Development of 19F-NMR chemical shift detection of DNA B-Z equilibrium using 19F-NMR.

Various DNA conformational changes are in correlation with biological events. In particular, DNA B-Z equilibrium showed a high correlation with translation and transcription. In this study, we developed a DNA probe containing 5-trifluoromethylcytidine or 5-trifluoromethylthymidine to detect DNA B-Z equilibrium using 19F-NMR. Its probe enabled the quantitative detection of B-, Z-, and ss-DNA based on 19F-NMR chemical shift change.

Limb salvage using bypass to the perigeniculate arteries.

OBJECTIVE: To describe bypass to perigeniculate vessels for limb salvage. DESIGN: Retrospective cohort study. MATERIAL AND METHODS: Between 1995 and 2009, 47 bypass procedures to perigeniculate collateral arteries were performed in 46 patients (15 women, 31 men; median age, 68 years). All patients presented with critical ischaemia (tissue loss in 87.5%, rest pain in 12.5%). Mean ankle brachial index was 0.27 ± 0.17. The site of distal anastomosis was the descending genicular artery (DGA) in 23 bypasses (1 bilateral) and the medial sural artery (MSA) in 24. Proximal anastomosis was to the external iliac artery in 2 cases, common femoral artery in 23 cases, superficial femoral artery in 8 cases, deep femoral artery in 8 cases, above-knee poplitaeal artery in 2 cases, and previous graft in 4 cases. RESULTS: There were four deaths during the immediate postoperative period. Mean follow-up duration was 27 months. Ten patients required major amputation. Mean ankle brachial index post-operatively was 0.60 ± 0.21. At 3 years, primary patency was 74.7 ± 7%, secondary patency was 83.4 ± 8%, and the limb salvage and survival rates were 73.5 ± 7% and 77.4 ± 7%, respectively. CONCLUSION: Bypass to perigeniculate arteries is a viable treatment option for critical limb ischaemia in selected patients.

Human toxocariasis: diagnosis, worldwide seroprevalences and clinical expression of the systemic and ocular forms.

Although human toxocariasis ranks among the most common zoonotic infections worldwide, it remains relatively unknown to the public. The causal agents are the nematode parasites Toxocara canis and T. cati, whose definitive hosts are dogs and cats, respectively. When embryonated eggs are accidentally ingested by humans, larvae hatch in the small intestine, penetrate the intestinal wall and migrate, via the bloodstream, to the liver, lungs, muscles, eye and central nervous system. Although most human infections are asymptomatic, two well-defined clinical syndromes are classically recognised: visceral larva migrans (a systemic disease caused by larval migration through major organs) and ocular larva migrans (a disease limited to the eyes and optic nerves). Two less-severe syndromes have recently been described, one mainly in children (covert toxocariasis) and the other mainly in adults (common toxocariasis). Here, the current laboratory diagnosis, epidemiology and main clinical features of both the systemic and ocular forms of human toxocariasis are reviewed. New developments in serological diagnosis are described, the available seroprevalence data are analysed, and the results of relevant clinical studies that have been published over the last decade are explored, to provide an updated overview of this neglected but highly prevalent human infection.

Visual dominance in amending the directional parameter of feedforward control.

The authors examined visual dominance between trials in which the movement program was amended (i.e., off-line processing). Weighting between visual and proprioceptive feedback was examined in a trial-by-trial analysis of the directional parameter of feedforward control. Eight participants moved a cursor to a target displayed on a computer screen by manipulating a hand-held stylus on a digitizing tablet. In the first 30 trials, the cursor followed the stylus movement (practice condition). In the next 30 trials, the directional error of the stylus movement was presented in the opposite direction (reversal condition). Subjects knew the presence and the nature of the reversal. In the last 10 trials, the reversal was withdrawn (transfer condition). Directional error of feedforward control was relatively small in the practice condition, and it increased gradually in 1 of 2 directions as trials proceeded in the reversal condition. Positive aftereffect was observed in the transfer condition. A constant increment of the directional error indicated that both visual and proprioceptive feedback are registered, with higher weight on vision, and that weighting between those inputs is determined automatically or is fixed without any strategic control.

Serum levels of immunoglobulins in children with recurrent otitis media.

Seventy-seven children with recurrent otitis media took part in the study at the Department of Otorhinolaryngology and Pediatrics of the University of São Paulo from February 1992 to March 1995. They were administered serum immunoglobulins and the relationship between recurrent otitis media and immunodeficiency was evaluated. No total IgG and IgM values were detected below the normal level; however, seven children with low levels of subclasses of IgG were detected and a below normal IgA value was detected in a child.

Human T-cell lymphotropic virus type 1 infection and ocular manifestations in São Paulo, Brazil.

