Subcutaneous graft of D1 mouse mesenchymal stem cells leads to the formation of a bone-like structure

Mesenchymal stem cells (MSC) are capable of both self-renewal and multi-lineage differentiation into mesoderm-type cells such as osteoblasts, chondrocytes, adipocytes and myocytes. Together the multipotent nature of MSCs and the facility to expand them in vitro make these cells ideal resources for regenerative medicine, particularly for bone reconstruction, and therefore research efforts focused on defining efficient protocols for directing their differentiation into the requisite lineage. Despite much progress in identifying mechanisms and factors that direct and control in vitro osteogenic differentiation of MSCs, a rapid and simple model to evaluate in vivo tissue formation is still lacking. Here, we describe the unique capacity of the murine bone marrow-derived D1 MSC cell line, which differentiates in vitro into at least three cell lineages, to form in vivo a structure resembling bone. This bone-like structure was obtained after subcutaneous grafting of D1 cells into immunocompetent mice without the need of neither an osteogenic factor nor scaffold material. These data allow us to propose this cell model as a tool for exploring in vivo the mechanisms and/or factors that govern and potentially regulate osteogenesis.     

Genomic organization, chromosomal assignment, and expression analysis of the mouse Suppressor of fused gene (Sufu) coding a Gli protein partner

Suppressor of fused (Sufu) is a negative regulator of the Hedgehog pathway both in Drosophila and vertebrates. Here, we report the genomic organization of the mouse Sufu gene (mSufu). This gene comprises 11 exons spanning more than 30 kb and encodes a protein with a putative PEST sequence. DNA-consensus sequences recognized by basic helix-loop-helix (bHLH) proteins, referred to as E-box motifs, are found in the 5′ flanking region. Analysis by single-strand conformation polymorphism and radiation hybrid positioned the Sufu locus to the distal end of mouse Chr 19 between D19Mit102 and D19Mit9, near the Fgf8 and dactylin genes. Mouse Sufu is expressed in various tissues, particularly in the nervous system, ectoderm, and limbs, throughout the developing embryo. Sufu binds with all three Gli proteins, with different affinities. This report, in conjunction with recent studies, points out the importance of Sufu in mouse embryonic development. Received: 14 January 2000 / Accepted: 31 March 2000     

Regulation of SMC traction forces in human aortic thoracic aneurysms

Smooth muscle cells (SMCs) usually express a contractile phenotype in the healthy aorta. However, aortic SMCs have the ability to undergo profound changes in phenotype in response to changes in their extracellular environment, as occurs in ascending thoracic aortic aneurysms (ATAA). Accordingly, there is a pressing need to quantify the mechanobiological effects of these changes at single cell level. To address this need, we applied Traction Force Microscopy (TFM) on 759 cells coming from three primary healthy (AoPrim) human SMC lineages and three primary aneurysmal (AnevPrim) human SMC lineages, from age and gender matched donors. We measured the basal traction forces applied by each of these cells onto compliant hydrogels of different stiffness (4, 8, 12, 25 kPa). Although the range of force generation by SMCs suggested some heterogeneity, we observed that: 1. the traction forces were significantly larger on substrates of larger stiffness; 2. traction forces in AnevPrim were significantly higher than in AoPrim cells. We modelled computationally the dynamic force generation process in SMCs using the motor-clutch model and found that it accounts well for the stiffness-dependent traction forces. The existence of larger traction forces in the AnevPrim SMCs were related to the larger size of cells in these lineages. We conclude that phenotype changes occurring in ATAA, which were previously known to reduce the expression of elongated and contractile SMCs (rendering SMCs less responsive to vasoactive agents), tend also to induce stronger SMCs. Future work aims at understanding the causes of this alteration process in aortic aneurysms.

     

Isolation and Characterization of Sex-Linked Female-Sterile Mutants in DROSOPHILA MELANOGASTER

