Anterolateral femoral bowing and loss of thigh muscle are associated with occurrence of atypical femoral fracture: Effect of failed tension band mechanism in mid-thigh.

BACKGROUND: The purpose of this study was to characterize anterolateral bowing of the femur using X-rays and muscular atrophy in the mid-thigh using computed tomography (CT) in patients with atypical femoral fractures (AFFs). We then compared the results with those of an intertrochanteric fracture to understand whether these measures act as causative factors of AFFs. METHODS: From January 2009 to December 2015, 37 patients with complete AFF and 12 patients with incomplete AFF were enrolled in this study. Lateral femoral bowing, anterior femoral bowing, cross-sectional area (CSA), and attenuation coefficient of thigh muscles in the AFF group are measured and compare with those in the intertrochanteric fracture group. RESULTS: Lateral and anterior femoral bowing in the AFF group were significantly higher than those in the intertrochanteric fracture group. The level of fracture was found to be significantly associated with lateral and anterior femoral bowing (r = 0.569, r2 = 0.324, p < 0.001; r = -0.530, r2 = 0.281, p < 0.001, respectively). Total CSA and CSA of anterior and medial compartments were significantly lower in the AFF group (p < 0.05). The attenuation coefficient of the total thigh muscle and all three compartments in the AFF group were significantly lower than those in the intertrochanteric fracture group (p < 0.05). CONCLUSIONS: This study demonstrated that anterolateral femoral bowing and loss of thigh muscle were highly associated with the occurrence of AFFs.

False-negative results using Neisseria gonorrhoeae porA pseudogene PCR - a clinical gonococcal isolate with an N. meningitidis porA sequence, Australia, March 2011.

The gonococcal porA pseudogene is a popular target for in-house Neisseria gonorrhoeae PCR methods. With this study we present two novel findings: the first case of an N. gonorrhoeae porA pseudogene PCR false-negative result caused by sequence variation, and in the same organism, the first description of a clinical N. gonorrhoeae strain harbouring an N. meningitidis porA sequence.

Functional role for the conformationally mobile phenylalanine 223 in the reaction of methylenetetrahydrofolate reductase from Escherichia coli.

The flavoprotein methylenetetrahydrofolate reductase from Escherichia coli catalyzes the reduction of 5,10-methylenetetrahydrofolate (CH(2)-H(4)folate) by NADH via a ping-pong reaction mechanism. Structures of the reduced enzyme in complex with NADH and of the oxidized Glu28Gln enzyme in complex with CH(3)-H(4)folate [Pejchal, R., Sargeant, R., and Ludwig, M. L. (2005) Biochemistry 44, 11447-11457] have revealed Phe223 as a conformationally mobile active site residue. In the NADH complex, the NADH adopts an unusual hairpin conformation and is wedged between the isoalloxazine ring of the FAD and the side chain of Phe223. In the folate complex, Phe223 swings out from its position in the NADH complex to stack against the p-aminobenzoate ring of the folate. Although Phe223 contacts each substrate in E. coli MTHFR, this residue is not invariant; for example, a leucine occurs at this site in the human enzyme. To examine the role of Phe223 in substrate binding and catalysis, we have constructed mutants Phe223Ala and Phe223Leu. As predicted, our results indicate that Phe223 participates in the binding of both substrates. The Phe223Ala mutation impairs NADH and CH(2)-H(4)folate binding each 40-fold yet slows catalysis of both half-reactions less than 2-fold. Affinity for CH(2)-H(4)folate is unaffected by the Phe223Leu mutation, and the variant catalyzes the oxidative half-reaction 3-fold faster than the wild-type enzyme. Structures of ligand-free Phe223Leu and Phe223Leu/Glu28Gln MTHFR in complex with CH(3)-H(4)folate have been determined at 1.65 and 1.70 A resolution, respectively. The structures show that the folate is bound in a catalytically competent conformation, and Leu223 undergoes a conformational change similar to that observed for Phe223 in the Glu28Gln-CH(3)-H(4)folate structure. Taken together, our results suggest that Leu may be a suitable replacement for Phe223 in the oxidative half-reaction of E. coli MTHFR.