OBJECTIVES: Although human T-cell lymphotropic virus type 1 (HTLV-1)-associated uveitis has been well recognized in Japan, related studies in Brazil are scarce. We performed a serologic survey for HTLV-1 infection among patients with uveitis and investigated the ocular findings in HTLV-1-asymptomatic carriers. METHODS: One hundred ninety serum samples from patients with uveitis of determined (n = 137) and undetermined origins (n = 53) being examined at the Uveitis Service, University of São Paulo, São Paulo, Brazil, underwent testing using HTLV enzyme-linked immunosorbent assay and discriminatory Western blots. One hundred five asymptomatic blood donors and/or their relatives who were seropositive for HTLV-1 (carrier group) and 105 age- and sex-paired blood donors who were seronegative for HTLV-1 (control group) underwent ocular evaluation. For the statistical analysis, chi2 test was used. RESULTS: Only 1 patient with uveitis was seropositive for HTLV- 1, and she belonged to the group with uveitis of undetermined origin. Results of tear films were evaluated in 52 carriers. The prevalence of a decreased tear break-up time was significantly higher in the carrier compared with the control group (P = .02). Two carriers had keratoconjunctivitis sicca. Three of the 105 carriers exhibited mild uveitis (cells in the vitreous, retinal and choroidal infiltrates, retinal vasculitis, and bilateral pars planitis). Retinal pigmentary changes were found in both groups (no statistical difference). CONCLUSIONS: Early tear abnormalities may be present in asymptomatic carriers, and mild uveitis may be found among them. The relatively low seroprevalence of HTLV-1 in the Brazilian population made it difficult to establish the real importance of HTLV-1-associated uveitis among our patients with uveitis.

Eye disease in a patient with rheumatoid arthritis.

We report the case of a 40-year-old woman with diffuse uveitis, sensorineural hearing loss and cerebrospinal fluid pleocytosis as features of Vogt-Koyanagi-Harada syndrome who developed symmetric polyarthritis and stiffness of small and large joints, in addition to rheumatoid arthritis. Although their target tissues are distinct, both diseases have a possible autoimmune origin strongly associated with HLA-DRB4.

HLA-DRB1*0405 is the predominant allele in Brazilian patients with Vogt-Koyanagi-Harada disease.

Vogt-Koyanagi-Harada (VKH) disease is a rare disorder affecting pigmented structures especially the eye and is the main cause of autoimmune non-infectious uveitis in the Brazilian population. The autoimmune target is believed to be the melanocyte. A strong association of VKH disease with HLA-DR4 in the Japanese population is well known. The same association, albeit with lower relative risks has been found in other populations. A secondary association to HLA-DR1 involving a sequence linked with susceptibility to Rheumatoid Arthritis has also been described. VKH disease is more common in non-Caucasian populations. Brazilian patients of varying ethnic origins have been typed for HLA class II antigens. Several of the features found in other population samples are present. Over half of the patients typed HLA-DR4 (20/37) and typing with sequence-specific oligonucleotides disclosed predominance of the DRB1*0405 allele with a relative risk of 11.76 over the general population. In addition, HLA-DR1 and DQ4 were also present, in patients both positive and negative for HLA-DR4. These results suggest that, as in other autoimmune diseases, multiple overlapping susceptibility factors encoded by the MHC complex contribute to the overall susceptibility for the disease, the major factor however, being the presence of the DRB1*0405 allele.

Imunidade celular ao antígeno S empacientes com uveíte endógena / Cellular immune responses to S-antigen in endogenous uveitis

A auto-imunidade retiniana desempenha um papel na etiopatogenia de várias uveítes endógenas. Estudos experimentais e ensaios clínicos têm demonstrado a importância de antígenos retinianos, como o antígeno S (AgS), näo somente na patogenia mas também na elaboraçäo de estratégias de imunoterapia. O presente trabalho visa analisar o perfil da imunidade celular in vitro ao AgS e a dois de seus peptídeos relevantes, denominados M e G, em uma populaçäo brasileira com diagnóstico de uveíte por doença de Behçet (DB) (n=19), doença de Vogt-koyanagi-Harada (DVKH) (n=27) e vasculite da retina (n=5) acompanhados no serviço de uveíte do Hospital das Clínicas da Faculdade de Medicina da USP. Pacientes com DB sem uveíte (n=17) e 16 controles normais foram também analisados ..

Advanced glycation end products induce expression of vascular endothelial growth factor by retinal Muller cells.

Recent studies have suggested that advanced glycation end products (AGEs) are involved in the development of diabetic complications. To assess the pathogenic role of AGEs and vascular endothelial growth factor (VEGF) in the development of retinal neovascularization in diabetic retinopathy, we investigated the effect of AGEs on induction of VEGF by retinal Muller cells and measured AGE and VEGF concentrations in the vitreous of patients with proliferative diabetic retinopathy (PDR) and nondiabetic patients. The expression of VEGF mRNA and the production of VEGF protein by cultured Muller cells were enhanced by the presence of AGEs. The vitreous concentrations of AGEs and VEGF were both elevated in patients with PDR compared with patients without diabetes (P < 0.01). There was a moderate positive correlation between the levels of crossline and VEGF (r=0.698, P < 0.01). Elevation of AGEs in the vitreous may promote intraocular neovascularization in diabetic retinopathy through production of VEGF from Muller cells.

