beta-N-acetylhexosaminidases in human cerebrospinal fluid and serum of patients with multiple sclerosis.

β-N-Acetylhexosaminidase activity and isoenzyme have been investigated in normal human cerebrospinal fluid and that of patients with multiple sclerosis. β-N-acetylhexosaminidase activity in normal cerebrospinal fluids has been resolved into five components. The major component was in a form that eluted from DEAE cellulose at the same salt concentration as hexosaminidase As, the isoenzyme previously identified in human serum. Cerebrospinal fluid from patients exhibited a different isoenzyme profile, showing a remarkable increase in a form having a pI which was more acidic than that of As. These changes have a potential use in the diagnosis and further biochemical characterization of multiple sclerosis.     

Calcium-alginate beads for the oral delivery of transforming growth factor-β1 (TGF-β1): stabilization of TGF-β1 by the addition of polyacrylic acid within acid-treated beads

The susceptibility of the gastrointestinal tract to the toxic effects of chemotherapeutic drugs remains a complication in chemotherapy. Recent studies have suggested that transforming growth factor-β₁ (TGF-β₁) can be used as a cytoprotectant against cell cycle specific drugs. This work describes the use of alginate beads as a potential oral delivery system for TGF-β₁ designed to release the drug in the lumen of the small intestine. TGF-β₁ encapsulation and extent of release from alginate beads approached 100% as determined by ¹²⁵I-labelled TGF-β₁. However, when assayed by ELISA and a growth inhibition assay, nearly all immunoreactivity and bioactivity was lost, apparently due to a very high affinity of the alginate for TGF-β₁. This limitation was overcome by two novel methods: (1) incorporation of selected polyanions within the alginate beads to ‘shield’ TGF-β₁ from interaction with alginate and (2) exposure of the alginate beads containing TGF-β₁ to 0.1 N HCl (acid treatment) to simultaneously reduce the molecular weight of the alginate and its ability to interact with TGF- β₁. If the beads were only acid treated, just 8% of the immunoreactivity ofTGF-β₁ was retained. If polyacrylic acid (90 kDa) was added to the beads, 50% of the immunoreactivity of TGF-β₁ was retained. However, when TGF-β₁ was released from acid-treated beads also containing polyacrylic acid, more than 80% of the TGF-β₁ remained immunoreactive and bioactive. The retained TGF-β₁ activity after release from the beads was found to continue to increase with increasing concentrations of polyacrylic acid, until a concentration was reached where beads would not form. The dramatic increase in retained TGF-β₁ activity is attributed to the ability of polyacrylic acid to shield TGF-β₁ from interaction with lower molecular fragments of alginate.     

Pentagalloylglucose, an antisecretory component of Paeoniae radix, inhibits gastric H, K-ATPase

We purified a compound with strong inhibitory effect on H⁺, K⁺-ATPase from Paeoniae radix, which has been used in Japan for the treatment of gastritis and peptic ulcers. The compound was identified as 1,2,3,4,6,-penta-o-galloyl-β- -glucose by proton nuclear magnetic resonance, carbon-13 nuclear magnetic resonance, and fast atomic bombardment mass spectrometry. The IC₅₀ of the compound for H⁺, K⁺-ATPase was 166 nmol/l. Kinetic analyses indicated that the inhibition of the enzyme by pentagalloylglucose was noncompetitive with respect to K⁺. Pentagalloylglucose had relatively weak inhibitory effects for Mg⁺-ATPase (IC₅₀: >10 μmol/l) and Na⁺, K⁺-ATPase (IC₅₀: 2.7 μmol/l). Pentagalloylglucose also inhibited the accumulation of [¹⁴C]aminopyrine in parietal cells that had been isolated from guinea pig stomach and stimulated by 10 μmol/l histamine (IC₅₀: 7.8 μmol/l) and 1 mmol/l dbc-AMP (IC₅₀: 10 μmol/l). These results suggest that pentagalloylglucose is a potent inhibitor of H⁺, K⁺-ATPase and may be responsible for inhibition of acid secretion by Paeoniae radix.     