S phase-specific proteolytic cleavage is required to activate stable DNA binding by the CDP/Cut homeodomain protein.

The CCAAT displacement protein (CDP), the homologue of the Drosophila melanogaster Cut protein, contains four DNA binding domains that function in pairs. Cooperation between Cut repeat 3 and the Cut homeodomain allows stable DNA binding to the ATCGAT motif, an activity previously shown to be upregulated in S phase. Here we showed that the full-length CDP/Cut protein is incapable of stable DNA binding and that the ATCGAT binding activity present in cells involves a 110-kDa carboxy-terminal peptide of CDP/Cut. A vector expressing CDP/Cut with Myc and hemagglutinin epitope tags at either end generated N- and C-terminal products of 90 and 110 kDa, suggesting that proteolytic cleavage was involved. In vivo pulse/chase labeling experiments confirmed that the 110-kDa protein was derived from the full-length CDP/Cut protein. Proteolytic processing was weak or not detectable in G(0) and G(1) but increased in populations of cells enriched in S phase, and the appearance of the 110-kDa protein coincided with the increase in ATCGAT DNA binding. Interestingly, the amino-truncated and the full-length CDP/Cut isoforms exhibited different transcriptional properties in a reporter assay. We conclude that proteolytic processing of CDP/Cut at the G(1)/S transition generates a CDP/Cut isoform with distinct DNA binding and transcriptional activities. These findings, together with the cleavage of the Scc1 protein at mitosis, suggest that site-specific proteolysis may play an important role in the regulation of cell cycle progression.

The high-potential flavin and heme of nitric oxide synthase are not magnetically linked: implications for electron transfer.

BACKGROUND: The homodimeric nitric oxide synthase (NOS) catalyzes conversion of L-arginine to L-citrulline and nitric oxide. Each subunit contains two flavins and one protoporphyrin IX heme. A key component of the reaction is the transfer of electrons from the flavins to the heme. The NOS gene encodes two domains linked by a short helix containing a calmodulin-recognition sequence. The reductase domain binds the flavin cofactors, while the oxygenase domain binds heme and L-arginine and additionally mediates the dimerization of the NOS subunits. We investigated the origin of the unusual magnetic properties (rapid-spin relaxation) of an air-stable free radical localized to a reductase domain flavin cofactor. RESULTS: We characterized the air-stable flavin in wild-type NOS, both in the presence and absence of calcium and calmodulin, the imidazole-bound heme complex of wild-type NOS, the NOS Cys415-->Ala mutant, and the isolated reductase domain. All preparations of NOS had the same flavin electron-spin relaxation behavior. No half-field transitions or temperature-dependent changes in the linewidth of the radical spin signal were detected. CONCLUSIONS: These data suggest that the observed relaxation enhancement of the NOS flavin radical is caused by the environment provided by the reductase domain. No magnetic interaction between the heme and flavin cofactors was detected, suggesting that the flavin and heme centers are probably separated by more than 15 A.

Exon/intron structure and alternative transcripts of the CUTL1 gene.

The human CUTL1 gene (Cut-like 1) is a candidate tumor suppressor gene located on chromosome 7 at band 22, a region that is frequently deleted in several human cancers. The gene spans at least 340kb and contains 33 exons. Synthesis of five different transcripts involves two promoter regions, two polyadenylation sites and seven alternative splicing events. The two polyadenylation sites are located at the ends of exons 24 and 33 and are separated by approximately 40kb. Transcription is initiated in two genomic regions, giving rise to alternate first exons which are spliced to a common exon 2. All transcripts contain exons 2 to 14, but differ in their 3' regions. Exon 14 can be spliced alternatively to the beginning or the middle of exon 15, or to exon 25, generating transcripts with exons 15 to 24 or exons 25 to 33. Moreover, exon 16 can be spliced out from the mature transcripts that contain exons 15 to 24. Overall, five distinct transcripts are generated as a result of alternative transcription initiation, splicing and polyadenylation. We discuss potential mechanisms by which alternate polyadenylation site usage may affect alternative splicing events and vice versa.