Preincubation of thymocytes with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) induces apoptosis in non-stimulated thymocytes.

1-(5-Isoquinolinesulfonyl1)-2-methylpiperazine hydrochloride (H-7), an inhibitor of protein kinases, has been shown to inhibit the thymocyte apoptosis induced by various apoptogenic agents. In the present study, when mouse thymocytes were pretreated with H-7, washed, and cultured for an additional time, apoptosis was induced depending on the preincubation time and the dose of H-7. The protein kinase C activity in the H-7-pretreated and -washed cells was not altered, suggesting that an alteration of a certain PKC isoform is related to both the triggering and the progression of apoptosis.

Effects on archery performance of manipulating metamotivational state and felt arousal.

This study investigated the effect of the four different combinations of metamotivational state and felt arousal level (telic-low, paratelic-low, telic-high, paratelic-high) on archery performance. Skilled, average, and novice archers (n = 28) were voluntary subjects, randomly divided into 2 groups, balanced for ability. Each group performed 2 out of the 4 experimental conditions. In this reversal theory-based field experiment, telic and paratelic metamotivational states and arousal level were manipulated prior to archery performance. The hypothesis that archery performance would be superior under telic-low arousal conditions was rejected, but evidence pointed to the possible importance of hedonic tone in performance. Post boc analysis showed differences between combined high (telic-low, paratelic-high) and combined low (telic-low, paratelic-high) hedonic-tone groups which fell short of significance. This observation is taken as justification for further exploration of the relationship between hedonic tone and sports performance.

Staurosporine-induced thymocyte apoptosis is inhibited by herbimycin A, a specific inhibitor of protein tyrosine kinase.

When mouse thymocytes were incubated with staurosporine at low doses (1-100 nM), increased tyrosine phosphorylation of proteins with a molecular weight of 31-34 kD in the detergent-soluble fraction was observed, together with an increase in internucleosomal DNA fragmentation. Incubation with staurosporine for 6 h was enough to trigger and progress of apoptosis. Herbimycin A, a specific inhibitor of protein tyrosine kinases, and cycloheximide, an inhibitor of protein synthesis, reduced the amount of the phosphotyrosine proteins, this being followed by the inhibition of DNA fragmentation and cell death, suggesting a close relationship between protein tyrosine phosphorylation and the induction of apoptosis by staurosporine.

Purification, characterization, and amino acid sequence of foxtail millet trypsin inhibitor III.

One of the major trypsin inhibitors of foxtail millet, Setaria italica, was purified from a seed extract to an electrophoretically homogeneous protein by methods including chromatofocusing and affinity chromatography. This inhibitor (FMTI-III) was shown to be specific and single-headed for trypsin. The molecular weight and the amino acid composition together with the above nature were identical with those of another major trypsin inhibitor (FMTI-II) previously purified from foxtail millet grain. Sequence analysis of FMTI-III indicated that the protein contains 67 amino acid residues, the sequence of which is the same as that of FMTI-II except for the replacement of the C-terminal glutamine by glutamic acid. This single amino acid substitution had no effect on inhibitor-enzyme association.

The complete amino acid sequence of a major trypsin inhibitor from seeds of foxtail millet (Setaria italica).

The complete amino acid sequence of a major trypsin inhibitor (FMTI-II) from seeds of foxtail millet (Setaria italica) was determined by analysis of peptides derived from the reduced and S-carboxymethylated protein by digestion with TPCK-trypsin and Staphylococcus aureus V8 protease. FMTI-II consists of 67 amino acid residues, including 10 half-cystine residues which are involved in 5 disulfide bridges in the molecule. The established sequence had a high degree of homology to Bowman-Birk type inhibitors from leguminous and gramineous plants. The trypsin reactive-site peptide bond in FMTI-II also appears to be Lys (16)-Ser (17) by comparison with these sequences.

Disposition of cefmetazole in healthy volunteers and patients with impaired renal function.

