[Assessment of cortical bone material distribution in medial column of proximal humerus with computed tomography].

Objective: To evaluate the cortical bone characteristics in the medial column of proximal humerus. Methods: A total of one hundred and three healthy adults who underwent shoulder computed tomography scanning in Tianjing Hospital were included in this study.The subjects were divided into three groups according to the age: group A (20-39 years), group B (40-59 years), and group C (>60 years). Cortical bone mapping (CBM) was used to analyze ordinary clinical CT scans using Stradwin 5.2 software.Colors thickness maps were created for each proximal humerus.The region of interest (ROI) 1-6 were set at three levels of the lateral and medial column of proximal humerus.Cortical thickness (CTh), cortical mass surface density (CMSD), and endocortical trabecular bone mineral density (ECTD) were assessed in the three slices in proximal metaphysis.The impact of age, gender to the cortical bone indices of medial column of proximal humerus were investigated with relative analysis.Cortical indices of the lateral and medial column were compared with independent samples t test. Results: In ROI 2 and 3, men had higher cortical bone values than women, and significant differences in ECTD and CMSD were found in ROI 2, 3 and ROI 2 (t=2.100, 2.238, 2.530, all P<0.05). The lineal regression analysis showed that all cortical indices in ROI 1-3 decreased significantly with age for both women and men (r(2)=0.042-0.248, all P<0.05). In group A-C, the medial columns had higher CTh and CMSD values than lateral sides in plane 1, although significant differences were found only in group A (t=3.696, 3.749, both P<0.05). The highest CTh, CMSD and ECTD of the medial compact bone was detected in ROI 1, followed by ROI 2 and 3 in group A (F=5.867, 6.776, 19.062, all P<0.05). The medial columns had approximately equivalent cortica indices values in ROI 1-3 in group B and C. Conclusion: It indicated that significant regional variation in all cortical parameters exists in the medial column of proximal humerus, and the indices are influenced by gender and age.

[Effects and related mechanism of flavone from Galium verum L on peroxide induced oxidative injury in human umbilical vein endothelial cells].

