Increased exopolysaccharide production in Lactococcus lactis due to increased levels of expression of the NIZO B40 eps gene cluster

Exopolysaccharides (EPS) play an important role in the rheology and texture of fermented food products. This is the first report demonstrating that homologous overexpression of a complete eps gene cluster in Lactococcus lactis leads to increased EPS production levels. A ninefold-elevated EPS plasmid copy number led to an almost threefold increase in the eps expression level, resulting in an almost fourfold increase in the NIZO B40 EPS production level. It was previously reported that increased EPS precursor levels did not influence NIZO B40 EPS production levels. However, the present results indicate that the maximal NIZO B40 EPS production level is limited by the activity level of the expression products of the eps gene cluster rather than by the level of EPS precursors.     

Real-time PCR based on SYBR-Green I fluorescence: An alternative to the TaqMan assay for a relative quantification of gene rearrangements, gene amplifications and micro gene deletions

Background

Real-time PCR is increasingly being adopted for RNA quantification and genetic analysis. At present the most popular real-time PCR assay is based on the hybridisation of a dual-labelled probe to the PCR product, and the development of a signal by loss of fluorescence quenching as PCR degrades the probe. Though this so-called 'TaqMan' approach has proved easy to optimise in practice, the dual-labelled probes are relatively expensive.

Results

We have designed a new assay based on SYBR-Green I binding that is quick, reliable, easily optimised and compares well with the published assay. Here we demonstrate its general applicability by measuring copy number in three different genetic contexts; the quantification of a gene rearrangement (T-cell receptor excision circles (TREC) in peripheral blood mononuclear cells); the detection and quantification of GLI, MYC-C and MYC-N gene amplification in cell lines and cancer biopsies; and detection of deletions in the OPA1 gene in dominant optic atrophy.

Conclusion

Our assay has important clinical applications, providing accurate diagnostic results in less time, from less biopsy material and at less cost than assays currently employed such as FISH or Southern blotting.     

Carotenoid accumulation in the psychrotrophic bacterium Arthrobacter agilis in response to thermal and salt stress

A psychrotrophic strain of Arthrobacter agilis, isolated from Antarctic sea ice, grows from 5 degrees C to 40 degrees C and in culture media containing 0-10% (w/v) NaCl. Maximum growth rate occurred at 30-35 degrees C with a drastic decline as the cultivation temperatures diverged. Adaptation to extremes of low temperature may be partially attributed to the production of the C-50 carotenoid bacterioruberin, and its glycosylated derivatives. Lowering of the cultivation temperature resulted in a concomitant increase in carotenoid production, which may contribute to membrane stabilisation at low temperature. Maximum biomass accumulation occurred at 5-30 degrees C with a tenfold reduction at 40 degrees C. Changes in growth rates were minimal in culture media containing 0-2% (w/v) NaCl at 10 degrees C while a gradual decrease in growth rates occurred at higher salinity. Biomass accumulation at different salinity followed a trend similar to that observed with different cultivation temperatures. Maximum biomass accumulation was observed in culture media containing 0-5% (w/v) NaCl with a tenfold reduction at 10% (w/v) NaCl. Carotenoid production also decreased as salinity increased.     

Comparative analysis of IFN-gamma B7.1 and antisense TGF-beta gene transfer on the tumorigenicity of a poorly immunogenic metastatic mammary carcinoma

