In Caenorhabditis elegans males, a row of epidermal precursor cells called seam cells generates a pattern of cuticular alae in anterior body regions and neural sensilla called rays in the posterior. The Hox gene mab-5 is required for two posterior seam cells, V5 and V6, to generate rays. In mab-5 mutant males, V5 and V6 do not generate sensory ray lineages but instead generate lineages that lead to alae. Here we show that two independent regulatory pathways can activate mab-5 expression in the V cells. First, the caudal homolog pal-1 turns on mab-5 in V6 during embryogenesis. Second, a Wnt signaling pathway is capable of activating mab-5 in the V cells during postembryonic development; however, during normal development Wnt signaling is inhibited by signals from neighboring V cells. The inhibition of this Wnt signaling pathway by lateral signals between the V cells limits the number of rays in the animal and also determines the position of the boundary between alae and rays.

Primary adrenal lymphoma (PAL) is an extremely rare entity and adrenal insufficiency is a common complication. Bilateral enlargement of adrenal glands should raise the suspicion of lymphoma, especially in patients with clinical or laboratory features of adrenal insufficiency. Most of these cases are highly aggressive tumors and should be treated with multiagent chemotherapy. The role of bilateral adrenalectomy and/or radiotherapy cannot be estimated. Our patient with PAL was an 80-year-old man who presented with clinical and laboratory features of adrenal insufficiency. Combination chemotherapy plus rituximab was unsuccessful and our patient died from progressive disease.

OBJECTIVE

Connective tissue growth factor (CTGF) stimulates extracellular matrix formation, fibrosis, and angiogenesis. It has a role in the pathogenesis of diabetic nephropathy and possibly in diabetic retinopathy (DR): in cultured retinal vascular cells CTGF is induced by VEGF-A. To further characterise this role the authors investigated CTGF expression in normal and diabetic human retina.

METHODS

CTGF expression patterns were studied by immunohistochemistry in the retina of eyes of 36 diabetic persons and 18 non-diabetic controls and compared with markers of endothelial cells (CD31, PAL-E), pericytes (NG2), astrocytes (GFAP), and microglia (CD45).

RESULTS

In the retina, distinct and specific staining of CTGF was observed in microglia, situated around or in close vicinity of retinal capillaries. In the control cases, sporadic staining of pericytes was also observed within the vascular wall. In contrast, in the retina of people with diabetes, CTGF staining in microglia was decreased and staining in pericytes was increased. This pattern of predominantly pericyte staining was observed in 20 out of 36 diabetic cases and in one out of 18 controls. The altered CTGF staining patterns in the diabetic cases did not correlate to staining of PAL-E, a marker of retinal vascular leakage associated with DR.

CONCLUSIONS

The study shows that CTGF is expressed in microglia in the normal retina whereas in a large subset of diabetic persons, CTGF expression shifts to microvascular pericytes. This altered CTGF expression pattern appears unrelated to manifest DR and may therefore represent a preclinical retinal change caused by diabetes. The results suggest a distinct, but as yet unidentified, role of CTGF in the pathogenesis of diabetic retinopathy.

NtMyb2 is a regulator of the tobacco retrotransposon Tto1 and the defense-related gene phenylalanine ammonia lyase (PAL), which are induced by various stress stimuli such as wounding or elicitor treatment. NtMyb2 is also induced by wounding or elicitor treatment and is regulated at the transcriptional level. In this study, mutational analysis of the promoter of NtMyb2 and gain-of-function analysis in vivo showed that the sequence AGATCCAA, named the AG-motif, is a cis-element sufficient to confer responsiveness to wounding and elicitor treatment. Furthermore, by using the south-western method, we cloned cDNAs encoding a GATA-type zinc finger protein, which can specifically bind to the AG-motif, named AG-motif binding Protein (AGP1). Domain analysis revealed that not only the GATA-type zinc finger region but also the downstream His2 motif of AGP1 is required for binding activity, showing that the AGP has a novel GATA-type zinc finger domain. AGP1 can activate expression from promoters containing the AG-motif in tobacco protoplasts, indicating that AGP1 is a positive regulator of NtMyb2. We also found that the AGP1 binding activity is highly enhanced by adenine methylation of the AG-motif by bacterial dam methylase.