The small acid soluble proteins (SASP α and SASP β) of Bacillus weihenstephanensis and Bacillus mycoides group 2 are the most distinct among the Bacillus cereus group

The Bacillus cereus group includes Bacillus anthracis, B. cereus, Bacillus thuringiensis, Bacillus mycoides and Bacillus weihenstephanensis. The small acid soluble spore protein (SASP) β has been previously demonstrated to be among the biomarkers differentiating B. anthracis and B. cereus; SASP β of B. cereus most commonly exhibits one or two amino acid substitutions when compared to B. anthracis. SASP α is conserved in sequence among these two species. Neither SASP α nor β for B. thuringiensis, B. mycoides and B. weihenstephanensis have been previously characterized as taxonomic discriminators. In the current work molecular weight (MW) variation of these SASPs were determined by matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for representative strains of the 5 species within the B. cereus group. The measured MWs also correlate with calculated MWs of translated amino acid sequences generated from whole genome sequencing projects. SASP α and β demonstrated consistent MW among B. cereus, B. thuringiensis, and B. mycoides strains (group 1). However B. mycoides (group 2) and B. weihenstephanensis SASP α and β were quite distinct making them unique among the B. cereus group. Limited sequence changes were observed in SASP α (at most 3 substitutions and 2 deletions) indicating it is a more conserved protein than SASP β (up to 6 substitutions and a deletion). Another even more conserved SASP, SASP α–β type, was described here for the first time.     

Detection of myelin basic protein in cerebrospinal fluid

Radioimmunoassay for myelin basic protein in cerebrospinal fluid is commonly used as a biochemical marker of demyelination in multiple sclerosis patients. A sensitive enzyme-linked immunosorbent assay for myelin basic protein has been recently developed, which can make a clinical evaluation of myelin basic protein in cerebrospinal fluid of patients with multiple sclerosis and other neurological diseases. Most multiple sclerosis patients with acute exacerbation had markedly high myelin basic protein. Longitudinal studies of multiple sclerosis patients showed that myelin basic protein in CSF increases rapidly in agreement with acute relapse and then rapidly declines and disappears. Significantly higher cerebrospinal fluid myelin basic protein levels in human T-cell lymphotropic virus Type I-associated myelopathy/tropical spastic paraparesis patients were also detected. This enzyme-linked immunosorbent assay system can be used routinely to measure myelin basic protein in cerebrospinal fluid as a useful diagnostic indicator, not only for central active demyelination as in multiple sclerosis but, also for spinal cord demyelination as in human T-cell lymphotropic virus Type I-associated myelopathy/tropical spastic paraparesis.     

A simple PCR-based genotyping method for M105I mutation of alpha-SNAP enhances the study of early pathological changes in hyh phenotype

α-SNAP is an essential component of the protein machinery responsible for membrane fusion events in different cell types. The hyh (hydrocephalus with hop gait) mouse carries a missense mutation in Napa gene that results in a point mutation (M105I) in α-SNAP protein. Homozygous animals for the mutant allele have been identified by the clinical and/or neuropathological phenotype, or by direct sequencing of PCR products. The aims of the present study were (i) to develop a high-throughput technique to genotype hyh mice, (ii) to correlate genotype-phenotype, and (iii) to analyze the earliest pathological changes of hyh mutant mice. As no restriction sites are affected by the hyh mutation, we resolved this problem by creating a BspHI restriction site with a modified (mismatch) polymerase chain reaction (PCR) primer in wild-type allele. This artificially created restriction site (ACRS)–PCR technique is a simple, rapid and reliable method to genotype hyh mice in a day-work procedure. Biochemical and histological analysis of genotyped hyh embryos at different developmental stages allowed us to identify and characterize the earliest brain pathological changes of the hyh phenotype, including the first signs of neuroepithelial disruption and neuronal ectopia. In addition, genotype-phenotype analysis of 327 animals confirmed that (i) hyh is a single-gene autosomal recessive disorder, and (ii) the disorder has 100% penetrance (i.e., the mutation was only present in affected mice). The genotyping method described here enhances the potentiality of hyh mouse as a unique in vivo model to study the role of membrane trafficking in different developmental and physiological processes.     

