Growth of periocular basal cell carcinomas.

BACKGROUND: Basal cell carcinomas (BCCs) are generally regarded as slow-growing tumours. There is a paucity of data on the rate of BCC growth and the impact of delayed excision. OBJECTIVES: To measure the growth rate and assess the impact of delayed excision on the growth of periocular BCC (pBCC). METHODS: Patients referred to an oculoplastic service for excision of pBCC were recruited. The tumour dimensions and patient demographic data were recorded at the first specialist appointment (FSA). Measurement of the pBCC was repeated when the patient attended for tumour excision by Mohs micrographic surgery (MMS). Correlation analyses were performed to determine whether the histological subtype and patient factors affected the pBCC growth rates. RESULTS: The study included 112 patients and 115 pBCCs. The primary ethnicity was European with Fitzpatrick type I and II skin. The mean size of the pBCC at FSA was 8 × 6 mm (range 6-12 × 4-8 mm) with a mean area of 68·5 mm(2). The average waiting time for MMS was 157 ± 87 days. The pBCCs grew at a mean rate of 11·2 mm(2) every 30 days. From the FSA to the MMS, a mean increase of 41·9 mm(2) was observed. Recurrent tumours, larger tumours at presentation and male sex were associated with a faster growth rate. CONCLUSIONS: Periocular basal cell carcinomas can grow rapidly, and many have aggressive histological subtypes. Rapid growth is more likely in recurrent tumours, larger tumours and in men.

Controlling chaos with weak periodic signals optimized by a genetic algorithm.

In the present study we develop a relatively novel and effective chaos control approach with a multimode periodic disturbance applied as a control signal and perform an in-depth analysis on this nonfeedback chaos control strategy. Different from previous chaos control schemes, the present method is of two characteristic features: (1) the parameters of the controlling signal are optimized by a genetic algorithm (GA) with the largest Lyapunov exponent used as an index of the stability, and (2) the optimization is justified by a fitness function defined with the target Lyapunov exponent and the controlling power. This novel method is then tested on the noted Rössler and Lorenz systems with and without the presence of noise. The results disclosed that, compared to the existing chaos control methods, the present GA-based control needs only significantly reduced signal power and a shorter transient stage to achieve the preset control goal. The switching control ability and the robustness of the proposed method for cases with sudden change in a system parameter and/or with the presence of noise environment are also demonstrated.

Resting metabolic rate in obese and nonobese Chinese Singaporean boys aged 13-15 y.

BACKGROUND: Previous studies investigating the hypothesis that a low resting metabolic rate (RMR) is a cause of obesity yielded discrepant findings. Two explanations for these findings are the use of imprecise methods to determine obesity and a failure to control for differences in fat mass (FM) and fat-free mass (FFM) when comparing RMR values. OBJECTIVE: This study tested the hypothesis that RMR is lower in obese than in nonobese boys (with the use of precise methods to quantify body fatness and with adjustment for differences in both FM and FFM). DESIGN: Forty Chinese Singaporean boys aged 12.8-15.1 y were recruited. Boys were classified as obese (n = 20) or nonobese (n = 20) on the basis of their adiposity index (ratio of FM to FFM: >0.60 = obese, <0.40 = nonobese) determined by dual-energy X-ray absorptiometry. RMR was determined by using indirect calorimetry. RMR values were compared by using both linear (analysis of covariance) and log-linear (analysis of covariance with log-transformed data) regression to control for differences in FM and FFM. RESULTS: Age, height, and FFM did not differ significantly between groups. Body mass was 13 kg greater and FM was 16 kg greater in the obese boys than in the nonobese boys (P < 0.001). After control for FFM and FM, RMR did not differ significantly between the groups. CONCLUSION: When body composition is appropriately controlled for, RMR does not differ significantly between obese and nonobese boys.

Serum lipids, serum insulin, plasma fibrinogen and aerobic capacity in obese and non-obese Singaporean boys.