The purpose of the experiments described was to identify X chromosome genes functioning mainly or exclusively during oogenesis. Two mutagenesis experiments were carried out with ethyl methane sulfonate. Following treatment inducing 60% lethals, 9% of the treated X chromosomes carried a female sterility mutation which did not otherwise seriously affect viability. Among —95 isolated mutants, 19 were heat-sensitive and 5 cold-sensitive. The mutants have been classified as follows: I (16 mutants; 12 complementation groups): the females laid few or no eggs; the defect concerned either ovulation or oogenesis. II (37 mutants; 18 complementation groups): the female laid morphologically abnormal eggs, often with increased membrane permeability. III A (13 mutants; at least 8 complementation groups): the homozygous females were sterile if mated to mutant males; their progeny (homo- and hemizygous) died at a late embryonic stage (11 mutants), at the larval stage (1 mutant) or at the pupal stage (1 mutant). However fertility was partly restored by breeding to wild-type males as shown by survival of some heterozygous descendants. III B (29 mutants; 22 complementation groups): the fertility of the females was not restored by breeding to a wild-type male. Most of the eggs of 13 of the mutants died at a late stage of embryogenesis. The eggs of the others ceased development earlier or, perhaps, remained unfertilized. The distribution of the number of mutants per complementation group led to an estimation of a total of about 150 X-linked genes involved in female fertility. The females of three mutants, heat-sensitive and totally sterile at 29°, produced at a lower temperature descendants morphologically abnormal or deprived of germ cells. Three other mutants not described in detail showed a reduction in female fertility with many descendants lacking germ cells. A desirable mutant which was not recovered was one with normal fertile females producing descendants which, regardless of their genotype, bore specific morphological abnormalities. The value of the mutants isolated for analysis of the complex processes leading to egg formation and initiation of development is discussed.     

Consequences of ions and pH on the supramolecular organization of sphingomyelin and sphingomyelin/cholesterol bilayers

For drug delivery purpose the anticancer drug S12363 was loaded into ESM/Chol-liposomes using either a pH or an ammonium gradient. Association between the drug and the liposome depends markedly on the liposome membrane structure. Thus, ESM and ESM/Chol bilayer organization had been characterized by coupled DSC and XRDT as a function of both cholesterol concentration and aqueous medium composition. ESM bilayers exhibited a ripple lamellar gel phase P(beta') below the melting temperature and adopted a L(beta)-like gel phase upon Chol insertion. Supramolecular organization of ESM and ESM/Chol bilayers was not modified by citrate buffer or ammonium sulfate solution whatever the pH (3< or = pH < or =7). Nevertheless, in ESM bilayer, ammonium sulfate salt induced a peculiar organization of head groups, leading to irregular d-spacing and weakly correlated bilayers. Moreover, in the presence of salts, a weakening of van der Waals attraction forces was seen and led to a swelling of the water layer.     

Noise Exposure and Hearing Impairment among Chinese Restaurant Workers and Entertainment Employees in Hong Kong

Background

Noise-induced hearing loss (NIHL) is a major concern in the non-manufacturing industries. This study aimed to investigate the occupational noise exposure and the NIHL among Chinese restaurant workers and entertainment employees working in the service industry in Hong Kong.

Methods

This cross-sectional survey involved a total of 1,670 participants. Among them, 937 were randomly selected from the workers of Chinese restaurants and 733 were selected from workers in three entertainment sectors: radio and television stations; cultural performance halls or auditoria of the Leisure and Cultural Services Department (LCSD); and karaoke bars. Noise exposure levels were measured in the sampled restaurants and entertainment sectors. Each participant received an audiometric screening test. Those who were found to have abnormalities were required to take another diagnostic test in the health center. The “Klockhoff digit” method was used to classify NIHL in the present study.

Results

The main source of noise inside restaurants was the stoves. The mean hearing thresholds showed a typical dip at 3 to 6 KHz and a substantial proportion (23.7%) of the workers fulfilled the criteria for presumptive NIHL. For entertainment sectors, employees in radio and television stations generally had higher exposure levels than those in the halls or auditoria of the LCSD and karaoke bars. The mean hearing thresholds showed a typical dip at 6 KHz and a substantial proportion of the employees fulfilled the criteria for presumptive NIHL (38.6%, 95%CI: 35.1–42.1%). Being male, older, and having longer service and daily alcohol consumption were associated with noise-induced hearing impairment both in restaurant workers and entertainment employees.

Conclusion

Excessive noise exposure is common in the Chinese restaurant and entertainment industries and a substantial proportion of restaurant workers and entertainment employees suffer from NIHL. Comprehensive hearing conservation programs should be introduced to the service industry in Hong Kong.     

