Reg-II Is an Exocrine Pancreas Injury-Response Product That Is Up-Regulated by Keratin Absence or Mutation

The major keratins in the pancreas and liver are keratins 8 and 18 (K8/K18), but their function seemingly differs in that liver K8/K18 are essential cytoprotective proteins, whereas pancreatic K8/K18 are dispensable. This functional dichotomy raises the hypothesis that K8-null pancreata may undergo compensatory cytoprotective gene expression. We tested this hypothesis by comparing the gene expression profile in pancreata of wild-type and K8-null mice. Most prominent among the up-regulated genes in K8-null pancreas was mRNA for regenerating islet-derived (Reg)-II, which was confirmed by quantitative reverse transcription-polymerase chain reaction and by an anti-Reg-II peptide antibody we generated. Both K8-null and wild-type mice express Reg-II predominantly in acinar cells as determined by in situ hybridization and immunostaining. Analysis of Reg-II expression in various keratin-related transgenic mouse models showed that its induction also occurs in response to keratin cytoplasmic filament collapse, absence, or ablation of K18 Ser52 but not Ser33 phosphorylation via Ser-to-Ala mutation, which represent situations associated with predisposition to liver but not pancreatic injury. In wild-type mice, Reg-II is markedly up-regulated in two established pancreatitis models in response to injury and during the recovery phase. Thus, Reg-II is a likely mouse exocrine pancreas cytoprotective candidate protein whose expression is regulated by keratin filament organization and phosphorylation.

Genetic analysis of Treponema denticola ATCC 35405 biofilm formation

Treponema denticola is a major aetiological organism implicated in periodontal disease. The interaction of T. denticola with other oral bacteria, in particular Porphyromonas gingivalis, in biofilm formation is thought to be an important step in the onset of periodontal disease. The interaction between T. denticola and P. gingivalis has been examined using a panel of T. denticola mutants and their effects on mixed biofilm formation tested in a static biofilm model. T. denticola ATCC 35405 did not form detectable biofilms on various inert surfaces. However, the spirochaete was demonstrated to form a biofilm with preattached P. gingivalis 381. T. denticola cfpA, which lacks the cytoplasmic filament, was unable to produce a mixed biofilm with P. gingivalis. A T. denticola flgE mutant which lacks the flagella hook protein and is therefore non-motile displayed a reduced, but readily detectable, ability to form a mixed biofilm as did the T. denticola mutant which does not possess the major outer sheath protein (Msp). The T. denticola lrrA mutant was only moderately defective in forming mixed biofilms with P. gingivalis. However, the T. denticola methyl-accepting chemotaxis protein (DmcA) did not appear to play a major role in mixed biofilm formation. In contrast, T. denticola lacking the PrtP protein for prolyl-phenylalanine-specific protease, showed an increased ability to form mixed biofilms and a prolonged viability in the biofilm.

Cytoplasmic Filament-Deficient Mutant ofTreponema denticola Has Pleiotropic Defects

ABSTRACT In Treponema denticola, a ribbon-like structure of cytoplasmic filaments spans the cytoplasm at all stages of the cell division process. Insertional inactivation was used as a first step to determine the function of the cytoplasmic filaments. A suicide plasmid was constructed that contained part of cfpA and a nonpolar erythromycin resistance cassette (ermF andermAM) inserted near the beginning of the gene. The plasmid was electroporated into T. denticola, and double-crossover recombinants which had the chromosomal copy of cfpAinsertionally inactivated were selected. Immunoblotting and electron microscopy confirmed the lack of cytoplasmic filaments. The mutant was further analyzed by dark-field microscopy to determine cell morphology and by the binding of two fluorescent dyes to DNA to assess the distribution of cellular nucleic acids. The cytoplasmic filament protein-deficient mutant exhibited pleiotropic defects, including highly condensed chromosomal DNA, compared to the homogeneous distribution of the DNA throughout the cytoplasm in a wild-type cell. Moreover, chains of cells are formed by the cytoplasmic filament-deficient mutant, and those cells show reduced spreading in agarose, which may be due to the abnormal cell length. The chains of cells and the highly condensed chromosomal DNA suggest that the cytoplasmic filaments may be involved in chromosome structure, segregation, or the cell division process in Treponema.

