The comparative anatomy of the folds, fossae, and adhesions around the duodenojejunal flexure in mammals.

BACKGROUND: Anatomical knowledge of the duodenojejunal flexure is necessary for abdominal surgeries, and also important for physiologic studies about the duodenum. But little is known about the anatomy of this region in mammals. Here, we examined comparative anatomy to understand the anatomical formation of the duodenojejunal flexure in mammals. MATERIALS AND METHODS: The areas around the duonenojejunal flexure were ob-served in mouse, rat, dog, pig, and human, and the anatomical structures around the duodenojejunal junction in the animals were compared with those in human. RESULTS: The superior and inferior duodenal folds, and the superior and inferior duodenal fossae were identified in all examined humans. In pig, the structures were not clearly identified because the duodenum strongly adhered to the retroperitoneum and to the mesocolon. In mouse, rat, and dog, only the plica duodenocolica, which is regarded as the animal counterpart of the superior duo-denal fold in human, was identified, and other folds or fossae were not observed, probably because the duodenum was not fixed to the parietal peritoneum in those animals. Transection of the plica duodenocolica could return the normally rotated intestine back to the state of non-rotation in rat. CONCLUSIONS: This study showed the anatomical similarities and dissimilarities of the duodenojejunal flexure among the mammals. Anatomical knowledge of the area is useful for duodenal and pancreatic surgeries, and for animal studies about the duodenum. (Folia Morphol 2018; 77, 2: 286-292).

New screening methods for probiotics with adhesion properties to sialic acid and sulphate residues in human colonic mucin using the Biacore assay.

AIMS: To determine the relationship between adhesive ability of probiotics and acidic residues in human colonic mucin, we developed a new screening method using Biacore to evaluate adherence of bacteria before and after sialic acid or sulphate residues were blocked or removed from mucin. METHODS AND RESULTS: Ten strains of lactobacilli and three strains of bifidobacteria isolated from human faeces were evaluated for their adhesive properties to soluble human colonic mucin (sHCM) using the Biacore binding assay. Three strains (Lactobacillus strain ME-522, Lact. gasseri ME-527 and Bifidobacterium bifidum MCC1092) showing significant adherence were selected. Decreased binding activities were observed after removing sialic acid of sHCM using sialidase. However, after removing the sulphate residue using sulphatase, the adhesion of ME-527 decreased; whereas the remaining two strains had increased adhesion. The adhesion of three probiotics significantly decreased after the sulphate residue was blocked by elution with barium chloride. CONCLUSIONS: A new evaluation method using the Biacore assay was developed to observe binding properties to the acidic residues of sHCM. Results indicated that there was a strong relationship between probiotic adhesion and acidic residues of sHCM. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report showing a screening method that quantitatively measures the binding between bacteria and acidic residues in sHCM using the Biacore binding assay; and provides a new method for the selection of probiotics in the future.

Chain-length determination mechanism of isoprenyl diphosphate synthases and implications for molecular evolution.

In the synthesis of isoprenoids, isoprenyl diphosphate synthases catalyze the consecutive condensation of isopentenyl diphosphate with allylic diphosphates to produce a variety of prenyl diphosphates with well-defined chain lengths. Site-directed mutagenesis in conjunction with X-ray crystallographic studies have identified specific amino acid residues responsible for chain-length determination. Simple combinations of these residues within a characteristic motif are not only sufficient to confer product specificities to all isoprenyl diphosphate synthases but represent structural features that reflect the enzyme family's evolutionary course.

Cell surface Lactobacillus plantarum LA 318 glyceraldehyde-3-phosphate dehydrogenase (GAPDH) adheres to human colonic mucin.

