Expression of a Truncated, Kinase-Defective TGF-β Type II Receptor in Mouse Skeletal Tissue Promotes Terminal Chondrocyte Differentiation and Osteoarthritis

Members of the TGF-β superfamily are important regulators of skeletal development. TGF-βs signal through heteromeric type I and type II receptor serine/threonine kinases. When over-expressed, a cytoplasmically truncated type II receptor can compete with the endogenous receptors for complex formation, thereby acting as a dominant-negative mutant (DNIIR). To determine the role of TGF-βs in the development and maintenance of the skeleton, we have generated transgenic mice (MT-DNIIR-4 and -27) that express the DNIIR in skeletal tissue. DNIIR mRNA expression was localized to the periosteum/perichondrium, syno-vium, and articular cartilage. Lower levels of DNIIR mRNA were detected in growth plate cartilage. Transgenic mice frequently showed bifurcation of the xiphoid process and sternum. They also developed progressive skeletal degeneration, resulting by 4 to 8 mo of age in kyphoscoliosis and stiff and torqued joints. The histology of affected joints strongly resembled human osteo-arthritis. The articular surface was replaced by bone or hypertrophic cartilage as judged by the expression of type X collagen, a marker of hypertrophic cartilage normally absent from articular cartilage. The synovium was hyperplastic, and cartilaginous metaplasia was observed in the joint space.

We then tested the hypothesis that TGF-β is required for normal differentiation of cartilage in vivo. By 4 and 8 wk of age, the level of type X collagen was increased in growth plate cartilage of transgenic mice relative to wild-type controls. Less proteoglycan staining was detected in the growth plate and articular cartilage matrix of transgenic mice. Mice that express DNIIR in skeletal tissue also demonstrated increased Indian hedgehog (IHH) expression. IHH is a secreted protein that is expressed in chondrocytes that are committed to becoming hypertrophic. It is thought to be involved in a feedback loop that signals through the periosteum/ perichondrium to inhibit cartilage differentiation. The data suggest that TGF-β may be critical for multifaceted maintenance of synovial joints. Loss of responsiveness to TGF-β promotes chondrocyte terminal differentiation and results in development of degenerative joint disease resembling osteoarthritis in humans.

     

Effects on breathing of carotid body denervation in neonatal piglets.

The purpose of these studies was to test the hypothesis that carotid chemoreceptor activity is necessary for postnatal maturation of the ventilatory control system. By using a lateral surgical access, 17 piglets were carotid body denervated (CBD) and 14 were sham denervated at 3-25 days of age. After surgery, there was no irregular breathing in any group. There was no significant hypoventilation when CBD was performed at less than 5 days of age (n = 5) and only a mild (arterial PCO(2) 5 Torr; P < 0.05) to moderate, transient (arterial PCO(2) 8 Torr; P < 0.5) hypoventilation in piglets denervated at 10-15 (n = 6) and 20-25 (n = 6) days of age, respectively. Three weeks after surgery, both breathing of a hypoxic gas mixture and jugular venous NaCN injections elicited a hyperpnea in the CBD piglets that was attenuated compared with that in sham CBD piglets. In the CBD piglets, there was no response to injections of NaCN in the carotid arteries, but there was a response to NaCN injected into the proximal descending aorta, suggesting the residual peripheral chemosensitivity was of aortic origin. Carotid chemoreceptor-intact piglets had carotid and aortic NaCN chemosensitivity by 2 days of age. The carotid response persisted for the 40 days of the study, but the aortic reflex persisted only until approximately 8 days of age. We conclude that 1) the major effect of CBD per se in neonatal piglets is age-dependent hypoventilation and 2) there is a high degree of plasticity in peripheral chemosensitivity in neonates that may contribute to minimizing the changes in breathing after CBD.     

The effect of dexamethasone treatment on the expression of the regulatory genes of ketogenesis in intestine and liver of suckling rats

The influence of the injection of dexamethasone on ketogenesis in 12 day old suckling rats was studied in intestine and liver by determining mRNA levels and enzyme activity of the two genes responsible for regulation of ketogenesis: carnitine palmitoyl transferase I (CPT 1) and mitochondrial HMG-CoA synthase. Dexamethasone produced a 2 fold increase in mRNA and activity of CPT I in intestine, but led to a decrease in mitt HMG-CoA synthase. In liver the mRNA levels and activity of both CPT I and mitt HMG-CoA synthase decreased. Comparison of these values with the ketogenic rate of both tissues following dexamethasone treatment suggests that mitt HMG-CoA synthase could be the main gene responsible for the regulation of ketogenesis in suckling rats. The changes produced in serum ketone bodies by dexamethasone, with a profile that is more similar to the ketogenic rate in the liver than that in the intestine, indicate that liver contributes more to ketone body synthesis in suckling rats. Two day treatment with dexamethasone produced no change in mRNA or activity levels for CPT I in liver or intestine. While mRNA levels for mitt HMG-CoA synthase changed little, the enzyme activity is decreased in both tissues.     