Although phenylpropanoids and flavonoids are common plant natural products, these major classes of biologically active secondary metabolites are largely absent from bacteria. The ubiquitous plant enzymes phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) are key biosynthetic catalysts in phenylpropanoid and flavonoid assembly, respectively. Until recently, few bacterial counterparts were known, thus reflecting the dearth of these plant natural products in bacteria. This review highlights our progress on the biochemical and genetic characterization of recently identified streptomycete biosynthetic pathways to benzoic acid and type III polyketide synthase (PKS)-derived products. The sediment-derived bacterium "Streptomyces maritimus" produces benzoyl-CoA in a plant-like manner from phenylalanine involving a PAL-mediated reaction through cinnamic acid during the biosynthesis of the polyketide antibiotic enterocin. All but one of the genes encoding benzoyl-CoA biosynthesis in "S. maritimus" have been cloned, sequenced, and inactivated, providing a model for benzoate biosynthesis not only in this bacterium, but in plants where benzoic acid is an important constituent of many products. The recent discovery that bacteria harbor homodimeric PKSs belonging to the plant CHS superfamily of condensing enzymes has further linked the biosynthetic capabilities of plants and bacteria. A bioinformatics approach led to the prediction that the model actinomycete Streptomyces coelicolor A3(2) contains up to three type III PKSs. Biochemical analysis of one of the recombinant type III PKSs from S. coelicolor demonstrated activity as a 1,3,6,8-tetrahydroxynaphthalene synthase (THNS). A homology model of THNS based upon the known three-dimensional structure of CHS was constructed to explore the structural and mechanistic details of this new subclass of bacterial PKSs.

The doubly labeled water method for measuring average daily metabolic rate (ADMR) in combination with resting metabolic rate (RMR) allows one to assess the relation between exercise and energy balance. Three topics were included in an analysis of available data: 1) the limits of energy turnover in relation to physical performance for the achievement of energy balance, 2) the effect of an exercise intervention on daily energy turnover and its components, and 3) the effect of exercise on body composition. In the general population, physical activity level (PAL: ADMR/RMR) ranges between 1.2 and 2.2-2.5. There is no sex difference in the level of physical activity. Higher PAL values can be maintained by training and supplementation of the diet with energy-dense, carbohydrate-rich formulas. Exercise training does not influence spontaneous activity except in the elderly. In sedentary subjects, exercise training does not influence RMR when body weight is maintained. An exercise-induced increase in ADMR is about twice the training load. Exercise induces an increase in fat-free mass, especially in women, and a decrease in fat mass. Women tend to preserve energy balance and consequently loss of fat mass is significantly less.

Expression patterns of chitinase transcripts induced by N-acetylchitooligosaccharide elicitor were analyzed by northern blot hybridization in order to reveal a signal transduction pathway leading to the activation of class I chitinase genes (Cht-1 and Cht-3), which may play an important role in producing N-acetylchitooligosaccharide elicitor. The transcription level of both genes was enhanced in response to N-acetylchitooligosaccharides larger than pentaose at subnanomolar concentrations. These structure and dose dependencies were consistent not only with those for a 75 kDa high-affinity binding protein for N-acetylchitooligosaccharide elicitor in the plasma membrane, but also with other series of cellular responses including phytoalexin production and the expression of elicitor-responsive genes (EL2, EL3). Therefore, the elicitor signal to evoke these cellular responses including the activation of the chitinase genes could be common and transmitted into cells through the 75 kDa protein. However, the signal transduction pathway for the activation of the chitinase gene appeared to diverge from those for the other elicitor-responsive genes shortly after the signal perception. It was shown that the induction of chitinase expression by N-acetylchitooligosaccharide would require protein phosphorylation, but not de novo protein synthesis. The oxidative burst was demonstrated not to be necessary for transcriptional induction of the all four elicitor-responsive genes (Cht, PAL, EL2, EL3) by N-acetylchitooligosaccharide.