The effect of three Korean traditional medicines on the growth rate of cultured human keratinocytes.

The effect of three different Korean Traditional Medicines (KTM) was studied on several functional parameters of adult human cells in culture. The cells were non-transformed strains of normal, skin epidermal cells (keratinocytes) from adult humans. Aqueous extracts of the herbal medicines were tested using two types of cell strains: one type was essential fatty acid deficient (EFAD) cells which grow rapidly in medium that was low in calcium and had no essential fatty acids; the second type was a cell strain grown in medium supplemented with essential fatty acid (EFA-supplemented). These cells had much slower, in vivo skin growth rates, and the fatty acid composition resembled that measured in epidermal biopsy tissue. The KTMs chosen for this study were tae-gang-hual-tang (for treating osteoarthritis), hual-ak-tang (for pain relief) and sip-zeon-tae-bo-tang (for fortifying immune systems). Because high proliferation rates usually correlate with skin inflammation and because many of the chemotactic agents mediating inflammatory response are modified fatty acids, this study focused on cell growth rate and membrane fatty acid composition as signals for the effects of the herbal medicines. By monitoring growth rate, these experiments measured both a stimulatory and a regulatory effect on the growth of keratinocytes. Some toxicity was seen at the highest doses of the KTMs. These effects were modeled mathematically, and the results showed varying effects on growth rate depending on dose and herbal recipe. The fitting parameters were discussed as they relate to biological function. The experimental design was also discussed and alternatives were suggested.

Antibiotic-induced lethal enterocolitis in hamsters: studies with eleven agents and evidence to support the pathogenic role of toxin-producing Clostridia.

Clindamycin-induced enterocolitis in hamsters was studied, using a tissue culture assay to detect clostridial toxin. It was found that animals with lethal enterocolitis had a cytopathogenic substance in cecal contents and blood that was neutralized by clostridial antitoxins. Cultures of the cecal flora yielded numerous species of clostridia, but only 1 organism was detected which produced a toxin which was cytopathic in tissue culture. This organism, Clostridium difficile, was consistently present in high concentrations, and the cell-free supernate of these strains caused enterocolitis if injected intracecally into hamsters. Ten additional antimicrobials were tested ih hamsters. Ampicillin, vancomycin, erythromycin, cephalosporins, and oral gentamicin caused lethal enterocolitis in most recipients, and all animals which died had evidence of clostridia toxin in cecal contents at necropsy. Tetracycline and metronidazole were well tolerated, and the animals given these antimicrobials had no evidence of the toxin. We conclude that toxin-producing clostridia are responsible for lethal enterocolitis due to a variety of antimicrobials in hamsters.

Ebselen pretreatment attenuates ischemia/reperfusion injury and prevents hyperglycemia by improving hepatic insulin signaling and ß-cell survival in gerbils.

