Liberibacter crescens Is a Cultured Surrogate for Functional Genomics of Uncultured Pathogenic 'Candidatus Liberibacter' spp. and Is Naturally Competent for Transformation.

'Candidatus Liberibacter' spp. are uncultured insect endosymbionts and phloem-limited bacterial plant pathogens associated with diseases ranging from severe to nearly asymptomatic. 'Ca. L. asiaticus', causal agent of Huanglongbing or citrus "greening," and 'Ca. L. solanacearum', causal agent of potato zebra chip disease, respectively threaten citrus and potato production worldwide. Research on both pathogens has been stymied by the inability to culture these agents and to reinoculate into any host. Only a single isolate of a single species of Liberibacter, Liberibacter crescens, has been axenically cultured. L. crescens strain BT-1 is genetically tractable to standard molecular manipulation techniques and has been developed as a surrogate model for functional studies of genes, regulatory elements, promoters, and secreted effectors derived from the uncultured pathogenic Liberibacters. Detailed, step-by-step, and highly reproducible protocols for axenic culture, transformation, and targeted gene knockouts of L. crescens are described. In the course of developing these protocols, we found that L. crescens is also naturally competent for direct uptake and homology-guided chromosomal integration of both linear and circular plasmid DNA. The efficiency of natural transformation was about an order of magnitude higher using circular plasmid DNA compared with linearized fragments. Natural transformation using a replicative plasmid was obtained at a rate of approximately 900 transformants per microgram of plasmid, whereas electroporation using the same plasmid resulted in 6 × 104 transformants. Homology-guided marker interruptions using either natural uptake or electroporation of nonreplicative plasmids yielded 10 to 12 transformation events per microgram of DNA, whereas similar interruptions using linear fragments via natural uptake yielded up to 34 transformation events per microgram of DNA.

Induction of innate immune gene expression following methyl methanesulfonate-induced DNA damage in sea urchins.

Sea urchins are noted for the absence of neoplastic disease and represent a novel model to investigate cellular and systemic cancer protection mechanisms. Following intracoelomic injection of the DNA alkylating agent methyl methanesulfonate, DNA damage was detected in sea urchin cells and tissues (coelomocytes, muscle, oesophagus, ampullae and gonad) by the alkaline unwinding, fast micromethod. Gene expression analyses of the coelomocytes indicated upregulation of innate immune markers, including genes involved in NF-κB signalling. Results suggest that activation of the innate immune system following DNA damage may contribute to the naturally occurring resistance to neoplastic disease observed in sea urchins.

Telomerase expression in human somatic cells does not induce changes associated with a transformed phenotype.

Expression of the human telomerase catalytic component, hTERT, in normal human somatic cells can reconstitute telomerase activity and extend their replicative lifespan. We report here that at twice the normal number of population doublings, telomerase-expressing human skin fibroblasts (BJ-hTERT) and retinal pigment epithelial cells (RPE-hTERT) retain normal growth control in response to serum deprivation, high cell density, G1 or G2 phase blockers and spindle inhibitors. In addition, we observed no cell growth in soft agar and detected no tumour formation in vivo. Thus, we find that telomerase expression in normal cells does not appear to induce changes associated with a malignant phenotype.

Reconstitution of human telomerase with the template RNA component hTR and the catalytic protein subunit hTRT.

The maintenance of chromosome termini, or telomeres, requires the action of the enzyme telomerase, as conventional DNA polymerases cannot fully replicate the ends of linear molecules. Telomerase is expressed and telomere length is maintained in human germ cells and the great majority of primary human tumours. However, telomerase is not detectable in most normal somatic cells; this corresponds to the gradual telomere loss observed with each cell division. It has been proposed that telomere erosion eventually signals entry into senescence or cell crisis and that activation of telomerase is usually required for immortal cell proliferation. In addition to the human telomerase RNA component (hTR; ref. 11), TR1/TLP1 (refs 12, 13), a protein that is homologous to the p80 protein associated with the Tetrahymena enzyme, has been identified in humans. More recently, the human telomerase reverse transcriptase (hTRT; refs 15, 16), which is homologous to the reverse transcriptase (RT)-like proteins associated with the Euplotes aediculatus (Ea_p123), Saccharomyces cerevisiae (Est2p) and Schizosaccharomyces pombe (5pTrt1) telomerases, has been reported to be a telomerase protein subunit. A catalytic function has been demonstrated for Est2p in the RT-like class but not for p80 or its homologues. We now report that in vitro transcription and translation of hTRT when co-synthesized or mixed with hTR reconstitutes telomerase activity that exhibits enzymatic properties like those of the native enzyme. Single amino-acid changes in conserved telomerase-specific and RT motifs reduce or abolish activity, providing direct evidence that hTRT is the catalytic protein component of telomerase. Normal human diploid cells transiently expressing hTRT possessed telomerase activity, demonstrating that hTRT is the limiting component necessary for restoration of telomerase activity in these cells. The ability to reconstitute telomerase permits further analysis of its biochemical and biological roles in cell aging and carcinogenesis.