Ascorbic acid (vitamin C) is required for health and, in particular, its supplementation has beneficial effects in some pathological conditions. There are conflicting reports regarding the usefulness of ascorbic acid in the treatment of dementia. In this study, we investigated the effects of acute, short- and long-term pre-training administration of ascorbic acid (60,120 mg/kg) on passive avoidance learning (PAL) and memory in rats. Retention test was done 24h after training. The results showed that acute injection of ascorbic acid had no significant effect on PAL. On the other hand, both in the short- and long-term ascorbic acid treated groups trials to acquisition were less than control group. Also, ascorbic acid prolonged the step-through latency (STL) and decreased the time spent in the dark compartment in retention test. Thus, it can be concluded that short- and long-term supplementation with ascorbic acid has facilitatory effects on acquisition and retrieval processes of passive avoidance learning and memory in rats.

Limited proteolysis is widely used in biochemical and crystallographic studies to determine domain organization, folding properties, and ligand binding activities of proteins. The method has limitations, however, due to the difficulties in obtaining sufficient amounts of correctly folded proteins and in interpreting the results of the proteolysis. A new limited proteolysis method, named protease accessibility laddering (PAL), avoids these complications. In PAL, tagged proteins are purified on magnetic beads in their natively folded state. While attached to the beads, proteins are probed with proteases. Proteolytic fragments are eluted and detected by immunoblotting with antibodies against the tag (e.g., Protein A, GFP, and 6xHis). PAL readily detects domain boundaries and flexible loops within proteins. A combination of PAL and comparative protein structure modeling allows characterization of previously unknown structures (e.g., Sec31, a component of the COPII coated vesicle). PAL's high throughput should greatly facilitate structural genomic and proteomic studies.

Ozone (O3), a major photochemical oxidant, induces leaf injury concomitant with salicylic acid (SA) synthesis. In pathogen-infected leaves, SA is synthesized via two pathways, involving phenylalanine or isochorismate. SA biosynthesis under O3 fumigation is not well understood. When we applied 14C-labeled benzoic acid (a precursor of SA in the pathway via phenylalanine) to O3-exposed tobacco leaves, it was effectively metabolized to SA. However, the activity and mRNA level of isochorismate synthase (ICS) were not increased. In contrast, ICS activity was increased in O3-exposed Arabidopsis thaliana L. These results suggest that SA is synthesized via benzoic acid from phenylalanine in O3-exposed tobacco leaves but via isochorismate in Arabidopsis. Ethylene is a plant hormone that promotes leaf damage in O3-exposed plants. During O3 exposure, transgenic plants with a phenotype of reduced O3-induced ethylene production accumulated less SA than did wild-type plants. O3 increased the activity of phenylalanine ammonia-lyase (PAL) and the transcript levels of the chorismate mutase (CM) and PAL genes in wild-type tobacco, but their induction was suppressed in the transgenic plants. These results indicate that ethylene promotes SA accumulation by regulating the expression of the CM and PAL genes in O3-exposed tobacco.

Ammonia lyases catalyse the reversible addition of ammonia to cinnamic acid (1: R=H) and p-hydroxycinnamic (1: R=OH) to generate L-phenylalanine (2: R=H) and L-tyrosine (2: R=OH) respectively (Figure 1a). Both phenylalanine ammonia lyase (PAL) and tyrosine ammonia lyase (TAL) are widely distributed in plants, fungi and prokaryotes. Recently there has been interest in the use of these enzymes for the synthesis of a broader range of L-arylalanines. Aminomutases catalyse a related reaction, namely the interconversion of α-amino acids to β-amino acids (Figure 1b). In the case of L-phenylalanine, this reaction is catalysed by phenylalanine aminomutase (PAM) and proceeds stereospecifically via the intermediate cinnamic acid to generate β-Phe 3. Ammonia lyases and aminomutases are related in sequence and structure and share the same active site cofactor 4-methylideneimidazole-5-one (MIO). There is currently interest in the possibility of using these biocatalysts to prepare a wide range of enantiomerically pure l-configured α-amino and β-amino acids. Recent reviews have focused on the mechanism of these MIO containing enzymes. The aim of this review is to review recent progress in the application of ammonia lyase and aminomutase enzymes to prepare enantiomerically pure α-amino and β-amino acids.