Indirect evidence for early widespread gray matter involvement in relapsing-remitting multiple sclerosis

Multiple sclerosis (MS) has traditionally been viewed as an inflammatory demyelinating white matter (WM) disease of the central nervous system. However, recent pathology and MRI studies have shown lesions in the gray matter (GM) as well. To ascertain the extent of GM involvement, we obtained with nonlocalizing proton MR spectroscopy the concentration of N-acetylaspartate (NAA), a metabolite found almost exclusively in neuronal cells, T2-lesion loads, and GM and WM fractions in the entire brain of 71 relapsing–remitting (RR) MS patients (51 women, 20 men, 25–55 years old) and 41 healthy controls (27 women, 14 men, 23–55 years old). The average whole-brain NAA (WBNAA) difference between the patients and the controls was −2.9 mM (−22%, P < 0.0001); range: +1.2 to −7.8 mM (+8% to −63%). The patients' median T2 lesion volume was 5.5 (range: 0.140–28) cm³. GM and WM comprised 50.4 ± 3.8% and 30.4 ± 5.0% (mean ± standard deviation), respectively, of the total brain volume in the patients; 53.8 ± 3.7% and 35.4 ± 4.7% in the controls. Because WM and GM constitute approximately 40% and 60% of the brain parenchyma, respectively, and the NAA concentration in the former is 2/3 of the latter, WBNAA loss greater than 40% × 2/3 = 27% cannot be explained in terms of WM (axonal) pathology alone and must include widespread GM (neuronal) deficits. Therefore, the concept of MS, even at its earlier stages, as a WM disease might need to be reexamined.     

Cystatin C, alias post-gamma-globulin: a marker for multiple sclerosis?

Cystatin C, alias post-gamma-globulin or gamma-trace protein, has been shown to be a potent inhibitor of cysteine proteinases; this protein is normally present in different biological fluids, but particularly so in cerebrospinal fluid. The concentration of cystatin C was determined by radial immunodiffusion in cerebrospinal fluid from patients affected with multiple sclerosis, patients affected with various neurological diseases and in controls; it was also determined in brain tissue from 2 patients affected with multiple sclerosis and 3 control brains. Cystatin C cerebrospinal fluid levels were undetectable or depressed in many multiple sclerosis cases and the median value differed significantly from the control one. Its low concentration in multiple sclerosis suggests that the regulation of cysteine proteinases is impaired in this disease; hence enhanced activity of cysteine proteinases could initiate, or increase the breakdown of myelin. Although it is perhaps a little premature to consider cystatin C as a marker for multiple sclerosis, this protein is nevertheless associated to demyelination; consequently its biochemical assay in cerebrospinal fluid is recommended as a complementary diagnostic tool.     

Lecithin cholesterol acyltransferase in human cerebrospinal fluid: reduced level in patients with multiple sclerosis and evidence of direct synthesis in the brain

Summary Several studies suggest that apolipoproteins and the low-density lipoprotein receptor are implicated in lipid transport in the brain. Given that cerebrospinal fluid has been reported to contain cholesteryl esterifying enzyme, and that lipid metabolism in the brain is abnormal in subjects with multiple sclerosis, we examined the cerebrospinal fluid from eight control subjects with a normal cerebrospinal fluid IgG index, and without active demyelinating disease, and from eight subjects (6 were diagnosed as having multiple sclerosis) with an increased IgG index and the presence of oligoclonal banding in the cerebrospinal fluid, for the presence of the enzyme lecithin cholesterol acyltransferase and apolipoprotein(a). None of the subjects demonstrated imapairment of their blood-cerebrospinal fluid barrier, as estimated by the cerebrospinal fluid/serum quotient of albumin. Lecithin cholesterol acyltransferase was detected in the cerebrospinal fluid of all control subjects, being 0.12±0.06 μg/ml (mean±SD) or about 2.2% of that in serum (5.4±1.4 μg/ml). The cerebrospinal fluid lecithin cholesterol acyltransferase index was 5.2±2.5, very similar to the cerebrospinal fluid index of apolipoprotein E, a protein known to be synthesized in the brain. Since lecithin cholesterol acyltransferase mRNA is also expressed in the brain, we can conclude that the protein is synthesized and secreted in the brain. The cerebrospinal fluid concentration of lecithin cholesterol acyltransferase in the subjects with active demyelinating disease or multiple sclerosis was only about one-half of that found in control subjects (0.06±0.02 μg/ml). Lipoprotein(a) concentration was below the sensitivity of the enzyme immunoassay used for the measurement (<40 ng/ml) in cerebrospinal fluid from both control subjects and those with demyelinating disease or multiple sclerosis.     