Differential effects of albumin on microglia and macrophages; implications for neurodegeneration following blood–brain barrier damage

Microglial activation by blood-borne factors following blood–brain barrier damage may play a significant role in subsequent neuropathogenesis of several neurodegenerative diseases. Exposure of primary cultured rat brain microglia to pure, fatty acid- and lipid-deficient rat serum albumin or fraction V, (fatty acid and lipid-containing rat serum albumin), caused inducible nitric oxide synthase (iNOS) expression, glutamate release, tumour necrosis factor alpha (TNFα) and transforming growth factor-beta1 release. iNOS expression was attenuated by the MAPK/extracellular signal-regulated kinase pathway inhibitor U0126 and the phosphorylated forms of extracellular signal-regulated kinase 1 and 2 were detectable in microglia treated with albumin or fraction V. Glutamate release was prevented by l -α-aminoadipate and glutathione levels in microglia rose on exposure to albumin. Conditioned medium from microglia exposed to albumin or fraction V was neurotoxic. Peripheral macrophages were resistant to the effects of albumin but both microglia and macrophages responded to lipopolysaccharide, which induced interleukin-1 beta and tumour necrosis factor alpha release, cyclooxygenase-2 and iNOS expression in both cell types, indicating a discrete desensitised pathway in macrophages for albumin which was not desensitised in microglia. Thus, exposure of microglia in the brain to albumin may contribute to neuronal damage following blood–brain barrier breakdown and point to resident microglia rather than infiltrating macrophages as therapeutic targets.     

Induction of apoptosis by protein kinase C pseudosubstrate lipopeptides in several human cells

Intracellular enzymes or receptors are interesting targets for thepharmacomodulation of cellular metabolism. We have previously shown thatmodification of relatively long peptides by a palmitoyl-lysine residue couldfacilitate their delivery into the cytoplasm of living cells. Severalpeptides containing pseudosubstrate sequences of protein kinase C (PKC) havebeen evaluated for their ability to modulate phosphorylation of modelsubstrate, neuronal morphology or tumor necrosis factor secretion. In thiswork we have evaluated the effect of palmitoyl-modified PKC-pseudosubstratepeptides on induction of apoptosis. We have established that these peptidesare able to induce apoptosis in different human cell types (primaryfibroblasts, T- and B-lymphocyte cell lines) as assessed by (terminal deoxynucleotidyl transferase dUTP nick-end labelling) and DNAfragmentation. In contrast, control peptides (non-lipidicPKC-pseudosubstrate peptides and irrelevant lipopeptides) had no or littleeffect on programmed cell death. This work highlights the pharmacologicalinterest of lipopeptides and argues in favor of the potential role of PKC(s)in the cell death machinery.     

Plasma B-esterase activities in European raptors

B-esterases are serine hydrolases composed of cholinesterases, including acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), and carboxylesterase (CbE). These esterases, found in blood plasma, are inhibited by organophosphorus (OP) and carbamate (CB) insecticides and can be used as nondestructive biomarkers of exposure to anticholinesterase insecticides. Furthermore, B-esterases are involved in detoxification of these insecticides. In order to establish the level of these enzymes and to have reference values for their normal activities, total plasma cholinesterase (ChE), AChE and BChE activities, and plasma CbE activity were determined in 729 European raptors representing 20 species, four families, and two orders. The diurnal families of the Falconiforme order were represented by Accipitridae and Falconidae and the nocturnal families of the Strigiforme order by Tytonidae and Strigidae. Intraspecies differences in cholinesterase activities according to sex and/or age were investigated in buzzards (Buteo buteo), sparrowhawks (Accipiter nisus), kestrels (Falco tinnunculus), barn owls (Tyto alba), and tawny owls (Strix aluco). Sex-related differences affecting ChE and AChE activities were observed in young kestrels (2-3-mo-old) and age-related differences in kestrels (ChE and AChE), sparrowhawks (AChE), and tawny owls (ChE, AChE, and BChE). The interspecies analysis yielded a negative correlation between ChE activity and body mass taking into account the relative contribution of AChE and BChE to ChE activity, with the exception of the honey buzzard (Pernis apivorus). The lowest ChE activities were found in the two largest species, Bonelli's eagle (Hieraaetus fasciatus) and Egyptian vulture (Neophron percnopterus) belonging to the Accipitridae family. The highest ChE activities were found in the relatively small species belonging to the Tytonidae and Strigidae families and in honey buzzard of the Accipitridae family. Species of the Accipitridae, Tytonidae, and Strigidae families were characterized by a BChE contribution that dominated the total ChE activity, while in the species of the Falconidae family, AChE activity dominated. With the exception of the barn owl, CbE activity (eserine-insensitive alpha-naphthyl acetate esterase [alpha-NAE] activity) in all species was almost absent or very low. The values obtained in this study for ChE, AChE, and BChE activities and the AChE:BChE ratios for buzzard, kestrel, barn owl, and tawny owl provide a good estimate of the normal values in free-living individuals of these European species. They can be used as a baseline to evaluate the effect of anticholinesterase insecticides in the field.