Cancer progression is attributed in part to immune evasion strategies that include lack of co-stimulation, down-regulation of cell surface MHC molecules, and secretion of immunosuppressive factors, such as transforming growth factor-beta (TGF-beta). Gene therapy has been employed to counter these mechanisms of immune evasion by transference of B7.1, IFN-gamma or antisense TGF-beta genes into tumor cells, resulting in cell surface expression of B7.1, upregulation of MHC class I and class II molecules, or elimination of tumor-derived TGF-beta, respectively. Although each of these transgenes has been shown to alter tumorigenicity in murine models, a direct comparison of their efficacy has not been performed. In this study, we have employed a very aggressive, poorly immunogenic and highly metastatic mammary model, 4T1, to compare the efficacy of B7.1, IFN-gamma and antisense TGF-beta gene transfer in stimulating an anti-tumor response. We demonstrate that both IFN-gamma and antisense TGF-beta gene expression significantly reduced the tumorigenicity of these cells compared to mock transduced cells, with IFN-gamma having a greater effect. In contrast, B7.1 gene transfer did not affect the tumorigenicity of 4T1 cells. The anti-tumor response directed against antisense TGF-beta-expressing 4T1 tumors was mediated by CD4+ and CD8+ T cells. However, CD8+ T cells, and not CD4+ T cells, appeared to mediate the anti-tumor response against IFN-gamma-expressing tumors. Treatment of tumor-bearing animals with IFN-gamma or antisense TGF-beta gene-modified tumor cell vaccines reduced the number of clonogenic metastases to the lungs and liver compared to treatment with mock-transduced cells. Finally, in a residual disease model in which the primary tumor was excised and mice were vaccinated with irradiated tumor cells, treatment of mice with vaccinations consisting of 4T1 cells expressing both antisense TGF-beta and IFN-gamma genes was the most effective in prolonging survival.     

Production of recombinant human type I procollagen trimers using a four-gene expression system in the yeast Saccharomyces cerevisiae

The expression of stable recombinant human collagen requires an expression system capable of post-translational modifications and assembly of the procollagen polypeptides. Two genes were expressed in the yeast Saccharomyces cerevisiae to produce both propeptide chains that constitute human type I procollagen. Two additional genes were expressed coding for the subunits of prolyl hydroxylase, an enzyme that post-translationally modifies procollagen and that confers heat (thermal) stability to the triple helical conformation of the collagen molecule. Type I procollagen was produced as a stable heterotrimeric helix similar to type I procollagen produced in tissue culture. A key requirement for glutamate was identified as a medium supplement to obtain high expression levels of type I procollagen as heat-stable heterotrimers in Saccharomyces. Expression of these four genes was sufficient for correct assembly and processing of type I procollagen in a eucaryotic system that does not produce collagen.     

New species of Anolis (Sauria: Iguanidae) from the north region of eastern Cuba

A new Anolis species of the Alpha section from the north region of eastern Cuba (Holguín province) is described. It differs from all Cuban species of Anolis in its green coloration with greenish gray bands on body, legs and tail, in having subtriangular mental scales as well as in other details of color and scutellation. This new species is most closely related to A. isolepis but it can be distinguished from both, A. i. isolepis and A. i. altitudinalis, by its coloration and pattern, the larger body size, the presence of smooth ventral scales (similar in size to the dorsal scales) and by the absence of enlarged postcloacal scales in the male.     

The genes for benzene catabolism in Pseudomonas putida ML2 are flanked by two copies of the insertion element IS1489, forming a class-I-type catabolic transposon, Tn5542

Two directly repeated sequences of the IS elements IS1489v1 and IS1489v2 flank the benzene dioxygenase (bedC1C2BA) and the cis-benzene dihydrodiol dehydrogenase (bedD) genes on the catabolic plasmid pHMT112 in Pseudomonas putida ML2, forming a Class-I-type composite transposon, Tn5542. Both IS1489v1 and IS1489v2 contain an identical 1371-bp open reading frame, tnpA, that is preceded by a possible ribosome binding site. The tnpA gene of IS1489v1 is bound by a pair of 40-bp imperfect inverted repeats while that of IS1489v2 is flanked only by the left inverted repeat. The tnpA gene codes for a putative 53-kDa polypeptide of 456 amino acids bearing similarity to transposases encoded on IS elements of P. alcaligenes, P. aeruginosa, P. stutzeri, and Serratia marcescens. The basic nature of the putative TnpA protein with a deduced pI of 8.93 is typical of IS-encoded transposases. Similar to other IS elements, an outward facing promoter was detected at the right end of IS1489v1. Experiments involving the suicide vector, pKNG101, failed to show transposition of Tn5542.     

