Antiadipogenesis and Osseointegration of Strontium-Doped Implant Surfaces.

The decreased bone density and increased marrow adiposity that occur with aging may influence the outcome of dental implants. Strontium (Sr), an anabolic agent for the treatment of osteoporosis, has an inhibitory effect on adipogenesis but favors osteogenesis of bone marrow-derived mesenchymal stem cells (BMSCs). However, little is known about the effects and mechanisms of local Sr release on adipogenesis during bone formation in aged bone. In this study, a potential dental implant material, Sr-doped titanium, was developed via a sandblasted, large-grit, and acid-etched (SLA) method combined with a hydrothermal process. The effects of Sr-SLA on initial adhesion, proliferation, intracellular redox state, and adipogenic differentiation of senescent BMSCs were investigated. The in vitro results showed that Sr-SLA promoted spreading of senescent BMSCs via upregulation of the gene and protein expression of integrin ß1. In addition, it was revealed that Sr-SLA could reduce intracellular oxidative stress by decreasing the levels of reactive oxygen species and oxygen radicals and increasing the content of glutathione peroxidase. More important, Sr-SLA suppressed lipid droplet production and adipokines expression via downregulation of transcription peroxisome proliferator-activated receptor γ (PPARγ) and signal transducer and activator of transcription 1, thus inhibiting adipogenesis. Finally, the Sr-SLA implants were implanted in tibiae of aged (18-mo-old) Sprague-Dawley rats for 2 and 8 wk. Histomorphometric analysis demonstrated that Sr-SLA implants significantly enhanced osseointegration, and the inhibition effect on marrow adipose tissue formation was moderate. All these results suggest that due to the multiple functions produced by Sr, antiadipogenesis capability and rapid osseointegration were enhanced by the Sr-SLA coatings, which have potential application in dental implantation in the aged population.

Hemerythrin is required for Aeromonas hydraphlia to survive in the macrophages of Anguilla japonica.

Survival in host phagocytes is an effective strategy for pathogenic microbes to spread. To understand the mechanisms of Aeromonas hydrophila survival within host macrophages, a library of mini-Tn10 transposon insertion mutants was constructed. The M85 mutant, whose survival in host macrophages was only 23.1% of that of the wild-type (WT) strain, was utilized for further study. Molecular analysis showed that a 756-bp open reading frame (ORF) (GenBank accession No. CP007576) in the M85 mutant was interrupted by mini-Tn10. This ORF encodes for a 183-amino acid protein and displays the highest sequence identity (99%) with the hemerythrin (Hr) protein of A. hydrophila subspecies hydrophila ATCC 7966. The survival of the WT, M85 mutant, and complemented M85 (Hr) strains were compared in host macrophages in vitro, and the results showed that M85 exhibited defective survival, while that of M85 (Hr) was restored. To investigate the possible mechanisms of A. hydrophila survival in host macrophages, the expression of Hr under hyperoxic and hypoxic conditions was evaluated. The results revealed that the expression of this protein was higher under hyperoxic conditions than under hypoxic conditions, which indicates that Hr protein expression is sensitive to O2 concentration. Hydrogen peroxide sensitivity tests further suggested that the M85 mutant was more sensitive to oxidative stress than the WT and M85 (Hr) strains. Taken together, these results suggest that the Hr protein may act as an O2 sensor and as a detoxifier of reactive oxygen species, and is required for A. hydrophila survival within host macrophages.

FlgN plays important roles in the adhesion of Aeromonas hydrophila to host mucus.

Adhesion to the host mucus is a crucial step in the early infection stage of pathogenic bacteria. To investigate the mechanisms of the adhesion of Aeromonas hydrophila to its host mucus, a mutant library was constructed using the mini-Tn10 transposon mutagenesis system. Of 276 individual colonies, the mutant strain with the most attenuated adhesion ability in this study was screened out and designated A77. Molecular analysis showed that a 414-bp sequence flanking mini-Tn10 in A77 had the highest identity (97%) with the bacterial flagellar protein gene flgN. A complemented strain flgN+ was constructed and the biological characteristics of the wild-type, mutant A77, and complemented flgN+ strains were investigated. The results showed that the decreased abilities of motility, adhesion to mucus, and biofilm formation in the mutant strain were partially recovered in the complemented flgN+ strain, which suggested that flgN plays an important role in the adhesion of A. hydrophila to its host.