Lettuce (Lactuca sativa) plants grown in a protective environment, similar to in vitro conditions, were acclimated in a growth chamber and subjected to water stress to examine the activation of genes involved in secondary metabolism and biosynthesis of antioxidants. The expression of phenylalanine ammonia-lyase (PAL), gamma-tocopherol methyl transferase (gamma-TMT) and l-galactose dehydrogenase (l-GalDH) genes involved in the biosynthesis of phenolic compounds, alpha-tocopherol and ascorbic acid, respectively, were determined during plant adaptation. These genes were activated in tender plants, grown under protective conditions, when exposed to normal growing conditions in a growth chamber. A large increase in transcript level for PAL, a key gene in the phenylpropanoid pathway leading to the biosynthesis of a wide array of phenolics and flavonoids, was observed within 1h of exposure of tender plants to normal growing conditions. Plant growth, especially the roots, was retarded in tender plants when exposed to normal growing conditions. Furthermore, exposure of both protected and unprotected plants to water stress resulted in the activation of PAL. PAL inhibition by 2-aminoindan-2-phosphonic acid (AIP) rendered these plants more sensitive to chilling and heat shock treatments. These results suggest that activation of secondary metabolism as well as the antioxidative metabolism is an integral part of plant adaptation to normal growing conditions in lettuce plants.

BACKGROUND

In LBP patients, the relationship between pain and physical activity remains unclear. Whereas a negative relationship between pain and self-reported physical activity was found, this relation disappeared in the case of overt behavioral data (e.g., accelerometer). Cognitive-behavioral models of the development of chronic pain suggest subgroups with signs of physical underuse and overuse.

OBJECTIVE

To examine if patients with pain-related adaptive, endurance and fear-avoidance coping differ in pain, self-reported physical function and overt physical activity 6 months after disc surgery.

METHODS

24 patients completed questionnaires (Von Korff chronic pain grade (CPG), Kiel pain inventory (KPI), Funktionsfragebogen Hannover-Rücken FFbH-R) and underwent an 8-h accelerometer assessment in their daily life (physical activity level (PAL), number of constant postures (CP)). The KPI differed between adaptive coping (AC) (N=9), fear avoidance coping (FAC) (N=1) and endurance coping (EC) (N=14).

RESULTS

In the whole group, pain intensity was negative related to self-reported physical activity whereas PAL and CP displayed no correlation with pain. EC patients showed significantly higher pain scores and lower self-reported physical functioning compared to AC but the same level of PAL and furthermore, a significantly higher number of CPs in daily life. The visual inspection of the FAC patient revealed also high pain, low physical functioning and low overt physical activity.

CONCLUSIONS

The assessment of pain-related coping modes yielded an important differentiation between subgroups of LBP patients 6 months after surgery. Endurance copers displayed signs of overuse in their daily behavior in spite of pain than adaptive copers. The one fear avoidance coper tends to do less physical activity in the sense of underuse.