Influence of ensiling time and inoculation on alteration of the starch-protein matrix in high-moisture corn.

The fates of hydrophobic zein proteins, which encapsulate corn starch to create vitreous endosperm, have not been investigated in high-moisture corn (HMC). To assess influences of ensiling time and inoculation on zein proteins in HMC, quadruplicate samples of 2 random corn hybrids (A and B), containing 25.7 and 29.3% moisture, were ground, inoculated with (I) or without 600,000 cfu/g of Lactobacillus buchneri 40788 (Lallemand Animal Nutrition, Milwaukee, WI), and ensiled for 0, 15, 30, 60, 120, and 240 d. Nutrient composition [crude protein (CP), starch, acid detergent fiber, and neutral detergent fiber], fermentation (pH, lactate, and acetate), and protein degradation markers (buffer-soluble CP, isopropanol-soluble CP, and NH(3)-N) were evaluated. At 0 and 240 d, α, γ, δ, and ß zein subunits were profiled using HPLC. Data were evaluated as a split-split plot using the PROC MIXED procedures of SAS. Ensiling time and inoculation decreased pH, and altered lactate and acetate contents of HMC. Lactate and acetate contents of A, AI, B, and BI at 240 d were 0.40, 0.32, 1.11, 0.73, and 0, 0.35, 0.30, and 0.87% of DM, respectively. Buffer-soluble CP in HMC increased from 1.5 to 2.0% of DM at 0 d to >4.0% of DM at 240 d. Inoculation had no effect on buffer-soluble CP but increased NH(3)-N content of HMC. Corn A contained more isopropanol-soluble CP than did corn B and peak areas for 6 α, and all γ and δ zein regions were greater for corn A. Ensiling (0 vs. 240 d) decreased all zein subunits with the exception of 2 α and 1 δ subunit. Ensiling decreased (42.2-73.2%) γ zeins, which are primarily responsible for cross-linking in the starch-protein matrix. Despite altering lactate and acetate contents, inoculation had no effect on degrading hydrophobic zein proteins in HMC. Data suggest that hydrophobic zein proteins in the starch-protein matrix of HMC are degraded by proteolytic activity over an extended ensiling time.

Serum TRAIL levels in patients with epithelial ovarian cancer or primary peritoneal cancer treated with neoadjuvant chemotherapy. A pilot study.

AIMS: The study attempted to evaluate the kinetics of changes in serum TRAIL levels as a potential predictive and prognostic factor in patients with epithelial ovarian cancer (EOC) or primary peritoneal carcinoma (PPC), eligible for an interval debulking surgery (IDS). MATERIAL AND METHODS: 17 patients with primary inoperable EOC or PPC in FIGO Stage IIIC or IV who underwent an exploratory operation were enrolled to the study. Serum TRAIL levels were determined by ELISA method (DIACLONE, Besancon Cedex, France) before and after two courses of neoadjuvant chemotherapy (NAC) based on paclitaxel and platinum analogue (cisplatin or carboplatin). The control group consisted of six healthy volunteers. The median difference in concentration of TRAIL (dTRAIL) between the initial marking and after two courses of NAC in each patient was 192 pg/ml and it was used for dichotomization of the test group. RESULTS: Suboptimal interval debulking surgery (IDS) was performed in 23.5% (4/17) and optimal IDS in 76.5% (13/17) patients. TRAIL concentration before chemotherapy did not differ significantly between patients with EOC or PPC [1426.96 +/- 321.06 pg/ml (mean +/- SD) (U = 26, p = 0.08)] and the control group [1160.40 +/- 256.39 pg/ml (mean +/- SD. After two courses of NAC serum TRAIL concentration level was 1247.49 +/- 378.46 pg/ml (mean +/- SD). The difference was significant (Z = 2.44, p = 0.0147). Statistical analysis showed that dTRAIL did not significantly influence either extent of IDS (U = 35, p = 0.0962) or time to progression (log-rank test, p = 0.1185), overall survival (log-rank test, p = 0.1973) and response to treatment according to RECIST criteria (U = 35.5, p = 0.9616). CONCLUSIONS: Serum TRAIL concentration levels changed significantly during NAC. However, it seems that the concentration of this protein has no critical value as a predictive or prognostic factor in patients with EOC or PPC.