OBJECTIVES: To compare blood lipids, lipoproteins, apoproteins, fibrinogen, insulin and aerobic capacity in obese and non-obese Chinese Singaporean boys. To examine relationships between blood metabolites, body composition and aerobic capacity in these groups. DESIGN: Cross-sectional. SUBJECTS: Forty Chinese Singaporean boys aged 13-15 y. Classified as obese (n=20) or non-obese (n=20) based on adiposity (fat mass/fat free mass): >0.60=obese, <0.40=non-obese. MEASUREMENTS: Body composition (dual energy X-ray absorptiometry), waist circumference, peak oxygen consumption (VO(2) peak), serum concentrations of total cholesterol, triacylglycerol, high density lipoprotein cholesterol (HDL-C), total cholesterol/HDL-C, apoproteins AI and B, lipoprotein(a), insulin and glucose. Plasma concentration of fibrinogen. RESULTS: Obese boys had significantly (P<0.01) higher (mean+/-s.d.) concentrations of serum triacylglycerol (1.51+/-0.65 vs 1.04+/-0.34 mmol/l), serum insulin (24.1+/-11.5 vs 12.3+/-4.45 mU/l) and plasma fibrinogen (4.01+/-0.54 vs 3.35+/-0.76 g/l) than non-obese boys. Within the non-obese group plasma fibrinogen concentration was significantly related to percentage body fat (r=0.546, P<0.05). VO(2) peak relative to body mass (ml/kg/min or ml/kg(-0.67)/min) was significantly (P<0.001) lower in obese compared to non-obese boys but absolute VO(2) peak (l/min), adjusted for fat-free mass via analysis of covariance, was higher in obese than non-obese boys (P<0.01). Partial correlations revealed that none of the blood metabolites were significantly related to VO(2) peak independent of body fatness. CONCLUSIONS: Obesity was related to elevated concentrations of serum triacylglycerol, serum insulin and plasma fibrinogen in Chinese Singaporean boys. These elevated concentrations did not appear to be associated with a lower aerobic capacity (independent of body fatness) in the obese.

G561 site-directed deletion mutant chitinase from Aeromonas caviae is active without its 304 C-terminal amino acid residues.

A G561 mutant of the Aeromonas caviae chitinase ChiA was made by PCR site-directed deletion mutagenesis in order to study the role of the 304 C-terminal amino acid residues of ChiA in the enzymatic hydrolysis of chitin. The recombinant ChiAG561 encoded on a 1.6-kb DNA fragment of A. caviae chiA was expressed in a heterologous Escherichia coli host using the pET20b(+) expression system. The His-Tag-affinity-purified recombinant ChiAG561 had a calculated molecular mass of 63,595 Da, which was consistent with the 67,000 Da estimated by SDS-PAGE. The G561 deletion mutant enzyme had the same optimum pH (6.5) as the full-length ChiA and a lower optimum temperature (37 degrees C instead of 42.5 degrees C). Biochemical properties of the recombinant ChiAG561 suggested that deletion of the 304 C-terminal amino acid residues of ChiA did not significantly affect ChiA enzyme activity. However, compared to the full-length ChiA, the mutant chitinase had a ten-fold higher relative activity with 4-methylumbelliferyl-N-N'-N"-triacetylchitotriose [4-MU-(GlcNAc)3] as a substrate, and higher rates of hydrolysis with both chitin and colloidal chitin substrates. Results obtained from this study suggest that the active region of A. caviae ChiA is located in the region before G561 of the protein molecule.

Prediction of percentage body fat from anthropometry and bioelectrical impedance in Singaporean and Beijing Chinese.

Body composition was measured in 205 male and female Beijing Chinese and in 148 male and female Singaporean Chinese, age 34 (mean) (range 18-68) years and body mass index (BMI) 22.3 (15.9-38.5) kg/m2. In Beijing Siri's two-compartment model based on densitometry was used as a reference technique and in Singapore Siri's three-compartment model based on densitometry and deuterium oxide dilution was used. In addition, body composition was predicted using equations based on anthropometry and bioelectrical impedance developed in Caucasian populations. Percentage body fat (BF%) predicted from BMI was systematically underestimated by about 1% in Beijing Chinese and by about 3.5% in Singaporean Chinese. The difference in bias (measured minus predicted BF%) between the two population groups could be explained by differences in frame size. The Durnin and Womersley equations for BF% based on skinfold thickness predicted BF% in the male and female Chinese groups adequately, with only a slight (less than 1% body fat) and not significant bias. The prediction of BF% based on the waist circumference (Lean's formula) resulted in an unbiased estimate of BF% in females (bias about 1% body fat), whereas in males the formula systematically underestimated BF% by 3.5-5%. Bioelectrical impedance underestimated BF% systematically by 3%, in males and females to about the same extent. The bias of all prediction formulas was positively correlated with the level of body fatness and, except for impedance, also negatively correlated with age. The negative association of the bias with age indicates that the age-related increase in body fatness is lower in Chinese than in Caucasians. It can be concluded of the studied prediction techniques that only the skinfold methodology using the equations of Durnin and Womersley give valid mean estimates for both Chinese males and females. The other techniques require the development of population-specific prediction formula.