Biochemical assay for amyloid β deposits to distinguish Alzheimer's disease from other dementias

Biochemical markers for Alzheimer's disease (AD) are of great value for precise diagnosis and in studies of the pathogenetic processes of this disease. A new biochemical assay allowing to differentiate AD from other forms of dementia is described. The assay is based on the extraction of amyloid β (Aβ) from milligram amounts of brain tissue by using 20% acetonitrile in 0.1% trifluoroacetic acid and its detection by Western blotting. The presence of the 4 kDa Aβ was demonstrated in all cases of AD (n=8) that were diagnosed by the independent histopathological examination of the postmortem tissues. No Aβ was found in tissue extracts from seven out of eight cases of other forms of dementia. In contrast to other biochemical techniques of Aβ detection in brain, the developed assay is simple; it does not require any special equipment and allows detection of Aβ using milligram amounts of brain tissue.     

N-Acetyl-β-d-glucosaminidase (NAG) and NAG isoenzymes in children with upper and lower urinary tract infections

The use of N-acetyl-β-d-glucosaminidase (NAG) to diagnose the site of urinary tract infection was studied in pediatric patients. Differentiation between upper and lower tract infections (UTI) was based on clinical grounds and on elevated erythrocyte sedimentation rate, C-reactive protein and fever. NAG excretion expressed as nmol · h⁻¹ · mg⁻ of urinary creatinine was higher in children with upper UTI (mean ± SE 906 ± 236) than in those with lower UTI (145 ± 23) or healthy children (151.6 ± 10) (p<0.01 by Duncan's test). In children with upper UTI, NAG excretion fell in parallel with the remission due to antibiotic treatment. This however was not seen in children treated with aminoglycosides. A specific and significant elevation (p < 0.01) of the B isoenzyme of NAG was documented in children with upper UTI but not in those with lower UTI (B form in upper UTI 49.2% ± 3.9 versus 21.9 ± 3.3 in lower UTI; healthy children 18.9 ± 3.4). The percentage of B isoenzyme excreted was high in two children with upper UTI but was low total NAG urinary excretion, suggesting that the quantification of isoenzymes offers further specificity in diagnosis. We conclude that the measurement of NAG and its isoenzymes in children with UTI provides useful information in the diagnosis of the site of infection.     

Cystic fibrosis α2-macroglobulin protease interaction in vitro

α₂-Macroglobulin was purified from plasma of five cystic fibrosis patients and five normal controls. SDS gel electrophoresis of native az-macroglobulin from cystic fibrosis patients and normal donors showed identical subunit molecular weights, as did trypsin cleavage products. Cystic fibrosis and control α₂-macroglobulins were indistinguishable by isoelectric focusing and exhibited appropriate shifts in isoelectric point following binding of trypsin. The trypsinbinding capacities of control and cystic fibrosis α₂-macroglobulins did not differ, nor did the esterolytic activity of the trypsin-α₂-macroglobulin complexes.     

Normal saline versus colloid solutions for induction of hypothermia: the effect of specific heat capacity on cooling

