The crystal structure of a class II fructose-1,6-bisphosphate aldolase shows a novel binuclear metal-binding active site embedded in a familiar fold

BACKGROUND: [corrected] Aldolases catalyze a variety of condensation and cleavage reactions, with exquisite control on the stereochemistry. These enzymes, therefore, are attractive catalysts for synthetic chemistry. There are two classes of aldolase: class I aldolases utilize Schiff base formation with an active-site lysine whilst class II enzymes require a divalent metal ion, in particular zinc. Fructose-1,6-bisphosphate aldolase (FBP-aldolase) is used in gluconeogenesis and glycolysis; the enzyme controls the condensation of dihydroxyacetone phosphate with glyceraldehyde-3-phosphate to yield fructose-1,6-bisphosphate. Structures are available for class I FBP-aldolases but there is a paucity of detail on the class II enzymes. Characterization is sought to enable a dissection of structure/activity relationships which may assist the construction of designed aldolases for use as biocatalysts in synthetic chemistry. RESULTS: The structure of the dimeric class II FBP-aldolase from Escherichia coli has been determined using data to 2.5 A resolution. The asymmetric unit is one subunit which presents a familiar fold, the (alpha/beta)8 barrel. The active centre, at the C-terminal end of the barrel, contains a novel bimetallic-binding site with two metal ions 6.2 A apart. One ion, the identity of which is not certain, is buried and may play a structural or activating role. The other metal ion is zinc and is positioned at the surface of the barrel to participate in catalysis. CONCLUSIONS: Comparison of the structure with a class II fuculose aldolase suggests that these enzymes may share a common mechanism. Nevertheless, the class II enzymes should be subdivided into two categories on consideration of subunit size and fold, quaternary structure and metal-ion binding sites.     

The fate of filial attachment in juvenile guinea pigs housed apart from the mother

Thirty-five-day-old juvenile guinea pigs exhibited higher levels of plasma cortisol following 60 min of exposure to a novel environment when tested alone than when in the presence of the mother. This effect occurred regardless of whether the offspring had been housed continuously with mother and littermates from birth until testing or had been rehoused with just littermates 1 or 2 days prior to testing. Offspring vocalized more when alone then when in the presence of the mother only if they had been housed continuously with her until testing. In addition, juvenile males reunited with the mother after 1 or 2 days directed sexual behavior toward her. The sexual behavior was not accompanied by a significant change in plasma testosterone levels, though there was an overall suppression of levels following isolation in the novel test environment. The results show that guinea pig mothers maintain the capacity to prevent their offspring from exhibiting a plasma cortisol stress response even when the offspring are well beyond weaning and have been housed apart from her for up to 2 days. This suggests the existence of filial attachment in juveniles under these conditions. Further, juvenile males appear capable of simultaneously directing filial attachment and sexual behavior toward the same female.     

Use of functional magnetic resonance imaging

It has been the goal of this article to provide the reader with a brief background of fMRI, a basic understanding of the techniques of fMRI, and, more importantly, the potential for clinical and experimental studies using fMRI. In contrast to the limited number of installed PET and MEG units, the large installed base of MR imaging scanners (over 1000 installed at least at 1.5 T in the United States) makes fMRI potentially widely available. Initial studies (both clinical and experimental) have been validated and are reproducible in mapping the sensorimotor and visual cortices. The areas of language lateralization and memory are still preliminary at best. As methods to reduce the effects of head motion (due to both bulk head motion and physiologically induced motion) arise, the reliability of fMRI should improve, allowing for more definitive identification of task activation.     