Reiterated transfer RNA genes of Xenopus laevis

The proportion of the Xenopus laevis genome complementary to “7 S” RNA, unfractionated transfer RNA and some selected aminoacyl-tRNAs, and the sequence complexity of these RNA species, have been determined by following the kinetics of RNA-DNA hybridization on filters under conditions of RNA excess at optimum rate temperature. For hybridization of aminoacyl-labelled tRNAs, conditions for optimum aminoacylation were first determined and, where necessary, aminoacyl-tRNAs were treated with nitrous acid to prevent discharge during annealing. Neither the extent nor rate of hybridization was affected by this treatment.“7 S” RNA, coded for by 580 genes per haploid complement of chromosomes, reacts like a single family of nucleotide sequences, whereas about 43 basic tRNA sequences are coded for by at least 7800 genes. If hybrids are not treated with RNase A, the apparent tDNA redundancy is some 23% greater but no more nucleotide sequences are detectable. Taken together, the results suggest that each tRNA sequence is, on average, 200-fold reiterated.The reiteration varies, however, for different aminoacyl-tRNAs. Thus, hybridization resolves only one valyl-tRNA which is coded for by 240 genes, but at least four leucyl-tRNA sequences can be distinguished by hybridization, each of which is on average 90-fold reiterated. Reiteration also varies for the two methionyl-tRNAs detectable both by hybridization and by reversed phase chromatography: tRNA₁^Met and tRNA₂^Met are 310- and 170-fold reiterated, respectively, and each is kinetically homogeneous. These saturation values are almost exactly additive and are not influenced by the presence of other tRNA species. Thus the results suggest that Xenopus tRNAs are no more heterogeneous than would be predicted by the genetic code, despite the high but variable multiplicity of tRNA cistrons.     

The legumain McPAL1 from Momordica cochinchinensis is a highly stable Asx-specific splicing enzyme

Legumains, also known as asparaginyl endopeptidases (AEPs), cleave peptide bonds after Asn/Asp (Asx) residues. In plants, certain legumains also have ligase activity that catalyzes biosynthesis of Asx-containing cyclic peptides. An example is the biosynthesis of MCoTI-I/II, a squash family-derived cyclic trypsin inhibitor, which involves splicing to remove the N-terminal prodomain and then N-to-C-terminal cyclization of the mature domain. To identify plant legumains responsible for the maturation of these cyclic peptides, we have isolated and characterized a legumain involved in splicing, McPAL1, from Momordica cochinchinensis (Cucurbitaceae) seeds. Functional studies show that recombinantly expressed McPAL1 displays a pH-dependent, trimodal enzymatic profile. At pH 4 to 6, McPAL1 selectively catalyzed Asp-ligation and Asn-cleavage, but at pH 6.5 to 8, Asn-ligation predominated. With peptide substrates containing N-terminal Asn and C-terminal Asp, such as is found in precursors of MCoTI-I/II, McPAL1 mediates proteolysis at the Asn site and then ligation at the Asp site at pH 5 to 6. Also, McPAL1 is an unusually stable legumain that is tolerant of heat and high pH. Together, our results support that McPAL1 is a splicing legumain at acidic pH that can mediate biosynthesis of MCoTI-I/II. We purport that the high thermal and pH stability of McPAL1 could have applications for protein engineering.     

