Limited expression of APRIL and its receptors prior to intestinal IgA plasma cell development during human infancy.

The absence of immunoglobulin A (IgA) in the intestinal tract renders young infants highly susceptible to enteric infections. However, mediators of initial IgA induction in this population are undefined. We determined the temporal acquisition of plasma cells by isotype and expression of T cell-independent (TI) and -dependent (TD) IgA class switch factors in the human intestinal tract during early infancy. We found that IgA plasma cells were largely absent in the infant intestine until after 1 month of age, approaching adult densities later in infancy than both IgM and IgG. The restricted development of IgA plasma cells in the first month was accompanied by reduced expression of the TI factor a proliferation-inducing ligand (APRIL) and its receptors TACI (transmembrane activator and calcium-modulator and cyclophilin ligand interactor) and B cell maturation antigen (BCMA) within isolated lymphoid follicles (ILFs). Moreover, both APRIL and BCMA expression strongly correlated with increasing IgA plasma cell densities over time. Conversely, TD mediators (CD40 ligand (CD40L) and CD40) were expressed within ILFs before 1 month and were not associated with IgA plasma cell generation. In addition, preterm infants had lower densities of IgA plasma cells and reduced APRIL expression compared with full-term infants. Thus, blunted TI responses may contribute to the delayed induction of intestinal IgA during early human infancy.

Cytosolic phospholipase A(2) regulates golgi structure and modulates intracellular trafficking of membrane proteins.

The Golgi complex and the trans-Golgi network are critical cellular organelles involved in the endocytic and biosynthetic pathways of protein trafficking. Lipids have been implicated in the regulation of membrane-protein trafficking, vesicular fusion, and targeting. We have explored the role of cytosolic group IV phospholipase A(2) (cPLA(2)) in membrane-protein trafficking in kidney epithelial cells. Adenoviral expression of cPLA(2) in LLC-PK(1) kidney epithelial cells prevents constitutive trafficking to the plasma membrane of an aquaporin 2-green fluorescent protein chimera, with retention of the protein in the rough endoplasmic reticulum. Plasma membrane Na(+)-K(+)-ATPase alpha-subunit localization is markedly reduced in cells expressing cPLA(2), whereas the trafficking of a Cl(-)/HCO(3)(-) anion exchanger to the plasma membrane is not altered in these cells. Expression of cPLA(2) results in dispersion of giantin and beta-COP from their normal, condensed Golgi localization, and in marked disruption of the Golgi cisternae. cPLA(2) is present in Golgi fractions from noninfected LLC-PK(1) cells and rat kidney cortex. The distribution of tubulin and actin was not altered by cPLA(2), indicating that the microtubule and actin cytoskeleton remain intact. Total cellular protein synthesis is unaffected by the increase in cPLA(2) activity. Thus cPLA(2) plays an important role in determining Golgi architecture and selective control of constitutive membrane-protein trafficking in renal epithelial cells.

Recycling of AQP2 occurs through a temperature- and bafilomycin-sensitive trans-Golgi-associated compartment.

The exo- and endocytotic pathway in which aquaporin-2 (AQP2) travels between the plasma membrane and intracellular vesicles is only partially characterized. It is known that the antidiuretic hormone vasopressin induces a translocation of AQP2 from an intracellular to a plasma membrane location, both in kidney collecting duct principal cells and in transfected epithelial cells. Here we provide evidence suggesting that while AQP2 shifts from an intracellular location to the cell surface in response to vasopressin, AQP2 also constitutively recycles through a similar pathway in transfected LLC-PK(1) cells even in the absence of hormonal stimulation. Incubating cells at 20 degrees C blocks AQP2 recycling in a perinuclear compartment, regardless of whether vasopressin is present. The H(+)-ATPase inhibitor bafilomycin A1 also blocks the recycling pathway of AQP2 in a perinuclear compartment adjacent to the Golgi in the presence and absence of vasopressin stimulation, indicating a role of vesicle acidification in both the constitutive and regulated recycling of AQP2. Colocalization of AQP2 with clathrin, but not with giantin, after both bafilomycin treatment and a 20 degrees C block suggests that the compartment in which recycling AQP2 is blocked may be the trans-Golgi, and not cis- and medial-Golgi cisternae.

Aquaporin 2 is a vasopressin-independent, constitutive apical membrane protein in rat vas deferens.