The prevention of ischemic injury to preserve both end-organ function and improve neurological recovery by the implementation of therapeutic hypothermia has been well established in the literature. However, not only the means by which body temperature is cooled but also the rate by which target temperature is attained remains an area of continued interest and research. The induction of therapeutic hypothermia to begin the process of body temperature lowering through the infusion of a cold solution intravenously into the body may be one variable that influences not only rapidity of cooling but also subsequent clinical outcome. In a recent issue of Critical Care, Skulec and colleagues compared the induction of therapeutic hypothermia by cold normal saline versus cold colloid solution containing hydroxyethyl starch in a porcine animal model of cardiac arrest, assessing both the rate of temperature change and target temperature achieved, in addition to changes in intracranial pressure.The use of therapeutic hypothermia (TH) in clinical medicine is no longer a rarity. A variety of methods have been implemented in an attempt to cool patients faster [1]. In a recent issue of Critical Care, Skulec and colleagues [2] evaluated the efficacy of cold normal saline as compared with a colloid solution containing hydroxyethyl starch in the induction of TH in survivors of cardiac arrest by using a porcine animal model of ventricular fibrillation (VF). A 20-minute infusion of either 45 mg/mL of 1°C cold normal saline or 45 mg/mL of 1°C cold colloid solution (Voluven, 6% hydroxyethyl starch 130/0.4 in normal saline; Fresenius Kabi AG, Bad Homburg, Germany) was administered following 5 minutes of VF and subsequent resuscitation. Comparison of pulmonary blood temperature and cerebral blood temperature demonstrated a statistically significant decrease in temperature in animals given normal saline (-2.1 ± 0.3 versus -1.6 ± 0.2°C, and -1.7 ± 0.4 versus -1.1 ± 0.3°C, P <0.05, respectively). Moreover, initiation of TH with normal saline resulted in accelerated cooling when compared with colloid solution (-91 ± 22 versus -68 ± 23°C/min, P = 0.046). The authors also performed a mechanical substudy evaluating the specific heat capacities of each fluid and demonstrated that normal saline resulted in statistically more significant cooling than the studied colloid solution (-7,155 ± 647 versus -5,733 ± 636°C/min, P = 0.008), and this is consistent with the above clinical in vivo findings.These results demonstrating the differential cooling of normal saline compared with a colloid solution containing hydroxyethyl starch both in vivo and experimentally by Skulec and colleagues are both novel academically and consistent with the understanding of specific heat capacity of solutions following the addition of solutes from thermodynamics. Briefly, the addition of a solute to a solution results in an increase in the number of particles that can absorb energy and release it as heat, in turn resulting in an increase in the temperature of the solute-solution combination relative to the solution alone when a constant amount of energy is applied [3]. As the authors demonstrated, the thermodynamic effect of adding solute to a solution resulted in statistically significant differences clinically with respect to hypothermia induction and temperatures attained, as well as rapidity of cooling [2].Interestingly, the intracranial pressure (ICP) was increased in animals treated with cold colloid solution containing hydroxyethyl starch as compared with normal saline solution (end infusion ICPs of 25 ± 5 and 16 ± 6 mm Hg, respectively), which persisted for up to 90 minutes following infusion. The association of increased ICP with hydroxyethyl starch was also demonstrated by Khan and colleagues, who evaluated the combined incidence of delayed cerebral ischemia, hydrocephalous requiring cerebrospinal fluid shunting, and rebleeding in patients following subarachnoid hemorrhage, and demonstrated a statistically significant increase in both the combined endpoint (odds ratio 3.1, 95% confidence interval 1.30 to 7.36, P = 0.01) and the incidence of hydrocephalous requiring cerebrospinal fluid shunting (odds ratio 6.1, 95% confidence interval 1.63 to 22.95, P = 0.004) [4]. The increases in both ICP and hydrocephalous requiring cerebrospinal fluid shunting in these two studies are most likely due to changes in cerebrospinal fluid absorption by the arachnoid microvilli related to hydroxyethyl starch.The use of colloid solutions containing hydroxyethyl starch for volume resuscitation of critically ill patients has been controversial, and the literature suggests both increased mortality and increased need for renal replacement therapy when compared with normal saline, and subsequently the US Food and Drug Administration issued a boxed recommendation to health-care professionals not to use hydroxyethyl starch-containing solutions in patients admitted to the intensive care unit or in patients with pre-existing renal dysfunction [5-7]. The ICP increase seen by Skulec and colleagues in animals treated with colloid solution may be, in part, the mechanism associated with increased mortality in patients treated with hydroxyethyl starch solutions. This study not only elucidates the decreased efficacy of colloid solutions containing hydroxyethyl starch compared with normal saline in the induction of TH in vivo but also provides insight into complications associated with hydroxyethyl starch and re-emphasizes the importance of specific heat capacity of the solution with respect to rapidity of cooling during TH induction.     

Diagnosis of Tay-Sachs disease using [H]N-acetylneuraminic acid labelled GM2 ganglioside as substrate

G_M2 ganglioside labelled with tritium in the N-acetylneuraminic acid moiety was prepared and used to measure β-hexosaminidase A activity in cultured human skin fibroblast extracts. The latter convert this substrate to the correspondingly labelled G_M3 ganglioside which can easily be separated from the substrate by thin-layer chromatography. No cleavage of the N-acetylneuraminic acid group was observed under our conditions.Two methods are described for the determination of G_M2-β-hexosaminidase A activity in fibroblasts. The application of these methods to the diagnosis of Tay-Sachs disease is discussed.     

Diagnostic value of urinary N-acetyl-β-d-glucosaminidase, its isoenzymes and the fractional excretion of sodium following renal transplantation

Daily total urine N-acetyl-β-d-glucosaminidase activity, isoenzyme profile and fractional excretion of sodium were measured in 13 consecutive renal transplant patients. Rejection episodes were clinically diagnosed in 12 patients, 11 of whom (92%) showed an increased enzymuria either before or during the onset of clinical signs. The ratio of the two major isoenzymes (A/B) fell during 10 episodes (83%) and in six of these (50%) increased levels of the minor isoenzyme forms were observed. Increased fractional excretion of sodium was associated with nine (75%) of the episodes. Increased fractional excretion of sodium with a raised total enzymuria accompanied by a reduced A/B ratio and an increased proportion of the minor isoenzyme forms occurred in eight (67%) of the rejection episodes. The use of these measurements in the diagnosis of episodes of acute rejection in renal transplantation is discussed.     