Comparative costs of family planning services and hospital-based maternity care in Turkey

The effect of dietary fats on the chemical composition and enzyme activities has been studied in intestinal brush border membranes (BBM) or rats. Animals were given commercial rat pellet diet (RP) or semisynthetic diet rich in either saturated [coconut oil (CCO))] or polyunsaturated [n-6, corn oil (CO) or n-3, fish oil (FO)] fat at the 10% level for 5 weeks. The membrane cholesterol/phospholipid ratio was augmented in CO- or RP-fed rats. There was an increase in level of saturated fatty acids in BBM from CCO- or FO-fed animals. n-3 polyunsaturated fatty acid content was raised in FO-fed rats, while the proportion of linoleic acid and arachidonic acid was enhanced in animals given a CO diet. Membrane fluidity was in the order of CCO < RP = CO < FO. The membrane hexose content was high (p < 0.05) in the CCO group. Hexosamines were elevated (p < 0.05) in CCO- or FO-fed rat brush borders. Membrane fucose was unaltered, while sialic acid content was elevated in CO- (p < 0.05) and FO- (p < 0.01) fed vs. CCO-fed rats. Lectin binding to brush borders corroborated these findings. The activities of alkaline phosphatase, sucrase and lactase were augmented (p < 0.001) in CCO-fed animals. Leucine-aminopeptidase and sucrase activities were depressed by FO feeding. The activities of PNP-beta-glycosidases were the highest in FO-fed rats. These results indicate that dietary fat quality markedly affects microvillus membrane lipid composition, glycosylation and enzyme functions in rat intestine.     

Synthesis and characterization of 8-methoxy-2''- deoxyadenosine-containing oligonucleotides to probe the syn glycosidic conformation of 2''-deoxyadenosine within DNA

BACKGROUND: Rectal motor activity is incompletely understood. The aim of this study was to characterize the patterns of rectal motor activity and to examine their diurnal variation and their relationships to proximal colonic activity and to meals. METHODS: We performed a 30-h ambulatory motility study by recording pressure activity at multiple sites in the colon in 18 normal subjects. RESULTS: During 288 h of recording, discrete bursts of tonic and phasic activity were seen in the rectum of all subjects at night and during the day lasting > or = 3 min, with a predominant frequency of 3 waves/min: periodic rectal motor activity (PRMA). Nocturnally, the number of cycles and the proportion of time occupied by this activity were greater (p < 0.001) and the inter-cycle interval was shorter (p < 0.008) compared with daytime, but the cycle duration was similar. Only 4 versus 5% (nocturnal vs daytime) of cycles propagated aborad, whereas 36 versus 14% (p < 0.01) propagated retrogradely, 16 versus 47% (p < 0.01) occurred simultaneously, and 44 versus 34% were confined to the rectum. There was considerable intra- and intersubject variability. PRMA was not related to meals or to anal motor activity, but 81% of nocturnal and 94% of daytime cycles occurred within 5 min of a motor event in the more proximal colon. CONCLUSIONS: PRMA is a characteristic feature of the normal rectum and is more frequent at night. The temporal association with motor events in the proximal colon suggests that PRMA is triggered by the arrival of stool or gas in the rectum. Because most cycles are either segmental or are propagated retrogradely, PRMA may serve as an intrinsic braking mechanism that prevents untimely flow of colonic contents, particularly during sleep.     

Ion channels in freshly isolated and cultured human bronchial smooth muscle cells

Voltage-gated ion currents were studied in human bronchial airway smooth muscle (ASM) cells. Proliferating or growth-arrested cells in culture were compared with freshly isolated cells. Three types of charybdotoxin (ChTX)-sensitive K+ channel were observed in all cell types, with conductances in symmetrical 140 mM KCl solutions ([Ca2+]i < 0.1 nM) of 206 +/- 14 pS (n = 32), 144 +/- 11 pS (n = 27) and 109 +/- 5 pS (n = 25). The relative proportion of each channel type differed substantially between the three groups of cells. In freshly isolated ASM cells large conductance K+ channels were represented almost entirely by a conductance of 206 pS, which was found in all twenty-three patches studied. In contrast, in most patches from proliferating cells the majority of channels had conductances of either 144 pS (14 of 21 patches) or 109 pS (8 of 21 patches). Cultured cells that had been growth arrested by serum depletion revealed the same set of channels as the proliferating cells, but the occurrence of the 109 pS channel was much more frequent (16 of 19 patches). As has been shown previously, 206 pS channels were active at a physiological membrane potential (-60 to -20 mV) even at a very low free [Ca2+]. The 144 pS channels could be recorded only at depolarized potentials (+80 to +100 mV), whereas 109 pS channels were active over a wide range of potentials (-60 to +100 mV), but only in the presence of GTP. In a proportion of cultured cells a tetrodotoxin-sensitive Na+ current and a hyperpolarization-induced inwardly rectifying K+ current were also observed (15 and 21%, respectively, of all cells examined). Neither of these currents were observed in freshly isolated cells. Whole-cell outward current in all groups was sensitive to tetraethylammonium, ChTX, and iberiotoxin, but not to 4-aminopyridine. In summary, it is clear that during proliferation there are considerable changes in the expression of ionic channels in ASM that have profound functional significance. In particular, these changes would tend to make the tissue more excitable, and may be of relevance to the proliferative process itself.     