The impact of body build on the relationship between body mass index and percent body fat.

OBJECTIVE: The objective of the study was to test the hypothesis that differences in the relationship between percent body fat (%BF) and body mass index (BMI) between populations can be explained (in part) by differences in body build. DESIGN: Cross-sectional, comparative study. SUBJECTS: 120 age, gender and BMI matched Singapore Chinese, Beijing Chinese and Dutch (Wageningen) Caucasians. MEASUREMENTS: From body weight and body height, BMI was calculated. Relative sitting height (sitting height/height) was used as a measure of relative leg length. Body fat was determined using densitometry (underwater weighing) in Beijing and Wageningen and using a three-compartment model based on densitometry and hydrometry in Singapore. Wrist and knee widths were measured as indicators for frame size and skeletal mass was calculated based on height, wrist and knee width. In addition, a slenderness index (height/sum of wrist and knee width) was calculated. RESULTS: For the same BMI, Singapore Chinese had the highest %BF followed by Beijing Chinese and the Dutch Caucasians. Singaporean Chinese had a more slender frame than Beijing Chinese and Dutch Caucasians. Predicted %BF from BMI, using a Caucasian prediction formula, was not different from measured %BF in Wageningen and in Beijing, but in Singapore the formula underpredicted %BF by 4.0 +/- 0.8% (mean +/- s.e.m.) compared to Wageningen. The difference between measured and predicted %BF (bias) was related to the level of %BF and with measures of body build, especially slenderness. Correction for differences in %BF, slenderness and relative sitting height, decreased the differences between measured and predicted values compared to the Dutch group from 1.4 +/- 0.8 (not statistically significant, NS) to -0.2 +/- 0.5 (NS) in Beijing and from 4.0 +/- 0.8 (P < 0.05) to 0.3 +/- 0.5 (NS) in Singapore (all values mean +/- s.e.m.). CONCLUSIONS: The study results confirm the hypothesis that differences in body build are at least partly responsible for a different relationship between BMI and %BF among different (ethnic) groups.

Cloning, sequencing, expression and characterization of the manganese superoxide dismutase gene from Vibrio alginolyticus.

The sodA gene coding for manganese superoxide dismutase from the marine microorganism Vibrio alginolyticus was cloned, sequenced and over-expressed in Escherichia coli using the pET20b (+) expression vector. The full-length gene was consisted of 603bp open reading frame, which encoded a polypeptide of 201 amino acid residues, with a calculated molecular weight of 22672Da. The deduced amino acid sequence of the sodA showed considerable homology to other Mn-SODs. The recombinant enzyme was efficiently purified from crude E. coli cell lysate by the metal ion affinity chromatography. The recombinant VAMn-SOD resisted thermo-denaturation up to 60 degrees C and was insensitive to inhibitors such as H2O2, NaN3 and diethyldithiocarbamic acid.

Site-directed mutagenesis of Asp313, Glu315, and Asp391 residues in chitinase of Aeromonas caviae.

Site-directed mutagenesis was used to explore the roles of amino acid residues involved in the activity of chitinase from Aeromonas caviae. Kinetic parameters for 4-methylumbelliferyl-N,N'-diacetyl-chitobiose or 4-methylumbelliferyl-N,N',N"-triacetylchitotriose hydrolysis were determined with wild-type and mutant chitinases. Chitinases with the mutations E315D (or Q) and D391E (or N) were severely impaired and had dramatically decreased kcat. However, the effect of the these mutations on the Km values were different. The function of the carboxyl group of Asp313 was partially replaced by the amide of Asn when the 4-methylumbelliferyl-N,N',N"-triacetylchitotriose substrate was used. Results indicated that Asp313, Glu315, and Asp391 might be the best candidates for the catalytic residues of chitinase A from Aeromonas caviae.