Extension of life-span by introduction of telomerase into normal human cells.

Normal human cells undergo a finite number of cell divisions and ultimately enter a nondividing state called replicative senescence. It has been proposed that telomere shortening is the molecular clock that triggers senescence. To test this hypothesis, two telomerase-negative normal human cell types, retinal pigment epithelial cells and foreskin fibroblasts, were transfected with vectors encoding the human telomerase catalytic subunit. In contrast to telomerase-negative control clones, which exhibited telomere shortening and senescence, telomerase-expressing clones had elongated telomeres, divided vigorously, and showed reduced straining for beta-galactosidase, a biomarker for senescence. Notably, the telomerase-expressing clones have a normal karyotype and have already exceeded their normal life-span by at least 20 doublings, thus establishing a causal relationship between telomere shortening and in vitro cellular senescence. The ability to maintain normal human cells in a phenotypically youthful state could have important applications in research and medicine.

Gradual phenotypic conversion associated with immortalization of cultured human mammary epithelial cells.

Examination of the process of immortal transformation in early passages of two human mammary epithelial cell (HMEC) lines suggests the involvement of an epigenetic step. These lines, 184A1 and 184B5, arose after in vitro exposure of finite lifespan 184 HMEC to a chemical carcinogen, and both are clonally derived. Although early-passage mass cultures of 184A1 and 184B5 maintained continuous slow growth, most individual cells lost proliferative ability. Uniform good growth did not occur until 20-30 passages after the lines first appeared. Early-passage cultures expressed little or no telomerase activity and telomeres continued to shorten with increasing passage. Telomerase activity was first detected when the telomeres became critically short, and activity levels gradually increased thereafter. Early-passage cultures had little or no ability to maintain growth in transforming growth factor-beta (TGFbeta); however, both mass cultures and clonal isolates showed a very gradual increase in the number of cells displaying progressively increased ability to maintain growth in TGFbeta. A strong correlation between capacity to maintain growth in the presence of TGFbeta and expression of telomerase activity was observed. We have used the term "conversion" to describe this process of gradual acquisition of increased growth capacity in the absence or presence of TGFbeta and reactivation of telomerase. We speculate that the development of extremely short telomeres may result in gradual, epigenetic-based changes in gene expression. Understanding the underlying mechanisms of HMEC conversion in vitro may provide new insight into the process of carcinogenic progression in vivo and offer novel modes for therapeutic intervention.

Antiproliferative effect of 4-thiouridylate on OCM-1 uveal melanoma cells.

PURPOSE: Brachytherapy is a well-established, effective treatment for uveal melanoma with a failure rate of 15%. The fatal consequence of unsuccessful treatments offers reason for improvement of the method. The authors propose using an apoptosis inducing agent locally, concomitantly with the well-established therapy, to sensitize the tumor cells. The authors propose a new nontoxic moderately active apoptosis inducing agent, 4-thio-uridylate (s4UMP), for this purpose. METHODS: OCM-1 uveal melanoma cells were treated with various concentrations of s4UMP and its effect was monitored by measuring the cell viability (MTT assay). The following apoptosis detecting methods were performed to reveal the mechanism of decreased cell viability: light microscopy, DNA fragmentation assay, determination of caspase 9 activity, and FACS analysis. RESULTS: The viability of uveal melanoma cells was decreased by 32%, 40%, and 9% after 24, 48, and 72 hours of treatment with 10 microg/mL (30 microM) s4UMP. The effect was not dose dependent; it rather followed a saturation-type inhibition and the cells at lower drug concentration recovered after 72 hours. Characteristic apoptotic cell morphology and DNA fragmentation was detected in treated cells. The caspase-9 was activated upon treatment showing maximal activity at 48 hours suggesting the induction of apoptosis. The annexin binding activity further verified the apoptogenic activity of s4UMP. CONCLUSIONS: Uveal melanoma, more than other solid tumors, is resistant to most of the chemotherapeutic protocols as indicated by the high mortality rate of metastatic disease. The authors showed that s4UMP, a naturally occurring nucleotide, could induce apoptosis in uveal melanoma cells, suggesting a potential supplementary therapeutic application of the compound.