Transient carotid artery occlusion causes ischemia/reperfusion (I/R) injury resulting in neuron and pancreatic ß-cell death with consequential post-stroke hyperglycemia, which can lead to diabetes and may accelerate the development of Alzheimer's disease. Antioxidants have been shown to protect against the I/R injury and destruction of neurons. However, it is unknown whether the protection against I/R injury extends to the pancreatic ß-cells. Therefore, we investigated whether treatment with ebselen, a glutathione peroxidase mimic, prevents neuronal and ß-cell death following I/R in gerbils susceptible to stroke. After 28 days post artery occlusion, there was widespread neuronal cell death in the CA1 of the hippocampus and elevated IL-1ß and TNF-α levels. Pretreatment with ebselen prevented the death by 56% and attenuated neurological damage (abnormal eyelid drooping, hair bristling, muscle tone, flexor reflex, posture, and walking patterns). Ischemic gerbils also exhibited impaired glucose tolerance and insulin sensitivity which induced post-stroke hyperglycemia associated with decreased ß-cell mass due to increased ß-cell apoptosis. Ebselen prevented the increased ß-cell apoptosis, possibly by decreasing IL-1ß and TNF-α in islets. Ischemia also attenuated hepatic insulin signaling, and expression of GLUT2 and glucokinase, whereas ebselen prevented the attenuation and suppressed gluconeogenesis by decreasing PEPCK expression. In conclusion, antioxidant protection by ebselen attenuated I/R injury of neurons and pancreatic ß-cells and prevented subsequent impairment of glucose regulation that could lead to diabetes and Alzheimer's disease.

Prognostic significance of molecular subtype in T1N0M0 breast cancer: Korean experience.

PURPOSE: Gene expression profiling studies have identified several breast cancer subtypes associated with markedly different clinical outcomes. In general, patients with stage I breast cancer have excellent outcomes. We assessed the clinicopathological characteristics and outcomes of patients with T1N0M0 breast cancer according to molecular subtype. METHODS: Seven hundred and sixty-two T1N0M0 breast cancer patients undergoing curative surgery between January 1990 and December 2007 were analyzed. Subtypes were classified according to hormone receptor (HR) and human epidermal growth factor receptor-2 (HER2) status as follows: HR+/HER2-, HR+/HER2+, HR-/HER2- (triple-negative, TN), and HR-/HER2+. RESULTS: The distribution of subtypes was HR+/HER2-, 56.6%; HR+/HER2+, 10.1%; TN, 20.1%; and HR-/HER2+, 13.3%. Marked differences were observed among subtypes in multifocality/multicentricity, histological grade, extensive intraductal components, p53 expression and the Ki-67 index. There were differences in recurrence-free survival and overall survival among patients with different molecular subtypes (log-rank p < 0.001 and 0.024, respectively). By multivariate analysis, lymphovascular invasion and classification of molecular subtype were independent predictors of recurrence (p = 0.003 and 0.043, respectively). The TN subtype showed significantly worse recurrence-free survival compared to the HR+/HER2- subtype (hazard ratio, 4.54; 95% confidence interval, 1.60-12.86; p = 0.004). CONCLUSION: Patients with T1N0M0 breast cancer, a group with generally favorable clinical outcomes, had prognoses that were associated with the molecular subtype. The TN subtype was an independent predictor for recurrence in patients with T1N0M0 breast cancer.

Adamantinoma of the appendicular skeleton in children.

The 260 cases of adamantinoma described in the world literature have been reviewed. Case histories of 60 patients up to the age of 16 years mirror those of adults. The mortality rate is 20% in children and adults. Wide excision with bone grafting is the ideal treatment for patients under 16 years.

Progression of epiphytic microflora in wheat and alfalfa silages as observed by scanning electron microscopy.

Wheat and alfalfa silages were examined by scanning electron microscopy and standard methods of microbial enumeration. Epiphytic microflora were present at levels of 10 to 10/g in the fresh-cut plants. This flora was initially observed microscopically primarily on the surfaces. After 4 days of fermentation, lactic acid bacteria were observed on the surface in high concentrations near open stomata and throughout the interior mesophyll air sac spaces. At 4 days, populations on interior surfaces were restricted to the exterior surfaces of the air sacs. After 8 days the mesophyllic cells showed marked deterioration, and bacteria were observed on their inner surfaces. At 32 days, the end of the fermentation, vascular bundles and epidermal cells remained intact whereas stomata and mesophyllic cells were collapsed and often contained microorganisms. It is concluded that the interior of the leaves offers substantial nutritional and environmental advantages to epiphytic flora and is an important if not major deterioration site in fermented products. Since little deterioration of exterior surfaces was observed, these sites may play a minor role in supplying nutrients for microbial growth.

