Evidence for the expression of native Mycobacterium tuberculosis phospholipase C: recognition by immune sera and detection of promoter activity

The genome of Mycobacterium tuberculosis H37Rv contains three contiguous genes (plc-a, plc-b and plc-c) which are similar to the Pseudomonas aeruginosa phospholipase C (PLC) genes. Expression of mycobacterial PLC-a and PLC-b in E. coli and M. smegmatis has been reported, whereas expression of the native proteins in M. tuberculosis H37Rv has not been demonstrated. The objective of the present study was to demonstrate that native PLC-a is expressed in M. tuberculosis H37Rv. Sera from mice immunized with recombinant PLC-a expressed in E. coli were used in immunoblots to evaluate PLC-a expression. The immune serum recognized a 49-kDa protein in immunoblots against M. tuberculosis extracts. No bands were visible in M. tuberculosis culture supernatants or extracts from M. avium, M. bovis and M. smegmatis. A 550-bp DNA fragment upstream of plc-a was cloned in the pJEM12 vector and the existence of a functional promoter was evaluated by detection of ß-galactosidase activity. ß-Galactosidase activity was detected in M. smegmatis transformed with recombinant pJEM12 grown in vitro and inside macrophages. The putative promoter was active both in vitro and in vivo, suggesting that expression is constitutive. In conclusion, expression of non-secreted native PLC-a was demonstrated in M. tuberculosis.

Functional differences between two morphologically distinct cell subpopulations within a human colorectal carcinoma cell line

The LISP-I human colorectal adenocarcinoma cell line was isolated from a hepatic metastasis at the Ludwig Institute, São Paulo, SP, Brazil. The objective of the present study was to isolate morphologically different subpopulations within the LISP-I cell line, and characterize some of their behavioral aspects such as adhesion to and migration towards extracellular matrix components, expression of intercellular adhesion molecules and tumorigenicity in vitro. Once isolated, the subpopulations were submitted to adhesion and migration assays on laminin and fibronectin (crucial proteins to invasion and metastasis), as well as to anchorage-independent growth. Two morphologically different subpopulations were isolated: LISP-A10 and LISP-E11. LISP-A10 presents a differentiated epithelial pattern, and LISP-E11 is fibroblastoid, suggesting a poorly differentiated pattern. LISP-A10 expressed the two intercellular adhesion molecules tested, carcinoembryonic antigen (CEA) and desmoglein, while LISP-E11 expressed only low amounts of CEA. On the other hand, adhesion to laminin and fibronectin as well as migration towards these extracellular matrix proteins were higher in LISP-E11, as expected from its poorly differentiated phenotype. Both subpopulations showed anchorage-independent growth on a semi-solid substrate. These results raise the possibility that the heterogeneity found in the LISP-I cell line, which might have contributed to its ability to metastasize, was due to at least two different subpopulations herein identified.

Standardization of an isolated pig heart preparation with parabiotic circulation: methodological considerations

In the present study we standardized an experimental model of parabiotic circulation of isolated pig heart. The isolated heart was perfused with arterial blood from a second animal as support and submitted to regional ischemia for 30 min, followed by total ischemia for 90 min and reperfusion for 90 min. Parameters for measurement of ventricular performance using different indices measured directly or indirectly from intraventricular pressure were defined as: maximum peak pressure, final diastolic pressure, pressure developed, first derivative of maximum pressure (dP/dt max), first derivative of minimum pressure (dP/dt min), systolic stress of the left ventricle (sigmas), and maximum elastance of the left ventricle. Isolated hearts subjected to regional and global ischemia presented significant worsening of all measured parameters. Less discriminative parameters were dP/dt max and dP/dt min. Elastance was the most sensitive parameter during the reperfusion period, demonstrating an early loss of ventricular function during reperfusion. The model proved to be stable and reproducible and permitted the study of several variables in the isolated heart, such as ischemia and reperfusion phenomena, the effects of different drugs, surgical interventions, etc. The model introduces an advantage over the classical models which use crystalloid solutions as perfusate, because parabiotic circulation mimics heart surgery with extracorporeal circulation.