Simple epithelial keratins are dispensable for cytoprotection in two pancreatitis models

Pancreatic acinar cells express keratins 8 and 18 (K8/18), which form cytoplasmic filament (CF) and apicolateral filament (ALF) pools. Hepatocyte K8/18 CF provide important protection from environmental stresses, but disruption of acinar cell CF has no significant impact. We asked whether acinar cell ALF are important in providing cytoprotective roles by studying keratin filaments in pancreata of K8- and K18-null mice. K8-null pancreas lacks both keratin pools, but K18-null pancreas lacks only CF. Mouse but not human acinar cells also express apicolateral keratin 19 (K19), which explains the presence of apicolateral keratins in K18-null pancreas. K8- and K18-null pancreata are histologically normal, and their acini respond similarly to stimulated secretion, although K8-null acini viability is reduced. Absence of total filaments (K8-null) or CF (K18-null) does not increase susceptibility to pancreatitis induced by caerulein or a choline-deficient diet. In normal and K18-null acini, K19 is upregulated after caerulein injury and, unexpectedly, forms CF. As in hepatocytes, acinar injury is also associated with keratin hyperphosphorylation. Hence, K19 forms ALF in mouse acinar cells and helps define two distinct ALF and CF pools. On injury, K19 forms CF that revert to ALF after healing. Acinar keratins appear to be dispensable for cytoprotection, in contrast to hepatocyte keratins, despite similar hyperphosphorylation patterns after injury.

Molecular cloning of a gene (cfp) encoding the cytoplasmic filament protein P59Nc and its genetic relationship to the snowflake locus of Neurospora crassa.

P59Nc is a 59-kD polypeptide associated with 8-10-nm diameter cellular filaments in normal Neurospora crassa strains. Abnormally sized and shaped bundles of these structures are present in N. crassa strains carrying mutations at the locus sn (snowflake). By using molecular cloning and restriction fragment length polymorphism (RFLP) segregation analysis strategies we show here that sn is not the genetic locus of P59Nc. Several P59Nc cDNAs were cloned from a N. crassa lambda GT11 library after immunoscreening with specific polyclonal anti-P59Nc antibodies. Additional longer cDNAs were obtained from a N. crassa cDNA-lambda ZAP library. When used as probes in Southern blots of total DNA from wild-type strains, multicent-2 (a multiple mutant strain), and snowflake mutants, the P59Nc cDNAs revealed comparable patterns of hybridizing bands for all of the restriction enzymes tested. Analysis of segregation of BclI and ClaI RFLPs, detected in the genomic region of the P59Nc gene (locus cfp: cellular filament polypeptide), among a set of strains designed for RFLP mapping, or among selected progeny of crosses involving a snowflake parent, respectively, indicate that (i) there is in N. crassa a single cfp locus positioned on the right arm of linkage group VII between the locus for and the proximal breakpoint of the translocation T(VII----I)5936; (ii) the sn mutations in the centromere region of chromosome I do not represent translocations of cfp; and (iii) the snowflake mutants possesses a normal copy of the P59Nc gene on their chromosomes VII.(ABSTRACT TRUNCATED AT 250 WORDS)

The cytoplasmic filament system in critical point-dried whole mounts and plastic-embedded sections.

High voltage electron microscopy of intact cells prepared by the critical point drying (CPD) procedure has become an important tool in the study of three-dimensional relationships between cytoplasmic organelles. It has been claimed that critical point-dried specimens reveal a structure that is not visible in sections of plastic-embedded material; it has also been claimed that this structure, in association with known cytoplasmic filaments, forms a meshwork of tapering threads ("microtrabecular lattice"). Alternatively, this structure might be a surface tension artifact produced during CPD. To test possible sources of artifacts during CPD, model fiber systems of known structure were used. It was found that traces of water or ethanol in the CO2 caused distortions and fusion of fibers in pure muscle actin, fibrin, collagen, chromatin, and microtubules that produce a structure very similar to the proposed "microtrabecular lattice." These structures were, however, well preserved if water and ethanol were totally excluded from the CO2. The same results were obtained with whole mounts of cultured cells. A "microtrabecular lattice" was obtained if some water or ethanol was present in the pressure chamber. On the other hand, when water or ethanol were totally excluded from the CO2 during CPD, cytoplasmic filaments were uniform in thickness similar to their appearance in sections of plastic-embedded cells. It is concluded that the "microtrabecular lattice" is a distorted image of the cytoplasmic filament network produced during CPD by traces of water or ethanol in the CO2.

Evidence for the lack of actin involvement in mitosis and in the contractile process in Spirostomum teres

Studies using actin decoration techniques and electron microscopy have failed to show the presence of actin in the ciliate Spirostomum teres. The internal and external membranes of dividing cells were made permeable by treatment with Triton. The myosin subfragment S-1 was introduced into the cells for incubation under conditions suitable for actin decoration. Arrowhead decoration of microfilaments was not observed within dividing micronuclei or in cytoplasmic filament bundles. However, using similar procedures, the brush border of mouse small intestine yielded clearly decorated microfilaments. A spectrophotometric DNase I inhibition assay specific for G-actin demonstrated that the level of actin in S. teres extracts was less than 0.06% of the total soluble protein and confirms the observations made using the decoration procedures. On the basis of this work, it appears that actin does not play a significant part in either mitotic chromosome movement or contractility in S. teres.