Adamantinoma of the appendicular skeleton--updated.

An updated review of adamantinoma of the appendicular skeleton now provides 195 well-documented cases from the world literature. An additional five new cases are added. Statistically, the tumor remains unusually prevalent in the tibia, but all other major limb bones have been involved, and involvement of several short bones rarely has been reported. The neoplasm is more commonly found in males, but higher earlier age incidence is found in females. The frequent history of preceding trauma may indeed be important in tumor formation. The histogenesis of the tumor is now considered to be epithelial in origin by ultrastructural and immunohistochemical methods. A high incidence of recurrence or metastases is found with inadequate cancer surgery. Known mortalities have indicated severe metastatic disease by aggressive-appearing cells. Previously, early amputation had provided good results, but wide excision or segmental resection with grafting techniques are equally successful. The recent work with allograft replacement of a widely excised segment has shown good early results.

Regulation of the soxRS oxidative stress regulon. Reversible oxidation of the Fe-S centers of SoxR in vivo.

SoxR protein, a transcriptional activator of the soxRS (superoxide response) regulon of Escherichia coli, contains autooxidizable [2Fe-2S] centers that are presumed to serve as redox sensors. In vitro transcription experiments previously demonstrated that only the oxidized form is active. Reduced SoxR was detected in overproducing strains by EPR spectroscopy of suspensions of intact cells. Oxidized Fe-S centers were determined by lysing the cells and treating them with the reducing agent sodium dithionite prior to EPR measurements. In uninduced cells, 90% of the SoxR was in the reduced form. Treatment with the redox cycling agents phenazine methosulfate or plumbagin was accompanied by reversible oxidation of the Fe-S centers. Mutant SoxR derivatives that were constitutively activated existed constitutively in an oxidized state. The results indicate the presence of a cellular pathway for countering the autooxidation of SoxR and confirm the hypothesis that induction of the regulon is mediated by a shift in the redox equilibrium of SoxR rather than by assembly of its Fe-S clusters.

Commensalistic Interaction Between Lactobacillus acidophilus and Propionibacterium shermanii.

Propionibacterium shermanii and Lactobacillus acidophilus were grown in batch mixed culture in a 5-liter fermenter under controlled conditions of pH 5.8 and 35 degrees C on a semisynthetic medium with glucose as an energy source. Cellular efficiencies and fermentation balances were developed for this pair and compared with P. shermanii grown in pure culture on glucose, lactate, and a mixture of these substrates and with L. acidophilus grown on glucose. P. shermanii had ATP yield coefficient values of 17 for each substrate alone but had an average value of 30 for substrate mixtures. Growth rates were similar for P. shermanii on glucose or lactate but higher cell yields were observed for glucose. P. shermanii used both lactate and glucose in mixed substrate until lactate was exhausted, and growth rates slowed thereafter. L. acidophilus had a similar ATP yield coefficient of 15 but produced lower cell yields than did P. shermanii on glucose. Mixed culture of both microorganisms on glucose resulted in much faster and nearly equal growth rates for both and no lactate accumulation in the medium. Acetic acid production rates per generation were lower in mixed culture, suggesting use by the growing culture. The cause of the synergistic effect was not determined but may be due to the rapid production and removal of lactate or CO(2) enhancement in mixed culture.

CCAAT displacement activity involves CUT repeats 1 and 2, not the CUT homeodomain.