Aquaporin 2 (AQP2), the vasopressin-regulated water channel, was originally identified in renal collecting duct principal cells. However, our recent description of AQP2 in the vas deferens indicated that this water channel may have extra-renal functions, possibly related to sperm concentration in the male reproductive tract. In this study, we have examined the regulation and membrane insertion pathway of AQP2 in the vas deferens. The amino acid sequence of vas deferens AQP2 showed 100% identity to the renal protein. AQP2 was highly expressed in the distal portion (ampulla) of the vas deferens, but not in the proximal portion nearest the epididymis. It was concentrated on the apical plasma membrane of vas deferens principal cells, and very little was detected on intracellular vesicles. Protein expression levels and cellular localization patterns were similar in normal rats and vasopressin-deficient Brattleboro homozygous rats, and were not changed after 36 h of dehydration, or after 3 days of vasopressin infusion into Brattleboro rats. AQP2 was not found in apical endosomes (labeled with Texas Red-dextran) in vas deferens principal cells, indicating that it is not rapidly recycling in this tissue. Finally, vasopressin receptors were not detectable on vas deferens epithelial cell membranes using a [(3)H]vasopressin binding assay. These data indicate that AQP2 is a constitutive apical membrane protein in the vas deferens, and that it is not vasopressin-regulated in this tissue. Thus AQP2 contains targeting information that can be interpreted in a cell-type-specific fashion in vivo.

Vasopressin regulated trafficking of a green fluorescent protein-aquaporin 2 chimera in LLC-PK1 cells.

Aquaporin 2 (AQP2) transfected into LLC-PK1 cells functions as a vasopressin-regulated water channel that recycles between intracellular vesicles and the plasma membrane upon vasopressin stimulation. The green fluorescent protein (GFP) of the jellyfish, Aequorea victoria, was used as an autofluorescent tag to monitor AQP2 trafficking in transfected LLC-PK1 cells. Two chimeras were constructed, one in which GFP was fused to the amino-terminus of AQP2 [GFP-AQP2(NT)] and the second in which it was fused to the carboxyl-terminus [AQP2-GFP(CT)]. The GFP-AQP2(NT) chimera trafficked in a regulated pathway from intracellular vesicles to the basolateral plasma membrane in response to vasopressin or forskolin stimulation of cells. In contrast, the AQP2-GFP(CT) chimera expressed in LLC-PK1 cells was localized constitutively on both apical and basolateral plasma membranes. The cellular location of this chimera was not modified by vasopressin or forskolin. Thus, while the GFP-AQP2(NT) chimera will be useful to study AQP2 trafficking in vitro, the abnormal, constitutive membrane localization of the AQP2-GFP(CT) chimera suggests that one or more trafficking signals exist on the carboxyl-terminus of the AQP2 protein.

Cellular mechanisms of aquaporin trafficking.

Aquaporins (AQPs) are a family of functionally important water channel proteins that are of special cell biological interest because of their diverse intracellular targeting and trafficking properties. AQPs have been found in many different cells and tissues. This short review summarizes recent work that addresses the regulation of AQP2 trafficking in response to vasopressin.

Protein kinase A phosphorylation is involved in regulated exocytosis of aquaporin-2 in transfected LLC-PK1 cells.

Vasopressin-dependent translocation of aquaporin-2 (AQP2) between intracellular vesicles and the plasma membrane has been demonstrated in vivo and in vitro. Furthermore, the vasopressin-induced increase in apical membrane water permeability of renal principal cells is dependent on a rise in intracellular adenosine 3',5'-cyclic monophosphate and activation of protein kinase A (PKA). To determine whether trafficking of AQP2 is dependent on PKA phosphorylation, we first examined the effect of the PKA-inhibitor N-(2[[3-(4-bromophenyl)-2-propenyl]-amino]-ethyl)-5-isoquinolinesulfonam ide (H-89) on AQP2 translocation in transfected LLC-PK1 cells. Vasopressin-induced membrane insertion of AQP2 was completely inhibited by pretreatment of the cells for 60 min with H-89. This reagent also caused a dense accumulation of AQP2 in the Golgi region. Next, LLC-PK1 cells were stably transfected with AQP2 cDNA in which the PKA phosphorylation site, Ser256, was replaced with alanine (S256A). S256A-AQP2 was not phosphorylated in vitro by PKA, and S256A-AQP2 was mainly localized to intracellular vesicles in the basal condition, similar to wild-type AQP2. However, after stimulation with vasopressin or forskolin, the cellular distribution of S256A-AQP2 remained unchanged. In addition, the usual vasopressin-induced increase in endocytosis seen in AQP2-transfected cells was not observed in S256A-AQP2-transfected cells. These results demonstrate that the Ser256 PKA phosphorylation site is possibly involved in the vasopressin-induced trafficking of AQP2 from intracellular vesicles to the plasma membrane and in the subsequent stimulation of endocytosis.

Functional evidence for a colorectal cancer tumor suppressor gene at chromosome 8p22-23 by monochromosome transfer.