OBJECTIVE: To investigate the effects of flavone from Galium verum L (FGVL) on hydrogen peroxide induced oxidative injury in human umbilical vein endothelial cells (HUVEC), and explore related mechanisms. METHODS: HUVEC were divided into five groups: control group (1640 complete medium), injured group (HUVEC treated with 100 µmol/L hydrogen peroxide for 4 h), FGVL group (HUVEC treated with 12.5 mg/L FGVL (group F1), 25.0 mg/L (group F2), 50.0 mg/L (group F3) for 24 h before hydrogen peroxide). The nitric oxide content was measured by nitric acid reductase method. The 6-keto-Prostacyclin-F1α (6-keto-PGF1α), thromboxane B2 (TXB2), interleukin(IL)-6 and IL-22 were determined by ELISA.mRNA expression of phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt) and endothelial nitric oxide synthase (eNOS) was detected by RT-PCR.Protein expression of p-Akt (ser(473)) and p-eNOS (ser(1177)) was determined by Western blot.Cell apoptosis was observed with fluorescence microscope after Hoechst33258 staining. RESULTS: (1) The contents of nitric oxide were significantly lower in the injured group than in the control group ((34.11±1.78) µmol/L vs. (74.81±2.93) µmol/L, P<0.05), which was significantly increased in group F2 ((41.86±2.32) µmol/L) and group F3 ((62.79±1.16) µmol/L) compared with injured group (both P<0.05). (2)The secretion level of 6-keto-PGF1α was significantly lower in the injured group ((44.84±3.87) ng/L) than in the control group ((82.38±3.98) ng/L, P<0.05), which was significantly increased in group F1 ((52.76±1.78) ng/L), FGVL 2 group which was(56.58±1.44) ng/L and FGVL 3 group which was(67.78±2.02) ng/L than that of injured group(all P<0.05). The secretion level of TXB2 was significantly higher in the injured group((43.37±3.96) ng/L) than in the control group ((25.56±1.75) ng/L, P<0.05), which was significantly reduced group F2 group ((32.41±1.68) ng/L) and group F3 ((28.23±2.15) ng/L) than that of injured group(both P<0.05). (3) The contents of IL-6 and IL-22 were significantly higher in the injured group ((539.74±11.63) ng/L) and ((23.70±3.05) ng/L, respectively) than in the control group ((288.67±19.52) ng/L) and ((23.70±3.05) ng/L, respectively, both P<0.05). The contents of IL-6 were significantly lower in group F1, F2 and F3 compared to that of injured group(all P<0.05). The contents of IL-22 were significantly lower in group F2 and F3 than that of injured group(both P<0.05). (4)The relative levels of PI3K mRNA and eNOS mRNA in injured group (0.68±0.09 and 0.22±0.03, respectively) were significantly lower compared to control group(0.81±0.12 and 0.63±0.11, respectively, both P<0.05), PI3K mRNA in group F2 (0.76±0.03) and group F3 (PI3K mRNA 0.83±0.06) as well as eNOS mRNA in group F1 (0.37±0.08), F2 (0.53±0.04) and F3 (0.56±0.09) than those of injured group(all P<0.05). The mRNA expression of Akt was similar among groups (P>0.05). (5) The relative levels of p-Akt (ser(473)) and p-eNOS (ser(1177)) in injured group (0.48±0.05 and 0.23±0.03, respectively) were significantly lower compared to control group (0.71±0.12 and 0.66±0.05, respectively, both P<0.05), which was up-regulated in group F1, F2 and F3 groups compared to injured group(all P<0.05). (6) The cell apoptosis rate in injured groups was significantly higher compared to control group which ((63.67±11.37)% vs. (4.67±1.15)%, P<0.05) which was significantly reduced in group F1((43.33±4.16)%), F2((18.33±4.93)%) and F3((15.67±2.08)%) compared to injured group(all P<0.05). CONCLUSION: The FGVL can reduce hydrogen peroxide induced oxidative injury in HUVEC by increasing the level of nitric oxide through PI3K/Akt/eNOS pathway.

A novel 3-base pair deletion of the CRYAA gene identified in a large Chinese pedigree featuring autosomal dominant congenital perinuclear cataract.

Congenital cataract is caused by reduced transparency of the lens resulting from metabolic disorders during the fetal period. The disease shows great heterogeneity both clinically and genetically. We identified a 4-generation ethnic Han Chinese family affected by autosomal dominant congenital perinuclear cataract. The patients underwent full clinical and ophthalmologic examinations to rule out any concomitant disorders. Blood samples were collected and genomic DNA was extracted. Potential mutations in the candidate gene alpha A crystallin (CRYAA) were screened. Prenatal diagnosis was then provided for a fetus of the affected proband by chorionic villus sampling. In all patients, DNA sequencing of the CRYAA gene revealed a novel 3-bp deletion mutation in exon 3 (c.246_248delCGC), which led to deletion of codon 117 encoding arginine (p.117delR) in the peptide chain. The same mutation was not found among unaffected and healthy individuals. Bioinformatic analysis revealed that although the c.246_248delCGC is an 'in-frame' mutation, removal of arginine resulted in a significant change in the protein structure. The fetus did not possess this mutation and was confirmed to be healthy at 1-year follow-up. A novel disease-causing mutation, c.246_248delCGC (p.117delR), of the CRYAA gene has been identified in a Chinese family with autosomal-type perinuclear congenital cataracts. This is also the first report of prenatal diagnosis of this type of congenital cataract.

Plasma interleukin-1beta, prolactin, ACTH and corticosterone responses to endotoxin after damage of the anterior hypothalamic area.