Quenching of the intrinsic fluorescence of bovine serum albumin by chlorpromazine and hemin

The binding of chlorpromazine (CPZ) and hemin to bovine serum albumin was studied by the fluorescence quenching technique. CPZ is a widely used anti-psychotic drug that interacts with blood components, influences bioavailability, and affects function of several biomolecules. Hemin is an important ferric residue of hemoglobin that binds within the hydrophobic region of albumin with high specificity. Quenching of the intrinsic fluorescence of bovine serum albumin (BSA) was observed by selectively exciting tryptophan residues at 290 nm. Emission spectra were recorded in the range from 300 to 450 nm for each quencher addition. Stern-Volmer graphs were plotted, and the quenching constant estimated for BSA solution titrated with hemin at 25ºC was 1.44 (± 0.05) x 10(5) M-1. Results showed that bovine albumin tryptophans are not equally accessible to CPZ, in agreement with the idea that polar or charged quenchers have more affinity for amino acid residues on the outer wall of the protein. Hemin added to albumin solution at a molar ratio of 1:1 quenched about 25% of their fluorescence. The quenching effect of CPZ on albumin-hemin solution was stronger than on pure BSA. This increase can be the result of combined conformational changes in the structure of albumin caused firstly by hemin and then by CPZ. Our results suggest that the primary binding site for hemin on bovine albumin may be located asymmetrically between the two tryptophans along the sequence formed by subdomains IB and IIA, closer to tryptophan residue 212.

Frequency of Werner helicase 1367 polymorphism and age-related morbidity in an elderly Brazilian population

Werner syndrome (WS) is a premature aging disease caused by a mutation in the WRN gene. The gene was identified in 1996 and its product acts as a DNA helicase and exonuclease. Some specific WRN polymorphic variants were associated with increased risk for cardiovascular diseases. The identification of genetic polymorphisms as risk factors for complex diseases affecting older people can improve their prevention, diagnosis and prognosis. We investigated WRN codon 1367 polymorphism in 383 residents in a district of the city of São Paulo, who were enrolled in an Elderly Brazilian Longitudinal Study. Their mean age was 79.70 ± 5.32 years, ranging from 67 to 97. This population was composed of 262 females (68.4%) and 121 males (31.6%) of European (89.2%), Japanese (3.3%), Middle Eastern (1.81%), and mixed and/or other origins (5.7%). There are no studies concerning this polymorphism in Brazilian population. These subjects were evaluated clinically every two years. The major health problems and morbidities affecting this cohort were cardiovascular diseases (21.7%), hypertension (83.7%), diabetes (63.3%), obesity (41.23%), dementia (8.0%), depression (20.0%), and neoplasia (10.8%). Their prevalence is similar to some urban elderly Brazilian samples. DNA was isolated from blood cells, amplified by PCR and digested with PmaCI. Allele frequencies were 0.788 for the cysteine and 0.211 for the arginine. Genotype distributions were within that expected for the Hardy-Weinberg equilibrium. Female gender was associated with hypertension and obesity. Logistic regression analysis did not detect significant association between the polymorphism and morbidity. These findings confirm those from Europeans and differ from Japanese population.

TP53 codon 72 polymorphism as a risk factor for cardiovascular disease in a Brazilian population

TP53, a tumor suppressor gene, has a critical role in cell cycle, apoptosis and cell senescence and participates in many crucial physiological and pathological processes. Identification of TP53 polymorphism in older people and age-related diseases may provide an understanding of its physiology and pathophysiological role as well as risk factors for complex diseases. TP53 codon 72 (TP53:72) polymorphism was investigated in 383 individuals aged 66 to 97 years in a cohort from a Brazilian Elderly Longitudinal Study. We investigated allele frequency, genotype distribution and allele association with morbidities such as cardiovascular disease, type II diabetes, obesity, neoplasia, low cognitive level (dementia), and depression. We also determined the association of this polymorphism with serum lipid fractions and urea, creatinine, albumin, fasting glucose, and glycated hemoglobin levels. DNA was isolated from blood cells, amplified by PCR using sense 5'-TTGCCGTCCCAAGCAATGGATGA-3' and antisense 5'-TCTGGGAAGGGACAGAAGATGAC-3' primers and digested with the BstUI enzyme. This polymorphism is within exon 4 at nucleotide residue 347. Descriptive statistics, logistic regression analysis and Student t-test using the multiple comparison test were used. Allele frequencies, R (Arg) = 0.69 and P (Pro) = 0.31, were similar to other populations. Genotype distributions were within Hardy-Weinberg equilibrium. This polymorphism did not show significant association with any age-related disease or serum variables. However, R allele carriers showed lower HDL levels and a higher frequency of cardiovascular disease than P allele subjects. These findings may help to elucidate the physiopathological role of TP53:72 polymorphism in Brazilian elderly people.