AIMS: To characterize the adhesion molecule of Lactobacillus plantarum LA 318 that shows high adhesion to human colonic mucin (HCM). METHODS AND RESULTS: The adhesion test used the BIACORE assay where PBS-washed bacterial cells showed a significant decrease in adherence to HCM than distilled water-washed cells. A component in the PBS wash fraction adhered to the HCM and a main protein was detected as a c. 40-kDa band using SDS-PAGE. Using homology comparisons of the N-terminal amino acid sequences compared with sequence databases, this protein was identified as glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The DNA sequence of LA 318 GAPDH was 100% identical to the GAPDH (gapB) of L. plantarum WCFS1. The purified GAPDH adhered to HCM. CONCLUSIONS: We found the adhesin of L. plantarum LA 318 to HCM in its culture PBS wash fraction. The molecule was identified as GAPDH. Because LA 318 possesses the same adhesin as many pathogens, the lactobacilli GAPDH may compete with pathogens infecting the intestine. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report showing GAPDH expressed on the cell surface of lactobacilli adheres to mucin suggesting L. plantarum LA 318 adheres to HCM using GAPDH binding activity to colonize the human intestinal mucosa.

Cell cycle and cell fate in the nervous system.

Recently, a number of molecules originally thought to have a primary role in cell determination have been shown to affect the cell cycle at specific check points, while other molecules discovered for their roles in the cell cycle progression are known to affect the determination and differentiation of neurons. These discoveries have led to a more detailed investigation of the complex molecular machinery that co-ordinates proliferation and differentiation.

The neuronal architecture of Xenopus retinal ganglion cells is sculpted by rho-family GTPases in vivo.

Dendritogenesis, axonogenesis, pathfinding, and target recognition are all affected in distinct ways when Xenopus retinal ganglion cells (RGCs) are transfected with constitutively active (ca), wild-type (wt), and dominant negative (dn) Rho-family GTPases in vivo. Dendritogenesis required Rac1 and Cdc42 activity. Moreover, ca-Rac1 caused dendrite hyperproliferation. Axonogenesis, in contrast, was inhibited by ca-Rac1. This phenotype was partially rescued by the coexpression of dn cyclin-dependent kinase (Cdk5), a proposed effector of Rac1, suggesting that Rac1 activity must be regulated tightly for normal axonogenesis. Growth cone morphology was particularly sensitive to dn-RhoA and wt-Cdc42 constructs. These also caused targeting errors, such as tectal bypass, suggesting that cytoskeletal rearrangements are involved in target recognition and are transduced by these pathways.

Isoprenyl diphosphate synthases.

Isoprenyl diphosphate synthases catalyze consecutive condensations of isopentenyl diphosphates with allylic primer substrates to form linear backbones for all isoprenoid compounds including cholesterol. These synthases are classified according to the final chain length of their end products and the stereochemistry of the newly formed double bonds. Mutagenesis and X-ray crystallography data have uncovered the basic catalytic and chain length determination mechanisms of E-isoprenyl diphosphate synthases and shed light on their possible evolutionary course. Although much less is known about the Z-isoprenyl diphosphate synthase family, successful cloning and subsequent crystallizations in the near future will no doubt bring more insight as researchers begin to unravel the essential components and precise reaction mechanisms of this cellular machinery.

High sialic acid reactivity of sugar chain structure Lewis-X in patients with mental retardation.

It has been reported that platelet-derived growth factor B-chains homodimer (PDGF-BB) improves learning function of mice, and that sugar chain structure Lewis-X of N-glycosylated glycoprotein promotes PDGF-BB secretion from platelets. Based on these findings, we assumed that learning dysfunction in some patients with mental retardation might be due to abnormality in PDGF-BB metabolism and/or Lewis-X structure. No difference in the reactivity of PDGF-BB and Lewis-X was found between the serum of patients with mental retardation and that of normals. But sialic acid reactivity of the Lewis-X fraction in some patients was remarkably higher than that in other patients and in normals. These findings suggest that sialic acids in the Lewis-X fraction may have a relation to one of the causes of learning dysfunction in these patients.

Cloning of a gene from Escherichia coli that confers resistance to fosmidomycin as a consequence of amplification.