Chromosome 8p is considered, from loss of heterozygosity analysis, to be a strong candidate for the location of a tumor suppressor gene inactivated in colorectal cancer. We have found a 53% (27 of 51) rate of allelic loss at the LPL locus on 8p22, with the smallest region of overlap of deletions including the region D8S258 to D8S277. Using microcell-mediated monochromosome 8 transfer into three colorectal cancer cell lines, SW480, SW620 and HT29, we have demonstrated a reduction of tumorigenicity in SW620 hybrids. Partial deletions of chromosome 8 in some SW620/8 hybrids further delineate the critical region(s) to 8p22-23. Hybrids of the colorectal cancer cell lines SW480 and HT29 containing chromosome 8 did not show suppression of tumorigenesis, but the H29/8 hybrid showed total suppression of soft agar clonicity. This indicates an alternate pathway of mutational progression in these three lines, despite the fact that SW480 was derived from the same patient as SW620.

Loss of heterozygosity on the long arm of chromosome 11 in colorectal tumours.

We have examined a series of human colorectal adenomas, carcinomas and cell lines derived from human colorectal cancer for loss of heterozygosity (LOH) on chromosome 11q22-23 by polymerase chain reaction (PCR) amplification of a microsatellite polymorphism of the dopamine D2 receptor (DRD2) locus. LOH was demonstrated in 5/30 (16.7%) adenomas and 23/68 (33.8%) carcinomas. Only 2/20 (10%) cell lines showed homozygosity which could potentially be as a consequence of LOH. This moderate level of loss in the tumour samples was probably not an underestimation as a result of excessive stromal contamination because high rates (68-77%) have been detected in the same samples on chromosomes 17 and 18. In contrast to a previous report, LOH in carcinomas at 11q22-23 occurred at a lower frequency and was not associated with Dukes' stage, degree of differentiation, mucin production or the location of the cancer. However, a significant association was found between LOH on chromosome 11 and chromosome 14. Thus, inactivation of any putative tumour-suppressor gene at 11q22-23 by LOH is not a very common event in the development of colorectal tumours, but may be biologically significant if accompanied by chromosome 14 deletions.

Phylogenetic and molecular characterization of a 23S rRNA gene positions the genus Campylobacter in the epsilon subdivision of the Proteobacteria and shows that the presence of transcribed spacers is common in Campylobacter spp.

The nucleotide sequence of a 23S rRNA gene of Campylobacter coli VC167 was determined. The primary sequence of the C. coli 23S rRNA was deduced, and a secondary-structure model was constructed. Comparison with Escherichia coli 23S rRNA showed a major difference in the C. coli rRNA at approximately position 1170 (E. coli numbering) in the form of an extra sequence block approximately 147 bp long. PCR analysis of 31 other strains of C. coli and C. jejuni showed that 69% carried a transcribed spacer of either ca. 147 or ca. 37 bp. Comparison of all sequenced Campylobacter transcribed spacers showed that the Campylobacter inserts were related in sequence and percent G+C content. All Campylobacter strains carrying transcribed spacers in their 23S rRNA genes produced fragmented 23S rRNAs. Other strains which produced unfragmented 23S rRNAs did not appear to carry transcribed spacers at this position in their 23S rRNA genes. At the 1850 region (E. coli numbering), Campylobacter 23S rRNA displayed a base pairing signature most like that of the beta and gamma subdivisions of the class Proteobacteria, but in the 270 region, Campylobacter 23S rRNA displayed a helix signature which distinguished it from the alpha, beta, and gamma subdivisions. Phylogenetic analysis comparing C. coli VC167 23S rRNA and a C. jejuni TGH9011 (ATCC 43431) 23S rRNA with 53 other completely sequenced (eu)bacterial 23S rRNAs showed that the two campylobacters form a sister group to the alpha, beta, and gamma proteobacterial 23S rRNAs, a positioning consistent with the idea that the genus Campylobacter belongs to the epsilon subdivision of the class Proteobacteria.

Mutagenesis of the paracrystalline surface protein array of Aeromonas salmonicida by endogenous insertion elements.

The tetragonal paracrystalline surface protein array (A-layer) of the fish pathogenic bacterium Aeromonas salmonicida is a virulence factor and bacteria which are unable to produce A-layer are attenuated in their ability to kill fish. Ten independent mutants of Aeromonas salmonicida which were unable to produce A-layer were isolated by growth at 30 degrees C. These mutants displayed either reduced synthesis of the A-layer subunit, synthesis of a truncated subunit, or complete loss of the ability to produce the subunit protein. Restriction mapping and analysis by polymerase chain reaction showed that the mutations had resulted from insertion of two different insertion sequence (IS) elements into different sites in the A-layer subunit gene (vapA) and its promoter. Sequence comparisons indicated that ISAS1 is unique among reported IS elements. It is 1223 bp long with imperfect terminal inverted repeats of 22 bp and insertion resulted in a duplicated 8 bp target sequence in vapA. ISAS1 expressed a 42,000 molecular weight (M(r)) protein in mini-cells. ISAS2 was 1084 bp long, expressed proteins of M(r) 38,000 and 39,000 in vitro, had imperfect 29 bp terminal inverted repeats and had duplicated a 3 bp target sequence. Sequence comparisons indicated that ISAS2 was also unique to A. salmonicida; however, the proteins encoded by ISAS2 showed strong homology to the putative transposases encoded by the IS30 family of IS elements. Southern analyses showed that both ISAS1 and ISAS2 were restricted to A. salmonicida strains A449 and A450 where they were present in low copy number. The ability of these two IS elements to mutate the ability of A. salmonicida to produce its paracrystalline surface array provides a novel method for the attenuation of virulence.