Genotoxicity of surface water samples from Meiliang Bay, Taihu Lake, Eastern China.

Taihu Lake is the third largest freshwater lake in China. Taihu Basin is one of the most densely populated and urbanized areas in this country. This area provides 15% of the GDP. Meiliang Bay is located in the north part of the Lake. It provides the municipal water source for Wuxi City. Some parts of the lake have been found to be highly polluted due to eutrophication for over a decade. Surface water (0-0.5 m) samples were collected from the Meiliang Bay by the aid of Global Position System (GPS) for positioning. Water samples were concentrated 5000 times with XAD-2 resin columns. A reverse mutation test using histidine-dependent Salmonella typhimurium strains was employed to assay the genotoxicity of the samples. The results showed that the sample from position 6 had the highest genotoxicity either in the case of activating with eucaryotic S9 system or without S9. The genotoxic effect included, at least, two different molecular mechanisms: nucleotide point substitution on DNA molecules and reading frame shifting caused by nucleotide insertion or deletion. The genotoxicity of the water body in Meiliang Bay, Taihu Lake should be kept in close monitoring.

Association of somatotropin (BST) gene polymorphism at the 5th exon with selection for milk yield in Holstein cows.

A selection project produced control cows from continuous matings with breed average bulls for predicted transmitting ability for milk (PTA-milk) in 1964 and select cows from matings to four of the highest PTA-milk bulls each year since 1964. Blood samples were collected in 1992 when milk yield difference of select and control line cows exceeded 3,800 kg of milk/305-day lactation. Genomic DNA from control (n = 49) and select (n = 101) cows was analyzed for the presence of variants associated with amino acid position 127 (leucine, AluI[+]; valine, AluI[-]) of bovine somatotropin (bST). Amplification of a 428 base-pair fragment of the bST gene from individual cows, subsequent restriction enzyme (AluI) digestion, and separation resulting fragments indicated three genotypes AluI(+/+), AluI(+/-), and AluI(-/-) in 110, 39, and 1 animal(s), respectively. Gene frequencies of leucine127 and valine 127 alleles were similar for control (0.867m 0.133) and select (0.861, 0.139) animals. United States Department of Agriculture-PTA values were compared between the two genotypes, AluI(+/+) and AluI(+/-). Estimated breeding value for milk (EBV-milk) and average yield deviation for milk (AYD-milk) were not associated with genotype for control animals. However, presence of the valine allele was correlated with decreased EBV-milk (P = 0.03) and AYD-milk (P = 0.16) in select animals and accounted for a decrease of approximately 170 kg of EBV-milk and 240 kg of AYD-milk.

Ileal symbiont intracellularis, an obligate intracellular bacterium of porcine intestines showing a relationship to Desulfovibrio species.

A new genus and species of obligate intracellular-bacteria found in porcine intestines are described. Growth on any bacteriological medium deprived of living cells has not been demonstrated. The organism has been grown intracellularly in cell culture. The 16S rRNA gene sequence data, DNA probe results, and microscopic observations provide evidence that these bacteria differ from those in other described genera and that they belong to the delta subdivision of the class Proteobacteria. We have amplified and sequenced the 16S ribosomal DNA of four preparations of the intracellular bacterium from pigs. For this, intracellular organisms were released and purified from the infected cells without culture techniques. After DNA purification, the polymerase chain reaction with primers complementary to highly conserved eubacterial sequences was used to amplify regions of 16S ribosomal DNA which were subsequently cloned (in some cases) and sequenced directly by standard techniques. The sequences obtained from each preparation were identical and were most similar to that of a sulfate-reducing proteobacterium, Desulfovibrio desulfuricans ATCC 27774 (91% similarity). An oligonucleotide probe complementary to a hypervariable region of the 16S rRNA sequence of the bacterium hybridized with intracellular organisms obtained from porcine intestines. The bacterium is a gram-negative, curved rod with tapered ends. It multiplies intracellularly in the cytoplasm of ileal epithelial cells by septation. The vernacular name Ileal symbiont (IS) intracellularis is proposed for this bacterium. The type strain of IS intracellularis is strain 1482/89 grown in cell culture from a pig affected by proliferative enteropathy. It is deposited in the National Collection of Type Cultures, Colindale, London, as NCTC 12656.