A gene conferring resistance to fosmidomycin (Fs) was cloned from the gene pool of a wild-type strain of Escherichia coli. The cloned DNA fragment was sequenced and shown to encode a putative polypeptide of 406 amino acids (aa) with a molecular weight of 43303. The gene mapped at 10.9 min on the E. coli chromosome and was designated fsr (fosmidomycin resistance). Maxicell analysis revealed that the Fsr protein migrated in sodium dodecyl sulfate-polyacrylamide-gel electrophoresis as a broad band of 35 kDa. A comparison between the aa sequence of Fsr and sequences in a protein database revealed 18% homology to the bacterial drug-export proteins that mediate resistance to tetracycline and chloramphenicol. Hydropathy analysis of the Fsr protein revealed twelve putative transmembrane segments. The degree of FsR of transformants depended on the number of copies of the plasmid that contained fsr. The levels of ubiquinone-8 and undecaprenyl phosphate in cells that harbored a high-copy-number plasmid that included fsr were almost the same as those in the cells without the plasmid. These results suggest that Fsr does not have any direct effect on the biosynthesis of isoprenoid in E. coli, and that the mechanism for FsR involves the efflux of the drug by a process that is facilitated by Fsr.

Kinetic studies on the prenyl chain elongation by undecaprenyl diphosphate synthase with artificial substrate homologues.

In the undecaprenyl diphosphate synthase reaction, an allylic substrate homologue, (2Z,6E,10E)-4-methyl-geranylgeranyl diphosphate was found to be a potent competitive inhibitor against the allylic primer, (2Z,6E,10E)-geranylgeranyl diphosphate. On the other hand, it acted as a strong noncompetitive inhibitor against isopentenyl diphosphate. On the basis of these facts, the topology of the substrate-binding sites as well as the reason why the synthase reaction with (E)-3-methyl-3-pentenyl diphosphate always stops completely at the first stage of condensation, yielding an allylic diphosphate with a methyl group at the 4-position, are discussed.

Protein design of geranyl diphosphate synthase. Structural features that define the product specificities of prenyltransferases.

Geranyl diphosphate synthase catalyzes the condensation of isopentenyl diphosphate with dimethylallyl diphosphate to give a C(10) compound, geranyl diphosphate, which is a precursor of all monoterpenoids. However, the gene has not been isolated from any organisms. To examine the possibility that geranyl diphosphate synthase has evolved from a common ancestor of the prenyltransferase family and to predict the active site structure, we tried to convert Bacillus stearothermophilus farnesyl diphosphate synthase to geranyl diphosphate synthase, according to our previous findings. Several mutated farnesyl diphosphate synthases that have single amino acid substitutions before the first aspartate-rich motif were constructed. A mutated enzyme that has the replacement of serine by phenylalanine at the fourth position before the motif exclusively produced geranyl diphosphate when dimethylallyl diphosphate was used as the primer, and hardly accepted geranyl diphosphate as a primer, indicating that this mutation causes the conversion to geranyl diphosphate synthase. This result supports the idea that the product specificities of all members of the E-prenyltransferase family are mainly defined by a few structural features: the amino acids at the fourth position and the fifth position before the first aspartate-rich motif, and the insertion of two amino acids in the motif. This suggests that natural geranyl diphosphate synthases might have an active site structure similar to that of the mutated enzyme.

Identification and characterization of geranylgeraniol kinase and geranylgeranyl phosphate kinase from the Archaebacterium Sulfolobus acidocaldarius.

Geranylgeranyl diphosphate is an important precursor of archaebacterial ether-linked lipids, and it has been thought that all of this compound is "de novo" synthesized by geranylgeranyl diphosphate synthase. We studied the phosphorylation of geranylgeraniol, which seems to be related to the salvage pathway of biosynthesis of archaebacterial ether-linked lipids, in the Archaebacterium Sulfolobus acidocaldarius. Activities of geranylgeraniol kinase and geranylgeranyl phosphate kinase were detected in a cell lysate of S. acidocaldarius. The two enzymes were easily separated by ultracentrifugation. The membrane fraction and the cytosolic fraction contained geranylgeraniol kinase activity and geranylgeranyl phosphate kinase activity, respectively. Geranylgeraniol kinase, which requires divalent cation such as Mg2+, Co2+, and Mn2+ and NTP (ATP, GTP, CTP, UTP), catalyzes monophosphorylation of (all-E)-geranylgeraniol to produce geranylgeranyl phosphate. (all-E)-Farnesol, (all-E)-hexaprenol, and (all-E)-octaprenol were also active substrates, though they were less effective than (all-E)-geranylgeraniol. However, neither geraniol nor (22E,6E,10Z,14Z,18Z,22Z,26Z,++ +30Z,34Z,38Z)-undecaprenol was active. This enzyme is extremely thermostable and its pH optimal is between 6.5 and 8.5. The Michaelis constants for (all-E)-geranylgeraniol and ATP are 27 nM and 650 microM, respectively.

