independent) mutations occur in residues involved in H2A-H2B dimer formation, dimer- tetramer association, and in the H2B N-terminus. The strongest and most pleiotropic Sin- mutation removed 20 amino acid residues from the H2B N-terminus. This mutation allowed active chromatin to be formed at the SUC2 locus in a snf5Delta mutant and resulted in hyperactivated levels of SUC2 mRNA under inducing conditions. Thus, the H2B N-terminus may be an important target of Swi-Snf in vivo. The GCN5 gene product, the catalytic subunit of several nuclear histone acetytransferase complexes that modify histone N-termini, was also found to act in conjunction with Swi-Snf. The phenotypes of double gcn5Deltasnf5Delta mutants suggest that histone acetylation may play both positive and negative roles in the activity of the Swi-Snf-remodeling factor.     

High-dose etoposide with granulocyte colony-stimulating factor for mobilization of peripheral blood progenitor cells: efficacy and toxicity at three dose levels

High-dose etoposide (2.0-2.4 g m(-2)) with granulocyte colony-stimulating factor (G-CSF) is an effective strategy to mobilize peripheral blood progenitor cells (PBPCs), although in some patients this is associated with significant toxicity. Sixty-three patients with malignancy were enrolled into this non-randomized sequential study. The majority (55/63, 87%) had received at least two prior regimens of chemotherapy, and seven patients had previously failed to mobilize following high-dose cyclophosphamide with G-CSF. Consecutive patient groups received etoposide at three dose levels [2.0 g m(-2) (n = 22), 1.8 g m(-2) (n = 20) and 1.6 g m(-2) (n = 21)] followed by daily G-CSF. Subsequent leukaphereses were assayed for CD34+ cell content, with a target total collection of 2.0 x 10(6) CD34+ cells kg(-1). Toxicity was assessed by the development of significant mucositis, the requirement for parenteral antibiotics or blood component support and rehospitalization incidence. Ten patients (16%) had less than the minimum target yield collected. Median collections in the three groups were 4.7 (2 g m(-2)), 5.7 (1.8 g m(-2)) and 6.5 (1.6 g m(-2)) x 10(6) CD34+ cells kg(-1). Five of the seven patients who had previously failed cyclophosphamide mobilization achieved more than the target yield. Rehospitalization incidence was significantly lower in patients receiving 1.6 g m(-2) etoposide than in those receiving 2.0 g m(-2) (P = 0.03). These data suggest that high-dose etoposide with G-CSF is an efficient mobilization regimen in the majority of heavily pretreated patients, including those who have previously failed on high-dose cyclophosphamide with G-CSF. An etoposide dose of 1.6 g m(-2) appears to be as effective as higher doses but less toxic.     

MgcRacGAP, a new human GTPase-activating protein for Rac and Cdc42 similar to Drosophila rotundRacGAP gene product, is expressed in male germ cells

In a search for new partners of the activated form of Rac GTPase, we have isolated through a two-hybrid cloning procedure a human cDNA encoding a new GTPase-activating protein (GAP) for Rho family GTPases. A specific mRNA of 3.2 kilobases was detected in low abundance in many cell types and found highly expressed in testis. A protein of the predicted size 58 kDa, which we call MgcRacGAP, was detected in human testis as well as in germ cell tumor extracts by immunoblotting with antibodies specific to recombinant protein. In vitro, the GAP domain of MgcRacGAP strongly stimulates Rac1 and Cdc42 GTPase activity but is almost inactive on RhoA. N-terminal to its GAP domain, MgcRacGAP contains a cysteine-rich zinc finger-like motif characteristic of the Chimaerin family of RhoGAPs. The closest homolog of MgcRacGAP is RotundRacGAP, a product of the Drosophila rotund locus. In situ hybridization experiments in human testis demonstrate a specific expression of mgcRacGAP mRNA in spermatocytes similar to that of rotundRacGAP in Drosophila testis. Therefore, protein sequence similarity and analogous developmental and tissue specificities of gene expression support the hypothesis that RotundRacGAP and MgcRacGAP have equivalent functions in insect and mammalian germ cells. Since rotundRacGAP deletion leads to male sterility in the fruit fly, the mgcRacGAP gene may prove likewise to play a key role in mammalian male fertility.     

Mitochondrial phylogeny of the European cyprinids: implications for their systematics, reticulate evolution, and colonization time

Two different mitochondrial genes, the cytochrome b and the 16S rDNA, support the same European cyprinid molecular phylogeny: the most basal subfamily is the paraphyletic Rasborinae, the Cyprininae are monophyletic, the Tincinae and Gobioninae are close to the Cyprininae or more basal lineages but not close to Leuciscinae or Alburninae, and the Leuciscinae are paraphyletic but can become monophyletic if we include the biphyletic alburninae and exclude the Phoxinini. The relationship of the Acheilognathinae remains obscure. Natural intergeneric and interspecific hybridizations are clearly demonstrated within the Leuciscinae, both from high bootstrap proportions and intermediate morphological features: Chondrostoma toxostoma and Rutilus rutilus, Scardinius erythrophthalmus and R. rutilus, and Leuciscus multicellus and Leuciscus soufia. Finally, the use of the nonsaturated and clockwise 16S mtDNA sequences have been used to infer from nonintrogressive taxa the time of the first European cyprinid cladogeneses. The estimation confirms the hypothesis of Alma?a and Banarescu that European cyprinid subfamilies started to diversify 35 mya and confirms the hypothesis of Bianco on the diversification of European leuciscines in the Mediterranean area during the late Messinian (6.5 to 5.3 mya).