Cloning and characterization of manganese superoxide dismutase gene from Vibrio parahaemolyticus and application to preliminary identification of Vibrio strains.

The sodA gene coding for manganese superoxide dismutase (Mn-SOD) from the marine microorganism Vibrio parahaemolyticus was cloned, sequenced, and overexpressed in Escherichia coli by use of the pET20b (+) expression vector. The full-length gene consisted of a 588-bp open reading frame and encoded a polypeptide of 196 amino acid residues, with a calculated molecular mass of 21,713 Da. The recombinant enzyme was efficiently purified from crude E. coli cell lysate by metal ion affinity chromatography. The recombinant VPMn-SOD resisted thermo-denaturation up to 60 degrees C and was insensitive to such inhibitors as EDTA, NaN3 and diethyldithiocarbamic acid. The specificity of V. parahaemolyticus Mn-SOD gene probe was analyzed by cross-species polymerase chain reaction to provide information for Vibrio strain identification.

Elastic modulus, permeation time and swelling ratio of a new porcine dermal collagen membrane.

The goal of a single step therapy has been an important consideration in the development of guided tissue regeneration devices. It would spare the patient from the need for repeated surgery and eliminate many problems associated with a non-resorbable barrier. Animal studies of a collagen membrane extracted from porcine dermis (PDCM), as conditioned by different concentrations of glutaraldehyde (GA), have shown it to be biocompatible and biodegradable (up to 9 wk). This in vitro study further investigated the physical properties of this membrane. A PDCM modified and cross-linked with various concentrations (0.01%, 0.05% and 3.00%) of GA was used. A similar control series was not conditioned. At least 4 specimens for each experimental condition were prepared. The elastic modulus (EM) was measured by a universal testing machine. In the permeability test, Al2O3 particles of different sizes (5-23 microns) were mixed with normal saline to make 5 v/v% suspension and the time needed for collecting 7.5 ml of the filtered suspension from 10 ml suspension was recorded. Swelling ratio (gamma) was also measured according to gamma = 1/Vf (volume fraction). Data were analysed using ANOVA and Tukey's LSD test. The EM (40.8 +/- 3.8 gf/mm2) for the GA conditioned membranes showed no significant difference but was greater (p < 0.05) than that of the control. There was a significant increase (100-300%) in the permeation time with GA concentration (control 0.168 vs. 3% GA 0.100). The results suggest that the physical properties of the GA conditioned PDCM (especially in 3%) may fit the clinical requirements of membrane materials used in guided tissue regeneration techniques.

Expression and characterization of the recombinant gene encoding chitinase from Aeromonas caviae.

The gene encoding a chitinase from Aeromonas caviae was cloned by PCR techniques. Its recombinant gene expression was performed using pET20b(+) in Escherichia coli BL21 (DE3). The recombinant chitinase with the extra 33 and 13 amino acids in its N- and C-termini, respectively, was purified to near homogeneity using His-Tag affinity chromatography. The recombinant chitinase was found to be present in both the culture medium and the cytoplasm. A single protein band on the native polyacrylamide gel was confirmed by both the activity staining and protein staining. The optimum pH and temperature of the recombinant chitinase were determined to be 6.25-6.5 and 42.5 degrees C, respectively. It was stable within the pH range of 5-7. Significant activity stimulation by Cu2+ and inhibition by Fe3+ and Hg2+ were observed. Detergents such as SDS and Triton X-100 strongly inhibited the enzyme activity. Substrates such as 4-methylumbelliferyl-N,N'-diacetylchitobioside and 4-methylumbelliferyl-N,N',N"-triacetylchitotriose were hydrolyzed by the recombinant chitinase; however, 4-methylumbelliferyl-N-acetylglucosaminide was not cleaved during the activity assay periods. When chitin power was suspended in buffer with the chitinase (pH 6.5 and 42.5 degrees C), N-acetylchitooligosaccharides [(GlcNAc)n, n = 1-4] were detected at 24 h.