Detection of bovine leukemia virus in cattle by the polymerase chain reaction.

Bovine leukemia virus (BLV) is widely distributed in U.S. cattle herds. It infects B lymphocytes and causes neoplastic disease in 5-10% of infected animals. Direct economic losses are incurred as a result of death, reduced milk production and condemnation at slaughter. Thus the identification of cattle infected with BLV is of significant concern to the U.S. cattle industry. For this reason, polymerase chain reaction (PCR) amplification was used to examine seropositive and seronegative cattle for the presence of BLV DNA in peripheral blood mononuclear cells. Using an amplification protocol able to detect 1 viral genome in 100,000 cells, BLV was not detected in 7 seronegative cattle in an infected herd. BLV sequences were detected in 13 of 18 seropositive animals with various levels of infection as determined by in vitro lymphocyte culture and electron microscopy. An active infection was demonstrated in one animal, based on the presence of viral RNA. These findings indicate that PCR is a sensitive method for the detection of BLV in cattle and provides new information regarding the dynamics of the infection.

Cloned DNA probes specific for the intracellular Campylobacter-like organism of porcine proliferative enteritis.

Although the etiology of porcine proliferative enteritis is not understood, the consistent presence of intracellular Campylobacter-like organisms (CLOs) in proliferating pig intestinal epithelial cells suggests that the organism is involved in the disease process. In order to obtain information about this organism, we generated and characterized specific DNA probes to the intracellular CLO which was purified without culturing. Intracellular CLOs were isolated from mucosa by homogenization, filtration, and absorption to wheat germ agglutinin-Sepharose. The DNA was purified, and a CLO genomic library was constructed. The specificity of recombinant plasmids was confirmed by both dot blot hybridization and Southern analysis of normal and diseased mucosa, as well as of a variety of Campylobacter species. Several of the CLO-specific probes hybridized with porcine mucosa obtained from pigs with proliferative enteritis but not with nondiseased mucosa. The probes hybridized equally with mucosa or DNA obtained from each of the two clinical forms of proliferative enteritis, i.e., proliferative hemorrhagic enteropathy and porcine intestinal adenomatosis. The CLO-specific probes failed to hybridize with any of the commonly isolated porcine Campylobacter species, including Campylobacter hyointestinalis, C. mucosalis, and C. coli. Therefore, the intracellular CLO of porcine proliferative enteritis may be an as yet unidentified or uncultured species.

Southern blot analysis of strain variation in Campylobacter mucosalis.

A panel of three DNA probes were derived at random from a genomic DNA library of Campylobacter mucosalis strain E8384-4. Each probe hybridized specifically to C. mucosalis DNA from bacteria fixed to nylon membranes. The probes did not hybridize to DNA from other Campylobacter species or to other bacteria even at 100-fold higher amounts. Each probe hybridized to all of 24 isolates of C. mucosalis which had been collected over time from different geographic locations. Southern blot analysis of selected C. mucosalis isolates was carried out to determine if the probes would be useful for differentiating among various isolates. It indicated that restriction fragment length polymorphisms (RFLPs) exist at the loci identified by our probes. These differences were used to characterize seven C. mucosalis isolates recovered from pigs in Minnesota. The results suggest that RFLP analysis may be a useful tool for epidemiological studies of C. mucosalis.

The patterns of stress response in patients undergoing thyroid surgery under acupuncture anaesthesia in China.