Cloning and sequence determination of cDNA encoding a second rat liver peroxisomal 3-ketoacyl-CoA thiolase.

3-Ketoacyl-coenzyme A thiolase (thiolase) catalyzes the final step of the fatty acid beta-oxidation pathway in peroxisomes. Thiolase is unique among rat liver peroxisomal enzymes in that it is synthesized as a precursor possessing a 26-amino acid (aa) N-terminal extension which is cleaved to generate the mature enzyme. To facilitate further examination of the synthesis, intracellular transport and processing of this enzyme, cDNA clones were selected from a lambda gt11 rat liver library using antiserum raised against peroxisomal thiolase. Upon sequencing several cDNA clones, it was revealed that there are at least two distinct thiolase enzymes localized to rat liver peroxisomes, one identical to the previously published rat liver peroxisomal thiolase (thiolase 1) [Hijikata et al., J. Biol. Chem. 262 (1987) 8151-8158] and a novel thiolase (thiolase 2). The THL2 cDNA possesses a single open reading frame of 1302 nucleotides (nt) encoding a protein of 434 aa (Mr 44790). The coding region of THL2 cDNA exhibits 94.6% nt sequence identity with THL1 and 95.4% identity at the level of aa sequence. Northern-blot analysis indicates that the mRNA encoding thiolase 2 is approx. 1.7 kb in size. The mRNA encoding thiolase 2 is induced approx. twofold upon treatment of rats with the peroxisome-proliferating drug, clofibrate. In contrast, the thiolase 1 mRNA is induced more than tenfold under similar conditions.

A photobleaching energy transfer analysis of CD8/MHC-I and LFA-1/ICAM-1 interactions in CTL-target cell conjugates.

The photobleaching energy transfer (pbFRET) technique is a fluorescence method to measure proximity relationships between molecules, especially cell surface proteins, labeled with fluorophore-conjugated monoclonal antibodies, on a pixel-by-pixel base using digital imaging microscopy. This technique enables analysis of inter- and intramolecular proximities at cell surfaces at physiological conditions. We have developed a pbFRET approach to measure intercellular proximities in order to access spatial organization of interacting proteins in the contact region of two 'communicating' cells. Two examples, as possible application areas of this approach, are presented here: interaction between CD8 and MHC-I molecules in point contacts and interaction between LFA-1 and ICAM-1 molecules in focal contacts of CTL-target conjugates. The geometry of these protein contacts based on our resonance energy transfer (RET) data is consistent with the observed blocking effects of monoclonal antibodies (directed against the interacting proteins) on the cytolytic activity of CTLs and suggest a critical role for CD8beta-subunit in signal transmission in peripheral T-lymphocytes.

Modification of membrane cholesterol level affects expression and clustering of class I HLA molecules at the surface of JY human lymphoblasts.

Recently we have found that class I HLA molecules, key elements of the antigen presentation system for CD8 + effector cells, show a clustered lateral distribution (homoassociation) at the surface of activated human T- and B-lymphocytes as well as virus-transformed T- and B-lymphoblasts, in contrast to a disperse distribution on resting human PBLs (Matk6 et al. (1994) J. Immunol. 152, 3353; Bene et al. (1994) Eur. J. Immunol. 24, 2115). Expression of beta2m-free HLA heavy chains and exogenous beta2m have been shown as potential regulation factors of HLA-I clustering, which in turn may affect cytotoxic activity of CD8+ effector cells. Here we report a study on the effect of plasma membrane-modification (by exogenous cholesterol and phosphatidylcholine) on the expression of free HLA heavy chains and beta2m-bound HLA-I molecules on JY human B-lymphoblasts. The modulating effect of these two treatments on the lipid fluidity of cells was demonstrated by fluorescence anisotropy of DPH lipid probe. The lateral clustering (association) of HLA-I molecules was detected by flow cytometric fluorescence resonance energy transfer (FCET) and digital imaging microscopic photobleaching energy transfer (pbFRET) methods, using flourescein-isothiocyanate (FITC) (donor)- and tetramethyl-rhodamine-isothiocyanate (TRITC) (acceptor)-labeled W6/32 or KE2 antibodies directed against intact HLA-I molecules. Cholesterol enrichment of the plasma membrane increased membrane fluidity and reduced the expression of heavy- and light-chain determinants of HLA-I molecules and free heavy chains (FHCs). This was accompanied with a higher degree of HLA-I clustering as shown by the enhanced intermolecular energy transfer efficiency. In contrast, cholesterol depletion resulted in membrane fluidization and increased expression of HLA-I epitopes. Our results suggest that both cholesterol level and lipid structure/fluidity of the plasma membrane in lymphoblastoid cells may also potentially regulate lateral organization and consequently the presentation efficiency of HLA-I molecules.