This report concerns the use of an animal model described by us [J Submicrosc Cytol Pathol 1995;27:83-89] to investigate neural and endocrine sites for endotoxin (ENDO, E. coli 055:B5, 200 microg/100 g body weight in saline intravenously) effects on immunomodulatory hormone and cytokine release. Plasma interleukin-1beta (IL-1beta), prolactin (PRL), ACTH and corticosterone responses to ENDO after neurotoxic damage of neurons residing in the anterior hypothalamic area (AHA) were studied in freely behaving male rats. Excitotoxic cell damage in the AHA was produced by bilaterally injecting N-methyl-DL-aspartate (NMA) in artificial cerebrospinal fluid (aCSF) into this brain site. Injections of comparable volumes of aCSF alone served as controls for brain damage associated with the treatment. In both experimental brain manipulations before ENDO challenge the rise in plasma IL-1beta concentrations in response to ENDO was reduced by 2-fold at 1 h and 3- to 5-fold at 3 h when compared to controls. Nevertheless, experimental and control brain manipulations did not modulate the expected rise in corticosterone concentrations after ENDO exposure which rose 5-fold above the baseline level in all animals. However, AHA manipulation did reduce plasma ACTH and prolactin concentrations differentially. Introduction of either NMA or the control injection of aCSF alone into AHA reduced plasma ACTH concentrations by 2-fold at 0.5 and 1 h after ENDO. However, there was a greater reduction in the rise of plasma PRL concentrations after ENDO found in NMA-treated groups versus rats receiving control aCSF. These results demonstrate that variable-size hypothalamic damage (a larger lesion produced in AHA by NMA treatment vs. a smaller lesion control after aCSF) can result in a differential blunting of PRL, IL-1beta and ACTH release into blood in the face of robust, unmodulated corticosterone increases. In summary, these findings revealed a consistent predominant influence of ENDO on adrenal release of corticosterone as a concomitant to differential IL-1beta, ACTH and PRL release after AHA cell loss. In conclusion, these results constitute further evidence for hypothalamic orchestration of a balance between immunotropic and immunosuppressive neuroendocrine-immune events during acute bacterial infection of mammals.

Identification of a cis-acting element in the rat alpha-fetoprotein gene and its specific binding proteins in F9 cells during retinoic acid-induced differentiation.

Mouse F9 embryonic teratocarcinoma stem cells can be induced to differentiate into visceral endoderm. Following retinoic acid (RA) treatment, alpha-fetoprotein (AFP), a differentiation marker, is expressed and secreted. The mechanism by which RA regulates AFP expression during differentiation is not clear. The relatively late induction of AFP indicates that the AFP gene may not be a primary target of RA activity during F9 cell differentiation. In this study, a CAT reporter plasmid containing the rat AFP 5'-regulatory region (-7040 to +7) adjacent to the CAT gene (pAFPCAT) was stably transfected into F9 cells and used to delineate a cis-acting element which associates with AFP gene activation. Similar spatial and temporal expression patterns between the transcriptional activity of the recombinant AFP gene and the endogenous AFP gene demonstrate that this stably transfected F9 system can be used to dissect both cis-elements and trans-acting factors responsible for RA-induced AFP expression. Using a series of deletion mutants of the pAFPCAT, the region between -2611 to -1855 was found to be important in AFP-induction. Subsequent analysis identified a functional sequence (-1905 to -1891, 5'-ACTAAAATGGAGACT-3') that differentially binds nuclear proteins from undifferentiated and differentiated F9 cells. This sequence, designed as differentiation-associated sequence (DAS) for its unique binding of a nuclear protein (DAP-II) that appears during RA-induced F9 differentiation, acts as a regulatory protein factor in AFP gene activation.

Resonant activations for a fluctuating barrier system driven by dichotomous noise and Gaussian white noise.

We consider the escape over a fluctuating barrier in the presence of a dichotomous noise and a Gaussian white noise. It is shown that the mean first passage time (MFPT) over the fluctuating barrier displays two resonant activations. One is the resonant activation of the MFPT as a function of the flipping rate of the fluctuating potential barrier; the other is the resonant activation of the MFPT as a function of the transition rate of the dichotomous noise. In addition, we find that the dichotomous noise can weaken the former resonant activation, but enhance the latter one. By further study, we find that, when the fluctuating potential barrier is driven by two or more dichotomous noises, there are three or more resonant activations.