Antipseudomonal activity of piperacillin/tazobactam: more than a decade of experience from the SENTRY Antimicrobial Surveillance Program (1997–2007)

We evaluated the susceptibility rates for piperacillin/tazobactam tested against Pseudomonas aeruginosa isolates from the Asia-Pacific (APAC), Europe (EU), Latin America (LA), and North America (NA) for 1997 to 2007. A total of 25 460 isolates were tested originating from APAC (4441), EU (7695), LA (4277), and NA (9047). All testing was performed by reference broth microdilution methods. The samples were collected from >110 medical centers and samples averaging >30 nations/year. For this analysis, results from 1997 to 2007, 1997 to 1999, 2005 to 2007, APAC, EU, LA, and NA were assessed against several broad-spectrum β-lactams, including cefepime, ceftazidime, imipenem, meropenem, and piperacillin alone, for a total of 12 agents overall. Using P. aeruginosa breakpoints (≤64 μg/mL), piperacillin/tazobactam had the broadest coverage (% susceptible) in 2 regions (EU, LA) and, overall, at 83.6% followed by meropenem (83.0%) > imipenem (79.7%) > piperacillin (79.5%) > cefepime (77.5%) > ceftazidime (75.8%). Other non–β-lactam activity results were ciprofloxacin at only 71.5% susceptible, but tobramycin and polymyxin B had higher susceptibility rates (81.0% and 99.5%, respectively). Trends toward piperacillin/tazobactam resistance were noted between 1997 to 1999 and 2000 to 2007 in APAC (−11.6% susceptibility), NA (−4.0%), and EU (−2.3%). LA susceptibility rates were lowest overall but actually increased recently by +2.9% (current rate, 79.4% susceptible). For β-lactamase inhibitor combinations, susceptibility rates were higher for piperacillin/tazobactam when compared in all regions with piperacillin alone (+2.6–7.1%) and greatest for LA isolates. In contrast, ticarcillin/clavulanate susceptibility rates were lower than ticarcillin tested alone in NA (−1.5%, antagonism), and this agent only inhibited 70.3% of isolates worldwide. In conclusion, piperacillin/tazobactam remained a very active β-lactam when tested in vitro against clinical isolates of P. aeruginosa found in the SENTRY Program (1997–2007). Trends toward slightly decreased susceptibility were noted in all regions over the last decade (except LA); only polymyxins had susceptibility rates at >90%. Resistance surveillance programs should be sustained to document emerging resistance patterns of old and newer agents for difficult-to-treat pathogens such as P. aeruginosa.     

The contribution of α1-antitrypsin to the total antitrypsin activity of human serum. a study of two phenotype pi oo individuals

Comparison of the levels of α₁AT, α₂-M, inter α-AT, C₁ inactivator and antiplasmin and global antitrypsin activity in a group of normal phenotype PI MM individuals, a group of normal individuals with phenotypes with intermediate α₁AT activities and two α₂-AT-deficient persons show that α₁AT contributes more than 90% of the total antitrypsin activity of normal plasma.AT III and fast reacting antiplasmin are shown to contribute to the remaining activity. It can be assumed that due to test conditions the antitrypsin activity of α₂-M is not assessed. C₁ inactivator and inter α₁-AT do not contribute to a perceptible extent to the overall antitrypsin activity estimated according to the method of Eriksson (Eriksson, S. (1965) Acta Med. Scand. 177, 1).     