The CCAAT displacement protein, the homolog of the Drosophila melanogaster CUT protein, contains four DNA-binding domains: three CUT repeats (CR1, CR2, and CR3) and the CUT homeodomain (HD). Using a panel of fusion proteins, we found that a CUT repeat cannot bind to DNA as a monomer, but that certain combinations of domains exhibit high DNA-binding affinity: CR1+2, CR3HD, CR1HD, and CR2HD. One combination (CR1+2) exhibited strikingly different DNA-binding kinetics and specificities. CR1+2 displayed rapid on and off rates and bound preferably to two C(A/G)AT sites, organized as direct or inverted repeats. Accordingly, only CR1+2 was able to bind to the CCAAT sequence, and its affinity was increased by the presence of a C(A/G)AT site at close proximity. A purified CCAAT displacement protein/CUT protein exhibited DNA-binding properties similar to those of CR1+2; and in nuclear extracts, the CCAAT displacement activity also required the simultaneous presence of a C(A/G)AT site. Moreover, CR1+2, but not CR3HD, was able to displace nuclear factor Y. Thus, the CCAAT displacement activity requires the presence of an additional sequence (CAAT or CGAT) and involves CR1 and CR2, but not the CUT homeodomain.

Recovery of Lactobacillus bulgaricus and Streptococcus thermophilus on Nine Commonly Used Agar Media.

Of the nine media tested, Eugon, Elliker's lactic agar, pH 6.8, and modified tryptic soy broth agars showed superior recovery of Lactobacillus bulgaricus and Streptococcus thermophilus strains.

The 3M Automatic Colony Counter in the Bacterial Evaluation of Manufacturing-Grade Milk.

Twenty-seven raw milk samples were analyzed for their bacterial quality by the Standard Plate Count (SPC) and the plate loop count (PLC). All resultant plates were counted by three different technicians either manually or with a 3M Brand Automatic Colony Counter, Model 630. Results emphasize the superiority of the SPC over the PLC for samples with high bacterial numbers. The automatic colony counter tended to underestimate the number of colonies on a plate. Correction factors for conversion of PLC counts to SPC and automatic to manual counts were calculated. It is recommended that the PLC and the automatic colony counter be used with appropriate correction factors for routine analysis of large numbers of samples.

Methods for quantitative vaginal flora studies.

Quantitative bacteriology of the vaginal flora was performed with two techniques: One was a quantitative loop of vaginal secretions and the second was duplicate swabs of the vaginal fornix with one swab for bacteriologic studies and the second to determine specimen weight. Both sampling methods were evaluated for accuracy in weight or volume measurements. Comparison of the two methods in eight volunteers showed reasonably good correlation although the duplicate swab technique gave somewhat higher mean counts and more bacterial species. Advantages and limitations of these methods for quantitative vaginal flora studies are discussed.

Role of Clostridium difficile in antibiotic-associated pseudomembranous colitis.

Tissue cultures were performed on stools from 189 patients to detect a cytopathic toxin which is neutralized by Clostridium sordellii antitoxin. Specimens satisfying these criteria were considered positive in the tissue culture assay. Stools from 26 of 27 patients with antibiotic-associated pseudomembranous colitis were positive and 16 of these specimens showed toxin titers of 10(-3) dilutions or greater. The tissue culture assay was positive with specimens from 9 of 63 patients with antibiotic-associated diarrhea without documented pseudomembrane formation. Stools from patients with neonatal necrotizing enterocolitis, ulcerative colitis, and healthy controls were uniformly negative in this assay. Cultures were performed on stools from 38 patients with antibiotic-associated diarrhea or colitis to detect clostridia which produce a cytopathic toxin in vitro. Clostridium difficile was recovered from 6 of 8 specimens which were positive in the tissue culture assay and 5 of 30 which were negative in this assay. C. sordellii was recovered in a single specimen. One hundred and nine clostridia strains were tested in the tissue culture assay and C. difficile was the only species which produced a cytopathic toxin. All strains of this organism were positive in the tissue culture assay and, in each instance, cytotoxicity was neutralized by C. sordellii antitoxin. These results indicate that C. difficile is the major cause of antibiotic-associated pseudomembranous colitis and offer an explanation for previous studies showing that the cytotoxin of stools from these patients is neutralized by C, sordellii antitoxin.