Escape over a fluctuating barrier with additive and multiplicative noise.

The mean first passage time (MFPT) over the fluctuating potential barrier is investigated in the presence of additive and multiplicative noises. It is shown that the MFPT over the fluctuating potential barrier displays a resonant activation (RA). The effect of the additive and multiplicative noises and the correlation between them on the RA is that the additive and multiplicative noises can weaken the RA; but the correlation between them can enhance it. The susceptibility of the RA to the multiplicative noise is far larger than that to the additive one. In addition, we find that the transition rate (i.e., the inverse of the MFPT) over the fluctuating potential barrier can be suppressed by the positive correlation and show a minimum as the function of the noises' strengths.

cAMP-induced morphological changes are counteracted by the activated RhoA small GTPase and the Rho kinase ROKalpha.

Dramatic transient changes resulting in a stellate morphology are induced in many cell types on treatment with agents that enhance intracellular cAMP levels. Thrombin fully protects cells from this inductive effect of cAMP through the thrombin receptor. The protective effect of thrombin was shown to be Rho-dependent. Clostridium botulinum C3 exoenzyme, which inactivates RhoA functions, abolished the ability of thrombin to protect cells from responding to increased cAMP levels. A constitutively activated RhoAV14 mutant protein also prevented cells from responding to cAMP. RhoA can be specifically phosphorylated at Ser-188 by the cAMP-activated protein kinase A (PKA). We demonstrate that RhoAV14A188, which cannot be phosphorylated by PKA in vitro, is more effective than RhoAV14 in preventing cells from responding to cAMP and in inducing actin stress fiber formation. This suggests that PKA phosphorylation of RhoA impairs its biological activity in vivo. ROKalpha, a RhoA-associated serine/threonine kinase can also prevent cells from responding to cAMP with shape changes. Phosphorylation of RhoA by PKA in vitro decreases the binding of RhoA to ROKalpha. These results indicate that RhoA and cAMP have antagonistic roles in regulating cellular morphology and suggest that cAMP-mediated down-regulation of RhoA binding to its effector ROKalpha may be involved in this antagonism.

Aberrant cell division and random FtsZ ring positioning in Escherichia coli cpxA* mutants.

In Escherichia coli, certain mutations in the cpxA gene (encoding a sensor kinase of a two-component signal transduction system) randomize the location of FtsZ ring assembly and dramatically affect cell division. However, deletion of the cpxRA operon, encoding the sensor kinase and its cognate regulator CpxR, has no effect on division site biogenesis. It appears that certain mutant sensor kinases (CpxA*) either exhibit hyperactivity on CpxR or extend their signalling activity to one or more noncognate response regulators involved in cell division.

Expression of constitutively active alpha-PAK reveals effects of the kinase on actin and focal complexes.

The family of p21-activated protein kinases (PAKs) appear to be present in all organisms that have Cdc42-like GTPases. In mammalian cells, PAKs have been implicated in the activation of mitogen-activated protein kinase cascades, but there are no reported effects of these kinases on the cytoskeleton. Recently we have shown that a Drosophila PAK is enriched in the leading edge of embryonic epithelial cells undergoing dorsal closure (N. Harden, J. Lee, H.-Y. Loh, Y.-M. Ong, I. Tan, T. Leung, E. Manser, and L. Lim, Mol. Cell. Biol. 16:1896-1908, 1996), where it colocalizes with structures resembling focal complexes. We show here by transfection that in epithelial HeLa cells alpha-PAK is recruited from the cytoplasm to distinct focal complexes by both Cdc42(G12V) and Rac1(G12V), which themselves colocalize to these sites. By deletion analysis, the N terminus of PAK is shown to contain targeting sequences for focal adhesions which indicate that these complexes are the site of kinase function in vivo. Cdc42 and Rac1 cause alpha-PAK autophosphorylation and kinase activation. Mapping alpha-PAK autophosphorylation sites has allowed generation of a constitutively active kinase mutant. By fusing regions of Cdc42 to the C terminus of PAK, activated chimeras were also obtained. Plasmids encoding these different constitutively active alpha-PAKs caused loss of stress fibers when introduced into both HeLa cells and fibroblasts, which was similar to the effect of introducing Cdc42(G12V) or Rac1(G12V). Significantly dramatic losses of focal adhesions were also observed. These combined effects resulted in retraction of the cell periphery after plasmid microinjection. These data support our previous suggestions of a role for PAK downstream of both Cdc42 and Rac1 and indicate that PAK functions include the dissolution of stress fibers and reorganization of focal complexes.