Flavonols, phenylalanine-derived secondary metabolites, have protective and regulatory functions in plants. In Arabidopsis thaliana, they are consecutively glycosylated at their 3-OH and 7-OH groups. UGT78D1 and UGT78D2 are the major flavonol 3-O-glycosyltransferases in Arabidopsis leaves. The ugt78d1 ugt78d2 double mutant, which was strongly compromised in the initial 3-O-glycosylation, showed a severe and specific repression of flavonol biosynthesis, retaining only one-third of the wild-type level. This metabolic phenotype was associated with a repressed transcription of several flavonol biosynthetic genes including the committed step chalcone synthase [(CHS) or TRANSPARENT TESTA 4 (TT4)]. Furthermore, the committed step of the upstream, general phenylpropanoid pathway, phenylalanine ammonia-lyase (PAL), was down-regulated in its enzyme activity and in the transcription of the flavonol-related PALPALPAL inhibition in a tt4 ugt78d1 ugt78d2 line. PAL activity was even enhanced in the flavonol synthase 1 mutant, which compromises the final formation of flavonol aglycones. The dependence of the PAL feedback inhibition on flavonols was confirmed by chemical complementation of tt4 ugt78d1 ugt78d2 using naringenin, a downstream flavonoid intermediate, which restored the PAL repression. Although aglycones were not analytically detectable, this study provides genetic evidence for a novel, flavonol-dependent feedback inhibition of the flavonol biosynthetic pathway and PAL. It was conditioned by the compromised flavonol-3-O-conjugation and a decrease in flavonol content, yet dependent on a residual, flavonol synthase 1 (FLS1)-related capacity to form flavonol aglycones. Thus, this regulation would not react to a reduced metabolic flux into flavonol biosynthesis, but it might prevent the accumulation of non-glycosylated, toxic flavonols.

The increasing demand of strawberry (Fragaria×ananassa Duch) fruits is associated mainly with their sensorial characteristics and the content of antioxidant compounds. Nevertheless, the strawberry production has been hampered due to its sensitivity to abiotic stresses. Therefore, to understand the molecular mechanisms highlighting stress response is of great importance to enable genetic engineering approaches aiming to improve strawberry tolerance. However, the study of expression of genes in strawberry requires the use of suitable reference genes. In the present study, seven traditional and novel candidate reference genes were evaluated for transcript normalization in fruits of ten strawberry cultivars and two abiotic stresses, using RefFinder, which integrates the four major currently available software programs: geNorm, NormFinder, BestKeeper and the comparative delta-Ct method. The results indicate that the expression stability is dependent on the experimental conditions. The candidate reference gene DBP (DNA binding protein) was considered the most suitable to normalize expression data in samples of strawberry cultivars and under drought stress condition, and the candidate reference gene HISTH4 (histone H4) was the most stable under osmotic stresses and salt stress. The traditional genes GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and 18S (18S ribosomal RNA) were considered the most unstable genes in all conditions. The expression of phenylalanine ammonia lyase (PAL) and 9-cis epoxycarotenoid dioxygenase (NCED1) genes were used to further confirm the validated candidate reference genes, showing that the use of an inappropriate reference gene may induce erroneous results. This study is the first survey on the stability of reference genes in strawberry cultivars and osmotic stresses and provides guidelines to obtain more accurate RT-qPCR results for future breeding efforts.

Tyrosine ammonia-lyase (TAL) is a recently described member of the aromatic amino acid lyase family, which also includes phenylalanine (PAL) and histidine ammonia-lyases (HAL). TAL is highly selective for L-tyrosine, and synthesizes 4-coumaric acid as a protein cofactor or antibiotic precursor in microorganisms. In this report, we identify a single active site residue important for substrate selection in this enzyme family. Replacing the active site residue His89 with Phe in TAL completely switched its substrate selectivity from tyrosine to phenylalanine, thereby converting it into a highly active PAL. When a corresponding mutation was made in PAL, the enzyme lost PAL activity and gained TAL activity. The discovered substrate selectivity switch is a rare example of a complete alteration of substrate specificity by a single point mutation. We also show that the identity of the amino acid at the switch position can serve as a guide to predict substrate specificities of annotated aromatic amino acid lyases in genome sequences.

OBJECTIVE

To determine the effect of progressive addition lenses (PALs) and single vision lenses (SVLs) on peripheral defocus in myopic children, and to compare the effect of myopic versus hyperopic peripheral defocus on foveal myopia progression.