Diapausing egg banks,lake size,and genetic diversity in the rotifer <Emphasis Type="Italic">Brachionus plicatilis</Emphasis> Müller (Rotifera,Monogononta)

The fish from the family Anostomidae represent one of the most important groups of freshwater ichthyofauna from South America, with species of high economical value. The migratory characteristic of some species, through the several Amazonian environments, takes them into waters with different physico-chemical characteristics. Cytogenetic studies on the Anostomidae demonstrate that these fishes have a conserved diploid number and karyotype macrostructure. So, to verify if this conservation occurs also in the genomic level, the current study aimed at a chromosomal comparative physical mapping, using 45S and 5S rDNA, of seven species of anostomids: Leporinus fasciatus, L. agassizi, L. friderici, L. trifasciatus, Rhytiodus macrolepis, Laemolyta taeniata, and Schizodon fasciatus, collected in different Amazonian environments. The results obtained corroborate the conservation of the karyotype macrostructure. However, significant differences were found in the distribution of heterochromatin and on the pair bearing the nucleolus organizer region. The staining of 45S and 5S rDNA by FISH highlighted, for four of the seven species, more than one chromosome pair bearing the site 45S. The 5S rDNA, although present in only one chromosome pair, varied in its chromosome and karyotype position. Thus, although the Anostomidae family has a conserved chromosomic macrostructure the use of molecular techniques revealed the presence of chromosomic translocation during the evolution of these fishes.     

Profile of Overweight and Obese People in a Mediterranean Region

Objective: To assess the prevalence of excess weight (obesity and overweight) and the related environmental risk factors in a Mediterranean population. Self‐perception of body weight, attitudes toward weight‐control behaviors, and the associated factors were also examined. Research Methods and Procedures: A cross‐sectional nutritional survey was carried out in the Balearic Islands between 1999 and 2000. A random sample (n = 1200) of the adult population (20 to 60 years old) was interviewed. Dietary questionnaires and a global questionnaire incorporating questions related to sociodemographic and lifestyle variables were utilized. Anthropometric measurements were also obtained. Results: Nearly one‐half of the population of the Balearic Islands is above the normal weight range (BMI > 25). The predictors of overweight and obesity in the Balearic Islands are to be over 40 years old, to be married, to form part of the lowest educational levels, and to have a sedentary lifestyle. A large percentage of excess‐weight individuals tend to underestimate their BMI, are not concerned about their weight status, tend to snack more often, and have never dieted. Discussion: Although the likely causes of the rise in obesity prevalence are difficult to elucidate from this study, we have identified the profile of individuals with excess weight in the Balearic Islands as well as their attitudes toward their body image.     

Deparaffination time: a crucial point in histochemical detection of tissue copper

The search for a sensitive histochemical method for revealing tissue copper has been the object of many workers in the past. In spite of multiple methods available, the occurrence in clinical practice of negative histochemical stains, even in cases with high copper levels demonstrated by quantitative methods is very high.This study was aimed at verifying the role of technical variations in the sensitivity of the Timm method and, in particular, the role of the dewaxing time of paraffin sections. To this end, 15 liver specimens, 10 from patients affected by Wilson's disease and 5 newborn livers were fixed in 10% formalin, paraffin embedded and routinaly processed. Four 4-micron sections from each case were rinsed in xylene for 10, 20, 60 min, and for 24 hrs. All sections were stained with Timm's method. In 13 out of the 15 liver biopsies utilized in this study, the sensitivity of Timm's method in revealing copper deposits in liver cells appeared to be dependent on the dewaxing time. In two other cases, reactivity of copper granules to Timm solution did not change significantly with the different deparaffination times. The best results were obtained by rinsing sections in xylene for 24 hrs, the worst in sections treated with xylen for 10 minutes. In particular, in five cases of Wilson's disease, Timm stain applied to sections following ten minutes of xylene were completely negative, while copper granules were clearly evidenced in the same section following an overnight bath in xylene. Our data show that an overnight bath of paraffin sections in xylene may completely change the sensitivity of Timm stain in revealing copper deposits in the liver, relaunching copper histochemistry in the diagnosis of copper-related liver diseases.     

Use of multivariate analyses to investigate the contribution of metal pollution to diatom species composition: search for the most appropriate cases and explanatory variables

The aim of this study was to elucidate how the spatial scale and the set of variables included influence our ability to detect the effects of different types of pollution on the biota. Using variance partitioning analysis, we assessed the individual importance of a set of environmental factors (eutrophication and organic pollution) versus metal level pollution on the community structure of diatom assemblages at different spatial scales. At regional scale, environmental factors did not explain more of the variance compared to the watershed study. The results of the watershed scale field survey indicate that diatom community composition was influenced by low metal concentrations, but this pattern was only observed by the inclusion of biofilm metal concentration data. We recommend the analysis of metal traces in the water phase and the biota (fluvial biofilms) as well as the use of the Diffusive Gradient in Thin films (DGT) technique to characterize low metal level pollution in freshwater systems. Handling editor: Judit Padisák