Study of simplified measures for malaria surveillance in the late consolidation phase in China.

This paper presents the results of a study on simplified surveillance methods conducted in 23 pilot counties in 11 provinces and municipalities in China where reside 15 million people and malaria control has been in the late consolidation phase. Two simplified surveillance Schemes (A and B) taking treatment of clinical cases as the main measure were implemented in 1992-1994. The rate of annual blood examination for case detection was 1.0% in pilot Scheme A, while in areas of scheme B it was 0.3%. The implementation of both Scheme A and Scheme B, simplified or without treatment of infection foci and management of mobile populations, acquired satisfactory effects against malaria. Consequently, malaria incidence was declining steadily, only a few indigenous and introduced cases were detected. The parasite rate in residents and the IFA positive rate in children were very low. The results of pilot studies and cost-effectiveness analysis indicated that Scheme B is effective, rational and economic, and can be implemented to replace the routine surveillance measures in areas where malaria has been at the late consolidation phase in China.

The human neuronal alpha 1-chimaerin gene contains a position-dependent negative regulatory element in the first exon.

alpha 1-Chimaerin mRNA, which encodes a neuron-specific GTPase-activating protein for the signal transduction molecule p21 Rac, is highly expressed in certain brain regions and neuronal cell lines. The promoter region of human alpha 1-chimaerin transcriptional unit contains no TATA box, Sp1-binding site or initiator motif. However, a CCAAT box located in the proximal promoter region is essential for promoter activity. We now describe a negative regulatory element in the 5' untranslated region of exon 1 of the human alpha 1-chimaerin gene. Deletion of this 70-bp region from the alpha 1-chimaerin minimal promoter increased the promoter activity 5- to 6-fold. The negative element can suppress heterologous thymidine kinase promoter activity in an orientation-independent manner when placed in its native position. However, its function is position-dependent. The presence of a putative factor in rat liver, HepG2 and SK-N-SH cell nuclear extracts but not in rat brain nuclear extract which interacts with this element suggests a possible role of the negative element in controlling the neuron-specific expression of alpha 1-chimaerin in vivo.

Selective up-regulation of alpha 1-chimaerin mRNA in SK-N-SH neuroblastoma cells by K+/-induced depolarisation.

The expression of alpha 1-chimaerin, which encodes a neuron-specific GTPase-activating protein for p21rac, is spatially and temporally regulated in vivo. In vitro, expression of the mRNA of both alpha 1-chimaerin and its alternative spliced form, alpha 2-chimaerin, was up-regulated when human neuroblastoma SK-N-SH cells underwent neuronal-type differentiation in a serum-free medium. KCl-induced membrane depolarisation also specifically up-regulated alpha 1-chimaerin mRNA expression in SK-N-SH cells at the transcriptional level. The up-regulation of alpha 1-chimaerin expression by membrane depolarisation is not an immediate early event, and occurs 3 h after KCl treatment. It does not require de novo protein synthesis. The increase in calcium influx via the L-type voltage-sensitive calcium channel as the result of depolarisation is a key event leading to the up-regulation of alpha 1-chimaerin mRNA. alpha 1-Chimaerin expression was also found to respond positively to the hypertonic osmolarity changes. These results suggest that in vivo expression of alpha 1-chimaerin, a potential signal transduction molecule, may be regulated by neuronal/synaptic activity.