CaMV 35S promoter directs β-glucuronidase expression in the laticiferous system of transgenic Hevea brasiliensis (rubber tree)

Hevea brasiliensis anther calli were genetically transformed using Agrobacterium GV2260 (p35SGUSINT) that harboured the β-glucuronidase (gus) and neomycin phosphotransferase (nptII) genes. β-Glucuronidase protein (GUS) was expressed in the leaves of kanamycin-resistant plants that were regnerated, and the presence of the gene was confirmed by Southern analysis. GUS was also observed to be expressed in the latex and more importantly in the serum fraction. Transverse sections of the leaf petiole from a transformed plant revealed GUS expression to be especially enhanced in the phloem and laticifers. GUS expression was subsequently detected in every one of 194 plants representing three successive vegetative cycles propagated from the original transformant. Transgenic Hevea could thus facilitate the continual production of foreign proteins expressed in the latex. Received: 14 February 1997 / Revision received: 16 August 1997 / Accepted: 20 July 1997     

Nuclear Envelope Protein Lem2 is Required for Mouse Development and Regulates MAP and AKT Kinases

The nuclear lamina, along with associated nuclear membrane proteins, is a nexus for regulating signaling in the nucleus. Numerous human diseases arise from mutations in lamina proteins, and experimental models for these disorders have revealed aberrant regulation of various signaling pathways. Previously, we reported that the inner nuclear membrane protein Lem2, which is expressed at high levels in muscle, promotes the differentiation of cultured myoblasts by attenuating ERK signaling. Here, we have analyzed mice harboring a disrupted allele for the Lem2 gene (Lemd2). No gross phenotypic defects were seen in heterozygotes, although muscle regeneration induced by cardiotoxin was delayed. By contrast, homozygous Lemd2 knockout mice died by E11.5. Although many normal morphogenetic hallmarks were observed in E10.5 knockout embryos, most tissues were substantially reduced in size. This was accompanied by activation of multiple MAP kinases (ERK1/2, JNK, p38) and AKT. Knockdown of Lem2 expression in C2C12 myoblasts also led to activation of MAP kinases and AKT. These findings indicate that Lemd2 plays an essential role in mouse embryonic development and that it is involved in regulating several signaling pathways. Since increased MAP kinase and AKT/mTORC signaling is found in other animal models for diseases linked to nuclear lamina proteins, LEMD2 should be considered to be another candidate gene for human disease.     

Prominent Vessel Sign on Susceptibility-Weighted Imaging in Acute Stroke: Prediction of Infarct Growth and Clinical Outcome

Background and Purpose

Predicting the risk of further infarct growth in stroke patients is critical to therapeutic decision making. We aimed to predict early infarct growth and clinical outcome from prominent vessel sign (PVS) identified on the first susceptibility-weighted image (SWI) after acute stroke.

Materials and Methods

Twenty-two patients with middle cerebral artery (MCA) infarction had diffusion-weighted imaging, SWI, MR angiography, and clinical evaluation using the National Institutes of Health Stroke Scale at 7–60 hours and 5–14 days after stroke onset. Late-stage clinical evaluation at 1 and 3 months used the modified Rankin Scale. The infarct area and growth were scored from 10 (none) to 0 (infarct or growth in all 10 zones) using the Alberta Stroke Program Early CT Score (ASPECTS) system.

Results

Infarct growth on the second MRI occurred in 13 of 15 patients with PVS on the first MRI and not in any patient without PVS (n=7; r=0.86, P<0.001). The extent of PVS was significantly correlated with infarct growth (r=0.82, P<0.001) and early-stage outcome (P=0.02). No between-group difference in late-stage clinical outcome was found.

Conclusion

PVS on the first SWI after acute MCA territory stroke is a useful predictor of early infarct growth. Extensive PVS within the large MCA territory is related to poor early-stage outcome and could be useful for clinical assessment of stroke.