CARACTERIZAÇÃO FÍSICO-QUÍMICA DA AMÊNDOA DA CASTANHA DE CAJU (Anacardium occidentale L.) CRUA E TOSTADA

Realizou-se a caracterização físico-química da amêndoa da castanha de caju (Anacardium occidentale L.) crua e tostada, identificando-se as alterações provocadas pelo processo de tostagem. As amêndoas da castanha de caju crua e tostada apresentaram pH pr��ximos à neutralidade. A composição centesimal da amêndoa crua apresentou os seguintes teores: umidade - 5,05 %, cinzas - 2,40 %, proteínas - 22,11 %, lipídios - 46,28 %, açúcares totais - 7,93 % e amido - 16,07 %. Para a amêndoa tostada os resultados foram: umidade - 1,18 %, cinzas - 2,43 %, proteínas - 21,76 %, lipídios - 48,35 %, açúcares totais - 8,23 % e amido - 17,30 %. A comparação destes resultados foi significativamente diferente nos níveis de lipídios, açúcares totais e amido, possivelmente em conseqüência da perda de água durante o processo de tostagem, pois quando estes foram comparados na base seca, os resultados passaram a não ter diferenças estatísticas.

Grace Anglican Church (St. Catharines, Ont.) collection

Grace Anglican Church was founded as a mission church of St. Thomas', St. Catharines. A large house, originally built and owned by the William and Maria McCalla family, was donated by Colonel R.W. Leonard for this purpose. The mission was dedicated on 29 June 1921. It was not until 1938 that Grace Church became an independent parish. A church building was constructed and opened on 28 November 1939. In April 1956 part of the church was damaged by fire, was rebuilt and enlarged.

Henry Hope & Sons of Canada Ltd. Letter, 1916

Henry Hope & Sons of Canada Ltd. were located at 45 King Street West, Toronto, Ont. at the writing of this letter in 1916. The company specialized in “weathertight casements in iron steel or gun-metal, unbreakable steel windows, fanlight opening-gear, leaded lights and stained glass, patent glass roofing, locks and door furniture, rainwater goods in cast lead and cast iron”. The letter is addressed to Mr. H.Y. [Harry Young] Grant, c/o Fenwick Farm, Lundy’s Lane, Niagara Falls, Ont. from R.W. Smith. The letter is in reply to a query about casement windows. Harry Young Grant (1860-1934), son of Sir James Alexander Grant and Maria Malloch of Ottawa, Ont. was a medical doctor specializing in the treatment of the eye, ear, nose and throat. After his retirement he became a member of the Niagara Parks Commission. He was married to Grace A. Smith, daughter of James R. Smith of Buffalo.

Niagara Parks Commission School of Gardening Graduation Program, 1956

The Niagara Parks Commission School of Gardening was organized in 1935 in order to help fill the Commission’s need for skilled gardeners to maintain the extensive parkland owned by the Commission. In 1959 the School was renamed the Niagara Parks Commission School of Horticulture. The name changed again in 1990 to the Niagara Parks Botanical Gardens and School of Horticulture to better reflect the development of the program.

Photograph - Baie Comeau, Last Railway Spike

A photograph of the "last spike in the Baie Comeau railway. Photo shows, left to right; A.I.Cunningham, managing engineer of the Ontario Paper Company driving the spike: V.G. Younghusband, vice president of the Foundation Company of Canada; R.W. Shaver, construction manager for the paper company; K.W. Bash, general superintendent, Foundation Company; and His Lordship the Bishop of Quebec.

Photograph - Thomas Woodruff and Julia Cleveland Woodruff at DeVeaux Hall

Black and white, 12 cm x 17 cm photograph, mounted on board, of DeVeaux Hall in St. Catharines, Ontario. It was built by Samuel DeVeaux Woodruff. Shown in this photo are Thomas Woodruff and Julia Cleveland Woodruff. This photo was described by R.W. Band in 1973 and is included.