Transcriptional analysis of the Aeromonas salmonicida S-layer protein gene vapA.

The vapA gene of Aeromonas salmonicida encodes the subunit of the surface protein array known as A-layer. Nucleotide sequence analysis of the 374 bp of DNA immediately upstream of vapA revealed two potential promoter sequences and other possible regulatory sequences. Sequencing and polymerase chain reaction analysis showed that the region was conserved in wild-type A. salmonicida. Primer extension and Northern (RNA) blot analysis showed that vapA transcription in A. salmonicida was directed predominantly by a distal promoter, P1, resulting in a 1.7-kb unit-length mRNA with an untranslated 181-nucleotide leader sequence which contained two predicted low-free-energy stem-loop structures. Northern analysis of cells grown at 15 degrees C showed that vapA transcript production peaked during the mid-log phase of growth (A600 = 0.25). At 15 degrees C, the half-life of the vapA mRNA was 22 min, while at 20 degrees C, the half-life was significantly shorter, 11 min. The amount of vapA transcript produced was reduced by growth in the presence of the DNA gyrase inhibitors nalidixic acid and novobiocin. Environmental factors such as growth temperature and atmospheric oxygen tension also affected the quantity of vapA mRNA. vapA transcript could not be detected in mutants which produced either low levels of full-length or truncated A protein or no detectable A protein.

Identification of the Escherichia coli lytB gene, which is involved in penicillin tolerance and control of the stringent response.

The Escherichia coli lytB gene, which is involved in penicillin tolerance and control of the stringent response, was identified as a previously described open reading frame designated orf316 located in the ileS-lsp operon (0.4 min on the linkage map).

Effect of heat denaturation of target DNA on the PCR amplification.

The polymerase chain reaction (PCR) and amplification of specific regions of DNA in vitro is a widely used and powerful technique, and the optimization of conditions used to maximize PCR product yield has received much attention. We have shown that lengthy denaturation times of template DNA ranging from 1 to 7 min at pH 7.0-8.0, that are often employed prior to the start of a PCR reaction, result in marked degradation of the template. This can result in a significant reduction in the yield of PCR products larger than 500 bp, by up to 99%. This effect was demonstrated for both complex genomic template DNA, and also for a 2691-bp linear piece of template DNA using both a rapid hot-air thermocycler and a conventional block thermocycler. This decrease in product yield is likely due to the increased degradation of the template or target DNA as a result of pre-amplification denaturation (PAD). We therefore recommend that when amplifying larger pieces of DNA, the template DNA should not be exposed to PAD prior to a PCR reaction, irrespective of the starting pH of the template solution.

Detection of Aeromonas salmonicida from fish by using polymerase chain reaction amplification of the virulence surface array protein gene.

A DNA-based assay was developed to detect Aeromonas salmonicida from infected fish by analyzing tissues, feces, and the tank water in which the infected fish were held. This analysis was done both by direct detection from samples and after a bacterial outgrowth step. Polymerase chain reaction (PCR) amplification of a 421-bp sequence from the 3' region of the surface array protein gene (vapA) of A. salmonicida provided a specific and sensitive method for the detection and identification of this important fish pathogen. The sensitivity of PCR detection of A. salmonicida directly from tissues was less than 10 CFU/mg. Furthermore, a detection level of 5 fg, equivalent to approximately 1 cell, was obtained by using purified chromosomal DNA as the template. This highly reproducible assay, which requires 45 min to complete, is therefore sensitive enough to be used as a noninvasive method for monitoring fish populations for the presence of carrier fish. Because the surface protein array (A-layer) is a virulence factor of A. salmonicida, PCR analysis with oligonucleotide primers directed at vapA can also be used to provide information on the potential virulence of a strain.

Prehistoric use of fur seals: evidence from the Olympic coast of Washington.

Archeological excavations on the Olympic Coast of Washington provide evidence that marine mammals, particularly fur seals, constituted a major source of food for inhabitants of this region for more than 2000 years. A recent change in the migratory pattern of the fur seals is suggested by the high percentage of mature males present in prehistoric populations.