Chain length distribution of the products formed in solanesyl diphosphate synthase reaction.

Factors that affect the termination of isoprenoid chain elongation catalyzed by prenyltransferase were investigated. The chain-length distribution of reaction products of solanesyl diphosphate synthase [EC 2.5.1.11] homogeneously purified from Micrococcus luteus changed dramatically according to the concentration of the complex formed between isopentenyl diphosphate and Mg2+ (IPP-Mg) in the reaction mixture. However, the concentration of the complex between farnesyl diphosphate and Mg2+ (FPP-Mg), the priming substrate for this synthase, did not affect the product distribution, provided that the concentration of IPP-Mg was maintained at a certain level. Thus, the level of IPP-Mg is decisive in affecting the chain length distribution of the products of the prenyltransferase reaction, and the Mg(2+)-dependent variability of product specificity so far observed can now be understood in terms of the effect of IPP-Mg concentration.

Identification of genes affecting lycopene formation in Escherichia coli transformed with carotenoid biosynthetic genes: candidates for early genes in isoprenoid biosynthesis.

Although isopentenyl diphosphate is a precursor of isoprenoids in Escherichia coli, the genes and enzymes involved in its biosynthesis have not been identified. Thus, we tried to isolate E. coli mutants deficient in the biosynthesis and their complementary genes by use of an artificial phenotypic screening system employing three carotenoid biosynthetic genes, crtE, crtB, and crtI. Cells were mutagenized with ethylmethanesulfonate, then transformed with a plasmid for expression of the carotenogenic genes. Mutants deficient in biosynthesis of isopentenyl diphosphate were expected to form white colonies, because they are unable to produce enough lycopene, whereas wild-type cells form red colonies. Among large numbers of red colonies, we identified 117 white colonies. Next, we transformed each mutant with an E. coli genomic library. Twenty-nine complementary genes that restore red color of host colonies were isolated. A homology search and further complementation study using subcloned genes revealed that the true complementary genes encode isopentenyl diphosphate isomerase, subunits of ATP synthase, enzymes of the Krebs cycle, some aldehyde dehydrogenases, phosphate acetyltransferase, and enzymes which relate to the biosynthesis of ubiquinones and menaquinones. Two unknown genes were also found, designated elb1 and 2, which may be involved in the early steps of isoprenoid biosynthesis.

Effects of random mutagenesis in a putative substrate-binding domain of geranylgeranyl diphosphate synthase upon intermediate formation and substrate specificity.

Archaeal geranylgeranyl diphosphate (GGPP) synthase catalyzes the consecutive condensation of isopentenyl diphosphate (IPP) with allylic diphosphates to produce GGPP with significant amounts of intermediates. To obtain information about the amino acids involved in the condensation and the release of intermediates, we randomly mutagenized two proximal regions, I and II, of the Sulfolobus acidocaldarius GGPP synthase gene and created two degenerate libraries, I and II, respectively. Regions I and II correspond to amino acid residues 170-173 and 166-168, respectively. The prenyltransferase activities of about 200 clones were analyzed using the in vivo red-white system and the conventional in vitro assay. Although, in library I, no mutated enzymes that failed to catalyze the formation of GGPP were found, as assayed with the red-white system, almost all the mutated enzymes exhibited weak GGPP synthesis activity, and many produced large amounts of intermediates. The formation of intermediates increased as the concentration of IPP was decreased or as the concentration of the allylic substrate was increased. These phenomena can be regarded as a reflection of the increased K(m) for IPP and the decreased affinity for products including intermediates. On the other hand, no mutants from library II showed such changes. These results suggest that the region from 170 to 173 is concerned in the recognition of both IPP and allylic diphosphates, and that the change in responsiveness to prenyl diphosphates causes a change in intermediate formation.

Recognition of allylic substrates in Sulfolobus acidocaldarius geranylgeranyl diphosphate synthase: analysis using mutated enzymes and artificial allylic substrates.