Subacute sclerosing panencephalitis manifesting as viral retinitis: clinical and histopathologic findings.

PURPOSE AND METHODS: To describe the clinical and histopathologic features of a patient with viral retinitis secondary to subacute sclerosing panencephalitis. RESULTS: The patient was a human immunodeficiency virus-negative intravenous drug abuser with an acute retinitis that later progressed to encephalitis despite aggressive treatment for possible viral, protozoal, bacterial, and rickettsial infections. The patient had many of the characteristic findings of subacute sclerosing panencephalitis, including a history of measles in early childhood, myoclonus, periodic complexes on electroencephalographic testing, persistently elevated serum and cerebrospinal fluid antimeasles immunoglobulin G (IgG) titers, and a cerebrospinal fluid oligoclonal IgG gammopathy. Ultrastructural examination demonstrated numerous filamentous microtubular intranuclear viral inclusions in the nuclear layers of the retina consistent with the measles virus. This case is unusual in that our patient developed subacute sclerosing panencephalitis later in life and because there was an 8-year period between presumed viral infections in the two eyes. CONCLUSIONS: An acute retinitis in an intravenous drug abuser is not always caused by human immunodeficiency virus-related infections; not all viral retinitis responds to therapy; and mortality as well as the usual morbidity may be associated with viral retinitis. One might consider the diagnosis of subacute sclerosing panencephalitis in a young person with an acute retinitis with little or no vitreal inflammation and lack of response to anticytomegalovirus and antitoxoplasmosis therapy.

The visually inattentive preterm infant.

A retrospective study was made of 16 premature infants who were visually inattentive despite normal eye findings and a lack of factors predisposing them to cerebral blindness. A comparison of this study group with other premature infants who were visually attentive revealed a much greater incidence of upper motor neuron disease and mental retardation in the study infants.

[Femoropatellar pain syndrome--conservative therapeutic possibilities]. / Femoropatelläres Schmerzsyndrom--konservative Therapiemöglichkeiten.

Patello-femoral knee pain is a frequent problem the general practitioners, rheumatologists and orthopedists are confronted with. Very often, no exact structural diagnosis can be made. Studies on the natural history of PFPS are lacking. Nevertheless, conservative treatment has been successful in most of the cases. In this respect, management being pertinent to each patient and problem is important. Four categories of frequently affected patients are discussed in this article: young people in their adolescence, well trained athletes, athletes at the end of their career and patients in the 'second half' of their life. The stages of soft-tissue healing in the course of treatment over a long time are of importance. In the sense of a secondary prevention, the intrinsic and extrinsic risk factors have to be considered as well. The taping technique offers an easy and effective treatment method. It usually pulls the patella medially, thereby 'unloading' the joint and the surrounding soft tissues. In an early stage, this results in an analgesic effect and allows an early functional load. In a later stage, the patella tape helps to prevent reinjury.

[Initial experience with ESWL therapy of pancreatic duct calculi]. / A pancreas vezetékkövek lökéshullám (ESWL) kezelésének kezdeti tapasztalatai.

Extracorporeal shock wave lithotripsy of pancreatic stones was performed in four patients with chronic pancreatitis and a dilated duct system harbouring stones 5-12 mm in diameter. The stones were disintegrated by shock waves using a Dornier lithotripter in one or more sessions. Disintegration of stones was achieved in 4/4 patients, initial (6-11 months) relief of pain in 3/4 patients, and total clearance of pancreatic duct in 3/4 patients. No complications were observed. In the first patient in whom ESWL was not completely successful, underwent an operation: a longitudinal pancreato-gastrostomy and the stones were found completely disintegrated. From these early data they conclude that ESWL of pancreatic duct stones is a provisional new alternative for surgery in the treatment of chronic pancreatitis.