Structure and transcription of genes within the beta-hbd-adh1 region of Clostridium acetobutylicum P262.

The 1.2-kb DNA fragment upstream of the linked beta-hbd (3-hydroxybutyryl-CoA dehydrogenase) and adh1 (NADPH-dependent alcohol dehydrogenase) genes from Clostridium acetobutylicum P262 was sequenced. The upstream region contained an open reading frame (ORFB) which was found to have 44% amino acid identity to the fixB gene products of Rhizobium and Azorhizobium. The beta-hbd and ORFB genes were expressed during the acidogenic and solventogenic phases. The beta-hbd gene was transcribed on a single mRNA species of 2.0 kb, whereas the ORFB gene was transcribed on two species of mRNA of 2.0 and 3.5 kb, respectively. The adh1 gene was induced or derepressed at the pH breakpoint before the onset of solventogenesis and was transcribed on a single species of mRNA of 2.4 kb.

Purification, characterization, and amino terminal sequence of xylose reductase from Candida shehatae.

D-Xylose is a major component of the carbohydrates derived from agricultural residues and forest products. Among more than two hundred known xylose-utilizing yeasts, only a few species are known to be able to ferment xylose anaerobically. Candida shehatae is one of such xylose-fermenting yeasts. Xylose reductase (E.C. 1.1.1.21) is a key enzyme responsible for xylose metabolism in xylose-utilizing as well as xylose-fermenting yeasts. In this paper, we report the development of a convenient and reliable procedure for the purification of xylose reductase from C. shehatae to near homogeneity. The amino acid composition and N-terminal sequence of the enzyme have also been analyzed. C. shehatae seems to contain only a single xylose reductase, but the enzyme has a dual coenzyme specificity for both NADPH and NADH. The enzyme is remarkably stable at room temperature and 4 degrees C.

Human hepatitis delta virus RNA subfragments contain an autocleavage activity.

Hepatitis delta virus (HDV) contains a single-stranded circular RNA genome of 1.7 kilobases. In this report we demonstrate that subfragments of HDV RNA can undergo autocatalytic cleavage. This cleavage requires at least 500 microM of Mg2+ or Ca2+, is not affected by varying the pH from 5.0 to 9.1, and occurs with RNA fragments as small as 133 nucleotides. The larger RNA fragments containing additional HDV sequences have a lower efficiency of cleavage. Deletion analysis at both ends of RNA subfragments suggested that the catalytic ability of HDV RNA resides in a stretch of no more than 117 nucleotides around the cleavage site. The cleavage occurs at the phosphodiester bond between nucleotides 688 and 689 on the HDV genomic map, generating a 5' fragment with a terminal uridyl 2',3'-cyclic monophosphate residue and a 3' fragment with a guanosyl residue with a 5'-hydroxyl group. The smallest autocleaving RNA does not contain the "hammerhead" sequence required for the autocleavage of other known self-cleaving RNA. The cleavage of HDV RNA occurs at a much faster rate, even at a very low Mg2+ concentration, than that of other "ribozymes." Thus, HDV RNA represents a distinct class of ribozyme.

Electroblotting onto glass-fiber filter from an analytical isoelectrofocusing gel: a preparative method for isolating proteins for N-terminal microsequencing.

A new method has been developed for the isolation of proteins for microsequencing. Proteins were separated by isoelectric focusing on polyacrylamide slab gels. Ampholytes in the gel were washed out with 3.5% (v/v) perchloric acid, and the proteins were electroblotted onto unmodified glass-fiber sheets. The immobilized proteins on the glass-fiber sheet were detected with Coomassie blue dye staining. The protein bands were then excised from the sheet and inserted into a gas phase sequenator for direct sequencing. They could also be extracted with sodium dodecyl sulfate buffer for molecular weight determination. Bovine serum albumin, beta-lactoglobulin A, and soybean trypsin inhibitor have been used as standard proteins for the test of this technique. Using this technique, we have determined the partial N-terminal sequence (26 residues) of an acidic (pI 5.6) glutathione S-transferase isolated from the chicken liver.