Promoter region of the transcriptional unit for human alpha 1-chimaerin, a neuron-specific GTPase-activating protein for p21rac.

alpha 1-chimaerin is a neuron-specific GTPase-activating protein for p21rac, a protein involved in morphological events. The mRNA is highly expressed in certain brain regions. It is also detected in cultured neuronal, but not in non-neuronal cells. As a first step towards understanding the mechanisms underlying this regulation, genomic clones containing the 5'-flanking region of the human alpha 1-chimaerin transcriptional unit were isolated and characterised. A cluster of multiple transcription start sites of alpha 1-chimaerin mRNAs was detected by primer-extension and S1-mapping analyses. The cluster was mapped to nucleotides -464 to -434 (relative to nucleotide A in the initiation codon) in genomic DNA. The 5'-proximal region contained no TATA box, initiator motif and Sp1-binding site. A 210-bp fragment with approximately 110 bp 5'-flanking sequence could function as a minimal promoter upon analysis using hybrid chloramphenicol acetyltransferase reporter constructs and transient transfection. Internal deletion and point-mutation experiments revealed that a GGCCAATC sequence located at nucleotides -519 to -512 was essential for alpha 1-chimaerin promoter activity. Mobility-shift assay showed the specific binding of nuclear factor(s) to this region, which was competed by the oligonucleotides corresponding to wild-type but not mutant forms. The data also suggest the existence of possible novel CCAAT-binding factor(s) interacting with the alpha 1-chimaerin CCAAT box binding site. A cell-type-preferred suppressor located in the 5'-distal region was found which may play a role in controlling neuron-specific expression of alpha 1-chimaerin mRNA. These findings of a specific promoter for alpha 1-chimaerin transcription will facilitate further studies on its neuronal-specific expression and function.

The distribution of Gossypium hirsutum chromatin in G. barbadense germ plasm: molecular analysis of introgressive plant breeding.

Cotton is unusual among major crop plants in that two cross-fertile species are widely cultivated for a common economic product, fiber. Both historical evidence and classical genetic studies suggest that many improved forms of Gossypium barbadense ("Sea Island", "Egyptian", and "Pima" cottons) may include chromatin derived from G. hirsutum. Using 106 restriction fragment length polymorphism (RFLP) loci well distributed across the cotton genome, we revealed the amount and genomic distribution of G. hirsutum chromatin in 54 G. barbadense collections from around the world. The average G. barbadense collection was comprised of 8.9% alleles apparently derived from G. hirsutum. Pima cultivars (7.3 %) had fewer G. hirsutum alleles than Sea Island (9.0%) or Egyptian (9.6%) cultivars. G. hirsutum alleles were not randomly distributed, as 57.5% of the total introgression observed was accounted for by five specific chromosomal regions that span less than 10% of the genome. The average length of an introgressed chromosome segment was [Symbol: see text] 12.9 cM. Overlap of introgressed chromatin in different breeding programs hints that retention of these G. hirsutum chromosomal segments may impart a selective advantage to G. barbadense genotypes. Although cluster analysis generally grouped germ plasm from common classes and/or breeding programs together, no 2 genotypes were identical - thus differences in the length and repertoire of introgressed chromosome segments also permit DNA fingerprinting of G. barbadense cultivars.

A detailed RFLP map of cotton, Gossypium hirsutum x Gossypium barbadense: chromosome organization and evolution in a disomic polyploid genome.

We employ a detailed restriction fragment length polymorphism (RFLP) map to investigate chromosome organization and evolution in cotton, a disomic polyploid. About 46.2% of nuclear DNA probes detect RFLPs distinguishing Gossypium hirsutum and Gossypium barbadense; and 705 RFLP loci are assembled into 41 linkage groups and 4675 cM. The subgenomic origin (A vs. D) of most, and chromosomal identity of 14 (of 26), linkage groups is shown. The A and D subgenomes show similar recombinational length, suggesting that repetitive DNA in the physically larger A subgenome is recombinationally inert. RFLPs are somewhat more abundant in the D subgenome. Linkage among duplicated RFLPs reveals 11 pairs of homoelogous chromosomal regions-two appear homosequential, most differ by inversions, and at least one differs by a translocation. Most homoeologies involve chromosomes from different subgenomes, putatively reflecting the n = 13 to n = 26 polyploidization event of 1.1-1.9 million years ago. Several observations suggest that another, earlier, polyploidization event spawned n = 13 cottons, at least 25 million years ago. The cotton genome contains about 400-kb DNA per cM, hence map-based gene cloning is feasible. The cotton map affords new opportunities to study chromosome evolution, and to exploit Gossypium genetic resources for improvement of the world's leading natural fiber.