METHODS

Eighty-four myopic children aged 6 to 11 years with spherical equivalent (SE) cycloplegic autorefraction between -0.75 diopters (D) and -4.50 D were randomly assigned to wear SVLs or PALs. Aberrometry measurements of the eye and spectacles were made centrally, 30° nasally, temporally, and superiorly, and 20° inferiorly on the retina using a Complete Ophthalmic Analysis System for Vision Research (COAS-VR). The association between peripheral defocus and the 1-year change in central myopia was investigated.

RESULTS

SVLs caused a hyperopic shift in peripheral defocus at all locations (all P ≤ 0.0003). PALs caused a myopic shift in peripheral defocus in three of four locations measured (all P ≤ 0.01) with the greatest shift superiorly due to the PAL addition (-1.04 ± 0.30 D). Superior retinal defocus when wearing either SVLs or PALs was associated with the 1-year change in central myopia. The adjusted 1-year change in central SE myopia was -0.38 D for children with absolute superior myopic defocus (n = 67) and -0.65 D for children with absolute superior hyperopic defocus (n = 17; difference = 0.27 D; P = 0.002).

CONCLUSIONS

PALs caused a myopic shift in peripheral defocus. Superior myopic defocus was associated with less central myopia progression. These data support the continued investigation of optical designs that result in peripheral myopic defocus as a potential way to slow myopia progression. (ClinicalTrials.gov number, NCT00335049.).

BACKGROUND

With the aid of assistive technology, some patients with amyotrophic lateral sclerosis (ALS) are able to live for several years past the lowest measurable level of function on the Amyotrophic Lateral Sclerosis Functional Rating Scale - Revised (ALSFRS-R), a widely used end-point in ALS assessment. There is a research need to monitor patient function at the end of life, particularly in the face of severe impairment or 'locked in syndrome'.

METHODS

We used an online community for people with ALS (PALS) (PatientsLikeMe) to construct and pilot a number of new items to add to the ALSFRS-R scale to improve its sensitivity at lower levels of physical function in patients with advanced ALS.

RESULTS

Ten new scale items were generated by a survey of PALS with advanced disease. These were added to the existing ALSFRS-R and data were received from 326 PALS at baseline with 169 PALS (52%) completing a 1-week test-retest and 218 PALS (67%) completing a 3-month retest [corrected].

CONCLUSIONS

Three new items were selected which conformed to the existing factor structure of the ALFRS-R. These relate to the ability to use fingers to manipulate devices, ability to show emotional expression in the face, and ability to get around inside the home. Real-world validation is the next step to assess the utility of the ALFRS-EX.

The Tol-Pal system of Escherichia coli is involved in maintaining outer membrane stability. Mutations in tolQ, tolR, tolA, tolB, or pal genes result in sensitivity to bile salts and the leakage of periplasmic proteins. Moreover, some of the tol genes are necessary for the entry of group A colicins and the DNA of filamentous bacteriophages. TolQ, TolR, and TolA are located in the cytoplasmic membrane where they interact with each other via their transmembrane domains. TolB and Pal form a periplasmic complex near the outer membrane. We used suppressor genetics to identify the regions important for the interaction between TolB and Pal. Intragenic suppressor mutations were characterized in a domain of Pal that was shown to be involved in interactions with TolB and peptidoglycan. Extragenic suppressor mutations were located in tolB gene. The C-terminal region of TolB predicted to adopt a beta-propeller structure was shown to be responsible for the interaction of the protein with Pal. Unexpectedly, none of the suppressor mutations was able to restore a correct association between Pal and peptidoglycan, suggesting that interactions between Pal and other components such as TolB may also be important for outer membrane stability.