The patterns of catecholamines (adrenaline and noradrenaline), peptide hormones (adrenocorticotropic hormone, antidiuretic hormone, beta-endorphin, growth hormone and prolactin), hydrocortisone (cortisol) and those of immunoglobulins (IgA, IgG and IgM) and total and differential leucocyte counts in the peripheral blood were investigated during and for 6 days after thyroid surgery in 20 patients (F/M: 18/2) performed under acupuncture anaesthesia, supplemented by small doses of pethidine (mean: 45.0 mg, s.d. 8.9). Throughout surgery the patients remained conscious. During surgery a significant increase in the level of catecholamines and the above-mentioned circulating hormones and a decrease of immunoglobulins were observed, whereas the leucocyte and differential counts demonstrated leucocytosis due to lymphocytosis, a decreased percentage of eosinophils and a remarkably reduced percentage of neutrophils. In the postoperative phase, levels of noradrenaline and beta-endorphin remained elevated, whereas the other circulating hormones gradually returned to normal values. Immunoglobulin levels and eosinophil counts returned to the preinduction values within 24 h, and those of neutrophil and lymphocyte counts within 2 days. Changes in number of monocytes and basophils could not be detected peri- and postoperatively.

Species-specific DNA probes for Campylobacter species isolated from pigs with proliferative enteritis.

Cloned, chromosomal DNA probes from porcine isolates of Campylobacter hyointestinalis and C. mucosalis were developed for the detection and identification of these putative swine enteric pathogens. High molecular weight chromosomal DNA from each species was used to construct genomic libraries in plasmids. Recombinants were selected which hybridized strongly to the homologous organism, but not to any other species of Campylobacter. Species-specific recombinants were labeled with phosphorus-32 and tested for sensitivity by dot blot hybridization to various dilutions of DNA and bacteria from each swine species, including C. hyointestinalis, C. mucosalis, C. coli and C. jejuni. Specificity was tested by hybridizing these probes against various strains of C. hyointestinalis or C. mucosalis, and against reference strains of all other described Campylobacter species. A C. hyointestinalis-specific probe and a C. mucosalis-specific probe were identified which were capable of detecting 1 ng of DNA or 10(4) cfu by bacterial spot blotting on nylon membranes. These probes hybridized to intestinal mucosal scrapings containing C. hyointestinalis and C. mucosalis obtained from pigs with proliferative enteritis, but not to material from normal pigs. Thus, cloned, chromosomal DNA probes may be useful in the detection and identification of bacteria involved in swine proliferative enteritis.

Acupuncture anaesthesia. Observations on its use for removal of thyroid adenomata and influence on recovery and morbidity in a Chinese hospital.

Acupuncture anaesthesia, supplemented by small doses of pethidine, was evaluated in 20 patients who had surgery for removal of a thyroid adenoma. There were significant increases in mean arterial pressure and respiratory rate during surgery, but no significant change in heart rate. The mean dose of pethidine given during surgery was 45 mg (SD 8.9). Postoperative recovery was rapid and complication free. Acupuncture anaesthesia did not provide complete analgesia, but was safe and preferable to general anaesthesia where there was a shortage of facilities.

Peripheral leukocytes in psoriasis.

The white blood cell (WBC) count and differential count, including the morphology and acid alpha-naphthyl acetate esterase (ANAE) activity of lymphocytes, in psoriatic patients of different types, stages, and extensiveness of skin lesions were analyzed. The number of total WBC and polymorphonuclear leukocytes was markedly increased in psoriasis erythrodermica and psoriasis pustulosa. Lymphocytopenia, especially T-lymphocytopenia, was noticed in all types of psoriasis. Antineoplastic drugs and immunosuppressants intensified the degree of T-lymphocytopenia. This might be the cause of recurrence and more recalcitrant course of the disease. Therefore, it is considered that these drugs should not be the first choice in the treatment of psoriasis.

Association between a satellite DNA sequence and the Responder of Segregation Distorter in D. melanogaster.

A large array of satellite DNA sequences are always associated with the Responder (Rsp) element of Segregation Distorter in D. melanogaster. In the appropriate genetic backgrounds, Rsp causes aberrant chromatin condensation in spermiogenesis, leading to dysfunction of sperm carrying Rsp, and meiotic drive. The repeat array is deleted or translocated to the Y chromosome whenever Rsp is. Moreover, the translocation of part of Rsp to Y is associated with the translocation of an incomplete repeat array. The number of repeats among 35 independently derived chromosomes correlates nearly perfectly with sensitivity to distortion. We hypothesize that this satellite repeat array represents Responder itself. Finally, the molecular structure of this locus is extremely variable, indicating a very active process of change.