cis-acting elements in 5'-flanking region of rat alpha-fetoprotein mediating retinoic acid responsiveness.

A distal RA responsive cis-acting element has been identified in the 5'-flanking region of the alpha-fetoprotein gene by transfection of different deletion mutants of AFP-CAT fusion gene. The retinoic acid receptor specifically binds to this RA responsive cis-acting element in mobility shift assays. Furthermore, this cis-acting element functions in exogenous TK promoter in transient cotransfection assays. This study suggests a role for the RA responsive cis-acting element in the RA induction of alpha-fetoprotein gene expression.

Effects of nitrate respiration on expression of the Arc-controlled operons encoding succinate dehydrogenase and flavin-linked L-lactate dehydrogenase.

Expression of sdhCDAB (encoding succinate dehydrogenase) and lctD (encoding the flavin-linked L-lactate dehydrogenase) is elevated aerobically and repressed anaerobically in Escherichia coli. The repression is initiated by autophosphorylation of the sensor protein ArcB, followed by phosphoryl group transfer to the regulator ArcA. ArcA-P, a global transcriptional regulator, then prevents sdh and lct expression. The stimulus for ArcB is not O2 deficiency per se. In vitro experiments showed that ArcB phosphorylation is enhanced by pyruvate, D-lactate, acetate, and NADH, the concentrations of which are likely to increase with the lack of an effective exogenous electron sink. In addition to their aerobic function, the two primary dehydrogenases also have roles in anaerobic nitrate respiration. Results presented here indicate that the increase of sdh and lct expression by nitrate depended on its chemical reduction, which in turn diminished the ArcA-P pool. Unexpectedly, a mutation in the fnr gene (encoding a global regulator involved in anaerobic metabolism) also alleviated the anaerobic repressions. Mutations in arcB or arcA were epistatic over that of fnr. Moreover, since this relief was counteracted by pyruvate in the growth medium, Fnr appears to affect formation of stimuli for ArcB. It is possible that Fnr also indirectly affects some of the other members of the arcA modulon, e.g., cyoABCDE (encoding the cytochrome o complex), cydAB (encoding the cytochrome d complex), and sodA (encoding the manganese-dependent superoxide dismutase).

Three overlapping lct genes involved in L-lactate utilization by Escherichia coli.

In Escherichia coli, the lct locus at min 80 on the chromosome map is associated with ability to grow on L-lactate and to synthesize a substrate-inducible flavin-linked dehydrogenase. Similar to that of the glpD-encoded aerobic glycerol-3-phosphate dehydrogenase, the level of induced enzyme activity is elevated by aerobiosis. Both of these controls are mediated by the two-component signal transduction system ArcB/ArcA, although sensitivity to the control is much more striking for L-lactate dehydrogenase. This study disclosed that the lct locus contained three overlapping genes in the clockwise order of lctD (encoding a flavin mononucleotide-dependent dehydrogenase), lctR (encoding a putative regulator), and lctP (encoding a permease) on the chromosomal map. These genes, however, are transcribed in the counterclockwise direction. No homology in amino acid sequence was found between aerobic glycerol-3-phosphate dehydrogenase and L-lactate dehydrogenase. A phi (lctD-lac) mutant was inducible by L-lactate but not D-lactate. Although the mutant lost the ability to grow on L-lactate, growth on D-lactate, known to depend on a different enzyme, remained normal.