We examined the substrate specificity of two mutated geranylgeranyl diphosphate synthases, I-9 and I-11, with respect to several artificial substrates. These mutated enzymes have replacements in the amino acid sequences from positions 170 to 173, which are thought to be a part of the putative substrate binding region. The wild-type enzyme catalyzes the condensation of IPP with a series of (2E)-3-methyl-2-alkenyl diphosphates to give products with carbon numbers between 14 and 21. On the other hand, the mutated enzymes show lower activities for artificial substrates with short alkyl chains than those of the wild-type enzyme though the carbon numbers of the products are similar to those in the case of the wild-type. The mutated enzyme I-11 never accepts artificial substrates shorter than C8. Analysis of additional mutated enzymes revealed that the characteristics of the mutated enzymes arise from a few substitutions within positions 171 to 173. These results indicate that the amino acids in the positions 171 to173 of the geranylgeranyl diphosphate synthase from Sulfolobus acidocaldarius are involved in recognition of short allylic substrates, such as dimethylallyl diphosphate, but not in recognition of the chain length of the products.

Use of cultured tubular cells isolated from human urine for investigation of renal transporter.

AIM: To facilitate the understanding of the transporter function of human renal tubular cells, we have developed a simple method using primary cultured proximal tubule (PT) cells isolated from voided urine. METHODS: PT cells grown to confluence on glass coverslips could be identified by parallel arrays of spindle cells and hemicyst formation. Brush-border gamma-glutamyl transpeptidase (gammaGTP) activity was histochemically identified. Apical membrane Na+/H+ exchanger (NHE) activity was measured by monitoring changes in intracellular pH (pHi) after an acid load in a single cell level using the pH-sensitive dye 2'7'-bis-(2-carboxyethyl)-5.6'carboxyfluorescein (BCECF). RESULTS: Amiloride and 5-(N-ethyl-N-isopropyl) amiloride (EIPA) inhibited the NHE activity with half-maximal inhibition values (IC50) of 15.3 and 4.0 microM, respectively. NHE-3 mRNA was detected by the RT-PCR technique in clonally proliferated PT cells. CONCLUSION: These results suggest that cultured PT cells isolated from human urine express amiloride-resistant NHE-3 activity on the apical membranes, which can be compared to functional properties of PT in vivo. Our experimental strategy offers a useful experimental approach to investigating human renal tubular transport function in vitro.

Cauda equina syndrome complicating pneumococcal meningitis.

A 14-month-old female with pneumococcal meningitis presented with flaccid paraplegia, saddle anesthesia, and bladder and bowel dysfunction. Magnetic resonance imaging of the spine demonstrated intense gadolinium enhancement of the cauda equina, whereas the conus medullaris appeared normal. This finding indicated that lumbosacral polyradiculopathy caused her symptoms.

An outbreak of allergy-like food poisoning.

Eight cases of allergy-like food poisoning resulting from the ingestion of yellowfin tuna, which had been kept in stock for 10 days prior to being cooked, are described. The main symptoms were headaches, facial flushing and palpitation. Samples of the ingested fish were analyzed for histamine content, and a high level of histamine was confirmed (310 mg/100 g of fish). Corticosteroids were given to 3 patients who exhibited dyspnea or persistent symptoms, while the remaining patients improved without medication. In situations where allergy-like clinical features are present after the ingestion of food, the possibility of allergy-like food poisoning should be recognized and included in a differential diagnosis.

Portal and mesenteric vein and inferior vena cava thrombosis associated with antiphospholipid syndrome.

We report a 48-year-old man with thrombosis of the portal and superior mesenteric vein and inferior vena cava associated with primary antiphospholipid syndrome (APS). Primary APS was diagnosed by a positive reaction with anticardiolipin antibody (aCL) and the absence of any evidence suggesting the presence of other disease states known to be associated with aCL. A coeliac angiography showed obstruction of the portal and superior mesenteric vein with prominent collaterals and cavernous transformation. Femoral vein angiography showed total obstruction of the external iliac vein and inferior vena cava, and dilation of the pelvic veins, with contrast medium in the lumbar vein. This case is noteworthy as a report of primary APS accompanied by extensive abdominal and pelvic venous thrombosis.