This paper aims to report the physical activity pattern and energy expenditure of adults aged 18 - 59 years in the Malaysian Adults Nutrition Survey (MANS) carried out between October 2002 and December 2003. The survey included 7349 adults representative of Peninsular Malaysia (Northern, Southern, Central and East Coast), as well as Sabah and Sarawak. A total of 6926 adults, comprising 3343 men and 3583 women, completed the physical activity section of the survey. Physical activity data was obtained using a physical activity questionnaire and 24-hour physical activity recall. Basal metabolic rate (BMR) was calculated from Ismail et al. (1998) predictive equations; total energy expenditure (TEE) was then estimated through factorial calculations based on time allocated and energy cost of various activities. Physical activity level (PAL) was calculated as the ratio of TEE to BMR. It was found that almost three-quarters of Malaysian adults traveled by passive modes of transportation. Only a third reported having ever-exercised, and an even smaller proportion of the population (14%) had adequate exercise. The population also spent the majority of their time (74% of the day) in sedentary activities, such as sleeping or lying down; doing light intensity activities (15% of the day), and doing moderate to vigorous intensity activities (10% of the day). Mean BMR and TEE was significantly higher amongst men than women, while mean PAL values were similar for men [1.6 (1.6 - 1.7)] and women [1.6 (1.6 - 1.6)]. More men (16%) were categorised as having active PAL compared to women (10%), while more women (43%) were categorised as having sedentary PAL compared to their male counterparts (37%). The present study provides the first in-depth report of the physical activity pattern, and national estimates of energy expenditure and physical activity levels of Malaysian adults, and concluded that Malaysian adults are generally sedentary. It is thus important that physical activity be further promoted and integrated into the lives of the population, preferably through various health promotion efforts as well as through the commitment of the authorities in providing a suitable environment for an active lifestyle.

Escherichia coli is used as a model organism for elucidation of menaquinone biosynthesis, for which a hydrolytic step from 1,4-dihydroxy-2-naphthoyl-coenzyme A (DHNA-CoA) to 1,4-dihydroxy-2-naphthoate is still unaccounted for. Recently, a hotdog fold thioesterase has been shown to catalyze this conversion in phylloquinone biosynthesis, suggesting that its closest homolog, YbgC in Escherichia coli, may be the DHNA-CoA thioesterase in menaquinone biosynthesis. However, this possibility is excluded by the involvement of YbgC in the Tol-Pal system and its complete lack of hydrolytic activity toward DHNA-CoA. To identify the hydrolytic enzyme, we have performed an activity-based screen of all nine Escherichia coli hotdog fold thioesterases and found that YdiI possesses a high level of hydrolytic activity toward DHNA-CoA, with high substrate specificity, and that another thioesterase, EntH, from siderophore biosynthesis exhibits a moderate, much lower DHNA-CoA thioesterase activity. Deletion of the ydiI gene from the bacterial genome results in a significant decrease in menaquinone production, which is little affected in ΔybgC and ΔentH mutants. These results support the notion that YdiI is the DHNA-CoA thioesterase involved in the biosynthesis of menaquinone in the model bacterium.

Matrix metalloprotease-9 (MMP-9; 92-kd type IV collagenase, gelatinase B) is regarded as important for degradation of the basement membrane and extracellular matrix during cancer invasion and other tissue-remodeling events. Expression of MMP-9 was analyzed in 22 cases of human ductal breast cancer by immunohistochemistry and in 8 of these cases also by in situ hybridization. For immunohistochemistry we used affinity-purified polyclonal antibodies as well as a MMP-9-specific monoclonal antibody (clone 6-6B). Three different stromal cell types with a positive MMP-9 immunoreaction were identified morphologically: neutrophils and macrophage-like cells in all cases and vascular cells in 16 of 22 cases. Double immunofluorescence with antibodies to CD68 conclusively demonstrated MMP-9 expression in macrophages. To identify the positive vascular cells, we employed antibodies to von Willebrand factor and PAL-E for identification of endothelial cells, high molecular weight melanoma-associated antigen for pericytes, and alpha-smooth muscle actin for vascular smooth muscle cells. Using conventional and confocal double immunofluorescence microscopy, colocalization of MMP-9 was seen with high molecular weight melanoma-associated antigen, the pericyte marker, whereas little or no coexpression was seen with alpha-smooth muscle actin. Virtually no coexpression was seen with the endothelial cell markers PAL-E and von Willebrand factor. In situ hybridization showed that MMP-9 mRNA colocalized with MMP-9 immunoreactivity in macrophages and vascular structures, whereas no MMP-9 mRNA was detected in neutrophils. No MMP-9 immunostaining or in situ hybridization signal was detected in cancer cells in any of the cases. Based on these results, it is concluded that MMP-9 in human breast cancer is located in tumor-infiltrating stromal cells, including neutrophils, macrophages, and vascular pericytes, and that the latter two cell types also produce this metalloprotease. We suggest that the MMP-9 produced in pericytes may play a role in extracellular matrix degradation during tumor angiogenesis.