Association of a novel polymorphism in the bovine PPARGC1A gene with growth, slaughter and meat quality traits in Brangus steers

The PPARGC1A gene (peroxysome proliferator-activated receptor-γ coactivator 1α gene) controls muscle fiber type and brown adipocyte differentiation; therefore, it is a candidate gene for beef quality traits (tenderness and fat content). Two SNPs (Single Nucleotide Polymorphisms) were identified within exon 8 by multiple alignment of DNA sequences obtained from 24 bulls: a transition G/A (SNP 1181) and a transversion A/T (SNP 1299). The SNP 1181 is a novel SNP, corresponding to a non-conservative substitution (AGT/AAT) that could be the cause of amino acid substitution (³⁶⁴Serine/³⁶⁴Asparagine). A Mismatch PCR method was designed to determine genotypes of 73 bulls and 268 steers for SNP 1181. Growth, slaughter and meat quality information were available for the group of steers. Allele A of SNP 1181 was not found in Angus. In 243 steers, no significant differences (P > 0.05) were found for either final live body weight, gain in backfat thickness in Spring, kidney fat weight, kidney fat percentage, Warner–Bratzler shear force at 7 days postmortem, intramuscular fat percentage or meat colour between genotype GG and AG. This SNP could be included in breed composition and population admixture analyses because there are marked differences in allelic frequencies between Bos taurus and Bos indicus breeds.     

Immunodiagnosis in cerebrospinal fluid of cerebral toxoplasmosis and HIV-infected patients using Toxoplasma gondii excreted/secreted antigens

Cerebral toxoplasmosis is the most common neurologic opportunistic infection in HIV-infected patients. Excretory–secretory antigens (ESA) are the majority of the circulating antigens in sera from hosts with acute toxoplasmosis, and their usefulness as antigens has been shown. This study considered whether it could find anti-ESA antibodies in cerebrospinal fluid (CSF) and whether these antibodies can be markers of active infection. Samples of CSF from 270 HIV-infected patients were analyzed and divided into 3 groups according to the presence or absence of active toxoplasmosis. Group I: 99 patients with cerebral toxoplasmosis; group II: 112 patients with other opportunistic neurologic diseases and seropositive for toxoplasmosis; and group III: 59 patients with other opportunistic neurologic diseases and seronegative for toxoplasmosis. Toxoplasma gondii ESA and a crude tachyzoite antigen were used as antigens using ELISA and immunoblotting. The statistical analysis was done using the F test and unpaired Student's t test. Crude tachyzoite antigen: mean ELISA-relative values ± standard error for CSF of groups I and II were 7.0 ± 0.27 and 3.9 ± 0.19, respectively. Variance analysis revealed that results of both groups of patients were statistically different (1.80, P = 0.0025). The difference between the mean results was 3.0 ± 0.3, and the Student's t test value was 9.41 (P = 0.0001). Samples from groups I and II were reactive by immunoblotting, with similar intensities. In ESA-ELISA, the mean for group I was 9.0 ± 0.39. Group II showed a mean value of 2.7 ± 0.12. Both groups were statistically different (9.16, P < 0.001). However, in ESA, the difference between the mean results was higher (6.2 ± 0.39) and the Student's t test value was 16.04 (P < 0.0001). Similar results were shown in immunoblotting where a CSF sample from group I reacted well with ESA, and the sample from a group II patient failed to do so. The mean ELISA-relative value of the control group (group III) was 0.5 ± 0.09 for the first antigen and 0.4 ± 0.22 for the second. ESA-ELISA and/or immunoblotting of CSF samples can be used for diagnosis of cerebral toxoplasmosis in association with clinical, serologic, and radiological information, thus providing a simple straightforward methodology, particularly suitable in countries with high prevalence of latent toxoplasmosis in the general population.     

Emergence of multiresistant variants of the community-acquired methicillin-resistant Staphylococcus aureus lineage ST1-SCCmecIV in 2 hospitals in Rio de Janeiro, Brazil

Usually, community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) is susceptible to a variety of non–β-lactam drugs. These isolates commonly display SCCmecIV and are associated with community-acquired infections. More recently, CA-MRSA has been isolated from health-care–associated diseases. We characterized MRSA isolates from 2 hospitals in Rio de Janeiro area to assess the entry of new lineages. The isolates were primary genotyped using a combination of molecular typing methods including SCCmec, restriction modification test, and Panton–Valentine leukocidin (PVL) detection. Pulsed-field gel electrophoresis was carried out for representatives of each lineages found. Disk diffusion test was performed as recommended by the Clinical and Laboratory Standards Institute. SCCmecIV was the predominant cassette mec detected. The most frequent MRSA lineage, a PVL nonproducer, was allocated in the CC1-SCCmecIV. It was found that 56% of these isolates were resistant to 3 or more non–β-lactam drugs. Multilocus sequence typing of a representative of the CC1 isolates supported our finds that multiresistant variants of a CA-MRSA lineage (ST1-SCCmecIV) emerged in this city.