Firearm-related hunting fatalities in North Carolina: impact of the 'Hunter Orange' Law

In the decade spanning 1983 through 1992, 68 people were killed by firearms while hunting in North Carolina (average of 1.66 fatalities/100,000 licenses issued). Of these, 58 deaths involved two parties, a shooter and a victim. In 22% of the incidents the victims were mistaken for game. During the 1987-1988 hunting season a "Hunter Orange" law was initially enforced. This law requires sportsmen to wear an article of bright orange clothing while hunting. After enactment of this law, a reduction in the incidence of hunters being killed because they were "mistaken for game" has proven statistically significant. The present study documents that legally mandating bright orange clothing has resulted in fewer firearms-related fatalities due to the victim's being mistaken for game while hunting. The North Carolina experience implies that governmental intervention can influence the incidence of accidental deaths during recreational hunting.     

Quantal duration of auditory memories

Neuronal responses in the caudomedial neostriatum (NCM) of adult zebra finches (Taeniopygia guttata) decreased upon repeated, unreinforced presentations of conspecific song, calls, or other complex sounds. This "stimulus-specific habituation" is a form of learning, and its spontaneous loss, a form of "forgetting." Spontaneous forgetting occurred only at narrowly defined times (2 to 3, 6 to 7, 14 to 15, 17 to 18.5, 46 to 48, or 85 to 89 hours after first exposure to a stimulus), determined by stimulus class, number of presentations, and interval between presentations. The first five forgetting times coincided with periods when gene expression and protein synthesis in NCM were required for maintenance of the longer lasting (85 to 89 hours) habituation. The number of successive episodes of gene expression induced by a stimulus, but occurring long after stimulus presentation, appears to determine the quantal duration of auditory memories.     

Transforming growth factor beta 1 down-regulates vascular endothelial growth factor receptor 2/flk-1 expression in vascular endothelial cells

Although the importance of the vascular endothelial growth factor (VEGF)/VEGF tyrosine kinase receptor (VEGFR) system in angiogenesis is well established, very little is known about the regulation of VEGFR expression in vascular endothelial cells. We have cloned partial cDNAs encoding bovine VEGFR-1 (flt) and -2 (flk-1) and used them to study VEGFR expression by bovine microvascular- and large vessel-derived endothelial cells. Both cell lines express flk-1, but not flt. Transforming growth factor beta 1 (TGF-beta 1) reduced the high affinity 125I-VEGF binding capacity of both cell types in a dose-dependent manner, with a 2.0-2.7-fold decrease at 1-10 ng/ml. Cross-linking experiments revealed a decrease in 125I-VEGF binding to a cell surface monomeric protein corresponding to Flk-1 on the basis of its affinity for VEGF, molecular mass (185-190 kDa), and apparent internalization after VEGF binding. Immunoprecipitation and Western blot experiments demonstrated a decrease in Flk-1 protein expression, and TGF-beta 1 reduced flk-1 mRNA levels in a dose-dependent manner. These results imply that TGF-beta 1 is a major regulator of the VEGF/Flk-1 signal transduction pathway in endothelial cells.