OBJECTIVE

To describe the relationship between graded levels of obesity and free-living energy expenditure in men and women in affluent societies.

METHODS

Analysis of 319 measurements of energy expenditure in adults aged 18-64 years. The variables analysed were: total energy expenditure (TEE, assessed by the doubly-labelled water method); measured basal metabolic rate (BMR); activity energy expenditure (AEE, derived as TEE-BMR); and physical activity level (PAL, derived as TEE/BMR). Results were analysed according to four categories of body mass index (BMI): < 25.0, 25.0-29.9, 30.0-35.0 and>> 35.0 kg/m2.

RESULTS

TEE increased steadily with increasing BMI (9.5 to 13.5 MJ/d in women, 12.9 to 17.5 MJ/d in men, ANOVA, P < 0.0001 for both sexes). BMR also increased (5.7 to 8.2 MJ/d in women, 7.2 to 11.6 MJ/d in men, P < 0.0001 for both). AEE increased steadily in women (3.8 to 5.3 MJ/d, P < 0.0003), but in men increased up to the third BMI category (5.7 to 7.5 MJ/d, n.s.) and then declined in the most obese group (5.9 MJ/d, n.s.). The increases in energy expenditure were not in direct proportion to body weight since, when expressed per kg, both TEE and AEE declined significantly with increasing BMI. PAL remained quite constant across the three lowest BMI groups, indicating similar levels of physical activity. There was a non-significant decrease in PAL in the most obese men and women.

CONCLUSIONS

This analysis confirms that habitual energy expenditure is substantially and progressively raised in obesity. It contradicts the claim, based on self-reported food intake, that obesity develops and is maintained in spite of very low levels of energy intake. The analysis suggests that, except in massive obesity, patterns of physical activity are quite similar at different levels of BMI. This does not exclude the possibility that an inactive lifestyle may be an important general risk factor for the development of obesity.

The spleen of the rat contains 5 well delineated compartments: a central and peripheral part of the periarteriolar lymphatic sheath (PALS), the marginal zone and the follicle with centre and corona. A study was made on the development of these compartments in correlation with the onset of immunocompetence of the spleen. The spleens of animals in the age range from 1-40 days were studied with the light and electron microscopes. Immunocompetence was assessed by measuring serum antibody levels 5 days after intravenous antigen administration. Comparable doses of paratyphoid vaccin (PTV) and sheep red blood cells (SRBC) were used: PTV as a thymus-independent, SRBC as a thymus-dependent antigen. At the first day of life few lymphocytes are present around small arterioles. The marginal zone is first present at 9 days of age. At 14 days of age the typical thymus-dependent area develops. Interdigitating cells, which seem to develop from monocytes, and lymphocytes are present in close apposition. Primary follicles were first seen on 20 days of age, follicle centres at 25 or 30 days of age. The appearance of "typical" dendritic cells coincided with the appearance of follicle centres. The first titre against PTV was seen after antigen administration at 9 days after birth, against SRBC after injection in 14 days old animals. As PTV is a thymus-independent antigen it needs only B-cells for a primary IgM response. The appearance of functional B-cells in sufficient numbers to give a measurable response therefore coincides with the appearance of the marginal zone. Although T-cells are present from birth, the response against SRBC is delayed until the thymus-dependent area has developed. Thus, in the first 2 weeks of life, T-cells are either immature, or they are not able to react, because their microenvironment is not yet adequate.