The disposition of cefmetazole was studied in 25 subjects with various degrees of renal function after a 1,000-mg, constant-rate, 30-min intravenous infusion of cefmetazole sodium. In six subjects with creatinine clearance (CLCR) of greater than 90 ml/min per 1.73 m2 (group 1), the terminal elimination half-life (t1/2 beta) was 1.31 +/- 0.54 h (mean +/- standard deviation), cefmetazole total body clearance (CLP) was 132.8 +/- 25.1 ml/min per 1.73 m2, and volume of distribution at steady state was 0.165 +/- 0.025 liter/kg. The fraction of dose excreted unchanged in the urine was 84.0% +/- 26.1%. Subjects with CLCRS of 40 to 69 (group 2, n = 6) and 10 to 39 (group 3, n = 6) ml/min per 1.73 m2 demonstrated prolongation of the t1/2 beta (3.62 +/- 1.06 and 5.93 +/- 1.81 h, respectively) and significant reductions in cefmetazole CLP (52.8 +/- 14.3 and 30.2 +/- 10.2 ml/min per 1.73 m2, respectively), compared with group 1. In seven subjects on chronic hemodialysis (group 4) studied during an interdialytic period, the cefmetazole t1/2 beta was increased to 24.10 +/- 8.12 h and the CLP was reduced to 6.8 +/- 2.1 ml/min per 1.73 m2. Cefmetazole CLP correlated positively with CLCR (r = 0.951, P less than 0.001): CLP = (1.181 . CLCR) -- 0.287. The disposition of cefmetazole was also assessed in six group 4 subjects during an intradialytic period. The t1/2 beta during hemodialysis (2.09 +/- 0.69 h) was significantly shorter than that observed during the interdialytic period. The hemodialysis clearance of cefmetazole was 86.1 +/- 20.1 ml/min, and the fraction of cefmetazole removed during hemodialysis was 59.8% +/- 5.9%. It is recommended that patients with renal insufficiency received standard doses of cefmetazole at extended intervals and patients on maintenance hemodialysis received standard doses after hemodialysis.

Effect of concomitant administration of piperacillin on the dispositions of netilmicin and tobramycin in patients with end-stage renal disease.

The effect of piperacillin administration on the dispositions of netilmicin and tobramycin was assessed in 12 chronic hemodialysis patients. Six subjects each received netilmicin (2 mg/kg) or tobramycin (2 mg/kg) alone and in combination with piperacillin (4 g every 12 h for four doses). Subjects also received a single dose of piperacillin (4 g) on a separate occasion. The serum concentration-versus-time profiles of netilmicin and tobramycin were biexponential. The terminal elimination half-life (t1/2 beta) of tobramycin was markedly reduced (59.62 +/- 25.18 [mean +/- standard deviation] versus 24.71 +/- 5.41 h) and total body clearance (CLP) was significantly increased in the presence of piperacillin (3.45 +/- 1.61 versus 7.16 +/- 1.64 ml/min). In contrast, the t1/2 beta (41.80 +/- 13.24 versus 40.07 +/- 10.37 h) and CLP (5.11 +/- 2.15 versus 5.55 +/- 2.32 ml/min) of netilmicin were not significantly altered when netilmicin was administered in combination with piperacillin. No change in the central or steady-state volume of distribution of netilmicin or tobramycin was observed. The disposition of piperacillin in hemodialysis patients was not altered in the presence of either aminoglycoside antibiotic. Although no adjustment in netilmicin dosing is required, tobramycin should be administered more frequently when given concomitantly with piperacillin to hemodialysis patients to avoid prolonged periods of subtherapeutic concentrations.

Steady-state pharmacokinetics of intravenous and oral ciprofloxacin in elderly patients.

The steady-state pharmacokinetics of ciprofloxacin were evaluated in nine elderly patients with lower respiratory tract infections after an intravenous dosage regimen of 200 mg every 12 h (n = 9) and an oral dosage regimen of 750 mg every 12 h (n = 6). Ciprofloxacin concentrations in serum and urine were measured by high-performance liquid chromatography. The peak concentration in serum, total body clearance (CLs), steady-state volume of distribution (Vss), and terminal elimination half-life after intravenous dosing were 3.5 +/- 0.8 micrograms/ml, 4.38 +/- 1.80 ml/min per kg, 1.6 +/- 0.6 liters/kg, and 5.8 +/- 2.4 h, respectively. The peak concentration in serum, time to peak concentration in serum, absorption lag time, and absolute bioavailability (F) after oral dosing were 7.6 +/- 2.2 micrograms/ml, 1.9 +/- 1.0 h, 0.4 +/- 0.5 h, and 7.7 +/- 24.2%, respectively. The elevated drug concentrations in serum samples from the elderly after oral dosing, compared with data obtained from younger subjects, appear to be a function of reduced CLs, renal clearance, and Vss. The increased F observed in some patients may be due to the effect of concomitant or proximate administration of tube feedings, medications which may alter gastric motility or acidity, or decreased first-pass metabolism. The results demonstrate that factors related to age and declining renal function, rather than infectious disease state, may be primary in determining alterations in pharmacokinetic parameters in the elderly. In elderly patients with normal renal function for their age, no dosage adjustment for intravenous or oral ciprofloxacin is necessary.